InteractiveFly: GeneBrief

Presenilin: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - Presenilin

Synonyms -

Cytological map position - 77C1--7

Function - surface protein of unknown function

Keywords - Notch pathway, component of γ secretase

Symbol - Psn

FlyBase ID: FBgn0019947

Genetic map position - 3-

Classification - presenilin-like

Cellular location - surface transmembrane protein

B>NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Hong, Y. G., Roh, S., Paik, D. and Jeong, S. (2017). Development of a reporter system for in vivo monitoring of γ-Secretase activity in Drosophila. Mol Cells 40(1): 73-81. PubMed ID: 28152299
The γ-secretase complex (see Presenilin) represents an evolutionarily conserved family of transmembrane aspartyl proteases that cleave numerous type-I membrane proteins, including the β-amyloid precursor protein (APP) and the receptor Notch. All known rare mutations in APP and the γ-secretase catalytic component, presenilin, which lead to increased amyloid β peptide production, are responsible for early-onset familial Alzheimer's disease. β-amyloid protein precursor-like (APPL) is the Drosophila ortholog of human APP. This study created Notch- and APPL-based Drosophila reporter systems for in vivo monitoring of γ-secretase activity. Ectopic expression of the Notch- and APPL-based chimeric reporters in wings results in vein truncation phenotypes. Reporter-mediated vein truncation phenotypes are enhanced by the Notch gain-of-function allele and suppressed by RNAi-mediated knockdown of presenilin. Furthermore, apoptosis was found to partly contribute to the vein truncation phenotypes of the APPL-based reporter, but not to the vein truncation phenotypes of the Notch-based reporter. Taken together, these results suggest that both in vivo reporter systems provide a powerful genetic tool to identify genes that modulate γ-secretase activity and/or APPL metabolism.
Kang, J., Shin, S., Perrimon, N. and Shen, J. (2017). An evolutionarily conserved role of presenilin in neuronal protection in the aging Drosophila brain. Genetics [Epub ahead of print]. PubMed ID: 28495961
Mutations in the Presenilin genes are the major genetic cause of Alzheimer's disease. Presenilin and Nicastrin are essential components of γ-secretase, a multi-subunit protease that cleaves Type I transmembrane proteins. The roles of Drosophila Presenilin (Psn) and Nicastrin (Nct) in the adult fly brain are unknown. To knockdown (KD) Psn or Nct selectively in neurons of the adult brain, multiple shRNA lines were generated. Using a ubiquitous driver, these shRNA lines resulted in 80-90% reduction of mRNA and pupal lethality, a phenotype that is shared with Psn and Nct mutants carrying nonsense mutations. Furthermore, expression of these shRNAs in the wing disc caused notching wing phenotypes, which are also shared with Psn and Nct mutants. Similar to Nct, neuron-specific Psn KD using two independent shRNA lines led to early mortality and rough eye phenotypes, which were rescued by a fly Psn transgene. Interestingly, conditional KD (cKD) of Psn or Nct in adult neurons using the elav-Gal4 and tubulin-Gal80ts system caused shortened lifespan, climbing defects, increases in apoptosis and age-dependent neurodegeneration. Together, these findings demonstrate that similar to their mammalian counterparts, Drosophila Psn and Nct are required for neuronal survival during aging and normal lifespan, highlighting an evolutionarily conserved role of Presenilin in neuronal protection in the aging brain.

Drosophila Presenilin was isolated on the basis of shared sequences with mammalian presenilins. Mutations in the two human presenilins, PS1 and PS2, and in another protein, the amyloid precursor protein (APP), are associated with early onset familial Alzheimer's disease (AD). Presenilins have two known functions: they affect the processing of ß-amyloid precursor protein and facilitate the activity of transmembrane receptors of the Notch family. Before discussing the role of presenilins in Notch processing, their influence on ß-amyloid protein processing will be described.

ß-Amyloid precursor protein (ß-APP) is a transmembrane protein that travels by way of the endoplasmic reticulum and Golgi to the cell surface and undergoes proteolytic processing. A set of ß-amyloid (Aß) peptides are generated from ß-APP by proteases known as the ß- and gamma-secretases. The ß-secretase cleavage occurs in the extracellular domain and the heterogeneous gamma-secretase cleavages occur in the transmembrane domain. Dominant mutations in either of two Presenilin genes appear to cause Alzheimer's disease by increasing the amount of the Aß42(43) fragment that is produced. A null allele of mouse Presenilin 1 appears selectively to reduce-gamma-secretase activity (DeStrooper, 1998). These observations indicate that presenilin either stimulates the activity of gamma-secretase, or is itself a component of gamma-secretase (Struhl, 1999 and references).

Evidence has been found of a role for Drosophila Psn in Notch processing. Notch acts as a transmembrane cell-surface receptor for intercellular signals during development. It has been proposed that signal transduction involves cleavage and transport of the Notch intracellular domain to the nucleus. Results from Drosophila and mammalian cells indicate that cleavage occurs in or near the transmembrane domain (Struhl, 1998; Schroeter, 1998; Lecourtois, 1998, and Kidd, 1998). In mammalian cells, at least one proteolytic event occurs in the extracellular domain during Notch transit to the cell surface (Logeat, 1998), and it has been suggested that ligand-binding might trigger additional extracellular proteolytic processing. Thus Notch proteins undergo proteolytic processing events that resemble the ß- and gamma-secretase cleavages of ß-APP. These parallels, as well as genetic studies of presenilin in C. elegans, indicate that the presenilins may promote proteolytic cleavage during receptor maturation or activation (Levitan, 1998)

To investigate the involvement of presenilin in proteolysis of Notch protein, an in vivo assay was used for ligand-dependent cleavage and nuclear access of the intracellular domain of Drosophila Notch. To identify Psn mutants, an examination was made of a collection of recessive-lethal mutations that map to the location identified for Psn and that cause a neurogenic phenotype in genetic mosaics (J. Jiang, C.-M. Chen and G. Struhl cited in Struhl, 1999). Two independent mutations, PSC1 anPSC2, contain lesions in Psn. Both alleles are predicted to cause premature termination and appear to be null alleles. To generate embryos with no Psn activity, both maternal and zygotic Psn activity were removed by generating Psn minus embryos derived from Psn minus germ cells. In both Psn minus and Notch minus embryos, clusters of neuroblasts segregate at the positions normally occupied by single neuroblasts, as revealed by Hunchback staining. Both Psn minus and Notch minus embryos also show extensive neural hyperplasia during subsequent development and die as pharate first-instar larvae lacking both dorsal and ventrical cuticle. In addition, the number of midline cells, as defined by the expression of Single-minded (Sim), is greatly reduced. Notch protein is found predominantly at the plasma membrane and at similar levels in both wild-type and Psn minus embryos. Hence, the profound developmental defects in Psn minus embryos appears to result from the absence of Notch signal-transducing activity, rather than from a marked decrease in Notch protein at the plasma membrane (Struhl, 1999).

The effect of Psn null mutation on nuclear access by the Notch intracellular domain was examined by using three Notch proteins in which the chimaeric transcription factor Gal4-VP16 (GV) was inserted in-frame into Notch just after the transmembrane domain. Nuclear access was assayed by UAS-lacZ expression. N+-GV3 functions like the wild-type Notch protein and the intracellular domain gains nuclear access and has signal transducing activity only in the presence of the ligand, Delta. NECN-GV3 contains a deletion that removes most of the extracellular domain and causes constitutive signal transducing activity and nuclear access in the absence of Delta. Nintra-GV3 lacks the extracellular and transmembrane domains and also displays ligand-independent nuclear access. The key difference between the two constitutively active forms is that NECN-GV3 retains the transmembrane and extracellular juxtamembrane domains, whereas Nintra-GV3 is a cytosolic protein (Struhl, 1999).

In Psn minus embryos, neither N+-GV3 nor NECN-GV3 has access to the nucleus, as indicated by the complete absence of ß-galactosidase (ß-Gal) expression. In contrast, the nuclear access of Nintra-GV3 is unaffected by the absence of presenilin activity. The N+-GV3 observation indicates that presenilin activity is normally required for the nuclear access of Notch intracellular domain. Furthermore, the observation that presenilin is needed for nuclear access of NECN-GV3, a constitutively active transmembrane form, but not for Nintra-GV3, a constitutively active cytosolic form, suggests that presenilin participates in the release of the intracellular domain from the plasma membrane. Only about 35 amino acids of the Notch extracellular juxtamembrane region remain in the NECN-GV3 protein. Thus, if there are specific signals required for presenilin-dependent cleavage, they are likely to be somewhere in this region or within the transmembrane domain (Struhl, 1999). Similar experiments by Y. Ye (1999) confirm these results.

Although these experiments demonstrate that presenilin is necessary for the ligand-dependent nuclear access of the intracellular domain of Notch, it is not known whether presenilin directly mediates proteolytic release of the intracellular domain or if it acts more indirectly, for example by activating a protease or mediating the protease's transit to the plasma membrane. These findings can be incorporated into a model of events involved in Notch signal transduction, in which ligand-binding activates Notch, thereby creating a substrate for presenilin-dependent release of the intracellular domain from the membrane. Although there are other possibilities, release could require the direct participation of presenilin in the proteolytic cleavage of Notch protein in or near the transmembrane domain. Presenilin may play an analogous role in the processing of ß-APP (Struhl, 1999). Experiments with mammalian Notch1 and PS1 show that the two proteins physically interact. The interaction predominantly occurs early in the secretory pathway, prior to Notch cleavage in the Golgi, because PS1 immunoprecipitation preferentially recovers the full-length Notch1 precursor. These results suggest that the genetic relationship between presenilins and the Notch signaling pathway derives from a direct physical association between these proteins in the secretory pathway (Ray, 1999).

What is the function of presenilin? Given the evidence that presenilin is required for processing of Notch and APP at transmembrane sites, there are a number of possibilities for the function of presenilin. One is that presenilin is required for the proper trafficking of Notch and APP to their protease(s), which may reside in an intracellular compartment. Another is that presenilin is required for the proper biogenesis or trafficking of the gamma-secretase, the protease thought to target APP. Another is that presenilin is an essential cofactor for gamma-secretase, and yet another is that presenilin itself is gamma-secretase. The current data cannot distinguish among these possible functions. Chan (1999) refers to Wolfe et al., (1999), who favor the idea that presenilin is itself gamma-secretase. Wolfe et al. focused on two transmembrane aspartate residues conserved in all known presenilins. Expression in cultured cells of presenilin constructs in which either of these two residues is mutated results in loss of presenilin activity as assayed by production of Aß, even though these cells express wild-type presenilin-1 endogenously. It was therefore concluded that these constructs act as dominant-negative presenilins. While wild-type presenilins are cleaved at a site in the cytosolic loop, the authors found that the aspartate mutants are not cleaved in this manner; they take this result as circumstantial evidence that this cleavage is mediated by presenilin-1 itself. They also point out that gamma-secretase has some characteristics of aspartyl proteases and speculate that the conserved aspartate residues may contribute to an active protease site in presenilin. Wolfe et al. further found that Aß can be produced by in vitro translation of an APP-derived construct in the presence of microsomes derived from wild-type cells but not when the microsomes are prepared from presenilin-1 mutant cells. It was reasoned that presenilin-1 is not required for trafficking of APP, as little or no vesicular trafficking is expected to occur in the in vitro microsomal preparations, and the authors suggest that presenilin is required in the same subcellular compartment in which gamma-secretase resides. An alternative possibility is that presenilin is required for the proper production, processing, or localization of gamma-secretase or an essential cofactor. Presenilin could also have multiple functions, including trafficking of APP, only one of which is required in microsomes. As Wolfe et al. point out, conclusive evidence that presenilin is gamma-secretase will require a purified, reconstituted system, which may be technically difficult to accomplish. A somewhat more tractable, but less conclusive, approach would be to determine whether the newly identified gamma-secretase inhibitors can bind directly to presenilin with the appropriate kinetics (Chan, 1999).

Even should the presenilins not have a role in trafficking, endocytosis appears to have an important role in the processing of APP and in the function of Notch. Inhibitors of endocytosis partially block the processing of APP by gamma-secretase in cultured cells. shibire, which encodes the Drosophila dynamin small GTPase and is required for endocytosis, appears to be required for activation of Notch upon ligand binding. Trafficking of APP and Notch could regulate accessibility to their protease(s). Hence, determining the subcellular compartments for various processing events may contribute importantly to understanding the events in Notch signaling and Aß production as well as their regulation. The normal function of the presenilins is still elusive, and many other questions remain to be answered. How do the mutations associated with AD affect presenilin function? What is the extent of overlap between the Notch and APP processing machineries? Other genes implicated in the Notch pathway might also have a role in APP processing. Are Notch and APP the only molecules that require presenilins for proteolysis? A careful analysis of the phenotypes of presenilin mutants may reveal defects not found in Notch mutants. By unraveling the connection between Notch and the presenilins, researchers may find answers to long-standing questions regarding both Notch signaling and the pathogenesis of Alzheimer's disease (Chan, 1999 and references).


Self-refinement of Notch activity through the transmembrane protein Crumbs: modulation of gamma-secretase activity

Cell interactions mediated by Notch family receptors have been implicated in the specification of tissue boundaries. Tightly localized activation of Notch is crucial for the formation of sharp boundaries. In the Drosophila wing imaginal disc, the Notch receptor is expressed in all cells. However, Notch activity is limited to a narrow stripe of cells along the dorsal-ventral compartment boundary, where it induces the expression of target genes. How a widely expressed protein becomes tightly regulated at the dorsal-ventral boundary in the Drosophila wing is not completely understood. This study shows that the transmembrane protein Crumbs is involved in a feedback mechanism used by Notch to refine its own activation domain at the Drosophila wing margin. Crumbs reduces the activity of the gamma-Secretase complex, which mediates the proteolytic intracellular processing of Notch. These results indicate a novel molecular mechanism of the regulation of Notch signal, and also that defects in Crumbs might be involved in similar abnormal gamma-Secretase complex activity observed in Alzheimer's disease (Herranz, 2006; full text of article).

Crumbs associates with the Stardust and DPATJ proteins through its short cytoplasmic tail to establish apical-basal cell polarity in the embryo. Expression of this cytoplasmic tail in a mutant background for crb is sufficient to partially rescue the failure in apical-basal cell polarity, indicating that the large extracellular domain of Crb is dispensable for this process. Three different observations indicate that the extracellular domain of Crb is required to attenuate Notch signalling and that the intracellular domain is dispensable. Mutant clones for a null allele of stardust (sdtXP96) are able to cover large areas of the wing without any overt phenotype when abutting the DV boundary or when running along the longitudinal veins. The crb mutant wing phenotype can be rescued when simultaneously expressing either full-length Crb or a truncated form of Crb lacking the whole intracellular tail (Crb-Extra-TM). Overexpression of Crb-Extra-TM leads to a mild downregulation of the Notch signalling pathway. In the adult wing, veins are thicker, resembling a Notch loss-of-function phenotype. In the wing imaginal disc, Crb-Extra-TM overexpression reduces the expression levels of Cut at the DV boundary, a target of Notch that requires high levels of Notch activity. Wg expression is not affected (Herranz, 2006).

Signalling centres along compartment boundaries are required to organize the growth and pattern of the surrounding tissue. However, too much of a signal has deleterious effects. The Notch signalling center organizes the growth and pattern of the developing wing primordium, partially through the secreted protein Wingless. Wingless activity contributes to limit Notch activity to cells immediately adjacent to the DV boundary. This study presents evidence that Notch also contributes to the refinement of its activation domain through its target gene crumbs. Crumbs attenuates Notch signalling by repressing the activity of the gamma-Secretase complex. Many loss-of-function mutations in the human homologue of Crumbs, CRB1, cause recessive retinal dystrophies, including retinitis pigmentosa. Given the fact that the gamma-Secretase complex also mediates the intracellular cleavage of the transmembrane protein APP, leading to accumulation of the Aβ peptide in plaques in AD, it is postulated that Crumbs may also be involved in modulating AD pathogenesis. The analysis indicates a role for the extracellular part of the Crb protein in this process. It is interesting to note that many mutations that give rise to retinal dystrophies are missense mutations that affect different EGF or LG domains of CRB1. Thus, molecular interactions mediated by the extracellular domain of Crb may be crucial in both types of disease (Herranz, 2006).

Protein Interactions

Requirements for Presenilin-dependent cleavage of Notch and other transmembrane proteins

Ligand binding to receptors of the LIN-12/Notch family causes at least two proteolytic cleavages: one between the extracellular and transmembrane domains, and the other within the transmembrane domain. The transmembrane cleavage depends on Presenilin, a protein also required for transmembrane cleavage of beta-APP. The substrate requirements for Presenilin-dependent processing of Notch and other type I transmembrane proteins in vivo has been assayed. Presenilin-dependent cleavage does not depend critically on the recognition of particular sequences in these proteins but rather on the size of the extracellular domain: the smaller the size, the greater the efficiency of cleavage. Hence, Notch, beta-APP, and perhaps other proteins may be targeted for Presenilin-mediated transmembrane cleavage by upstream processing events that sever the extracellular domain from the rest of the protein (Struhl, 2000).

Evidence suggests that Presenilin is a component of a general mechanism that cleaves type I transmembrane proteins in the transmembrane domain, provided that they have a relatively small extracellular domain. Little if any processing occurs when the extracellular domain is greater than 200-300 amino acids. However, as the size of the extracellular domain is reduced incrementally, progressive increases in the efficiency of processing is attained, with proteins having very small extracellular domains (<50 amino acids) exhibiting similar, if not higher, levels of processing to those of full-length Notch in response to ligand. These results support the view that ligand activates LIN-12/Notch proteins by inducing a cleavage of the extracellular domain close to the membrane. Consistent with this proposal, recent biochemical studies indicate that mammalian Notch proteins undergo just such a cleavage event in response to ligand. Although this cleavage could activate the receptor in a number of different ways, these findings indicate that the resulting reduction in the size of the extracellular domain should suffice to convert the remainder of the protein into a substrate for Presenilin-dependent cleavage. Hence, the hypothesis is favored that ligand activates Notch by severing the extracellular domain from the rest of the receptor, a process described as 'ectodomain shedding' for other transmembrane proteins (Struhl, 2000).

Presenilin-dependent processing of betaAPP provides a second example of a possible link between ectodomain shedding and Presenilin-dependent cleavage. betaAPP initially contains a large extracellular domain of approximately 600 amino acids, and the full-length protein is not believed to be a substrate for Presenilin-dependent cleavage. However, full-length betaAPP is a target for cleavage by beta-secretase, a transmembrane aspartyl protease, which cuts at a site around 25 amino acids amino-terminal to the transmembrane domain. This initial cleavage is thought to be responsible for shedding the extracellular domain and for rendering the transmembrane domain susceptible to the Presenilin-associated gamma-secretase activity (Struhl, 2000).

The finding that proteins with diverse transmembrane domains can all be processed in a Presenilin-dependent fashion provided that the extracellular domain is small raises the possibility that such transmembrane cleavages can be viewed as relatively general and indiscriminate scavenging events that allow a cell to clear residual, truncated proteins from the membrane. Such a role could account for the transmembrane cleavages that generate the beta-amyloid peptides, which have no known function in normal cell physiology. However, in the case of LIN-12/Notch receptors, it appears that this cleavage mechanism has been incorporated as a critical step in signal transduction (Struhl, 2000).

Presenilin-dependent cleavage has been implicated in transduction of the unfolded protein response (UPR), which depends on the release and nuclear import of the cytosolic domain of the UPR receptor. Hence, activation of the UPR receptor, like that of Notch, may depend on processing events that cause ectodomain shedding and thereby target the remainder of the receptor for Presenilin-dependent cleavage. It is suggested that LIN-12/Notch proteins and the UPR receptors may belong to a general class of receptors that are activated by ectodomain shedding and which transduce signals by a mechanism involving Presenilin-dependent release of the intracellular domain from the rest of the receptor. It is possible that beta-APP also belongs to this class of receptors, since there is evidence that the intracellular domain of beta-APP interacts via an adaptor protein with a transcription factor (Struhl, 2000).

The mechanism by which ligand might induce dissociation of the Notch extracellular domain from the rest of the protein remains uncertain. Evidence has been found that the Presenilin-dependent cleavage of full-length Notch does not occur in shibire mutant embryos, which are defective in endocytosis due to reduced activity of Dynamin. In contrast, truncated forms of Notch that lack virtually the entire extracellular domain appear to be cleaved in these embryos. These results indicate that the Presenilin-dependent cleavage is not inherently dependent on endocytosis. Instead, endocytosis may be required for upstream events that are necessary to shed the ectodomain and hence to target the rest of the receptor for transmembrane cleavage. For example, endocytosis of the transmembrane ligand Delta in the signaling cell bound to Notch on the receiving cell might expose the Notch extracellular domain to cleavage. Alternatively, Notch may undergo extracellular processing in response to Delta while both proteins are on the cell suface, but endocytosis by the receiving cell might be required to dissociate the cleaved ectodomain from the rest of the receptor (Struhl, 2000).

In mammals, Notch proteins are cleaved at an extracellular Furin site (termed S1) close to the transmembrane domain during their trafficking to the cell membrane. As a consequence, the mature receptor is a heterodimer composed of two components: (1) a large extracellular domain and (2) the remainder of the receptor consisting of a short extracellular stub, the transmembrane domain, and the intracellular domain. In principle, interactions with ligand could activate the receptor by disrupting the association between these two components, causing the ectodomain to be separated from the rest of the protein by displacement rather than by proteolysis. Alternatively, ligand might induce shedding by triggering cleavage at a second site (S2) between the Furin cleavage and the transmembrane domain, a possibility directly supported by biochemical studies of Notch activation in mammalian cell culture. In the case of Drosophila, there is evidence that the mature Notch protein on the cell surface is not normally processed by Furin to form a heterodimer. If the Furin-mediated S1 cleavage does not occur in Drosophila Notch, ectodomain shedding would presumably depend on a ligand-induced S2 cleavage in order to convert the receptor into a substrate for the transmembrane cleavage (referred to as the S3 cleavage), which requires Presenilin (Struhl, 2000).

Recent biochemical evidence in mammals suggests that the metalloprotease TACE can execute the S2 cleavage of Notch in response to ligand. Genetic data in C. elegans and Drosophila suggest that a related metalloprotease, Kuzbanian/SUP-17, is essential for LIN-12/Notch signaling. However, there are conflicting biochemical data concerning whether Kuzbanian cleaves Notch or its ligands, complicating interpretation of whether it plays a direct role in executing the S2 cleavage. The nature of the event that precipitates the S2 cleavage is not known, but genetic evidence in C. elegans raises the possibility that ligand-induced oligomerization is involved (Struhl, 2000).

One determinant of whether a protein is a substrate for Presenilin-dependent cleavage appears to be the size of the extracellular domain. How might the size of the extracellular domain be assayed by the Presenilin-dependent cleavage mechanism? One possibility is that the cleavage mechanism requires the assembly of an active processing complex in close proximity to the transmembrane domain of the substrate. Although Presenilin has been reported to associate with Notch proteins as they move from the endoplasmic reticulum to the cell surface, the presence of a large extracellular domain may interfere sterically with the assembly of the complete complex or with the proteolytic activity of the complex. Another possibility is that the cleavage mechanism recognizes a free amino terminus in close proximity to the transmembrane domain, a condition that may be more likely when the extracellular domain is small. Both of these possible mechanisms are compatible with the finding that there is a progressive decline in cleavage efficiency as the size of the extracellular domain is increased incrementally (Struhl, 2000).

A second factor appears to be the primary sequence of the transmembrane domain. Although all of the transmembane domains tested can be cleaved in a Presenilin-dependent fashion, the amount of cleavage varies. The transmembrane domains of Notch, beta-APP, and Sevenless all appear to be cleaved efficiently, whereas those of Torso, Delta, and GlycophorinA are less efficiently cleaved. Similarly, substitution or deletion of a conserved valine located immediately downstream of the likely S3 cleavage site reduces, but does not eliminate, cleavage in mammalian tissue culture, and evidence has been found in Drosophila. for a reduction in the efficiency of cleavage of such mutated or deleted forms of Notch. These findings suggest that Presenilin-dependent processing may be limited to some extent by the conformational state of the transmembrane domain, a property that is likely to depend on the primary sequence. Nevertheless, it remains striking that many different transmembrane domains, each with a distinct primary sequence, can be cleaved in a Presenilin-dependent fashion. Hence, the protease activity does not appear to require recognition of specific primary sequences (Struhl, 2000).

A third variable that appears to influence substrate specificity is the potential for oligomerization. The transmembrane domain of Glycophorin A, which dimerizes avidly in the membrane, is a relatively poor substrate, whereas a single amino acid substitution, which is expected to severely reduce dimerization of this transmembrane domain, renders it a better substrate for Presenilin-dependent cleavage. Similarly, the presence of an extracellular dimerization domain, a leucine zipper, severely reduces the efficiency of Presenilin-dependent cleavage compared to a control protein that carries a mutated and inactive zipper. Hence, the Presenilin-dependent cleavage reaction appears to work better on isolated monomeric proteins. It is not clear why oligomerization reduces the efficiency of Presenilin-dependent cleavage. One possibility is that the cleavage mechanism depends on the assembly of a protease complex that wraps around a single, isolated transmembrane domain. Another possibility is that oligomerization effectively increases the size of the extracellular domain. The inhibitory effect of oligomerization on Presenilin-dependent cleavage might also be important for stabilizing single-pass transmembrane proteins that normally have short extracellular domains, such as the zeta and eta chains of the T cell receptor CD3 signaling complex. Ligand may intially activate the receptor by inducing oligomerization, but cleavage of the ectodomain may in turn generate truncated proteins that can no longer oligomerize, helping to convert them into substrates for Presenilin-dependent cleavage (Struhl, 2000).

Finally, the amino-to-carboxyl polarity of the transmembrane domain of a protein may also govern whether it is a substrate for Presenilin-mediated cleavage. Notch, betaAPP, and the other proteins assayed are all type I transmembrane domains with amino-to-carboxyl polarity oriented in the extracellular-to-intracellular direction. In contrast, the first transmembrane domains of sterol regulatory element-binding proteins (SREBPs), which are cleaved in response to changes in sterol abundance, have the opposite polarity and do not appear to be Presenilin dependent. There is evidence that Presenilin-dependent cleavage depends on an aspartyl protease activity, perhaps Presenilin itself, and not the S2P metalloprotease, which appears responsible for the transmembrane cleavage of SREBPs. Perhaps these different proteolytic activities reflect distinct mechanisms involved in cleaving type I and type II transmembrane proteins (Struhl, 2000).

Presenilin affects Arm/beta-Catenin localization and function in Drosophila

Both loss of expression and overexpression of Presenilin suggested a role for this protein in the localization of Armadillo/beta-catenin. In blastoderm stage Presenilin mutants, Arm is aberrantly distributed, often in Ubiquitin-immunoreactive cytoplasmic inclusions predominantly located basally in the cell. These inclusions are not observed in loss of function Notch mutants, suggesting that failure to process Notch is not the only consequence of the loss of Presenilin function. Human presenilin 1 expressed in Drosophila produces embryonic phenotypes resembling those associated with mutations in armadillo; embryos exhibit reduced Armadillo at the plasma membrane; this is likely due to retention of Armadillo in a complex with Presenilin. The interaction between Armadillo/beta-catenin and Presenilin 1 requires a third protein, which may be delta-catenin. These results suggest that Presenilin may regulate the delivery of a multiprotein complex that regulates Armadillo trafficking between the adherens junction and the proteasome (Noll, 2000).

Arm at the cell membrane is associated with E-cadherin, and the continued expression and function of E-cadherin are dependent on the presence of functional Arm. E-cadherin is encoded by the shotgun gene, and shotgun mutant embryos develop poorly formed cuticles due to a loss of cell adhesion. It was reasoned that, if the increased level of cytoplasmic Arm observed in the presence of overexpression of hPS1 correlates with a depletion of the membrane-associated pool of Arm, then overexpression of hPS1 should result in a shotgun-like cuticle phenotype. Indeed most hPS1 embryos that survive long enough to secrete cuticle exhibit phenotypes that resemble the loss of E-cadherin function. In embryos that develop more cuticular elements, additional phenotypes include loss of head structures, variable degrees of segmental fusion, and a 'dorsal open' phenotype. In a small subset of animals (~10%) that survive to secrete cuticle, a weak/moderate wingless-like cuticle phenotype is observed, and this phenotype can be correlated with a failure to maintain Engrailed expression (Noll, 2000).

Because Drosophila PS appears to have a role in the Notch pathway, embryos expressing hPS were labeled with Fas III to assess the integrity of the ventral ectoderm and to look for any indication of hypertrophy of the nervous system. When the ectodermal cells associated with the ventral midline were examined there was evidence of some disruption in patterning as indicated by the meandering of the midline, but the epidermis was found to be intact. Further, when the Fas III-positive cells in the nervous system of these same embryos were visualized the number and location of Fas III-positive cells were found to be normal. This indicates that the nervous system hypertrophy at the expense of ventral ectodermal cell fates that is found as a result of disruption in the Notch signaling pathway does not occur when UAS-hPS1 is expressed. Since Fas III can be found at the cell membrane, the localization of Fas III was examined. Compared to wild-type, there was no obvious mislocalization of Fas III as a result of UAS-hPS1 expression. This suggests that the mislocalization of Arm as a result of UAS-hPS1 expression is not due to some nonspecific effect, but rather reflects a specific interaction been Arm and hPS1 (Noll, 2000).

The resemblance to shotgun mutations of the cuticle phenotypes associated with hPS1 expression strongly implicates dysfunction in adhesion and cytoskeletal organization. To investigate the basis of this phenotype further, phalloidin staining, used to reveal the distribution of actin, was carried out in these embryos at stages before cuticle deposition. UAS-hPS animals that make it through cellularization usually fail to initiate and/or complete dorsal closure, a phenotype resembling that observed in some Arm mutations. At the stage when an accumulation of actin in the peripheral nervous system is clearly apparent, dorsal closure should be approaching completion. However, in the hPS1 animals, the leading edge cells do not undergo the proper change in shape, and there is no accumulation of actin along the dorsal most edge of the cells. Cells all along the dorsoventral axis of the epidermis fail to stretch and take on the proper thin, cuboidal shape. Alterations in the normal distribution of actin due to expression of hPS1 can also be observed in blastoderm stage embryos, concurrent with the mislocalization of Arm to the cytoplasm. In hPS1 animals, the overall amount of actin present at the membrane is greatly reduced, resulting in a thin, spotty phalloidin staining pattern in some regions of the embryo and often in a complete degeneration of the membrane structure in other regions. In areas where hexagonal arrays are not present, large patches of intense phalloidin staining are evident (Noll, 2000).

It is unknown whether the interaction between PS1 and beta-catenin is direct or requires additional factor(s). To address this issue, different mouse beta-catenin deletion constructs were prepared and tested for interaction with PS1. PS1 failed to coimmunoprecipitate with beta-catenin antibodies and beta-catenin did not coimmunoprecipitate with PS1 antibodies. Taken together, these observations suggest that some cofactor or posttranslational modification is required for beta-catenin to bind to PS1, and this cofactor is not present in the yeast two-hybrid or coimmunoprecipitation assays. Because delta-catenin binds directly to PS1, could delta-catenin outcompete beta-catenin from an in vivo complex with PS1 in CHO cells? The minimal delta-catenin interactive fragment requires both the last four Arm repeats and a portion of the carboxy terminal sequence just beyond the Arm repeats. Expression of the delta-catenin DEco fragment (residues 828-1127) almost completely displaces beta-catenin from the PS1 complex. These experiments support the hypothesis that beta-catenin interacts with the hydrophilic loop of PS1 via a third protein, which competes with the DEco fragment of delta-catenin for binding to PS1. The most parsimonious explanation for these observations is that beta-catenin associates with the complex via full-length delta-catenin itself, but is unable to do so in the presence of the DEco fragment alone. If this were the case, then full-length delta-catenin would not be expected to compete beta-catenin from the PS1 complex, as the DEco fragment of delta-catenin did. Furthermore, it should be possible to demonstrate a direct interaction between delta-catenin and beta-catenin. Indeed, in the presence of full-length delta-catenin, beta-catenin is retained in the PS1 complex, suggesting that delta-catenin is capable of mediating beta-catenin association with PS1 (Noll, 2000).

Thus, embryos derived from presenilin germline clone females exhibit mislocalization of Armadillo. These embryos contain cytoplasmic inclusions that are both Arm and Ubiquitin immunoreactive, suggestive of a failure to target Arm to a degradative pathway. A role for PS in regulating the degradation of proteins is suggested by other PS interactions. sel-12, the C. elegans ortholog of PS, interacts with sel-10, a member of the Cdc4p family which targets proteins for Ubiquitin-mediated turnover. Furthermore, the fly ortholog of Cdc41p, Slimb, may target beta-catenin for Ubiquitin/proteasome degradation. Also, the LEF/beta-catenin complex is thought to be affected in its translocation to the nucleus by mutations in PS. A genetic relationship between Drosophila PS and Arm is also suggested by a genetic modifier screen for mutations that can suppress the armadillo mutant phenotype. Together these observations implicate PS in a complex with beta-catenin as a means to target beta-catenin and possibly its cargo for degradation or other functions at remote sites in the cell. Thus the Presenilin/ beta-catenin complex may serve as an endoplasmic reticular staging platform for complex assembly and targeting to a variety of cellular destinations including the proteosome (Noll, 2000).

Before beta-catenin joins alpha-catenin and arrives at the plasma membrane, it forms a 'preadhesion' complex with Cadherin that is required for ER exit and membrane delivery of the complex. The delivery of Arm requires that the cell specify a polar trafficking route to the site of the adherens junctions at the apical part of the cell during blastoderm stages. The fact that many of these inclusions are located basally suggests impaired apical trafficking in the absence of PS. Although apparently reduced, sufficient Arm does reach the adherens junctions in these embryos so they do not develop an early adhesion defect phenotype. Instead, the phenotype includes a neurogenic defect thought to be related to the role of PS in cleaving Notch to generate an active product. The reports of the close resemblance between the Notch and PS phenotypes suggested a highly restricted function for PS: enhancement of Notch function by facilitating Notch cleavage. However, loss of Notch does not produce the Arm inclusions observed with loss of PS. This finding suggests a broader function for PS that extends beyond its role in Notch processing (Noll, 2000).

One site of residence for beta-catenin is in a complex with Axin, APC, and GSK3beta where it mediates regulation of Wnt signaling. Although quantitatively less frequent than the shotgun phenotype among embryos expressing hPS1, phenotypes that resemble the loss of wingless activity were occasionally observed, suggesting that binding to hPS1 also successfully competes beta-catenin away from its signaling pool. In conjunction with the evidence that PS is involved in Notch activation by releasing its cytoplasmic domain these findings suggest another link between the Wingless and Notch pathways. Previous studies reported genetic interactions between wg and N and direct interactions between these pathways via Dishevelled, as well as isolation of wg mutations in screens for genetic modifiers of Notch and vice versa. PS is primarily localized in the ER, but cleavage of Notch occurs either at or close to the cell surface. Transit of PS to the region of the adherens junction could resolve the contradiction between previous views regarding the location of PS in the endoplasmic reticulum and the cleavage of Notch either in or near the plasma membrane. PS is associated with two proteins -- beta-catenin and delta-catenin -- whose destination is the adherens junction. Both Notch and Wingless have also been reported in the region of the adherens junction. A large regulatory complex associated with PS may cleave Notch leaving the released cytoplasmic fragment to translocate to the nucleus and activate transcription or prevent transcription by binding through its carboxy terminus to Dsh. Dishevelled may independently localize to intracellular junctions through its discs large homology (DHR) region or utilize the PS complex to direct the Notch cytoplasmic fragment toward a degradative pathway (Noll, 2000 and references therein).

Alternatively, members of the PS complex such as beta-or delta-catenin may regulate the inhibitory interaction between Notch and Dishevelled. Because both of the putative substrates for PS (Notch and the amyloid precursor protein) transit through the ER, PS-associated proteins may serve in the ER to prevent premature cleavage. If PS cleaves Notch, it is curious that the expression of hPS1 does not induce a Notch activation phenotype. Neither was a Notch activation phenotype reported when the endogenous Drosophila PS was overexpressed. Significant inhibitory controls must be present that either prevent Notch cleavage or prevent the activity of the Notch active fragment. The interactions of beta-catenin with other proteins are complex and numerous. PS1 may delimit the components of the beta-catenin complex at specific cellular locales and allow it to discriminate among potential binding partners. In the case of the beta-catenin/APC complex, GSK3beta can phosphorylate both proteins. Although a direct association of GSK3beta with beta-catenin could not be demonstrated in vitro, it has been observed that Axin simultaneously and directly binds to APC, beta-catenin, and GSK3beta. The binding of all three proteins to axin may coordinate beta-catenin down-regulation by bringing these proteins into proximity (Noll, 2000).

GSK3beta binds PS1 between residues 259 and 298 of the fragment that is N-terminal after endoproteolytic cleavage. This site differs from the delta-catenin binding site on hPS1, which spans residues 319 to 371. Thus PS may coordinate the entry of both beta-catenin and GSK3beta into the complex with APC and Axin. Alternatively, PS may coordinate the trafficking routes of beta-catenin as it assembles and shifts large multicomponent protein complexes to diverse destinations in the cell (Noll, 2000).

Presenilin-mediated transmembrane cleavage is required for Notch signal transduction in Drosophila

The cleavage model for signal transduction by receptors of the LIN-12/Notch family posits that ligand binding leads to cleavage within the transmembrane domain, so that the intracellular domain is released to translocate to the nucleus and activate target gene expression. The familial Alzheimer's disease-associated protein Presenilin is required for LIN-12/Notch signaling, and several lines of evidence suggest that Presenilin mediates the transmembrane cleavage event that releases the LIN-12/Notch intracellular domain. However, doubt was cast on this possibility by a report that Presenilin is not required for the transducing activity of NECN, a constitutively active transmembrane form of Notch, in Drosophila. This finding has been reassessed and it has been shown instead that Presenilin is required for activity of NECN for all cell fate decisions examined. These results indicate that transmembrane cleavage and signal transduction are strictly correlated, supporting the cleavage model for signal transduction by LIN-12/Notch and a role for Presenilin in mediating the ligand-induced transmembrane cleavage (Struhl, 2001).

The classic Notch-mediated neurogenic interaction occurs during embryonic development, so that some cells in the 'proneural' portion of the ventral ectoderm segregate as neuroblasts, while the others remain in the ectoderm and eventually differentiate into the ventral epidermis. The absence of Notch activity results in neural hyperplasia at the expense of the epidermis, whereas constitutive Notch activity suppresses neuroblast segregation so that all ectodermal cells differentiate as epidermis (Struhl, 2001 and references therein).

Early neuroblast segregation can be readily visualized by the expression of the transcription factor Hb. During wild type development, the initial rounds of neuroblast segregations generate a stereotyped pattern of three anteroposterior columns of Hb-expressing neuroblasts on each side of the ventral midline. Early neural segregations also appear normal in embryos in which N+ is ubiquitously expressed from a transgene. In contrast, embryos lacking Notch activity form a broad swath of Hb-expressing neuroblasts in place of the normal pattern of three columns, whereas embryos in which constitutively activated forms of Notch (NECN or Nintra) are ubiquitously expressed, are found to lack Hb expression (Struhl, 2001 and references therein).

Embryos lacking maternal and zygotic Presenilin activity, referred to as PS- embryos, resemble Notch- embryos. This phenotype results from the absence of Notch signal transducing activity rather than from a marked decrease in Notch protein levels at the plasma membrane. The ability of N+, NECN, and Nintra to suppress neuroblast formation has been examined in PS- embryos. Ubiquitous expression of N+ or NECN fails to suppress neuroblast segregations, so that such embryos appear indistinguishable from PS- embryos. In contrast, ubiquitous expression of Nintra in PS- embryos efficiently suppresses neuroblast segregations, as it does in otherwise wild-type embryos (Struhl, 2001).

The intracellular domains of N+-GV3 (wild type N) and NECN-GV3 do not gain access to the nucleus in PS- embryos, in contrast to Nintra-GV3, which appears to have ready access. Thus, Notch nuclear access in PS- embryos appears to correlate with Notch transducing activity: Nintra has access and retains constitutive transducing activity, whereas NECN and N+ lack access and show no evidence of transducing activity (Struhl, 2001).

Notch activity is required in several distinct processes during the development of the wing imaginal disc. The eponymous Notch phenotype is a notched wing, a consequence of reduced Notch-mediated signaling across the dorsoventral compartment boundary. Notch-mediated signaling also regulates classic neural/ectodermal decisions that control the pattern of mechanosensory bristles on the mesonotum (the dorsal portion of the fuselage of the adult thorax). Finally, Notch signaling is required to resolve thin stripes of wing vein cells from initially broader stripes of 'prevein' tissue, a process essential for normal vein development. The consequences of expressing NECN and Nintra in genetically marked clones of PS- cells for each of these processes was examined. In all cases, in the absence of Presenilin, Nintra retains constitutive transducing activity, whereas NECN shows no evidence of transducing activity (Struhl, 2001).

Activation of Notch signaling across the dorsoventral compartment boundary in wing imaginal discs induces a thin stripe of 'edge cells' that straddle the boundary to express the target genes Cut and Wingless (Wg). Cut is a transcription factor that is required for differentiation of the edge cells and Wg is a morphogen that controls growth and patterning of the wing, including specification of the mechanosensory bristles that decorate the wing margin. Clones of cells that lack either Notch or Presenilin activity fail to express either Cut or Wg along the presumptive wing margin. The loss of Cut expression can be visualized in discs by antibody staining; furthermore, in adults, the loss of Wg signaling can be readily assayed morphologically by the presence of large wing notches. Conversely, clones of cells that express constitutively active forms of Notch, such as Nintra or NECN, ectopically express both Cut and Wg wherever they arise within the wing blade primordium. Ectopic expression of Wg in turn induces the formation of ectopic sensory mother cells (SMCs) in neighboring wing tissue and also causes ectopic wing outgrowths (Struhl, 2001).

Clones of PS- cells that express NECN or Nintra, as well as a nuclearly localized form of Green fluorescent protein and the Yellow protein (which both allow adult structures to be genetically marked), were generated early during wing disc development by using the MARCM (Mosaic analysis with a repressible cell marker) technique and their effects on Cut expression and growth in the wing blade were assayed. PS- clones expressing NECN that straddle the dorsoventral compartment boundary fail to express Cut. In addition, these clones are associated with severe notching of the adult wing, consistent with loss of Wg signaling. These phenotypes indicate that the constitutive activity of NECN in the developing wing depends on Presenilin activity (Struhl, 2001).

In contrast, the constitutive activity of Nintra does not require Presenilin activity. Clones of PS- cells that express Nintra autonomously express Cut. In addition, they are associated with two phenotypes that indicate that they ectopically express Wg: (1) they induce ectopic wing margin bristles in neighboring wild-type cells; (2) they are associated with bulges in the disc epithelium suggesting excessive wing growth, a possibility confirmed by the behavior of the clones in the adult wing where they are associated with large outgrowths of wing tissue and ectopic rows of margin bristles formed by wild-type cells adjacent to the clone (Struhl, 2001).

During the development of the mesonotum, small 'proneural clusters' of ectodermal cells undergo Notch-mediated interactions so that one cell within the cluster becomes an SMC, whereas the others remain ectodermal. In the absence of Notch or Presenilin function, all cells of the cluster choose the SMC fate, so that a cluster of neurons is produced at the expense of the epidermis. Conversely, the constitutive activity of NECN or Nintra prevents any cell from choosing the SMC fate, thereby suppressing bristle formation. All of the SMCs can be marked by the expression of the smc-Z reporter gene, and a subset of these also expresses Cut. Presenilin activity is essential for the constitutive transducing activity of NECN during SMC specification. Clones of PS- cells expressing NECN that arise within the mesonotum primordium cause clusters of SMCs to form in place of a single SMC. In contrast, no SMCs appear to segregate within clones of PS- cells expressing Nintra or clones of PS- cells expressing NECN which also carry the rescuing Tubulinalpha1-PS+ transgene. Thus, the constitutive transducing activity of NECN in this context also depends on Presenilin (Struhl, 2001).

Cells of initially broad 'provein' regions undergo Notch-mediated cell-cell interactions so that some cells become vein cells whereas the others become intervein cells. In the absence of Notch or Presenilin function, most or all provein cells become vein cells, so that the wing veins are abnormally thick; conversely, constitutive activation of the Notch pathway suppresses vein cell formation. Clones of PS- cells that express NECN can contribute to the adult wing blade, provided that they do not cross the wing margin where Notch signal transduction is essential for activating Wg. Such clones cause a thickened vein phenotype indicating a failure of Notch signal transduction in the provein cells. Because Nintra-expressing PS- cells as well as Tubulinalpha1-PS+ NECN-expressing PS- cells strongly activate Wg expression and cause outgrowths composed primarily of surrounding, wild-type wing cells, whether they have the ability to differentiate as vein cannot readily be assessed. Nevertheless, the finding that NECN-expressing PS- cells form abnormally thickened veins indicates that Presenilin is essential for NECN transducing activity in this context as well (Struhl, 2001).

Nicastrin is required for Presenilin-mediated transmembrane cleavage

The transmembrane glycoprotein Nicastrin was identified in a complex with the multipass membrane protein Presenilin. Presenilin mediates transmembrane cleavage of single-pass transmembrane proteins with short extracellular domains, including the ligand-activated form of the receptor Notch and beta-amyloid precursor protein (beta-APP). Transmembrane cleavage of Notch is essential for signal transduction, and transmembrane cleavage of beta-APP generates pathogenic amyloid peptides implicated in Alzheimer's disease. This study investigates the requirement for Nicastrin in Presenilin-mediated transmembrane cleavage. In Drosophila, loss of Nicastrin activity blocks the accumulation of Presenilin associated with the apical plasma membrane, abolishes Presenilin-dependent cleavage of the transmembrane domains of Notch and beta-APP, and abrogates Notch signal transduction (Chung, 2001).

Nicastrin is required for gamma-secretase cleavage of the Drosophila Notch receptor

Drosophila nicastrin mutations have been isolated by systematic lethal mutagenesis screening. nicastrin mutants exhibit defective cell fate specifications at all stages of development, similar to what has been observed in Notch and Presenilin mutants. Biochemical analysis of Notch proteolysis reveals that loss of either nicastrin or Presenilin activity affects the same step of Notch proteolysis that is blocked by a peptidomimetic gamma-secretase inhibitor compound. Consistent with these observations, Nicastrin is essential for signaling from a membrane-tethered form of constitutively activated Notch, but dispensable for signaling from a nuclearly localizing, nonmembrane-bound Notch intracellular domain. Using RNA interference (RNAi) in Drosophila S2 cells, it has been shown that absence of Nicastrin function is accompanied by a loss of mature Presenilin protein. Expression of human Presenilin in this assay is upregulated when endogenous Drosophila Presenilin is inactivated, similar to results seen with expression of heterologous Presenilins in transfected mammalian cells and transgenic mice. On the basis of these results and the known binding properties of Nicastrin, it is suggested that Nicastrin may bind gamma-secretase substrates and recruit them into the Presenilin-containing complex, which might be an obligatory step in stabilizing Presenilin during assembly of the active proteolytic complex (Hu, 2002).

aph-1 and pen-2 are required for Notch pathway signaling, gamma-Secretase cleavage of ßAPP, and Presenilin protein accumulation

Presenilins are components of the gamma-secretase protein complex that mediates intramembranous cleavage of ßAPP and Notch proteins. A C. elegans genetic screen revealed two genes, aph-1 (Drosophila homolog: anterior pharynx defective 1) and pen-2, encoding multipass transmembrane proteins, that interact strongly with sel-12/presenilin and aph-2/nicastrin (see Drosophila Nicastrin). Human aph-1 and pen-2 partially rescue the C. elegans mutant phenotypes, demonstrating conserved functions. The human genes must be provided together to rescue the mutant phenotypes, and the inclusion of presenilin-1 improves rescue, suggesting that they interact closely with each other and with presenilin. RNAi-mediated inactivation of aph-1, pen-2, or nicastrin in cultured Drosophila cells reduces gamma-secretase cleavage of ßAPP and Notch substrates and reduces the levels of processed presenilin. aph-1 and pen-2, which, like nicastrin, are required for the activity and accumulation of gamma-secretase (Francis, 2002).

To study the roles of aph-1 and pen-2 in APP and Notch cleavage, γ-secretase activity assays were developed in Drosophila tissue culture cells, similar to nuclear access assays that accurately report presenilin-dependent processing in Drosophila. To confirm that the Drosophila cell assays accurately measure γ-secretase-like activity, Dmel2 cells and developing flies were tested for sensitivity to previously described γ-secretase inhibitors. When applied to Drosophila embryos and larvae at intermediate concentrations, compound E (Seiffert, 2000) causes adult wing notching and rough eye phenotypes characteristic of Notch mutants and a presenilin hypomorphic mutant. Compound E also induces glp-1-like sterility when applied to developing hop-1 mutant C. elegans. These results demonstrate that this selective γ-secretase inhibitor interferes with Notch signaling in vivo in Drosophila and C. elegans. In Dmel2 cell assays, compound E inhibits membrane-tethered APP C99-GV reporter activity by 2- to 3-fold but has no effect on the non-membrane-bound APP C59-GV reporter activity. Similar reporter inhibition was obtained with two additional γ-secretase inhibitors. Membrane-tethered NECN-GV reporter activity is inhibited by compound E at very similar concentrations to C99-GV, while NINTRA-GV reporter activity is unaffected (Francis, 2002).

To determine whether the γ-secretase activity observed in Dmel2 cells produces the same cleavage products as the human enzyme, secreted Aβ peptides released from the C99-GV substrate were measured using specific Aβ40 and Aβ42 ELISA assays. Aβ40 and Aβ42 are both detected, and, as in human cell supernatants, Aβ40 is approximately 10-fold more abundant than Aβ42. Production of both peptides is inhibited by compound E to levels below the limit of detection in ELISA assay conditions. The ability of compound E to completely inhibit Aβ production in this assay contrasted with the maximum 2- to 3-fold inhibition of NECN-GV and C99-GV reporter gene activity observed, suggesting that residual reporter gene activity may be a result of presenilin-independent nuclear access of the GV activator in Dmel2 cells. As described for mammalian presenilins, high concentrations of compound E cause an increase in ~50 kDa full-length presenilin levels, but the strong inhibition of Aβ production observed in this assay cannot be explained simply by reduction in presenilin CTF levels. These data confirm that Dmel2 cells have an endogenous γ-secretase activity with pharmacological and substrate cleavage properties similar to human γ-secretase (Francis, 2002).

RNAi was used to determine whether γ-secretase activity in Dmel2 cells is presenilin dependent. RNAi of Drosophila psn strongly reduces endogenous PSN C-terminal fragment (CTF) protein levels. psn RNAi also reduced secreted Aβ40 and Aβ42 peptides by 85% and decreased C99-GV and NECN-GV reporter gene activity by 50%–70% but had no effect on the intracellular domain reporters C59-GV or NINTRA-GV. The APP cleavage reporter, Notch cleavage reporter, and Aβ ELISA assays thus each demonstrate presenilin dependence and provide independent measures of γ-secretase function (Francis, 2002).

RNAi of Drosophila aph-1, pen-2, or nicastrin (nct) results in reduction of secreted Aβ40 and Aβ42 and reduction in reporter gene activity elicited by both C99-GV and NECN-GV, but not by C59-GV or NINTRA-GV. Inhibition in each case is approximately as strong as that observed for RNAi of presenilin itself. In addition, RNAi of aph-1 and pen-2, like that of nct, leads to strong reductions in PSN CTF protein levels, with no buildup of detectable full-length PSN. pen-2 RNAi is slightly less efficient and more variable in these assays than aph-1 or nct RNAi, in contrast to the identical behavior of pen-2 and aph-1 in all C. elegans in vivo genetic assays. The difference in efficiency of pen-2 versus aph-1 RNAi in Drosophila cells may thus reflect incomplete inactivation of pen-2 by RNAi rather than a functional difference between the two genes. These data reveal that aph-1 and pen-2, like nct, are necessary for γ-secretase activity and for the accumulation of processed presenilin protein in Drosophila cells (Francis, 2002).

Using a genetic approach in C. elegans, aph-1 and pen-2, two new, phylogenetically conserved members of the Notch pathway have been identified. aph-1 and pen-2 have very similar functions in C. elegans, since their individual mutant phenotypes and their genetic interaction phenotypes with aph-2, hop-1, and sel-12 are indistinguishable. aph-1 and pen-2 are required for all lin-12- and glp-1-mediated signaling events examined, and no phenotypes were observed in the aph-1 or pen-2 mutants that would suggest functions other than in lin-12/glp-1-dependent processes. It is concluded that aph-1 and pen-2 define new obligate members of the Notch signaling pathway (Francis, 2002).

The sel-12 enhancer screen identified only these two genes with strong effects on germline presenilin activity. Three other related genetic screens were performed for presenilin pathway components and additional alleles were isolated of aph-1, pen-2 and aph-2, but no additional genes were found with similar strong phenotypes. If other genes that are essential for presenilin function in C. elegans exist, they may be masked by genetic redundancy or may have additional functions and associated phenotypes that obscure their contributions to Notch signaling. These genetic studies suggest that aph-1, pen-2, and aph-2 define a set of genes that is unique in the strength and specificity of their interactions with the presenilin genes (Francis, 2002).

The lin-12(gf) suppression tests and Drosophila cell culture experiments on defined γ-secretase substrates presented in this study demonstrate that aph-1 and pen-2 are required for Notch and βAPP cleavages, specifically at the presenilin-mediated γ-secretase/S3 step. It was also found that aph-1 and pen-2, like nct, are required for accumulation of processed presenilin protein. Although this requirement could be the simple explanation for the loss of γ-secretase activity upon depletion of aph-1 and pen-2, recent studies have suggested that there may be separable requirements for nicastrin in Notch signaling and in accumulation or maintenance of presenilin protein. Similarly, cell culture data suggest that pen-2 RNAi can have stronger inhibitory effects on γ-secretase activity than on presenilin protein levels, suggesting that, as is the case for nicastrin, γ-secretase activity may be lost more rapidly than PSN-CTF stability or accumulation upon pen-2 depletion. Although the mechanism is not clear, accumulation of processed presenilin in Drosophila cells depends on aph-1, pen-2, and nct (Francis, 2002).

Despite their extensive functional similarities with aph-2, aph-1 and pen-2 behave distinctly from aph-2 in certain genetic interaction tests. (1) aph-2 mutants strongly interact with hop-1, but not with sel-12 mutants, to produce the glp-sterile phenotype, while aph-1 and pen-2 mutants exhibit sterility with sel-12, but not with hop-1 mutants. (2) aph-1 and pen-2 mutants or RNAi do not show additive phenotypes with each other, while aph-2 RNAi interacts with both aph-1 and pen-2 mutants to give partially penetrant sterility. (3) Rescue of either aph-1 or pen-2 is dependent on coexpression of human aph-1 and pen-2 genes together, but not on nicastrin, whereas aph-2 is rescued by human nicastrin alone. APH-2/nicastrin is a type 1 glycosylated transmembrane protein, while aph-1 and pen-2 are predicted to be polytopic integral membrane proteins, suggesting a basis for functional differences between aph-2 and the aph-1 and pen-2 genes. It is suggested that aph-1 and pen-2 interact closely with each other in the same process, perhaps at the same step, but in a role somewhat distinct from that of aph-2/nicastrin, to facilitate presenilin activity (Francis, 2002).

In rescue experiments it was found that human aph-1 and pen-2 are corequired for rescue. APH-1 and PEN-2 thus cooperate functionally, consistent with a model in which the two proteins associate directly. The APH-1 and PEN-2 proteins are highly conserved, but sequence divergence between the C. elegans and human APH-1 and PEN-2 proteins might interfere with functional cross-species APH-1/PEN-2 interactions and account for the corequirement observed for aph-1 and pen-2 mutant rescue. Cross-species interactions between a putative APH-1/PEN-2 unit and presenilins can occur because haph-1 and hpen-2 together are able to confer low-level rescue of pen-2 mutants in the absence of PS1 and because human PS1 or PS2 alone is able to rescue sel-12 mutants. However, coinjection of human PS1 together with haph-1 and hpen-2 improves the efficiency of pen-2 rescue, suggesting that the putative APH-1/PEN-2 protein complex also interacts with presenilin, and these interactions are more functional with intraspecific forms. APH-1 and PEN-2 are good candidates to be regulators and/or components of the high-molecular weight γ-secretase complex (Francis, 2002).

The role for γ-secretase in proteolysis of Notch, APP, and other cell surface transmembrane proteins suggests a site of action at the cell surface or in endocytic compartments. Some presenilin is detected in these locations, although most is found in ER and Golgi compartments. PEN-2::GFP expression in adult C. elegans, like SEL-12::GFP, is found primarily in internal membrane compartments, consistent with a model in which PEN-2 interacts with APH-1 and presenilin early in the secretory pathway. Endogenous APH-2 protein in early C. elegans embryos, in contrast, is localized primarily to the plasma membrane. In aph-1 and hop-1; sel-12 mutant embryos, APH-2 localizes instead in a perinuclear staining pattern consistent with ER/Golgi localization. Transport of APH-2/nicastrin to the cell surface is thus dependent on presenilins and APH-1. One model for APH-1 and PEN-2 function would be to facilitate trafficking of APH-2 to the cell surface, which in turn promotes trafficking of presenilin. However, genetic data favor a model in which APH-1 and PEN-2 together interact directly with presenilin or a presenilin/nicastrin complex to promote maturation and accumulation of the complex. It will be important to examine expression of endogenous proteins in the same cell types to determine to what extent APH-1 and PEN-2 colocalize with presenilins and nicastrin (Francis, 2002).

aph-1, pen-2, and aph-2/nicastrin define a core set of genes essential for the activity and accumulation of presenilin-dependent γ-secretase complexes. Several mechanisms could explain the failure to stably accumulate processed presenilin protein in the absence of APH-1, PEN-2, and APH-2/nicastrin proteins, including an inability to assemble, mature, transport, or stabilize the γ-secretase complex. If APH-1 and PEN-2 proteins interact directly with each other and with presenilin, these interactions could be transitory during early steps in γ-secretase complex assembly or may persist in the mature enzyme complex. Further studies of APH-1 and PEN-2 function should enhance the understanding of the γ-secretase enzyme and improve opportunities for the design of selective Alzheimer's disease therapeutics (Francis, 2002).

The role of presenilin cofactors in the gamma-secretase complex

Mutations in presenilin genes account for the majority of the cases of the familial form of Alzheimer's disease (FAD). Presenilin is essential for gamma-secretase activity, a proteolytic activity involved in intramembrane cleavage of Notch and ß-amyloid precursor protein (APP). Cleavage of APP by FAD mutant presenilin results in the overproduction of highly amyloidogenic amyloid 42 peptides. gamma-Secretase activity requires the formation of a stable, high-molecular-mass protein complex that, in addition to the endoproteolysed fragmented form of presenilin, contains essential cofactors including nicastrin, APH-1 (also known as Pen-1 or Presenilin stabilization factor) and PEN-2. However, the role of each protein in complex formation and the generation of enzymatic activity is unclear. Drosophila APH-1 (Aph-1) is shown to increase the stability of Drosophila presenilin (Psn) holoprotein in the complex. Depletion of PEN-2 by RNA interference prevents endoproteolysis of presenilin and promotes stabilization of the holoprotein in both Drosophila and mammalian cells, including primary neurons. Co-expression of Drosophila Pen-2 with Aph-1 and nicastrin increases the formation of Psn fragments as well as gamma-secretase activity. Thus, APH-1 stabilizes the presenilin holoprotein in the complex, whereas PEN-2 is required for endoproteolytic processing of presenilin and conferring gamma-secretase activity to the complex (Takasugi, 2003).

Presenilin is essential for gamma-secretase cleavage, which releases amyloid ß-peptide (Aß) and the intracellular domain of Notch by intramembranous proteolysis of ß-amyloid precursor protein (APP) and Notch, respectively. Presenilin mediates gamma-secretase function by forming a highly stable protein complex of high relative molecular mass (high-Mr) together with a set of cofactor proteins. In addition to nicastrin (NCT), a type I single-pass membrane glycoprotein, two additional putative presenilin cofactors have been identified: APH-1, a multi-transmembrane protein coded by a gene whose deletion leads to hypoplasia of the anterior pharynx in Caenorhabditis elegans, was found to be a Notch pathway member possibly involved in presenilin function; aph-1 was also identified as one of the presenilin enhancer genes (pen-1) together with pen-2, which codes for a double-membrane-spanning protein. NCT, APH-1 and PEN-2 are required for gamma-secretase function and accumulation of presenilin fragments, although an understanding of the differential roles of each cofactor in the formation of the high-Mr presenilin protein complex, and whether NCT, APH-1 and PEN-2 represent the principal presenilin cofactors to confer gamma-secretase activity, has remained elusive (Takasugi, 2003).

Drosophila S2 cells was stably transfected with Aph-1, and a significant increase in the levels of endogenous Psn holoprotein was found. This is markedly enhanced by expression of Aph-1 and Drosophila Nct, but is not observed in cells transfected with Nct alone. The levels of Psn fragments involved in the active form of gamma-secretase are not altered in either case. Stably co-transfected S2 cells overexpressing Aph-1 and Nct were treated with cycloheximide (CHX) to block total cellular protein synthesis, and the stability of Psn and other proteins was examined. In mock-transfected S2 cells (transfected with an empty vector alone) only small amounts of Psn holoprotein were detectable; these were rapidly degraded within about 4 h of CHX treatment, however, fragments of Psn were relatively more abundant and highly stable, in a manner similar to that of mammalian presenilin. In contrast, Psn holoprotein levels were significantly increased by overexpression of Aph-1 or of Aph-1 and Nct, and remained highly stabilized. Levels of Aph-1 and Nct in co-transfected S2 cells were also highly stable during the period of CHX treatment, suggesting that Psn (including holoprotein) forms a highly stabilized protein complex together with Aph-1 and Nct under these conditions. To gain support for this hypothesis, CHAPSO-solubilized membrane fractions of S2 cells stably transfected with Aph-1 and Nct were separated by glycerol velocity gradient centrifugation. Psn holoprotein in cells overexpressing Aph-1 and Nct was fractionated totally in high-Mr ranges of 232-440K together with Psn fragments. This was in contrast to the exclusive low-Mr distribution of short-lived Psn holoproteins in S2 cells in the absence of Aph-1 overexpression. Furthermore, most of the Aph-1 and Nct proteins were found in the high-Mr fractions. Taken together, these data support the hypothesis that Aph-1 represents the major 'stabilizing' cofactor of Psn that promotes the key step of stable high-Mr complex formation. However, the level of A secretion from cells with or without co-expression of Nct and Aph-1 was not significantly different, suggesting that some additional factors are needed for the upregulation of gamma-secretase activity (Takasugi, 2003).

To examine the role of PEN-2 (another putative cofactor of presenilin that harbours two membrane-spanning domains) in the formation and function of the gamma-secretase complex, double-stranded RNA-mediated interference (RNAi) experiments were performed. Unlike the results from Aph-1 or Nct RNAi, treatment of S2 cells with Pen-2 RNAi led to loss of fragment forms of Psn and accumulation of Psn holoprotein. Application of CHX to S2 cells treated with Pen-2 RNAi showed that the accumulated Psn holoprotein is highly stabilized and is recoverable in high-Mr fractions. In contrast to the result of the Pen-2 RNAi experiment, combinations of Pen-2 and Aph-1 or Pen-2 and Nct RNAi abolished the accumulation of either fragment or holoprotein forms of Psn. gamma-Secretase activities, as determined by an in vitro Aß generation assay using recombinant APP C100 (carboxy-terminal 99 amino acid residues of human APP) tagged with Flag, Myc and His as a substrate, were totally abolished by every combination of RNAi that diminished the Psn fragment. These data provide genetic evidence to support the hypothesis that Nct and Aph-1 act upstream of Pen-2 in the formation of the gamma-secretase complex. The accumulation of Psn holoprotein by Pen-2 RNAi was also observed in BG2 cells, a Drosophila neuronal cell line derived from primary cultures of larval central nervous system (Takasugi, 2003).

To examine further whether PEN-2 has a similar role in the formation of the gamma-secretase complex in mammalian cells, PEN-2 RNAi was performed by transfection of small interfering RNA (siRNA). Knockdown of PEN-2 expression in human HeLa cells results in the accumulation of highly stabilized presenilin 1 (PS1, also known as PSEN1) holoproteins accompanied by a decrease in the levels of PS1 fragments as well as of gamma-secretase activity, as evidenced by the accumulation of APP C83. It was confirmed that RNAi depletion of PEN-2 causes an accumulation of PS1 holoproteins in mammalian neuronal cells. Overexpression of human PEN-2 in Pen-2 RNAi-treated Drosophila S2 cells rescued the diminution in Psn fragments and attenuated the accumulation of Psn holoproteins (Takasugi, 2003).

Transient overexpression of Pen-2 was performed in S2 cells stably transfected with Aph-1 and Nct; an increase in the accumulation of Psn fragments was observed. To determine whether Pen-2, Aph-1, Nct and Psn function by directly binding to each other, co-immunoprecipitation studies were performed in CHAPSO-solubilized membrane fractions of S2 cells stably co-transfected with the three cofactors; any two of the components among Psn (holoprotein as well as fragments), Nct, Aph-1 or Pen-2 were found to be co-immunoprecipitated. In addition, Pen-2 was fractionated in the high-Mr ranges together with Psn, Nct or Aph-1 by glycerol velocity gradient centrifugation, suggesting that they function together and form a protein complex. Finally, it was found that secretion of Aß from S2 cells, as well as the total cellular gamma-secretase activity as determined by an in vitro Aß generation assay using recombinant APP C100 was significantly increased by the co-transfection of Pen-2, indicating that gamma-secretase activity is augmented by triple co-expression of Aph-1, Nct and Pen-2. Taken together, these data indicate that Pen-2 is a cofactor that is required for the final step of presenilin complex maturation after presenilin is stabilized in a holoprotein form and incorporated into a high-Mr protein complex, conferring gamma-secretase activities and leading to endoproteolysis of presenilin (Takasugi, 2003).

By combining RNAi inactivation and stable overexpression of cofactor proteins of Psn in Drosophila S2 cells, it was possible to dissect the process of high-Mr protein complex formation and the activation of gamma-secretase function of presenilin. Overexpression of Aph-1 is sufficient to elicit the stabilization and high-Mr complex formation of endogenous presenilin in a holoprotein form, although this high-Mr protein complex harbouring presenilin holoprotein is still inactive with respect to gamma-secretase. The final maturation step of the presenilin complex, which consists of presenilin fragments and demonstrates gamma-secretase activity, may then be mediated by the additional function of Pen-2 through directly interacting with the Psn-Aph-1-Nct complex1. This model is supported by the following observations: (1) Pen-2 RNAi facilitates the accumulation of stabilized Psn holoprotein, as is seen with Aph-1 overexpression; (2) Psn polypeptide accumulation is totally abolished by RNAi-mediated inactivation of Nct or Aph-1 expression; and (3) co-expression of Pen-2, Nct and Aph-1 renders the stabilized Psn holoprotein into the gamma-secretase-active fragment form. The latter result also suggests that APH-1, NCT and PEN-2 represent the set of major presenilin cofactors sufficient for the formation of the gamma-secretase complex; a large protease complex reminiscent of the proteasome that executes the cleavage of peptide bonds buried within the lipid bilayer. Further efforts to reconstitute gamma-secretase activity from recombinant proteins of the presenilin and the three cofactors in vitro will be needed to elucidate the configuration and function of presenilin complex and to develop therapeutic approaches to Alzheimer's disease based on inhibition of gamma-secretase (Takasugi, 2003).

The intracellular domain of the Frazzled/DCC receptor is a transcription factor required for commissural axon guidance

In commissural neurons of Drosophila, the conserved Frazzled (Fra)/Deleted in Colorectal Cancer (DCC) receptor promotes midline axon crossing by signaling locally in response to Netrin and by inducing transcription of commissureless (comm), an antagonist of Slit-Roundabout midline repulsion, through an unknown mechanism. This study shows that Fra is cleaved to release its intracellular domain (ICD), which shuttles between the cytoplasm and the nucleus, where it functions as a transcriptional activator. Rescue and gain-of-function experiments demonstrate that the Fra ICD is sufficient to regulate comm expression and that both γ-secretase proteolysis of Fra and Fra's function as a transcriptional activator are required for its ability to regulate comm in vivo. These data uncover an unexpected role for the Fra ICD as a transcription factor whose activity regulates the responsiveness of commissural axons at the midline and raise the possibility that nuclear signaling may be a common output of axon guidance receptors (Neuhaus-Follini, 2015).

This study has identify the Fra ICD as a transcription factor that regulates the expression of comm, a key modulator of axonal responsiveness at the midline. γ-secretase proteolysis of Fra releases its ICD, which is capable of nuclear translocation and is sufficient to promote midline crossing and regulate comm expression in rescue and gain-of-function assays in vivo. The conserved P3 motif within the Fra ICD functions as a transcriptional activation domain and this activity is required for Fra's regulation of comm expression. Thus, in addition to its canonical role signaling locally to regulate growth cone dynamics, Fra functions as a transcription factor to regulate axonal responsiveness at the midline (Neuhaus-Follini, 2015).

comm is expressed in commissural neurons with exquisite temporal specificity. How might the transcriptional activity of the Fra ICD be regulated to contribute to comm's expression pattern? γ-secretase proteolysis is typically the second cleavage event in a proteolytic cascade, preceded by ectodomain shedding. Indeed, previous pharmacological experiments suggest that DCC's ectodomain is shed as a result of metalloprotease cleavage and that this proteolytic event is required for subsequent γ-secretase-dependent processing. Metalloprotease-dependent ectodomain shedding is often ligand-dependent, while subsequent γ-secretase processing depends on the shape of the membrane-tethered metalloprotease cleavage product. For example, metalloprotease-dependent shedding of the Notch ectodomain is stimulated by the binding of Notch ligands, and the subsequent γ-secretase cleavage of the membrane-tethered ICD is constitutive. As Fra regulates comm independent of Netrins, Fra ectodomain shedding may occur in response to the binding of a different ligand. Alternative ligands for DCC have been identified, including the vertebrate- specific proteins Draxin and Cerebellin 4. In addition, the secreted protein MADD-4 physically associates with the C. elegans ortholog of Fra/DCC, UNC-40, and guides sensory neurons and muscle arms in an UNC-40-dependent manner. The function of the Drosophila ortholog of MADD-4, Nolo, has not been investigated, nor has its ability to bind to Fra (Neuhaus-Follini, 2015).

It seems unlikely that the transcriptional activity of the Fra ICD is controlled at the level of nuclear localization. When Fra ICDDP3 (lacking a NES) was expressed in the commissural EW neurons in vivo, it accumulates in the nucleus at the earliest developmental stages that can be observed, suggesting that the Fra ICD is constitutively imported into the nucleus. Nuclear accumulation of full-length Fra ICD (with a NES) was only observed occasionally, implying that after the Fra ICD translocates to the nucleus, it is rapidly exported. The fact that Fra's NES and activation domain are both encoded by P3 raises the possibility that when Fra is engaged in transcriptional activation, the association of co-activators with P3 might prevent it from associating with nuclear export machinery, coupling Fra's nuclear activity to its nuclear retention (Neuhaus-Follini, 2015).

The finding that Fra's ability to regulate comm expression depends on its function as a transcriptional activator seems to imply that the Fra ICD can associate with chromatin, but the Fra ICD does not contain an obvious DNA-binding domain. A Neo DNA-binding domain has not been identified either, but chromatin immunoprecipitation experiments have demonstrated that the Neo ICD associates with chromatin in vitro. The Fra ICD's DNA-binding activity and specificity probably arise from associations between the Fra ICD and DNA-binding partners, as is the case with Notch. The Notch ICD has no DNA-binding activity of its own and associates with DNA as part of a complex including an obligate CSL (CBF1/ RBPjk, Su(H), Lag-1) DNA-binding partner. If the Fra ICD can associate with multiple DNA-binding proteins, it might allow the Fra ICD to regulate the expression of many different target genes, depending on which of its DNA-binding partners are expressed in particular cell types or developmental contexts (Neuhaus-Follini, 2015).

The observation that a structurally intact P3 is required for Fra-dependent transcription suggests that P3 plays another role in Fra's transcriptional output besides its function as an activation domain. One possibility is that P3 is required for Fra's association with chromatin, perhaps by functioning as a binding interface for Fra's DNA-binding co-factors. This idea is supported by the observation that FraE1354A antagonizes midline crossing in both fra mutants and heterozygotes, while FraDP3 has only a mild effect. Perhaps the ICD of FraE1354A inhibits midline crossing by occupying chromatin sites that are normally targets of both Fra and other transcriptional activators that act in a parallel pathway; the ICD of FraDP3 would not have this effect if P3 is required for Fra's association with chromatin. FraE1354A is not likely to be inhibiting endogenous Fra in rescue experiments, as fra3 is either a strong hypomorphic or null allele. This model predicts that Fra has other transcriptional targets in EW neurons that are relevant for commissural axon guidance. It will be informative to identify additional transcriptional targets of Fra both in embryonic commissural neurons and in other cell types. In the retina, R8 photoreceptor axons have targeting defects that are much milder in Netrin mutants than in fra mutants, raising the possibility that the Netrin-independent output of Fra signaling in this system might be through the transcriptional pathway that this study has identified (Neuhaus-Follini, 2015).

Cleavage of axon guidance receptors has been shown to regulate the activities of these receptors in a number of different ways. Degradation of axon guidance receptors can provide temporal control of axonal sensitivity to guidance cues. In vertebrates, this mode of regulation controls axonal responsiveness to members of the class 3 family of secreted Semaphorins (Sema3s), which signal repulsion through Neuropilin (Nrp)/ Plexin (Plex) co-receptors. Calpain proteolysis of PlexA1 in pre-crossing spinal commissural neurons reduces their sensitivity to Sema3B, which is expressed in the ventral spinal cord as these axons are growing toward the ventral midline. ADAM metalloprotease cleavage of Nrp1 reduces the sensitivity of proprioceptive sensory axons to Sema3A allowing them to terminate in the ventral spinal cord, where Sema3A expression is high. In addition, γ-secretase proteolysis of DCC in vertebrate motor neurons inhibits their responsiveness to midline-derived Netrin, preventing them from ectopically projecting toward the midline (Neuhaus-Follini, 2015).

Proteolytic processing has also been implicated as a requisite step in local repulsive Robo signaling in Drosophila. The Robo ectodomain is cleaved by the ADAM metalloprotease Kuzbanian and this proteolytic event is required for Robo's ability to transduce repulsive signals in vivo and for Slit-dependent recruitment of effectors of local Robo signaling in vitro. As γ-secretase-dependent intramembrane proteolysis is typically constitutive following ectodomain shedding, and occurs subsequent to metalloprotease processing of the human Robo1 receptor, it is likely that Drosophila Robo is cleaved to produce a soluble ICD. The observation that Robo proteolysis is required for local Slit-Robo signaling does not exclude the possibility that the Robo ICD may also have a nuclear function that contributes to axon guidance, but this possibility has not yet been explored (Neuhaus-Follini, 2015).

Proteolysis has also been identified as a regulator of contact- mediated axonal repulsion. Eph receptors signal repulsion in response to their transmembrane ephrin ligands; ephrins can also function as receptors, signaling repulsion in response to Eph binding. Metalloprotease and subsequent γ-secretase cleavage of both Ephs and ephrins have been demonstrated, providing a mechanism through which adhesive interactions can be broken to allow for repulsive signaling. The importance of this mode of regulation for axon targeting has not yet been established in vivo and a recent study using an EphA4 variant that is insensitive to metalloprotease cleavage suggests that EphA4 proteolysis is not required for EphA4-dependent motor axon targeting (Neuhaus-Follini, 2015).

This study has identified a new way in which axon guidance receptor proteolysis can influence axon responsiveness to guidance cues. γ-secretase-dependent processing of Fra releases its ICD, which translocates to the nucleus, where it functions as a transcription factor to regulate the guidance of commissural axons. It is proposed that the ability to signal from the nucleus may be a common property of axon guidance receptors and may serve as a general mechanism through which axon guidance receptors regulate their own activities or the activities of other proteins. Human Robo1 is processed by sequential metalloprotease and γ-secretase cleavage and its ICD localizes to the nucleus in vitro. It remains to be seen whether the ICDs of Ephs and ephrins, which are cleaved by γ-secretase, and of Plexins, which are proteolytically processed, but have not yet been identified as γ-secretase substrates, translocate to the nucleus as well. It will also be interesting to determine whether the ICDs of Fra and other axon guidance receptors signal from the nucleus to regulate aspects of neuronal morphogenesis and function besides axon pathfinding. Finally, recent work indicating that the cleaved C terminus of the Drosophila Wnt receptor Frizzled translocates to the nucleus and contributes to the establishment of postsynaptic structures by regulating RNA export serves as a reminder that the trafficking of cell surface receptor fragments to the nucleus may allow these fragments to signal not only by regulating transcription, but in other ways as well (Neuhaus-Follini, 2015).



High levels of Presenilin mRNA are present during oogenesis in the nurse cells and the developing oocyte. Psn is also uniformly distributed in blastoderm stage embryos; this probably represents maternal mRNAs which have accumulated during oogenesis. When the segmentation of the embryo is apparent, the Psn hybridization signal is detected in a striped pattern. In third instar larval imaginal discs, Psn expression is restricted to the eye-antennae and the leg discs. In the eye-antennal disc, Psn mRNA accumulates in the precursor of the postoccipital sensilla of the antennae and in areas of the eye disc corresponding to progenitors of the ocellus and the ocellar bristles. In the leg discs Psn is restricted to the posterior compartment, specifically in areas that give rise to the adult tibia and femur. This dynamic expression pattern for Psn suggests that it may have multiple roles during development (Boulianne, 1997 and Marfany, 1998).

Drosophila Presenilin is widely expressed throughout oogenesis, embryogenesis, and imaginal development, and generally accumulates at comparable levels in neuronal and nonneuronal tissues. During cellularization, Psn is present in all cells of the embryo, including pole cells, and appears to be enriched near cell membranes. During gastrulation, Psn expression is fairly ubiquitous but higher levels are apparent in ectoderm relative to mesoderm. Double immunolabeling with Notch antibodies reveals that Presenilin and Notch are coexpressed in many tissues throughout Drosophila development and display partially overlapping subcellular localizations, supporting a possible functional link between Presenilin and Notch (Ye, 1998).

To examine the processing as well as the cellular and subcellular distribution of Psn during development, polyclonal antibodies were raised to the N- and C-terminal domains of Psn. Psn is found to be ubiquitously expressed throughout development. The high levels of expression observed in 0-2 hr embryos appear to result from large maternal contributions. In wild-type extracts the majority of endogenous Psn protein is proteolytically cleaved to an ~25 kDa N-terminal fragment and an ~35 kDa C-terminal fragment. However, full-length Psn protein (~60 kDa) can be detected in transgenic flies that overexpress Psn from a heatshock promoter or by the GAL4/UAS system.. The fact that presenilins are processed in worms, flies, and vertebrates suggests that the cellular machinery involved in this processing must be highly conserved between species. Using immunocytochemical techniques, Psn is widely distributed during development, although some tissues, including the larval CNS, express higher levels of Psn. Within the CNS, Psn is mainly found in the axons and cell bodies of neurons and primarily within the cytoplasm (Guo, 1999).

Larval and Adult

During oogenesis, Psn is expressed mainly in nurse cells and follicle cells throughout oogenesis. Vesicular staining is seen throughout the anterior germarium region of each ovariole and is limited to nurse cells and follicle cells of late-stage egg chambers. Psn protein is hardly detected within the oocyte in a stage 11 egg chamber: this may simply reflect poor antibody penetration of this tissue. High levels of Psn are seen near membranes of the follicle cells and nurse cells, whereas lower levels are detected in the cytoplasm of the nurse cells. In nurse cells, membrane staining appears to be confined to the apical membranes and pronounced Psn accumulation is seen around the nuclei (Ye, 1998).

Psn is primarily localized in the cytoplasm of immature photoreceptor neurons and is preferentially localized near the apical membranes of the imaginal columnar epithelial cells. Psn localization at the apical membranes of imaginal wing discs results in prominently stained disc surface folds in histological preparations. Psn is expressed at high levels in cells that give rise to the interommatidial bristles of the adult eye. Psn is expressed at a very high level in certain regions of the larval and pupal brain. Psn expression is enhanced in part of the optic lobe anlagen, presumably in undifferentiated neurons that give rise to optic lobe structures of the adult brain. Intense Psn expression in the developing medulla, an optic lobe structure, is readily visible as an intensely labeled crescent-shaped structure proximal to the pupal eye disc. In most cases, Psn colocalizes with Notch, but there exceptions. For example, although both proteins are found near the cell membranes of most tissues, the Psn level in the cytoplasm is relatively higher than Notch, especially in larval discs and the pupal retina. Largely nonoverlapping vesicular staining for both proteins is seen in the larval eye disc. In general, Notch is more highly restricted to the apical cell region and more tightly associated with cell membranes, clearly outlining the apical profiles of the cells when visualized by confocal optical sectioning. In contrast, Psn is detected at more equivalent levels both in the cytoplasm and at the cell membrane, such that Psn immunostaining appears to be more diffuse and graded than Notch in the same cell types (Ye, 1998).

A novel interaction between hedgehog and Notch promotes proliferation at the anterior-posterior organizer of the Drosophila wing

Notch has multiple roles in the development of the Drosophila melanogaster wing imaginal disc. It helps specify the dorsal-ventral compartment border, and it is needed for the wing margin, veins, and sensory organs. Evidence is presented for a new role: stimulating growth in response to Hedgehog. This study shows that Notch signaling is activated in the cells of the anterior-posterior organizer that produce the region between wing veins 3 and 4, and strong genetic interactions are described between the gene that encodes the Hedgehog pathway activator Smoothened and the Notch pathway genes Notch, presenilin, and Suppressor of Hairless and the Enhancer of split complex. This work thus reveals a novel collaboration by the Hedgehog and Notch pathways that regulates proliferation in the 3-4 intervein region independently of Decapentaplegic (Casso, 2011).

This article shows activation of N signaling at the wing AP organizer by defining with cellular resolution the expression patterns of N protein and N pathway reporters in relation to the AP organizer, and dependence on Hh signaling is shown. Strong interactions are also shown between hh- and N-signaling pathways, and it is confirmed that the activation of N signaling is necessary for the normal growth of the AP organizer. This work uncovers a previously unknown activity of the Hh pathway in mitogenesis at the AP organizer: the activation of N signaling. These results are surprising in that they show that the roles of N signaling in the growth of the wing are not limited to the function of the DV organizer and a general growth-promoting function in the wing: N signaling also induces growth downstream of hh at the AP organizer (Casso, 2011).

N is essential for the cells that give rise to the DV margin, veins, and sensory organs of the wing, and its expression is elevated in the progenitors that produce these structures. The DV margin progenitors, which transect the wing disc in a band that is orthogonal to the Hh-dependent AP organizer, express wg in response to N. These wg-expressing cells function as a DV organizer, and several lines of evidence suggest that the AP and DV organizers function independently: Hh signaling along the AP axis is not N-dependent, N signaling along the DV axis is not hh-dependent, and targets regulated by the AP and DV organizers are not the same. The findings reported in this study show that, separately from its roles elsewhere in the wing disc, N signaling has an essential mitogenic role in the cells of the AP organizer region (Casso, 2011).

While N can stimulate growth by inducing expression of wg (as it does in the DV organizer), hyper-activation of N signaling near the AP border of the wing pouch causes overgrowth that is independent of wg. wg is not normally expressed along the AP axis, but this study found that N signaling is activated at the AP compartment border in late third instar discs, pupal discs, and pupal wings. Through vn expression, Hh signaling at the AP compartment border increases expression of Dl flanking the organizer, and Hh signaling activates N in the 3-4 intervein region. While a role for Ser at the AP organizer has not been directly investigated, Ser expression in the wing disc is very similar to that of Dl, with high levels of Ser in the vein 3 and 4 primordia as well as along the DV border. The results show that growth of the 3-4 intervein region, long known to be dependent on Hh, is also dependent on Hh-induced activation of N (Casso, 2011).

Expression of N pathway reporters and components and genetic interactions support this model of regulation of the intervein region. The reporters Su(H)lacZ and E(spl)m-α-GFP express at the AP border in a Hh-dependent manner. Elevated levels of N protein expression on the anterior side of the AP border require Vn signaling. This N region is flanked by Dl expression in the vein 3 and vein 4 primordia; Dl expression is known to be dependent upon expression of the Hh target vn. Genetic interactions between smo RNAi and N and between smo RNAi and N pathway components [e.g., the Psn intramembrane protease, which activates N; the Su(H) transcriptional co-activator; the Su(dx) E3 ubiquitin ligase, which monitors levels of N protein; and the E(spl) complex of N transcriptional targets] also indicate a functional link between the Hh and N systems (Casso, 2011).

The model for the role of N in the 3–4 intervein region is consistent with previous reports of expression patterns of the E(spl) genes E(spl)m8, M-β, and M-α. Ectopic expression of HLH-mδ and m8 rescues smo RNAi. Although HLH-mδ does not appear to be expressed in the AP organizer in a wild-type wing because the E(spl) genes are thought to have partially overlapping functions, the fact that mδ phenocopies the rescue by m8 reinforces the conclusion that the function of the E(spl) genes is critical to inducing growth at the AP organizer. Importantly, these findings show that the cells that activate N are the anterior cells of the AP organizer and are not associated with development of veins in pupal wings. Vein 4 develops within the posterior compartment and in many cases has posterior cells between it and the AP border. Since activation of these reporters was never observed extending into posterior territory, their expression correlates better with the position of the AP organizer than with vein/intervein territories at the stages that were examined. It should be noted that no single readout currently available marks all tissues in which N is activated. The E(spl) genes, for example, express in a variety of spatial and temporal patterns in response to N, and these patterns are only partially overlapping. The possibility cannot be excluded that N signaling is also activated along the stripe of Dl expression in the vein 3 primordium or that signaling could be occurring in the entire broad stripe of elevated N expression in the AP organizer. No changes were seen in proliferation using a direct readout such as phosphohistone staining of mitotic cells to visualize increases or decreases in growth at the AP organizer. These proliferation assays mark cell cycle progression at a single time point in fixed tissues, and the changes that were seen in the adult wing could be due to one or two fewer cell division cycles occurring over the course of days of development (Casso, 2011).

The findings indicate a link between the Hh and N pathways and suggest a model in which the domain of N activation at the AP border [manifested by Su(H)lacZ expression] is a consequence both of flanking cells that express high levels of Dl and of Hh signaling. The proposed role for Hh signaling is multifaceted: Hh is required for vn expression, which is itself required for high levels of Dl expression in the vein 3 stripe and the vein 4 stripe and for N expression at the AP organizer. Although whether Dl expression in veins 3 and 4 activates N signaling has not been directly tested, vn function is necessary for N activation, and the reciprocal relationship between cells expressing high levels of Dl and neighboring cells expressing high levels of N is well established (Casso, 2011).

Interactions between the Sonic hedgehog (SHH) and N signaling pathways have been identified previously in vertebrates. Particularly noteworthy for their relevance to the interactions that were found in the Drosophila wing disc are the increased expression of the Serrate-related N ligand, Jagged 1, in the mouse Gli3Xt mutant; reduced expression of Jagged1 and Notch2 in the cerebella of mice with reduced SHH signaling; regulation of the Delta-related ligand, DNER, by SHH in Purkinje neurons and fetal prostate; activation of N signaling in neuroblastomas in Ptch+/– mice with elevated SHH signaling; and Notch2 overexpression in mice carrying an activated allele of smo. These studies establish a positive effect of SHH signaling on the N pathway, consistent with the current data (Casso, 2011).

In Drosophila, there have been several reports of interactions between the N and Hh pathways. In the wing pouch, for example, expression levels of the Hh targets ptc, ci, col, and en are markedly lower at the intersection of the AP and DV borders than elsewhere in the AP organizer. This repression is mediated by wg. In addition, N and col function together to determine the position of wing veins 3 and 4. However, loss of function of either col or vn did not show interactions with smo RNAi (Casso, 2011).

N functions in two types of settings. One is associated with binary fate choices; it involves adjacent cells that adopt either of two fates on the basis of the activation of N signaling in one cell and inactivation in the other. In these settings, activation of N not only induces differentiation in a designated cell, but also blocks activation of N in the neighbors. The second type of setting does not induce a binary fate choice, but instead activates the pathway at the junction of two distinct cell types. N pathway activation at the DV border in the wing is one example; in this setting, N is activated in a band that straddles the DV border and the N ligands Dl and Ser signal from adjacent domains from either the dorsal (i.e., Dl) or the ventral (i.e., Ser) side. Activation of N in the 3-4 intervein region at the AP border appears to be of this second type: it occurs adjacent to regions of elevated Dl expression at the apposition of anterior and posterior cell types. There is no apparent binary fate choice in this region of the wing (Casso, 2011).

In ways that are not understood well, development of the 3-4 intervein region is controlled differently from other regions of the wing pouch. Whereas Hh induces expression of Dpp, and Dpp orchestrates proliferation and patterning of wing pouch cells generally, Dpp does not have the same role in the 3-4 intervein cells. For these cells, Hh appears to control proliferation and patterning directly. For example, the lateral regions of wings that develop from discs with compromised Dpp function are reduced, but their central regions, between veins 3 and 4, are essentially normal. Downregulation of Dpp activity and repression of expression of the Dpp receptor appears to be the basis for this insensitivity. In contrast, partial impairment of Hh signal transduction that is insufficient to reduce Dpp function (such as in fu mutants or in the smo RNAi genotypes that were characterized) results in wings that are normal in size and pattern except for a small or absent 3-4 intervein region. Since the 3-4 intervein cells divide one to two times in the early pupa during disc eversion and wing formation, the direct role of Hh in regulating these cells may be specific to this post-larval period. N signaling has a well-described mitogenic function in the wing. Ectopic signaling causes hyper-proliferation, while clones that impair the activation of the pathway reduce growth. The current findings indicate that Hh regulates proliferation of cells in the 3-4 intervein region at least in part by activating N signal transduction (Casso, 2011).

The idea that this model promotes is that Hh-dependent activation of N at the AP organizer is stage- and position-specific. This model is consistent with the complex pattern of N expression and activation in the wing, since different pathways may regulate N in different locations. It is also consistent with the proposed role of N regulating the width and position of veins 3 and 4, since the processes that establish the veins and control proliferation of the intervein cells need not be the same, even if they are interdependent. The temporal specificity that this study describes represents an example of how complex patterns are generated with a limited number of signaling pathways -- in this case by using N signaling for different outcomes at different times and in different places. Throughout larval development, Dpp regulates proliferation and patterning in the wing disc. In the pupal wing, Dpp takes on a new instructive vein-positioning function. There is no evidence that Hh regulates Dpp in the pupal wing, and moreover, the cells that had produced Dpp at the AP organizer no longer do so and no longer function as a AP organizers. These data show that N also takes on a new role during late larval and pupal stages: functioning at the AP organizer to regulate growth in response to Hh signaling (Casso, 2011).


Clones of Psn minus cells in the wing cause scalloping and vein thickening, reflecting failures in well defined signaling events that depend on Notch, such as the specification of Wingless-secreting margin cells along the dorso-ventral compartment boundary and the lateral inhibition of vein differentiation (Struhl, 1999).

Genetic characterization of cytological region 77A-D harboring the Presenilin gene of Drosophila

A systematic lethal mutagenesis of the genomic region uncovered by Df(3L)rdgC-co2 (cytological interval 77A-D) has been carried out to isolate mutations in the single known Presenilin (Psn) gene of Drosophila. Because this segment of chromosome III has not previously been systematically characterized, inter se complementation testing of newly recovered mutants was carried out. A total of 79 lethal mutations were isolated, representing at least 17 lethal complementation groups, including one corresponding to the Psn gene. Fine structure mapping of the genomic region surrounding the Psn transcription unit by transgenic rescue experiments allowed the localization of two of the essential loci together with Psn within an ~12-kb genomic DNA region. One of these loci, located 3' to Psn, encodes a Drosophila protein related to the yeast 60S ribosomal protein L10 precursor. It was also determined which of the newly recovered lethal mutant groups corresponded to previously isolated lethal P-element insertions, lethal inversion breakpoints, and/or lethal polo gene mutants. Point mutations were identified in all five recovered Psn alleles, one of which results in a single amino acid substitution G-E at a conserved residue in the C-terminal cytoplasmic tail of the protein, suggesting an important functional role for this Presenilin C-terminal domain. In addition, some viable mutations were recovered in the screen, including new alleles of the clipped and inturned loci (Lukinova, 1999).

Sequence analysis confirms that all five Psn mutants bear lesions in the Psn gene as deduced from the genomic DNA rescue experiments. PsnC4, PsnS3, PsnI2, and PsnK2 are predicted to encode prematurely truncated Presenilins, in agreement with genetic studies suggesting that these mutant Psn alleles are likely to represent strong or complete loss-of-function mutants. PsnB3 is predicted to cause an amino acid substitution at a conserved residue of the C-terminal region of Presenilin, directing attention to this portion of the molecule. Most Alzheimer's disease-associated mutations in the human Presenilins occur in either transmembrane domain 2 (TM2) or in an N-terminal stretch of the large hydrophilic loop between TM6 and TM7; only one recently discovered mutation maps to the C-terminal tail of Psn1. In the case of PsnB3, a single amino acid substitution (G516E) at a conserved position 26 residues before the C-terminus results in a complete or nearly complete loss of Presenilin function. The affected glycine residue presumably plays an important role in normal Presenilin function, perhaps as part of a protein-protein interaction site or by contributing to the conformation of a functionally important C-terminal Presenilin domain. The significance of the C-terminal tail of Presenilin family members for the normal biological functions of these proteins will require further genetic and molecular analysis (Lukinova, 1999 and references therein).

Apoptotic activities of wild-type and Alzheimer's disease-related mutant Presenilins in Drosophila melanogaster

Larvae mutant for Psn secrete a pupal case and complete the last stages of larval development but do not form any adult structures: instead, they collapse into a homogeneous oily mass within the pupal case. This zygotic lethal null phenotype closely resembles that observed for Suppressor of Hairless [Su(H)], which encodes an effector of the Notch signaling pathway. (Ye, 1999).

Formation of the dorsal/ventral (D/V) boundary along the presumptive wing margin requires Notch activity, and may be visualized by the expression of specific reporter genes in the margin zone of the larval wing disc. Expression of cut-lacZ along the wing margin is abolished in Psn mutant wing discs. Similarly, margin zone expression of a wingless-lacZ construct and a vestigial (vg) D/V enhancer-lacZ transgene is absent in the Psn mutant wing disc, whereas lacZ expression outside the wing pouch persists in the mutants. Expression of a vg quandrant enhancer-lacZ transgene, which is specific to the non-margin zones of the wing pouch, is also completely absent in the Psn mutant wing disc. Expression of these wingless and vestigial gene reporters along the wing margin and in the pouch region is directly dependent on Notch signaling activity. The vg D/V enhancer contains a critical binding site for the Su(H) protein. The absence of margin structures in the Psn mutant wing disc, together with the overall reduction in size of the mutant wing pouch region, is reminscent of conditional Notch mutant and Su(H) null mutant phenotypes (Ye, 1999).

Histochemical characterization of Psn mutant wing discs using the early SOP markers achaete-lacZ and scabrous-lacZ reveals clusters of supernumerary SOP cells arising at certain normal SOP locations, as seen in typical neurogenic mutants. Lateral inhibition within these proneural cell clusters is severely impaired in Psn mutant discs, resulting in enlarged SOP territories and increased proneural achaete-lacZ and scabrous-lacZ activity. Embryos derived from females that lack both maternal and zygotic Psn activity display a Notch-like lethal hyperplasia of the embryonic nervous system, and the maternal phenotype is only weakly rescued by wild-type paternal zygotic Psn function (Ye, 1999).

Mutant human presenilins cause early-onset familial Alzheimer's disease and render cells susceptible to apoptosis in cultured cell models. Loss of presenilin function in Drosophila increases levels of apoptosis in developing tissues. Moreover, overexpression of presenilin causes apoptotic and neurogenic phenotypes resembling those of Presenilin loss-of-function mutants, suggesting that presenilin exerts a dominant negative effect when expressed at high levels. In Drosophila S2 cells, Psn overexpression leads to reduced Notch receptor synthesis affecting levels of the intact ~300-kD precursor and its ~120-kD processed COOH-terminal derivatives. Presenilin-induced apoptosis is cell autonomous and can be blocked by constitutive Notch activation, suggesting that the increased cell death is due to a developmental mechanism that eliminates improperly specified cell types. A genetic model is described in which the apoptotic activities of wild-type and mutant presenilins can be assessed, and it is found that Alzheimer's disease-linked mutant presenilins are less effective at inducing apoptosis than wild-type presenilin (Ye, 1999).

Although the Alzheimer's disease-linked Psn mutations (Psn referring to human presenilin) are not complete loss-of-function alleles, it is not clear if they are partial loss-of-function or gain-of-function mutations. All Alzheimer's disease-linked mutations occur at amino acid residues that are conserved in the Drosophila Psn protein, allowing these mutations to be introduced into the fly protein and their apoptotic effects assessed in transgenic animals. Four missense mutations, N141I, M146V, L235P, and E280A were tested in this manner. The N141I mutant of PS2 has been shown to induce more apoptosis in certain cell types, compared with wild-type PS2, and may thus represent a gain-of-function mutation. Both the M146V and E280A mutants of PS1 increase the ratio of Abeta42/Abeta40 by increasing the level of the more neurotoxic and amyloidogenic Abeta42 cleavage product of amyloid precursor protein. The L235P mutation has been identified in a family with an onset of Alzheimer's disease as early as age 29, and may therefore represent a particularly severe mutant form of presenilin. In addition, a truncated Psn protein named D-ALG3, consisting of the most COOH-terminal 100 amino acids, two loop constructs consisting of either the long or short variable hydrophilic loop, and transmembrane domain 7 (TM7) following the loop were also included in this analysis. These constructs were chosen because D-ALG3 resembles truncated mammalian PS proteins that confer resistance to cell death in PC12 cells, and the loop may be a cytoplasmic domain required for protein-protein interactions (Ye, 1999).

The apoptotic activities of mutant presenilins were assessed by the following criteria: their ability to generate a rough eye phenotype in flies bearing two copies of each mutant transgene and GMR-GAL4; their ability to modify the eye phenotype of GMR-GAL4, 2X UAS-Psn+14 flies; and their ability to modify the eye phenotype of flies expressing the Drosophila death-domain protein Reaper under GMR promoter control. At least five independent transgenic lines were analyzed for each construct. Only the M146V substitution produced a rough eye phenotype in two independent transgenic lines, out of the five analyzed. The remaining mutations and Psn fragments fail to produce rough eye phenotypes when expressed under GMR-GAL4 control, but all four Alzheimer's disease-associated mutants enhance the rough eye phenotype of GMR-GAL4, 2X UAS-Psn+14 flies. M146V and N141I also enhance the phenotype of flies bearing GMR-reaper. Although the ALG-3 fragment of PS2 inhibits programmed cell death in PC12 cells, the equivalent segment of Drosophila Psn, D-ALG3, instead weakly enhances the GMR-GAL4, 2X UAS-Psn+14 phenotype, indicating that it may possess weak apoptotic activity. The two loop variant fragments possess no modifying activity in these transgenic assays (Ye, 1999).

To gain further insight into the mechanism of Psn-induced cell death, an attempt was made to suppress this apoptosis by coexpression of Drosophila cell death inhibitors (DIAP1 or DIAP2) or the baculoviral survival factor p35. DIAP1 and DIAP2 are Drosophila homologs of baculoviral inhibitor of apoptosis proteins (IAPs), and can block programmed cell death induced by proapoptotic factors or mutations. Baculoviral p35 protein inhibits caspases and thus blocks apoptosis in many species. In the presence of any of these antiapoptotic proteins, the Psn-induced rough eye phenotype is largely suppressed, as revealed by the more regular external eye surface, the more normal trapezoidal pattern and orientation of R1-6 photoreceptor cells, and a restoration of the pigment cell lattice between ommatidia. The observation that Psn-induced rough eye phenotypes are suppressed by coexpressing either DIAP1, DIAP2, or p35 confirms that increased cell death is the main cause of the rough eye phenotype. Psn-induced programmed cell death may thus be mediated by the conserved caspase pathway of apoptosis or, alternatively, it may be circumvented by inhibition of this pathway (Ye, 1999).

The ability of wild-type and, to a lesser extent, mutant forms of Drosophila Psn to induce low levels of apoptosis similar to the apoptotic levels seen in developing imaginal tissues of Psn loss-of-function mutants suggests that the apoptotic effects of Psn may be a secondary consequence of reduced or dominant-negative Psn activity during developmental patterning. In C. elegans and mice, presenilin proteins have been shown to facilitate Notch signaling, and worms or mice lacking presenilin activity display typical Notch or lin-12/glp-1 loss-of-function phenotypes. Similarly, flies lacking functional Psn gene activity exhibit embryonic neurogenic phenotypes and imaginal disc phenotypes that are characteristic of impaired Notch signaling. Moreover, elevated levels of apoptosis have been noted previously in wing imaginal discs of flies having the partial loss-of-function heteroallelic Notch genotype Nts/N55e11; neurA101/+. These observations raise the possibility that the apoptotic effects of Psn overexpression may be due to a primary interference with Notch signaling, followed by elimination of cells that have not adopted their proper cell fate by a normal corrective mechanism of developmentally controlled apoptosis (Ye, 1999).

The in vivo genetic model for Psn-mediated apoptosis allowed for an examination of the potential involvement of Notch signaling in the apoptotic response, an important issue that has not been possible to assess in the widely used mammalian cell culture assays for Psn-induced apoptosis. First, it was determined if the UAS-Psn constructs that cause apoptosis in the Drosophila eye when driven by GMR-GAL4 are able to produce Notch pathway phenotypes in other tissues when expressed using suitable GAL4 driver constructs. Several GAL4 driver lines that are active in the wing and cuticle anlagen are indeed capable of producing adult Notch-like phenotypes in the wing blade and thorax, including wing margin notching, vein thickening, ectopic wing margin bristles, ectopic wing vein campaniform sensilla, ectopic thoracic macrochaetae, and missing thoracic microchaetae. These phenotypes are consistent with the notion that Psn overexpression leads to dominant-negative effects, since Psn loss-of-function mutants exhibit similar Notch-like phenotypes. To determine if Psn-induced apoptosis might be an indirect effect of reduced Notch activity, apoptosis was analyzed in imaginal wing discs of the conditional temperature-sensitive Notch mutant Nts1. Increased levels of programmed cell death are spatiotemporally correlated with progressive loss of Notch activity as visualized by reduced wing-pouch-specific expression of the Notch target gene reporter vg(quadrant enhancer)-lacZ and expansion of proneural cell clusters positive for ac-lacZ expression in the presumptive notum region, suggesting that developmental patterning defects caused by reducing Notch activity directly lead to elimination of affected cells by apoptosis. The next step involved testing whether reduction in the dosage of the wild-type Notch gene or coexpression of constitutively activated Notch is able to suppress or enhance Psn-related apoptotic phenotypes. The rough eye phenotype of GMR-GAL4, 2X UAS-Psn+14 is strongly enhanced in an N54l9 mutant background bearing only one functional copy of the Notch gene. Apoptosis caused by either Psn overexpression or removal of Psn gene function is also dramatically suppressed by coexpression of constitutively activated Notch in the retina. These studies show that genetic removal or overexpression of Psn in developing Drosophila tissues is able to induce Notch-like phenotypes, as well as apoptosis, and that when genetic methods are used to compensate for effects on Notch signaling, the levels of apoptosis are dramatically reduced. These results, together with the observed correlation between impaired Notch signaling and high levels of developmental apoptosis, offer a potential explanation of Psn-mediated apoptosis as a developmental response to a primary failure in cellular patterning events requiring Psn activity for proper Notch synthesis or signaling (Ye, 1999).

To further elucidate the molecular mechanism underlying the Psn overexpression phenotypes, Notch processing and trafficking was examined in S2 cells cotransfected with Psn and Notch. When expressed before Notch induction, wild-type Psn and the various Psn mutants lead to reduced Notch protein levels, affecting both the full-length and the processed COOH-terminal fragments of Notch. In agreement with genetic results, the biochemical effect appears to be more pronounced for wild-type Psn than for the mutant forms. The expression of a nonmembrane-bound control protein, Suppressor of Hairless, is not affected by either the wild-type or the mutant Psn proteins. The effect on Notch synthesis is unlikely to be due to increased protein degradation, because ectopic expression of the same Psn construct in S2 cells after Notch induction has no detectable effect on Notch protein levels. In addition, live cell surface immunostainings reveal that Notch protein is trafficked and inserted into the cell membrane normally in spite of the reduced protein levels caused by Psn overexpression (Ye, 1999).

Thus, the proapoptotic activity of different Alzheimer's disease-linked mutant variants has been assessed and it has been found that these variants possess less apoptotic activity than wild-type presenilin, consistent with the idea that this class of presenilin mutations represents partial loss-of-function mutations in nematodes and transgenic mice. In contrast to previous studies showing that Alzheimer's disease-associated missense mutations enhance the ability of PS1 and PS2 to induce cell death and that the Psn COOH-terminal ALG-3 fragment blocks the proapoptotic activity of PS2 , the corresponding missense mutants of fly Psn induce less cell death than wild-type Psn, and the Drosophila Psn COOH-terminal D-ALG3 fragment appears to induce apoptosis only weakly in vivo. In view of the dominant-negative role that is suggested for overexpressed Psn, the reduced apoptotic activity of these mutant variants may reflect a decreased ability to interfere with endogenous presenilin function or a more rapid clearance of misfolded mutant proteins from the ER/Golgi compartment. Analogous partial loss-of-function effects on Abeta secretion of human mutant presenilins may be relatively weak under physiological conditions, but may contribute to gradual deterioration of brain tissue during the aging process and eventually trigger disease onset when neuronal loss reaches a certain threshold (Ye, 1999).

Drosophila presenilin is required for neuronal differentiation and affects Notch subcellular localization and signaling

Recent studies have suggested a role for presenilins in the Notch-signaling pathway, but their specific function within this pathway remains unclear. The Drosophila Presenilin gene and protein have been characterized and their interaction with Notch has been studied in both mutants and transgenics. The Drosophila Psn protein is proteolytically cleaved and broadly expressed during development with the highest levels in neurons within the larval CNS. Mutations in Drosophila Psn genetically interact with Notch and result in an early pupal-lethal phenotype characterized by defects in eye and wing development and incomplete neuronal differentiation within the larval CNS. Moreover, processing of Notch in the Golgi by the furin protease is unaffected in Psn mutants and Notch is present and may even accumulate on the plasma membrane of neuroblasts in the larval CNS of Psn mutants. In contrast, overexpression of Psn in transgenics causes Notch to accumulate in the cytoplasm. Taken together, these results indicate that Drosophila Psn is required for proper neuronal differentiation and may regulate the subcellular localization of Notch proteins within cells, necessary for Notch accumulation and subsequent signaling capabilities (Guo, 1999).

To examine the function of Psn, a series of overlapping deletions was generated by imprecise P-element excision of P4, a homozygous viable P-element insertion located ~1 kb 3' from the Psn gene. Six deletions were generated, three of which remove portions of the Psn coding region (PSW20, DpsW6, and PSW11). All of the deletions fail to complement one another, and none produce any detectable Psn protein as determined by Western blot analysis on extracts from mutant larvae. The largest deletion (PSW20) deletes the Psn gene as well as portions of the lipoic acid synthase and the 50 S ribosomal protein L15 genes and is embryonic lethal. PSW11 and PSW6 that delete Psn and the 50S ribosomal protein L15 genes are lethal during the second larval instar, and these larvae are small and grow at a much slower rate (Guo, 1999).

In addition to the deletion mutants in Psn two EMS alleles, PS30 and PS46 have been characterized. These EMS alleles were originally identified in a lethal screen over the deficiency Df(3L)ri-79c/TM3 that uncovers the Psn locus and has break points at 77B-C;77F-78A. Both EMS lines fail to complement each other as well as the two Psn deletions tested, PSW6 and PSW11. To determine the molecular basis of these mutations, both PS30 and PS46 were sequenced. To date, no mutation within the coding region of PS30 could be detected and alterations in surrounding regulatory sequences are being sought. A single missense mutation was found in PS46, consisting of a proline to leucine substitution at amino acid 507 within the highly conserved C-terminal domain of Psn (Guo, 1999).

To address the function of PS during development, a test was made of the phenotype in flies of the genotype PS46/PSW6 or PS46/PSW11, that is, flies that are mutant for Psn but wild-type for lipoic acid synthase and hemizygous for the 50 S ribosomal protein L15 gene. These mutants are early pupal lethal; the third instar larvae form a pupal case, but no adult structures develop. When the phenotype of the mutants is examined during the late third larval instar stage, the mutant larvae have underdeveloped eye and wing imaginal discs. Although the eye imaginal disc forms, it fails to undergo proper neuronal differentiation. The wing imaginal disc is also smaller with the most severe defects observed in the region that will give rise to the wing blade. Furthermore, Wingless expression, which is normally detected in two domains within the developing disc, is disrupted in Psn mutant discs. In contrast, both the antennae and leg imaginal discs appeared normal. Both the lethality as well as the wing and eye phenotypes could be rescued using a Psn transgene driven by a heatshock promoter. To determine whether there was any evidence of neuronal differentiation within the eyes and optic lobes of these pupal lethal mutants, the expression of two neuronal markers, Elav and Cut, that are broadly expressed in neurons during postembryonic development, were examined. In wild-type eye-antennae imaginal discs of third larval instars, Elav is expressed in photoreceptor neurons. Cut is absent from the eye disc at this stage but is broadly expressed in the antennae disc. At later stages, Cut can be detected in cone cells and sensory organ precursors that give rise to interommatidial bristles. Wild-type discs stain positively for Elav (eye) and Cut (antennae), whereas mutant eye imaginal discs appear small and underdeveloped and fail to express Elav. Furthermore, although both Cut and Elav could be detected in the developing optic lobes, the highly organized, laminar pattern of expression that is normally found in wild-type optic lobes fails to develop in Psn mutants. This suggests that Psn is required for eye and wing imaginal disc development, and although it is not required for the development of the optic lobes, they fail to differentiate properly without it (Guo, 1999).

To examine the link between Psn and Notch, the ability of Psn mutants to interact genetically with Notch was examined. Specifically, each Psn mutant was crossed to several alleles of Notch and dosage-sensitive interactions were sought in transheterozygotes. An examination was made of the ability of the point mutant PS46 and the deletions PSW6, PSW11, and PSW20 to interact with the deficiency Df(1)N-8/In(1)dl-49, y1 Hw1 m2 g4. These interactions give rise to dominant notching of the wing blade. Transheterozygotes containing one copy of the Notch deficiency and one copy of the PS46 mutation or a Psn deletion all exhibit an enhanced wing phenotype. In addition, both Psn alleles enhance the phenotype of Nnd-3, a loss-of-function allele of Notch that gives rise to a thickened wing vein phenotype at 29°C. Specifically, transheterozygotes between Nnd-3 and Psn alleles have enhanced thickening of wing veins and notches at the tips of the wing similar to what is observed in stronger Notch alleles. This suggests that Psn mutations reduce Notch signaling within cells. In agreement with this, Psn alleles suppress the interrupted wing vein phenotype observed in the gain-of-function NAx-1 allele. Finally, Psn mutants also enhance the phenotype of the Delta allele Dl[7] that is characterized by thickened wing veins and small deltas located near cross-veins. Specifically, transheterozygotes between Dl[7] and Psn mutants exhibit a greater extent of thickening and deltas at the wing veins. Taken together, these results indicate that Psn mutations reduce Notch signaling and support a role for Psn in the Notch-signaling pathway (Guo, 1999).

To gain insight into the mechanism underlying Psn and Notch interactions, immunocytochemical studies were performed to examine the subcellular distribution of Notch within the larval CNS of Psn mutants. Using an antibody that recognizes the Notch intracellular domain, it has been found that Notch is expressed at high levels within neuroblasts throughout the proliferative centers in the developing optic lobes and at somewhat lower levels in neuroblasts within the thoracic ganglia of third larval instar CNS. Within wild-type neuroblasts, Notch is distributed throughout the cytoplasm and on the plasma membrane. In contrast, the overall distribution of Notch within the optic lobes is disrupted in Psn mutants, and the protein levels are reduced in the cytoplasm and appear to be preferentially retained on the plasma membrane of specific neuroblasts. To distinguish between primary effects of Psn on Notch localization versus secondary defects caused by altered differentiation of Psn mutants, the distribution of Notch protein within neuroblasts of second instar larvae was examined. At this stage, Notch is expressed in small groups of cells that consist of a large neuroblast surrounded by smaller ganglia mother cells. Within these groups of cells Notch appears throughout the cytoplasm and at high levels at the plasma membrane in regions of contact between the neuroblast and its progeny. There is little, if any, accumulation of Notch at the plasma membrane in regions that contact the remaining surrounding cells. In contrast, Notch distribution is altered with less in the cytoplasm and more staining seen over the entire plasma membrane of neuroblasts in Psn mutants. In agreement with this observation, processing of Notch, found to give rise to a functional heterodimeric receptor on the cell surface, is unaffected in Psn mutants (Guo, 1999).

The reciprocal experiment was performed to determine whether overexpression of Psn also affects the subcellular distribution of Notch. Specifically, the GAL4/UAS system was used to target expression of Psn to specific subsets of cells within a developing tissue. Specifically, a pannier-GAL4 line was used to drive expression of UAS-Dps in regions of the wing imaginal disc that will give rise to the adult notum and subsets of neurons within the eye imaginal disc or a cut-GAL4 line that drives expression in the presumptive wing margin. Notch protein is found to specifically accumulate within Psn-expressing cells and appears to be primarily localized within the cytoplasm. In contrast, neighboring cells that do not overexpress Psn show no change in Notch expression or distribution. Notch specifically accumulates in Psn-overexpressing cells but not in the adjacent cells (internal control) that only express basal levels of Psn. Taken together, these results show Psn does not affect the ability of Notch to undergo furin-dependent cleavage within the Golgi and to accumulate on the plasma membrane. Rather, Psn may be affecting the subcellular distribution of Notch and later cleavage events leading to defects in cell signaling (Guo, 1999).

In the absence of ligand interaction, proteolytic processing of Notch does not occur. Alternatively, Psn could be required for internalization of the receptor-ligand complex and subsequent proteolytic processing because it is not known where within the cell these cleavage events occur. Although presenilin in mammals is thought to reside predominantly in the endoplasmic reticulum (ER) and Golgi, consistent with possible roles in early stages of protein processing, some evidence suggests that it may transiently reach the plasma membrane where it could participate in endocytic processes. In fact, in Drosophila, Psn has been shown to accumulate within vesicular structures throughout the cytoplasm, including the ER and Golgi, and is also found at the apical regions of cells in some tissues. A role for presenilin in protein processing or trafficking has been suggested previously on the basis of its subcellular localization and its ability to affect the processing of APP in vertebrates. APP, like Notch, encodes a single-pass transmembrane protein found at the plasma membrane that is known to undergo several cleavage steps giving rise to two secreted peptides, Abeta(40) and Abeta(42-43). Recent studies in transgenic mice have shown that presenilins can affect the processing of both Notch and APP by mechanisms that are unknown. Current models suggest that presenilins may directly cause cleavage of Notch and APP or, alternatively, affect the ability of secretases to cleave. Alternatively, presenilins may play a more general role in regulating the subcellular distribution of transmembrane proteins like APP and Notch within the cell, thereby physically altering their ability to interact with proteases necessary for their proper signaling functions (Guo, 1999 and references therein).

A screen for mutations that suppress the phenotype of Drosophila armadillo, the ß-catenin homolog identifies Pen

During development, signaling pathways coordinate cell fates and regulate the choice between cell survival or programmed cell death. The well-conserved Wingless/Wnt pathway is required for many developmental decisions in all animals. One transducer of the Wingless/Wnt signal is Armadillo/ß-catenin. Drosophila Armadillo not only transduces Wingless signal, but also acts in cell-cell adhesion via its role in the epithelial adherens junction. While many components of both the Wingless/Wnt signaling pathway and adherens junctions are known, both processes are complex, suggesting that unknown components influence signaling and junctions. A genetic modifier screen was carried out to identify some of these components by screening for mutations that can suppress the armadillo mutant phenotype. Twelve regions of the genome were identified that have this property. From these regions and from additional candidate genes tested, four genes were identified that suppress arm: dTCF, puckered, head involution defective (hid), and presenilin. The interaction with hid, a known regulator of programmed cell death, was further investigated. The data suggest that Wg signaling modulates Hid activity and that Hid regulates programmed cell death in a dose-sensitive fashion (Cox, 2000).

While evaluating the effectiveness of this screen, a variety of candidate genes, including some that map within noninteracting Deficiencies, were tested. Heterozygosity for one of these, Presenilin, strongly suppresses arm. Both ßcat and other Arm repeat proteins such as delta-catenin associate with Presenilins in vivo. The function of this interaction remains confusing. Wild-type Presenilin stabilizes ßcat; this stabilization is abrogated by missense mutations in presenilin, and presenilin missense mutant cells from mutant patients have less nuclear ßcat. These data support a role for Presenilins as positive regulators of Wnt signaling via Arm/ßcat. In contrast, it has been reported that overexpression of wild-type Presenilin destabilizes ßcat; ßcat is stabilized in both Presenilin1 null fibroblasts or if Presenilin1 mutations are overexpressed, while a Wnt-responsive promoter is downregulated by Presenilin overexpression. These data support a conclusion that is the opposite of the one above, in which wild-type Presenilins negatively regulate Wnt signaling. It has been suggested that the presenilin-ßcat complex includes cadherins, in contravention of most other data. The genetic data are most consistent with a model in which Presenilins negatively regulate Wg signaling either directly or indirectly by binding Arm/ßcat or by influencing adherens junction assembly. Clearly much work remains to differentiate between the different possible mechanisms (Cox, 2000 and references therein).

Drosophila Nicastrin is essential for the intramembranous cleavage of Notch

To determine where Nicastrin (Nct) acts in the Notch signal transduction pathway, advantage was taken of transgenic Notch constructs that bypass some of the processing steps required for signaling. (1) hs-NFL was used as a control; it expresses full-length Notch protein under the control of a heat shock promoter. Like endogenous Notch, the protein from this construct requires both the ligand-induced S2 cleavage [this involves cleavage of the extracellular domain of Notch to produce a transient form of the receptor called NEXT (Notch extracellular truncation)] and the Psn-dependent S3 cleavage (an intramembranous cleavage to release the intracellular domain of Notch, which translocates to the nucleus, where it acts as a transcriptional activator in association with Suppressor of Hairless protein. (2) hs-NECN is a deletion of the extracellular domain of Notch beyond the S2 cleavage site, and therefore mimics NEXT. This protein signals independently of ligand, but still requires the S3 cleavage to release the intracellular domain of Notch from the membrane. (3) hs-NICD expresses the intracellular domain of the receptor and requires neither ligand, the S2 nor S3 cleavages, for signaling. The expression of hs-NFL has no effect on the development of wild-type embryos, whereas hs-NECN and hs-NICD disrupt germ band retraction because their ligand-independent signaling overactivates the Notch pathway. To test which of these constructs requires Nct for signaling, each was expressed in embryos that lack both maternal and zygotic Nct activity. hs-NECN and hs-NFL have no effect on the neurogenic phenotype of nct null embryos. In contrast, hs-NICD expression strongly rescues the nct phenotype: most embryos form patches of normal cuticle, and in some cases, the wild-type cuticular pattern is almost completely restored. Thus, the nct null mutation blocks Notch signaling after the S2 cleavage but before the release of the intracellular domain from the membrane, indicating that Nct is required for the S3 cleavage, as is Psn (López-Schier, 2002).

Presenilins and the human Nct associate with Notch during its passage through the secretory pathway, raising the possibility that they also function in earlier steps in Notch processing. Since the experiments above only reveal the last step at which these proteins are required, the behavior of Notch protein was followed in the follicle cells, where the downregulation of Notch in response to Delta binding can be visualized. Notch accumulates on the apical side of these cells until stage 7 of oogenesis, when Delta signals to trigger its proteolysis. Thus, Notch disappears from the apical membrane of cells that contact wild-type germline cells, whereas high levels of apical Notch persist in follicle cells that contact Delta germline clones, and this can be detected with antibodies against both the extracellular (NECD) and intracellular domains (NICD) of the protein. This reduction of apical Notch staining in response to Delta still occurs in Su(H) mutant follicle cell clones, in which Notch signaling in the nucleus is blocked. Therefore the disappearance of most Notch from the apical membrane appears to be a direct consequence of ligand-induced processing, and is not due to downregulation of the receptor in response to the activation of the signaling pathway. In nct and psn mutant follicle cells, apical NECD staining disappears at stage 7 as it does in wild-type, indicating that the ligand-dependent S2 cleavage and subsequent degradation of NECD occur normally. Unlike wild-type cells, however, mutant cells accumulate a processed form of Notch that can be stained with the anti-NICD antibody, and this is concentrated at the apical side of the cells and in intracellular clusters that may be endocytic vesicles. Thus, nct or psn mutant cells can transport Notch to the plasma membrane and process it to form a functional receptor that binds Delta and undergoes the S2 cleavage, indicating that both proteins are specifically required for the S3 cleavage, but not for any earlier steps in the pathway (López-Schier, 2002).

The processed form of Notch that accumulates on the apical side of nct and psn mutant follicle cells presumably corresponds to NEXT, which is the membrane-tethered product of the S2 cleavage and the substrate for the S3 cleavage. To test this possibility, Western blots of extracts from wild-type and embryos lacking maternal and zygotic Nct or Psn were probed with the anti-NICD antibody. In addition to the 300 kDa band that corresponds to the uncleaved form of Notch, nct and psn null embryos accumulate a 120 kDa species that is barely detectable in the wild-type extracts. This band migrates at the expected size of NEXT and NICD, which cannot be distinguished on these gels as they differ by only 3 kDa. This band is unlikely to be NICD, however, because the results above demonstrate that nct and psn block the S3 cleavage (López-Schier, 2002).

To investigate whether Nct is required for the localization or stability of Psn, nctagro clones of varying sizes were generated in the follicular epithelium. In wild-type follicle cells, Psn protein shows a punctate distribution in the cytoplasm that may correspond to the endoplasmic reticulum, and it localizes at the cell cortex. When nct clones are analyzed early in oogenesis, the mutant cells show a normal distribution of Psn, and this is also the case for small clones in late-stage egg chambers. In contrast, very large clones in late-stage egg chambers show a strong reduction in the levels of Psn. Thus, Nct seems to be required for the long-term stability of Psn in these cells. Since small nct clones show a completely penetrant Notch loss-of-function phenotype but have normal levels of Psn, this effect of Nct on Psn stability is unlikely to cause the defect in Notch signaling in these cells (López-Schier, 2002).

During an analysis of follicle cell clones, an additional function of Nct and Psn in the organization of the submembranous spectrin cytoskeleton was discovered. In wild-type cells, ßHeavy-spectrin (ßH-spectrin) associates with alpha-spectrin to form tetramers that localize to the apical membrane, while the basolateral spectrin cytoskeleton is composed of ß-spectrin/alpha-spectrin complexes. In psn and nct follicle cell clones, ßH-spectrin does not localize to the apical membrane. In contrast, ß-spectrin localization to the basolateral membrane is unaffected in mutant clones. The apical localization of ßH-spectrin requires its association with alpha-spectrin and vice versa, and the distribution of alpha-spectrin was therefore examined in follicle cell clones. Surprisingly, both psn and nct mutant cells show an increase in the amount of cortical alpha-spectrin, with the highest levels on the apical side. alpha-spectrin therefore appears to be recruited to the apical membrane of mutant cells by a novel mechanism that does not require its association with either ß subunit, neither of which localizes apically in these cells (López-Schier, 2002).

To test whether the mislocalization of alpha- and ßH-spectrin in nct and psn mutants is a consequence of the defect in Notch signaling, the localization of both proteins was examined in follicle cell clones of a null allele of Notch (N55e11) and in wild-type cells that abut Delta mutant germline clones. ßH-spectrin is recruited normally to the apical membrane in both cases, while alpha-spectrin localizes uniformly around the cell cortex at the same level as in wild-type cells. These results indicate that Psn and Nct have a novel function independent of their role in Notch signaling that somehow affects the organization of the spectrin cytoskeleton (López-Schier, 2002).

The absence of apical ßH-spectrin in nct and psn mutant cells suggested that their apical-basal polarity might be disrupted, and the localization of a variety of other polarity markers was therefore examined. The overall polarity of the cells is unaffected in either nct or psn mutant clones, since Coracle, Neurotactin, DE-Cadherin, Armadillo (ß-catenin), alpha-catenin, and Notch itself are localized to the proper membrane domains. However, DE-Cadherin, Armadillo, and alpha-catenin accumulate to much higher levels in mutant cells than in wild-type. These three proteins are components of the Cadherin adhesion complex, and are enriched at the sites of adherens junction formation at the apical margins of the cell. Their overaccumulation in mutant cells may therefore be linked to the loss of apical ßH-spectrin and the apical enrichment of alpha-spectrin, although the nature of this link remains unclear (López-Schier, 2002).

Previous studies have shown that almost all Psn is associated with the ER and intermediate compartment and that there is little or no protein at the plasma membrane, where APP and Notch reside. This discrepancy, which has been called the 'spatial paradox,' raises the question of where in the cell the S3 cleavage occurs. One possible solution to this paradox is suggested by the observation that small amounts of Psn can be coimmunoprecipitated with Notch at the cell surface. Both Nct and Psn have been shown to interact with Notch in the secretory pathway, and a minor fraction of the S3 protease complex could therefore be transported to the plasma membrane through binding to its future substrate. A second possibility is that the active protease resides in an intracellular compartment, and that the products of S2 cleavage of Notch are internalized and transported to this site. This analysis provides strong evidence for the first model. Since Nct and Psn are required for the S3 cleavage of Notch, the substrate for this cleavage, NEXT, accumulates in mutant cells. Most NEXT remains closely associated with the apical membrane, arguing against the existence of a major transport pathway to an intracellular compartment. Although a small amount of NEXT is found in intracellular clusters, these do not correspond to the major sites of Psn localization, and may be endocytic vesicles. Furthermore, recent data indicate that endocytosis is not required for the S3 cleavage of NEXT, because a membrane-tethered Notch derivative lacking the extracellular domain can signal normally in shibire mutant embryos, in which Dynamin-dependent endocytosis is blocked. Taken together, these results strongly suggest that S3 cleavage occurs predominantly at the plasma membrane (López-Schier, 2002).

Follicle cell clones mutant for either Nct or Psn have a more severe phenotype than that seen in Notch or Delta mutants, indicating that both proteins must have at least one additional function in these cells that is independent of their role in Notch signaling. One aspect of this phenotype is the overaccumulation of the components of the adherens junctions, DE-Cadherin, Armadillo, and alpha-catenin, and this is probably related to the fact that both alpha-catenin and the Armadillo ortholog ß-catenin associate with Psn in mammalian cells. Although neither is required for the activity of the S3 protease or gamma-secretase, loss of Psn leads to an overaccumulation of ß-catenin in Drosophila embryos and mouse epithelial cells. The precise function of Psn in ß-catenin regulation is unknown, but the overexpressed protein in Drosophila psn mutant embryos is associated with polyubiquitin-positive cytoplasmic inclusions, suggesting that Psn is required in some way to regulate Armadillo degradation. Psn also regulates the turnover of DE-Cadherin and alpha-catenin. Furthermore, Nct is necessary for this function, suggesting that it requires the formation of the high molecular weight protease complex. Since Psn is thought to mediate the proteolysis of membrane proteins, one possibility is that Psn is recruited to DE-Cadherin by binding to the catenins, and cleaves DE-Cadherin to trigger degradation. Alternatively, Psn could regulate the turnover of the catenins in some other way, and their overaccumulation in psn and nct mutants might then lead to the stabilization of Cadherin complexes at the membrane (López-Schier, 2002).

nct and psn mutants also disrupt the organization of the spectrin cytoskeleton in follicle cell clones. This phenotype is particularly puzzling, because the mutants have opposite effects on the two subunits of apical spectrin: ßH-spectrin disappears from the apical membrane, whereas alpha-spectrin accumulates all round the cortex of the cell, but particularly at the apical side. In wild-type cells, the apical localization of alpha-spectrin normally requires its association with ßH-spectrin to form (alphaßH)2 tetramers. Its apical localization in mutant cells must therefore occur by a different mechanism from normal, and this may sequester it in a form that cannot bind ßH-spectrin, which would account for the failure of the latter to localize. However, alpha-spectrin must still tetramerize with ß-spectrin, because it shows wild-type localization to the basolateral domain. It is unclear whether the defect in the spectrin cytoskeleton is related to the overaccumulation of Cadherin complexes, but it is intriguing that alpha-spectrin accumulates at sites where DE-Cadherin, Armadillo, and alpha-catenin are most enriched in nct and psn mutant cells. Thus, this localization and stabilization of alpha-spectrin could be mediated through binding to some component of these abnormal adhesion complexes. It is also possible, however, that the Nct/Psn complexes have multiple functions in the follicle cells that independently regulate Notch signaling and the spectrin cytoskeleton and Cadherin adhesion complexes (López-Schier, 2002).

The tumor suppressor gene l(2)giant discs is required to restrict the activity of Notch to the dorsoventral boundary during Drosophila wing development

During the development of the Drosophila wing, the activity of the Notch signalling pathway is required to establish and maintain the organizing activity at the dorsoventral boundary (D/V boundary). At early stages, the activity of the pathway is restricted to a small stripe straddling the D/V boundary, and the establishment of this activity domain requires the secreted molecule Fringe (Fng). The activity domain will be established symmetrically at each side of the boundary between Fng-expressing and non-expressing cells. Evidence is presented that the Drosophila tumor-suppressor gene lethal giant discs (lgd), a gene whose coding region has yet to be identified, is required to restrict the activity of Notch to the D/V boundary. In the absence of lgd function, the activity of Notch expands from its initial domain at the D/V boundary. This expansion requires the presence of at least one of the Notch ligands, which can activate Notch more efficiently in the mutants. The results further suggest that Lgd appears to act as a general repressor of Notch activity, because it also affects vein, eye, and bristle development (Klein, 2003).

It has also been observed that wingless (wg) is expressed ectopically in the pouch of lgd mutants during wing development. Similar phenotypes are observed, if the Notch pathway is ectopically activated during wing development, raising the possibility that the lgd mutant phenotype could stem from the ectopic activation of the Notch pathway. The Notch pathway is indeed ectopically active in lgd mutants, and hyperactivation as well as ectopic activation of the pathway accounts for the lgd phenotype during wing development. In lgd mutants, the expression of Notch target genes along the D/V boundary is expanded, indicating that Lgd is required for the restriction of Notch activity to the D/V boundary. Furthermore, the mutant phenotype of lgd is suppressed by concomitant loss of Presenilin or Suppressor of Hairless function, indicating that the mutant phenotype is caused by the activation of the Notch pathway. Evidence is provided that the activity of fng and Serrate seem to be dispensable in lgd mutant wing disc and that Delta can activate Notch efficiently enough to maintain its activity during wing development. The presented results indicate that the negative regulation of Notch by Lgd is not restricted to wing development and occurs during several other developmental processes, such as vein, eye, and bristle development, suggesting that Lgd suppresses the activity of the Notch pathway in a variety of developmental processes (Klein, 2003).

Loss of lgd function leads to an overgrowth of the imaginal discs, clearly noticeable in the wing region of the wing disc, which becomes enlarged and flat (Bryant, 1971). wg expression is normally restricted to the D/V boundary of the wing pouch. In lgd mutants, wg is activated ectopically in a much broader domain that extends into the wing pouch. In addition, lgd mutant wing discs often develop a second wing pouch in the region of the anlage of the scutellum. Similar phenotypes are caused by gain-of-function alleles of N (for example, Abruptex) and are also observed upon expression of the activated intracellular form of Notch, Nintra, or expression of Notch ligands, such as Dl. The ectopic activation of wg can already be observed in early third instar wing discs and precedes the visible morphological changes that occur at later stages. The deficiency Df(2L) FCK-20 deletes the lgd locus, allowing the classification of the relative strength of the available alleles. The phenotype is always variable, but the overall phenotype of lgdd7 and lgdd10 in homozygotes and in trans over Df(2L)FCK-20 is very similar, indicating that these two alleles are strong, probably amorphic alleles. lgdd4 and lgdd1 are weaker alleles. All alleles display a qualitatively similar phenotype over the deficiency as in homozygotes, indicating that the observed phenotype is probably caused by the loss-of-function of the lgd gene (Klein, 2003).

The similarity between the loss of lgd function and ectopic N activation suggests that the phenotype of lgd could be caused by ectopic activation of the Notch pathway. To examine this possibility, the expression of E(spl)m8, cut, Dl, and Ser was monitored as well as the activity of the vg-boundary enhancer (vgBE) in mutant wing discs. The expression of all these markers is initiated in cells at the D/V boundary in a Notch-dependent manner. The vgBE is initially expressed along the D/V boundary of the wing, but late in the third instar, it is activated in an additional stripe along the anteroposterior compartment boundary (A/P boundary), which is also dependent on Notch activity. Both domains depend on the presence of a single Su(H) binding site in the enhancer. Similarly, the expression of cut and E(spl)m8 is initiated in cells at the boundary by the Notch-pathway, and E(spl)m8 is also dependent on the presence of Su(H) binding sites in its promoter. As described above, the expression of Dl and Ser is more complex but always dependent on the activity of Notch in cells at the D/V boundary. In lgd mutant wing discs, the vgBE as well as cut, Dl, Ser, and E(spl)m8 are activated ectopically within the wing pouch. The activation of the vgBE is dependent on the presence of the Su(H) binding site in the enhancer, since a version lacking it shows no ectopic activity in the mutants. As in the case of wg, the expression of the vgBE is already expanded in early third larval wing discs. Altogether, these results show that the loss of lgd function leads to the ectopic expression of Notch target genes. This suggests that the Notch pathway is ectopically activated in lgd mutants (Klein, 2003).

All tested Notch-target genes are ectopically activated in lgd mutant wing discs or lgd mutant cell clones. The ectopic activation of Notch target genes as well as the observed overproliferation of lgd mutants is abolished in lgd;Psn double mutants. In addition, Notch target gene expression is also abolished in Psn or Su(H) mutant clones generated in lgd mutant wing imaginal discs. These data suggest that the Notch pathway becomes ectopically active in the absence of lgd function. Furthermore, the fact that Delta alone seems to provide sufficient Notch activity to sustain wing development in lgd mutants indicates that the pathway can be activated more efficiently in the mutant background. The activation of Notch is a consequence of loss of lgd function also in other developmental processes, such as bristle, leg, and wing vein development. Thus, the presented data make lgd a good candidate gene that regulates activity of the Notch pathway during adult development of Drosophila (Klein, 2003).

Identification of genes involved in Drosophila melanogaster geotaxis, a complex behavioral trait

Identifying the genes involved in polygenic traits has been difficult. In the 1950s and 1960s, laboratory selection experiments for extreme geotaxic behavior in fruit flies established for the first time that a complex behavioral trait has a genetic basis. But the specific genes responsible for the behavior have never been identified using this classical model. To identify the individual genes involved in geotaxic response, cDNA microarrays were used to identify candidate genes and fly lines mutant in these genes were assessed for behavioral confirmation. The identities of several genes that contribute to the complex, polygenic behavior of geotaxis have thus been determined (Toma, 2002).

Pioneering experiments on Drosophila melanogaster and Drosophila pseudoobscura investigated the nature of the genetic basis for extreme, selected geotaxic behavior. These experiments constituted the first attempt at the genetic analysis of a behavior. Selection and chromosomal substitution experiments successfully showed that there is a genetic basis for extreme geotaxic response in flies and, by implication, for behavior in general. These experiments also added to understanding of the role of variation in phenotypic evolution and selection. Despite their seminal contributions in behavioral genetics, population genetics and the study of selection, by their nature these experiments could not identify specific genes (Toma, 2002 and references therein).

These results highlight both the success and the limitation of behavioral selection experiments. Although selection results tend to be representative of the natural interactions of genes that produce behavior and can demonstrate that a trait has a genetic basis, they do not pinpoint specific genes that influence the trait. This is partly due to the involvement of many genes and the relatively minor role of each in complex polygenic phenotypes -- a problem that is especially acute for the intrinsically more variable phenotypes that are associated with behavior. The advent of cDNA microarray technology offers an easily generalized strategy for detecting gene expression differences and can complement other means of identifying the genes that underlie complex traits. An expression difference may occur in a gene that is not itself polymorphic, but that gene may contribute to the realization of the phenotypic difference (Toma, 2002).

As a starting point for identifying genes that affect a complex trait, the selected, established Hi5 and Lo extreme geotaxic lines were examined for changes in gene expression between strains of Drosophila melanogaster subjected to long-term selection and isolation. A two-step approach was used: (1) the differential expression levels of mRNAs isolated from the heads of Hi5 and Lo flies was determined using cDNA microarrays and real-time quantitative PCR (qPCR); (2) a subset of the differentially expressed genes was independently tested for their influence on geotaxis behavior by running mutants for these genes through a geotaxis maze. It was reasoned that some of the differences in gene expression between strains might be related to phenotypic differences and that it should therefore be possible, at least in part, to reconstruct the phenotype with independently derived mutations in some of the differentially expressed genes (Toma, 2002).

The findings indicate that differences in gene expression can be used to identify phenotypically relevant genes, even when no large, single-gene effects are detectable by classical, quantitative genetic analysis. Three of the four genes implicated by microarray and qPCR measurements caused differences in geotaxis, whereas none of the six control genes had an effect. Only those genes that had larger differences in expression according to the microarrays, or that were significantly different according to qPCR results (cry, Pdf and Pen), significantly changed geotaxis scores. The converse was not true, because altered geotaxis behavior did not always accompany larger differences in mRNA levels, as shown by pros, although this might reflect the sensitivity of pros to aspects of the genetic context. All of the genes tested for which there was little or no difference in mRNA levels between the selected Hi5 and Lo lines also showed no influence on geotaxis behavior (Toma, 2002).

The directionality of behavioral and mRNA differences proved to be consistent with predictions that were based on expression levels. Homozygous null mutants of Pdf and cry showed a significant increase in geotaxis score, which is consistent with a lower level of expression of these genes in Hi5 relative to Lo. Similarly, the heterozygous Pen mutant showed a significant downward shift in geotaxis score, which is consistent with a lower level of Pen expression in Lo relative to Hi5. Thus, the change in behavior of the tested mutants corresponds to the direction predicted by differences in transcript level in the selected Hi5 and Lo lines (Toma, 2002).

Whereas the cry, Pen and female Pdf mutants produced the anticipated effect on behavior, the magnitude of behavioral effect was smaller than in the original selected lines. This probably reflects the difference between the aggregate effect of an ensemble of genes in the selected lines as opposed to the individual effect of a single mutant gene in a neutral background. In addition, their relatively small effects are exactly the results that one would predict in a polygenic system such as geotaxis behavior, in which many genes have small contributions to the overall phenotype. The three genes identified in this study would not have been predicted on the basis of their previously defined functions (Toma, 2002).

These results show that the two separate approaches to behavioral genetics -- the classical Hirschian quantitative analysis of genetic architecture and the modern Benzerian approach of single-gene mutant analysis -- are complementary and can be unified. This study used the results of a Hirschian approach of laboratory selection for natural variants to identify single gene differences, such as one would find in a Benzerian approach. The results are consistent with the suggestion that naturally occurring variants in behavior correspond to mild lesions in pleiotropic genes (Toma, 2002).

Finally, the results show that differences in gene expression identified by cDNA microarray analysis can be used as a starting point for narrowing down the numbers of candidate genes involved in complex genetic processes. Such an approach is analogous, as well as complementary, to the current method of mapping quantitative trait loci to large chromosomal intervals and then making educated guesses about which genes within those intervals may be involved in the trait (Toma, 2002).

The combination of selection, with its ability to exaggerate natural phenotypic variation, and global analysis of differences in gene expression by cDNA microarray analysis offers a promising approach to previously intractable molecular analyses of behavior. The geotaxis genes that were identified might have been the direct targets of selection, or they might be downstream of the direct targets. Additional studies using the Hi5 and Lo selected lines will be required to distinguish between these possibilities and to determine the causal role that these genes have in the context of the selected lines (Toma, 2002).

This study has gone from the selection of a 'laboratory-evolved' behavioral phenotype, to screening for mRNA differences, to partially reconstituting the phenotype using mutants. This shows the feasibility of combining genomic and classical genetic approaches for the breakdown and partial reassembly of an artificially selected behavioral trait (Toma, 2002).

Modeling clinically heterogeneous Presenilin mutations with transgenic Drosophila

To assess the potential of Drosophila to analyze clinically graded aspects of human disease, a transgenic fly model was developed to characterize Presenilin (PS) gene mutations that cause early-onset familial Alzheimer's disease (FAD). FAD exhibits a wide range in severity defined by ages of onset from 24 to 65 years. PS FAD mutants have been analyzed in mammalian cell culture, but conflicting data emerged concerning correlations between age of onset and PS biochemical activity. Choosing from over 130 FAD mutations in Presenilin-1, 14 corresponding mutations at conserved residues were introduced in Drosophila Presenilin (Psn) and their biological activity in transgenic flies was assessed by using genetic, molecular, and statistical methods. Psn FAD mutant activities were tightly linked to their age-of-onset values, providing evidence that disease severity in humans primarily reflects differences in PS mutant lesions rather than contributions from unlinked genetic or environmental modifiers. This study establishes a precedent for using transgenic Drosophila to study clinical heterogeneity in human disease (Seidner, 2006).

Presenilin is an evolutionarily conserved polytopic membrane protein that is part of the multisubunit γ-secretase complex responsible for intramembranous cleavage of several transmembrane proteins, including APP, Notch, Delta, DCC, ErbB4, N-Cadherin, and E-Cadherin. Familial Alzheimer's disease (FAD) mutations in Presenilin-1 (PS1) and PS2 alter proteolytic processing of APP to generate more toxic Aβ42 peptides that accelerate amyloid plaque formation in brain tissues. PS mutations also contribute to neurodegeneration and cognitive decline through amyloid-independent mechanisms, involving altered regulation of receptor signaling and intracellular kinase pathways (Seidner, 2006).

The Drosophila APP ortholog, APPL, lacks homology to APP within the Aβ peptide region, and loss of APPL produces subtle behavioral deficits that are difficult to measure quantitatively. A more suitable substrate to monitor Presenilin (Psn) FAD mutant activity in Drosophila is the Notch receptor, the most extensively characterized fly γ-secretase substrate. Psn is required throughout Drosophila development for Notch signaling, and a wide variety of Notch-related phenotypes exist that range from severe embryonic lethality to specific defects in adult tissues. This phenotypic range is amenable to characterizing variable degrees of function among different Psn FAD mutants. In addition, genetic and molecular reagents are available to characterize Notch biochemical cleavage and subsequent target-gene activation in Drosophila (Seidner, 2006).

Previous studies have successfully used transgenic mice and C. elegans to assess the genetic properties of human PS FAD mutants, and one study suggested a potential difference between two FAD variants. However, quantitative comparison between FAD mutants was not practical because of heterologous expression methods and difficulties controlling transgene copy numbers. Two studies in mammalian cell culture showed little or no relationship between average ages of onset and Aβ42 secretion levels, contrary to one study reporting a strong correlation (Seidner, 2006).

This study assessed PS FAD mutant function in Drosophila by expressing 14 FAD-linked mutant psn transgenes in animals lacking endogenous psn function. The mutations were selected on the basis of the following criteria: (1) they span the entire protein, (2) relatively large numbers of families and affected individuals have been identified for most mutations, and (3) their ages of onset range from 24 to 65 years, or are possibly asymptomatic (E318G and F175S). Thirteen of the mutations affect residues that are conserved in fly Psn, and the remaining one alters a conservatively substituted residue. To achieve physiological expression of the transgenes, a 1.5 kb promoter of the endogenous psn gene was defined, termed PEPC (Presenilin endogenous promoter cassette), . At the larval-pupal transition, loss of psn function causes highly uniform lethality that is fully rescued by a wild-type PEPC-psn transgene but not by wild-type human PS1 or PS2 transgenes driven by PEPC. To circumvent this technical problem with human PS transgenes, each FAD mutation was engineered into the fly psn gene (Seidner, 2006).

Examining transgenic lines expressing PEPC-psn FAD mutants in a psn null genetic background, eight distinct phenotypic categories were defined that represent a graded series in order of increasing rescue ability, as follows: (1) prepupal lethal, (2) late prepupal lethal, (3) pupal lethal, (4) pharate lethal, (5) severe neurogenic/adult lethal, (6) moderate neurogenic/adult viable, (7) weak neurogenic/adult viable, and (8) morphologically normal. Quantitative analysis of survival rates, incidence of dorsoscutellar bristle duplications, wing notching, wing vein defects, and other morphological features was performed to assign each transgenic line to the appropriate rescue category (Seidner, 2006).

The phenotypic categories are consistent with an ordered series attributable to progressive increases in Psn-dependent Notch activation, but it was important to verify that they do not instead represent the effects of Psn on other substrates. In the canonical Notch pathway, ligand binding to the Notch receptor leads to ectodomain removal and subsequent γ-secretase-mediated intramembranous cleavage of Notch. The liberated intracellular domain, termed NICD, translocates to the nucleus and participates directly in transcriptional regulation of target genes, including the Enhancer of split m7 (E(spl)m7) gene in Drosophila. Activation of Notch signaling within proneural cell clusters destined to give rise to adult sensory organs results in restricted expression of the proneural marker scabrous (sca). Visualization of sca expression in the larval imaginal wing disc with a sca-lacZ transgene provides evidence for a progressive range of Notch signaling with different PEPC-psn transgenes (Seidner, 2006).

Biochemical evidence for progressively increasing Psn function was obtained by immunoblot analysis of Notch cleavage products across the spectrum of PEPC-psn FAD phenotypes. Representative PEPC-FAD transgenic lines of the different categories exhibit partial-to-complete failure in γ-secretase-mediated NICD production. Interestingly, although transgenic lines belonging to categories 2–4 exhibit significant levels of biological rescue activity, they do not produce levels of NICD detectable by immunoblot analysis. These results demonstrate that genetic assays such as these are valuable for studying low-level or tissue-specific aspects of γ-secretase substrate cleavage that might not be amenable to biochemical analysis (Seidner, 2006).

To determine whether the PEPC-psn FAD phenotypic series is similarly correlated with Notch target-gene activation, semiquantitative RT-PCR was used to monitor activation of the E(spl)m7 gene. Normalizing transcriptional activation to two control genes, a progressive increase in E(spl)m7 transcript levels was observed across categories 1-8, confirming that they represent graded increases in γ-secretase-dependent Notch activation. To verify this relationship, a temperature-sensitive Notch allele termed Nts1 was used. Incremental levels of Notch activity obtained by raising Nts1 flies at different temperatures produced phenotypes matching those seen in the Psn FAD mutant series, but were independent of manipulations involving endogenous or transgenic Psn. Levels of E(spl)m7 transcriptional activation seen in these Nts1 phenotypic classes resembled closely those observed in the corresponding PEPC-psn FAD classes, confirming the progressive increase in Notch target-gene activation across this phenotypic spectrum. Taken together, these results validate the use of the phenotypic criteria to characterize varying degrees of PEPC-psn FAD transgene activity toward the endogenous γ-secretase substrate Notch (Seidner, 2006).

A limitation of transgenic analysis in Drosophila is that the transgenes are inserted at essentially random genomic locations, leading to variations in expression as a result of local position effects at different sites. These effects are a confounding factor in comparisons of relative degrees of rescue function, which reflects properties of the primary lesion in each Psn mutant protein as well as the expression level of each transgenic insertion. Therefore, from four to 16 independent insertions were generated for each transgene, and each insertion was scored with multiple quantitative morphological criteria and assigned to the appropriate phenotypic category. Visual inspection of the results suggests a positive overall trend between increasing age of onset in human FAD pedigrees and increasing biological activity for these FAD mutants in transgenic Drosophila (Seidner, 2006).

Statistical tests demonstrate an overall correlation across the dataset as a whole, but the correlation is not smooth. Most FAD mutants appear to cluster in groups separated by discontinuities, with a few mutants that are noticeably different from nearby mutants. Mutants having similar ages of onset are, in general, not statistically different from one another. Nevertheless, the mutants can be statistically grouped into three distinct classes: strong (L166P, L173W, P436Q, V272A, and L235P), intermediate (M146L, M139V, H163R, E280A, A246E, and G206A), and weak (A79V, F175S, and E318G). Strong mutants differ most clearly from the weak ones in terms of function, whereas the intermediate group shows more modest differences compared to either of these two flanking groups. Pairwise comparisons of aggregated mutant groups confirmed these classes. The strong mutant group is significantly different from both the intermediate and weak groups. Despite its age of onset of ~60, the A79V mutant is more similar to the weak mutants than the intermediate ones, as confirmed by pairwise comparisons of aggregated intermediate and weak groups with A79V assigned to either the intermediate or the weak group (Seidner, 2006).

Similarly, two mutants, L235P and P436Q, are less functional in transgenic flies than might be predicted on the basis of their ages of onset, whereas another mutant, M139V, is somewhat more functional than expected. Whether these disparities reflect inadequate age-of-onset data, modifier effects in the corresponding human pedigrees, or an unknown feature of the fly assay system is not clear. Finally, the E318G and F175S mutants, proposed to be either very weak pathogenic mutants or functionally normal polymorphisms, are statistically indistinguishable from the wild-type transgene in the genetic assay, consistent with the idea that they are nonpathogenic polymorphisms. Overall, the results support the assertion that disease severity in early-onset Alzheimer's disease is primarily determined by PS mutant lesion type as opposed to unlinked genetic or environmental modifiers, as was also deduced from a study of Aβ secretion levels in PS FAD mutant transfected cells (Seidner, 2006).

A few other interesting patterns emerge from these comparisons. Mutants for which relatively few independent lines were obtained, most notably H163R, fail to show significant differences when compared to other mutant groups. On the basis of the complete dataset, it is estimated that ~10 independent lines of a given mutant are required to obtain statistically useful data. This problem might be circumvented by employing a 'knock in' strategy to precisely replace the endogenous psn gene with FAD variants, an approach that is not yet reliable in Drosophila. Additionally, one source of experimental noise is that transgenes occasionally insert into locations where they are poorly expressed or damaged during insertion, as is evident from a few instances involving the 'asymptomatic' F175S and E318G mutant and wild-type transgenes. Normalization of functional read-outs for each transgene insertion relative to its mRNA or protein expression level in the appropriate transcript null or protein null psn mutant background should reduce this noise and lead to further refinement of the statistical data (Seidner, 2006).

These findings establish the validity of using transgenic Drosophila or other heterologous organisms to evaluate clinically heterogeneous aspects of human diseases with a clearly defined genetic etiology. Transgenic Drosophila offer several advantages to augment more traditional clinical assessments as well as transgenic mouse models. Transgenic flies are relatively inexpensive and rapid to produce, large numbers of independent lines can be easily generated, and limitless numbers of progeny for each line can be examined under controlled genetic conditions. These features of the assay might make it useful for obtaining a rapid estimate of approximate disease severity for new PS1 and PS2 mutations, especially for those with limited pedigree data, small numbers of affected individuals, or suspected environmental or genetic confounding factors. Although the primary goal of this study was to assess an array of genetically diverse FAD mutant variants in a more standardized genetic background, the transgenic flies characterized could be used to study the effects of suspected modifiers or search for new modifiers of PS function. The transgenic lines could also be combined with APP-expressing transgenes to investigate more directly the role of PS FAD mutants in APP cleavage, amyloid-peptide accumulation, and neurotoxicity in a fly model. The correlation observed between the effects of different FAD mutations on Drosophila Notch signaling and human disease onset underscores recent proposals that in addition to APP processing, more global perturbations in pathways involving other γ-secretase substrates should be considered in early-onset Alzheimer's disease. Finally, the results offer encouragement that additional transgenic Drosophila models might be developed to investigate clinical heterogeneity in other human diseases (Seidner, 2006).

Antioxidant proteins TSA and PAG interact synergistically with Presenilin to modulate Notch signaling in Drosophila

Alzheimer's disease (AD) pathogenesis is characterized by senile plaques in the brain and evidence of oxidative damage. Oxidative stress may precede plaque formation in AD; however, the link between oxidative damage and plaque formation remains unknown. Presenilins are transmembrane proteins in which mutations lead to accelerated plaque formation and early-onset familial Alzheimer's disease. Presenilins physically interact with two antioxidant enzymes thiol-specific antioxidant (TSA) and proliferation-associated gene (PAG) of the peroxiredoxin family. The functional consequences of these interactions are unclear. In the current study a presenilin transgene was expressed in wing and sensory organ precursors of the fly. This caused phenotypes typical of Notch signaling loss-of-function mutations. While expression of TSA or PAG alone produced no phenotype, co-expression of TSA and PAG with presenilin led to an enhanced Notch loss-of-function phenotype. This phenotype was more severe and more penetrant than that caused by the expression of Psn alone. In order to determine whether these phenotypes were indeed affecting Notch signaling, this experiment was performed in a genetic background carrying an activated Notch (Abruptex) allele. The phenotypes were almost completely rescued by this activated Notch allele. These results link peroxiredoxins with the in vivo function of Presenilin, which ultimately connects two key pathogenetic mechanisms in AD, namely, antioxidant activity and plaque formation, and raises the possibility of a role for peroxiredoxin family members in Alzheimer's pathogenesis (Wangler, 2011).

The underlying genetic heterogeneity of Alzheimer's disease has made understanding the pathogenesis of the disease difficult without the aid of model genetic organisms. It is clear that plaque formation and oxidative damage are key pathogenetic mechanisms in AD. The presence of early oxidative stress in mice with Presenilin mutations lends further support for the idea that plaque formation and oxidative damage are connected in AD. However, how these two pathogenetic processes are linked is not known. This study used Drosophila as a model system to explore a protein-protein interaction between presenilin and two peroxiredoxin family members TSA and PAG, which may provide a link between oxidative stress and plaque formation in AD (Wangler, 2011).

There is genetic and biochemical evidence that peroxiredoxin family members play a role in the pathogenesis of AD. There are six peroxiredoxin family members in humans. Of these genes PRDX1 (PAG) was implicated in physical interaction with human Presenilin-1 and is primarily expressed in glia. PRDX2 (TSA) is globally expressed in the brain and has increased expression in the frontal cortex of patients with AD and is implicated in Parkinson’s disease and amyotrophic lateral sclerosis . Another family member PRDX6 has been more directly implicated in AD by linkage studies. The PRDX6 protein displays markedly increased expression in glia surrounding amyloid plaques in AD brain. Overall these studies support a role for some of these family members in AD pathogenesis. Furthermore, they demonstrate an increase in the expression of PRDX family members in AD brain, an effect which the data suggest could impact the function of Presenilin. These observations suggest the use of a study of models of the clinically relevant pathogenetic mechanism (Wangler, 2011).

One advantage of the Drosophila wing as an assay system is the ability to make use of a wealth of knowledge and an array of genetic tools for analyzing wing development. For example, this study observed phenotypes with overexpression of Psn which recapitulated Notch loss-of-function, and a substantial increase was observed in the penetrance and severity of these phenotypes when human TSA and PAG were co-expressed with Psn. This led to the hypothesis that TSA and PAG enhance the dominant negative effect of overexpressed Psn. Whether this enhancement was mediated by the Notch pathway was tested by suppressing this effect with an activated allele of Notch. Since Psn acts in conjunction with three other components that comprise γ- secretase, it is likely that overexpression of Psn acts in a dominant negative fashion to inhibit Notch signaling by altering the normal stoichiometry of γ-secretase subunits. The results suggest that association of Psn with TSA or PAG aggravates this effect of Psn overexpression. Interestingly, this seemed to occur with the human peroxiredoxin isoforms in the presence of the Drosophila Psn transgene. While the portion of the PSEN1 protein which interacts with TSA and PAG is only partially conserved with Psn, these observations suggest a functionally significant effect across species. Furthermore, an exacerbation of phenotype was seen in response to overexpression of peroxiredoxin antioxidant enzymes, conditions that one might expect to provide oxidative protection. One possible explanation for these findings is that by associating with Psn, TSA and PAG further reduce the complete intact γ-secretase complex. Alternatively, Notch inactivation could be a result of either degradation or abnormal processing of the endogenous presenilin in response to the expression of Psn along with the mammalian transgenes. Either possibility could have obvious disease relevance, as it seems clear that increased expression of these proteins occurs in AD brain, and the current data suggest that such an increase in expression could have an impact on Presenilin function and therefore indirectly could influence plaque formation (Wangler, 2011).

While these results support a role for peroxiredoxins PAG and TSA in modifying the Notch inactivation phenotype caused by Psn overexpression, it a direct role for these proteins in the processing of amyloid precursor protein, or in the pathogenesis of AD cannot be infered. Loss-of-function studies are needed to determine the mechanism of peroxiredoxin function and whether there is a clear role for endogenous peroxiredoxin in Notch signaling or amyloid cleavage. Further experiments in mammalian cells will be needed to determine whether peroxiredoxin family members are indeed involved in amyloid processing. Nonetheless, this study has demonstrated that Drosophila provides an effective system for testing the in vivo relevance of AD related protein-protein interactions. As the wing assay for Psn activity has proven robust in validating a suspected protein-protein interaction with Presenilin, it should be amenable to screening for new unknown functionally interacting partners of Psn (Wangler, 2011).


Phenotypic effect of Presenilin mutation in C. elegans

In addition to its role in cell fate decisions in non-neuronal tissues, presenilin activity is required in terminally differentiated neurons in vivo. Mutations in the Caenorhabditis elegans presenilin genes sel-12 and hop-1 result in a defect in the temperature memory of the animals. This defect is caused by the loss of presenilin function in two cholinergic interneurons that display neurite morphology defects in presenilin mutants. The morphology and function of the affected neurons in sel-12 mutant animals can be restored by expressing sel-12 only in these cells. The wild-type human presenilin PS1, but not the FAD mutant PS1 A246E, can also rescue these morphological defects. Since lin-12 mutant animals display similar morphological and functional defects as presenilin mutants, it is suggested that presenilins mediate their activity in postmitotic neurons by facilitating Notch signaling. These data indicate cell-autonomous and evolutionarily conserved control of neural morphology and function by presenilins (Wittenburg, 2000).

sel-12 mutants display a highly penetrant defect in their ability to sense and/or memorize temperature. Wild-type C. elegans display strong preference for their growth temperature, and can memorize it and store the information for several hours, suggesting a neuronal plasticity. This behaviour can be studied with a simple experimental model. When placed in a radial thermal gradient on the agar surface of a petri dish, wild-type animals migrate to their preferred temperature, and then move in isothermal circles. In contrast, sel-12 mutant animals have lost the ability to perform isothermal tracks. Most animals are non-responsive to the temperature gradient and moved randomly on the plate (athermotactic behavior), and 10% of the remaining animals moved to colder temperatures than the wild-type (cryophilic behaviour). These results indicate that sel-12 mutants may have defects in the neural circuit for thermotaxis (Wittenburg, 2000).

The neurons necessary for thermotaxis have been studied extensively by mutational analyses and laser ablation studies. Temperature input activates the two AFD sensory neurons, which synapse extensively onto the two AIY interneurons. Chemical signals from AIY and AIZ (synaptic partners that represent the four central integrating interneurons), in turn, regulate postsynaptic inter- and motor neurons that control the motor response. The morphology of the AFD, AIZ and AIY neurons was carefully examined in sel-12 animals using green fluorescent protein (GFP) reporter constructs, and no obvious defects in AFD and AIZ neurons were seen. However, defects in the morphology of AIY neurons are seen. In wild-type animals, the processes of both AIY neurons extend anteriorly from the cell bodies along the ventral cord, run around the nerve ring and meet and terminate at the dorsal midline. In adult sel-12 mutants the AIY cell bodies are correctly positioned in the head ganglion. However, the AIY axons often grow too far anteriorly before turning and fasciculating in the nerve ring, and/or do not stop growth at the dorsal side of the nerve ring, but turn posteriorly, sometimes extending up to the midbody region. In addition, short extra neurites often emerge directly from the cell soma or branch off the primary process. Behavioral defects are far more penetrant than the morphological defects, indicating that sel-12 animals may also have more subtle defects in the AIY neurons than can be visualized with GFP constructs. This is not unprecedented, because mutations in other axonal guidance genes often lead to highly penetrant behavioural defects with a much lower penetrance of morphological defects than is visible by light microscopy (Wittenburg, 2000).

To confirm that the observed defects were due to a loss of sel-12 activity, sel-12 mutants were transformed with a sel-12 complementary DNA under the control of the sel-12 promoter. This construct rescued the egg-laying defect, the thermotaxis behaviour of sel-12(ar171) and the neurite morphology defect. To determine whether sel-12 activity is required cell-autonomously or non-cell-autonomously, the ttx-3 promoter was used to express sel-12 cDNA exclusively in AIY9. sel-12(ar131) and sel-12(ar171) mutants that expressed ttx-3::sel-12 from an extra-chromosomal array were analyzed. All transgenic animals still showed the fully penetrant egg-laying defect typical of sel-12 mutants, but expression of sel-12 solely in AIY restored isothermal tracking. In addition, the morphology of the AIY neurons was indistinguishable from wild-type. Together, these data indicate that SEL-12 activity in AIY is required cell-autonomously for correct neurite connectivity and for the only known function of this neuron (Wittenburg, 2000).

During Caenorhabditis elegans hermaphrodite development, the anchor cell induces the vulva and the uterine pi cells whose daughters connect to the vulva, thereby organizing the uterine-vulval connection. Both the initial selection of a single anchor cell during the anchor cell vs. ventral uterine precursor cell decision and the subsequent induction of the pi cell fate by the anchor cell are mediated by the lin-12 gene. Members of the presenilin gene family can cause early onset Alzheimer's disease when mutated and are also required for LIN-12/Notch signaling during development. In C. elegans, mutation of the sel-12-encoded presenilin results in pi cell induction defects. By contrast, other lin-12-mediated cell fate decisions occur normally in sel-12 mutants due to the redundant function of a second C. elegans presenilin called HOP-1. The sel-12 egg-laying defect is partially rescued by expression of the sel-12 gene in the pi cells. sel-12-mediated pi cell fate specification provides a useful system for the analysis of presenilin function at single cell resolution (Cinar, 2001).

Presenilin is an essential component of the LIN-12/Notch signaling pathway and also plays a critical role in the genesis of Alzheimer's disease. Previously, a screen for suppressors of the egg-laying defective phenotype caused by partial loss of presenilin activity in Caenorhabditis elegans identified a number of new spr genes that are potentially involved in the regulation of LIN-12/Notch signaling or presenilin activity. The molecular identity of two spr genes, spr-1 and spr-5, is reported in this study. Genetic analysis indicates that loss of spr-1 elevates lin-12/Notch gene activity in many different cell fate decisions, suggesting that spr-1 is a negative regulator of LIN-12/Notch signaling. Sequence analysis revealed that spr-1 is an ortholog of human CoREST (Drosophila homolog: CoREST), a known corepressor. SPR-1 is localized to the nucleus and acts in a cell-autonomous manner; furthermore, human CoREST can substitute for SPR-1 in C. elegans. spr-5 encodes a homolog of p110b, another known member of the CoREST corepressor complex. These results suggest that the CoREST corepressor complex might be functionally conserved in worms, and the potential role of SPR-1 and SPR-5 in the repression of transcription of genes involved in, or downstream of, LIN-12/Notch signal transduction is discussed (Jarriault, 2002).

Mutations in presenilin genes impair Notch signalling and, in humans, have been implicated in the development of familial Alzheimer's disease. A reduction of the activity of the Caenorhabditis elegans presenilin sel-12 results in a late defect during sex muscle development. The morphological abnormalities and functional deficits in the sex muscles contribute to the egg-laying defects seen in sel-12 hermaphrodites and to the severely reduced mating efficiency of sel-12 males. Both defects can be rescued by expressing sel-12 from the hlh-8 promoter that is active during the development of the sex muscle-specific M lineage, but not by expressing sel-12 from late muscle-specific promoters. Both weak and strong sel-12 mutations cause defects in the sex muscles that resemble the defects found in lin-12 hypomorphic alleles, suggesting a previously uncharacterized LIN-12 signalling event late in postembryonic mesoderm development. Together with a study indicating a role of lin-12 and sel-12 during the specification of the picell lineage required for proper vulva-uterine connection, these data suggest that the failure of sel-12 animals to lay eggs properly is caused by defects in at least two independent signalling events in different tissues during development (Eimer, 2002b).

Regulation of presenilin transcription in C. elegans

Presenilins are part of a protease complex that is responsible for the intramembraneous cleavage of the amyloid precursor protein involved in Alzheimer’s disease and of Notch receptors. In C. elegans, mutations in the presenilin sel-12 result in a highly penetrant egg-laying defect. spr-5 was identified as an extragenic suppressor of the sel-12 mutant phenotype. The SPR-5 protein (Drosophila melanogaster CG17149, accession No. AAF49051) has similarity to the human polyamine oxidase-like protein encoded by KIAA0601 that is part of the HDAC-CoREST co-repressor complex. Suppression of sel-12 by spr-5 requires the activity of HOP-1, the second somatic presenilin in C. elegans. spr-5 mutants derepress hop-1 expression 20- to 30-fold in the early larval stages when hop-1 normally is almost undetectable. SPR-1, a C. elegans homolog of CoREST, physically interacts with SPR-5. Moreover, down-regulation of SPR-1 by mutation or RNA interference also bypasses the need for sel-12. These data strongly suggest that SPR-5 and SPR-1 are part of a CoREST-like co-repressor complex in C. elegans . This complex might be recruited to the hop-1 locus controlling its expression during development (Eimer, 2002a).

The mechanism of the derepression is not clear, but might affect chromatin structure and remodelling. Another known suppressor of the sel-12 Egl defect, spr-2, encodes a protein with similarity to the Set/TAF-Iß oncoprotein found in the INHAT (inhibitor of acetyltransferase) complex which is able to inhibit histone acetyltransferase (HAT) activities by p300/CBP and PCAF through histone masking. Genetically, spr-2 behaves similarly to spr-5 and was also shown to be dependent on hop-1 activity. However, in spr-2 mutants, hop-1 transcription does not increase, suggesting that spr-2 and spr-5 function through separate mechanisms. Interestingly, both proteins might be associated with chromatin complexes (Eimer, 2002).

The proteins that are most similar to SPR-5, except for T08D10.2, a C. elegans paralogue, are the human KIAA0601/ p110b and a predicted protein from D. melanogaster encoded by CG17149. All those proteins share regions of similarity with FAD-dependent PAOs. The mutation in spr-5(by113) results in an exchange of a conserved glycine residue to arginine at position 423. This allele is phenotypically indistinguishable from the deletion alleles, suggesting that the G423R point mutation interferes with an essential function of the protein. Interestingly, in the maize PAO crystal structure, this position is located in the FAD-binding domain, close to the FAD-binding pocket. The human KIAA0601 protein was found to be an integral component of the CoREST co-repressor complex. This may indicate that the mutation interferes with an enzymatic function of SPR-5 required for the repressor activity of such a complex (Eimer, 2002).

In addition to the PAO, other HDAC-CoREST complex components are HDAC1, HDAC2 and the SANT domain protein CoREST. CoREST, together with REST/NRSF (RE1 silencing transcription factor/neural-restrictive silencing factor), acts to repress neuronally expressed genes in non-neuronal cells. However, REST can act through multiple deacetylase complexes, only one of them being CoREST. The existence of a CoREST complex in C. elegans is corroborated further by the fact that screens also identified a mutant of spr-1/CoREST. The similar phenotypes of spr-5 and spr-1 suggest a similar function of both encoded proteins in the repression of early hop-1 transcription. Co-immunoprecipitation experiments also strongly indicate that SPR-1 and SPR-5 proteins interact in C. elegans , as was shown previously for their human homologs. Furthermore, in additional screens, two other sel-12 suppressors, spr-3 and spr-4, have been identified whose closest human homolog is REST. Therefore, mutations in at least four proteins similar to components of the CoREST-HDAC complex are able to suppress sel-12 by up-regulating the activity of the second presenilin, hop-1 (Eimer, 2002).

The FAD-binding motif of SPR-5 is well conserved and places SPR-5 in the superfamily of FAD-dependent oxidases. It is probably inactive in the spr-5(by113) mutant. The amino acid sequence identities between different members of this superfamily are normally quite low and range between 20% and 30%. In contrast to the FAD domain, the substrate recognition domain is not conserved among various members of this family and, therefore, different oxidative reactions are catalysed by individual amine oxidases. Although different substrates are bound, the overall three-dimensional structures of the substrate recognition domains of PAOs are strikingly similar to those of monoamine oxidases. It is possible, therefore, that despite its divergent substrate recognition domain, SPR-5 might have retained a comparable enzymatic activity. However, in the absence of functional data, one can only speculate about a function for this class of PAO (Eimer, 2002).

A number of different proteins with enzymatic activities have been identified recently in HDAC complexes regulating transcriptional repression. For example, both HAT and HDAC complexes are involved in controlling transcriptional regulation mediated by the Notch intracellular domain. However, the data clearly show that SPR-1 and SPR-5 do not regulate lin-12/Notch signaling directly. A recently discovered family of co-repressor proteins, the C-terminal binding proteins (CtBPs), exhibits similarity to dehydrogenase enzymes. CtBP adopts different conformations dependent on the cofactor bound (NAD+ or NADH), modulating its affinity for partner proteins and, thus, the level of repression. It is possible that, upon FAD binding, a similar mechanism is induced in the SPR-5/PAO in the CoREST complexes. It has been suggested that KIAA0601 could, in principle, have an enzymatic activity that involves the oxidation of amines or amino groups, such as for example the methylation of lysine or arginine side chains on modified histone tails. The methylation of lysine residues in the histone tails has been shown to modulate the interaction of repressor complexes with tails of specific regulatory sequences (Eimer, 2002).

In summary, the data strongly indicate that spr-5 encodes a PAO-like factor that is part of a transcriptional repressor complex similar to the human CoREST complex. This study describes a target gene that is controlled genetically by a CoREST-associated PAO. C. elegans SPR-5, most probably in a complex with CoREST/SPR-1, regulates the repression of hop-1 presenilin at early developmental stages. The presence of two homologous proteins of each component of the CoREST complex in C. elegans indicates that there may exist more co-repressor complexes of this type in the nematode, only one of them being involved in hop-1 regulation. Based on the dsRNAi experiment, it is possible that additional CoREST complexes might function in other regulatory processes not related to hop-1 (Eimer, 2002).

Mutations in presenilin genes are associated with familial Alzheimer's disease in humans and affect LIN-12/Notch signaling in all organisms tested so far. Loss of sel-12 presenilin activity in Caenorhabditis elegans results in a completely penetrant egg-laying defect. In screens for extragenic suppressors of the sel-12 egg-laying defect, mutations have been isolated in at least five genes. spr-3 and spr-4, which encode large basic C2H2 zinc-finger proteins have been cloned and characterized. Suppression of sel-12 by spr-3 and spr-4 requires the activity of the second presenilin gene, hop-1. Mutations in both spr-3 and spr-4 de-repress hop-1 transcription in the early larval stages when hop-1 expression is normally nearly undetectable. Since sel-12 and hop-1 are functionally redundant, this suggests that mutations in spr-3 and spr-4 bypass the need for one presenilin by stage-specifically de-repressing the transcription of the other presenilin. Both spr-3 and spr-4 code for proteins similar to the human REST/NRSF (Re1 silencing transcription factor/neural-restrictive silencing factor) transcriptional repressors. Since other Spr genes encode proteins homologous to components of the CoREST co-repressor complex that interacts with REST, and the INHAT (inhibitor of acetyltransferase) co-repressor complex, these data suggest that all Spr genes may function through the same mechanism that involves transcriptional repression of the hop-1 locus (Lakowski, 2003).

Although SPR-3 and SPR-4 do not have clear mammalian homologs, they may be performing a similar function as known transcriptional repressors. The C2H2 zinc-finger factor REST mediates repression of neuronal genes in non-neuronal cells, by recruiting the co-repressor complexes Sin3 and CoREST. Both of these co-repressor complexes contain multiple proteins, including histone deacetylases, and presumably repress transcription in part by removing activating acetyl groups from histones H3 and H4 at the target locus. It is possible that SPR-3 and SPR-4 may also function by recruiting conserved co-repressor complexes to the hop-1 locus. Three other Spr genes, spr-1, spr-2 and spr-5, encode proteins similar to components of known co-repressors. SPR-2 is a member of the Nucleosome Assembly Protein (NAP) family and is most similar to the human oncogene SET. Human SET has been purified as part of the INHAT co-repressor complex, which helps to repress transcription by binding to histones and masking them from being acetyltransferase substrates for p300/CBP and PCAF. Upregulation of SET also inhibits demethylation of methylated DNA and may integrate the epigenetic states of DNA and associated histones. SPR-5 has been shown to encode a polyamine oxidase-like protein most similar to a known component of the CoREST co-repressor complex. The core CoREST complex contains only six proteins. spr-1 encodes a homolog of the MYB domain-containing protein CoREST, an additional component of the CoREST co-repressor complex. SPR-1 and SPR-5 interact biochemically in vitro and in vivo. This suggests that a similar complex is present in C. elegans and functions to repress hop-1 transcription. Interestingly, CoREST has been found to associate with at least two large, basic C2H2 zinc-finger proteins, ZNF217 and REST, and may be a general co-repressor complex that is recruited to different loci in different cell types by binding to different C2H2 zinc-finger proteins. Recent work also suggests that CoREST may interact with components of the SWI-SNF complex and may be involved in silencing of chromosomal regions (Lakowski, 2003).

Regulation of presenilin activity

Mutations in two related genes, PS1 and PS2, account for the majority of early onset cases of familial Alzheimer's disease. PS1 and PS2 are homologous polytopic membrane proteins that are processed endoproteolytically into two fragments in vivo. The fate of endogenous PS1 and PS2 has been examined after overexpression of human PS1 or PS2 in mouse N2a neuroblastoma cell lines and human PS1 in transgenic mice. Remarkably, in N2a cell lines and in brains of transgenic mice expressing human PS1, accumulation of human PS1 derivatives is accompanied by a compensatory, and highly selective, decrease in the steady-state levels of murine PS1 and PS2 derivatives. Similarly, the levels of murine PS1 derivatives are diminished in cultured cells overexpressing human PS2. To define the minimal sequence requirements for 'replacement' familial Alzheimer's, disease-linked and experimental deletion variants of PS1 were expressed. These studies revealed that compromised accumulation of murine PS1 and PS2 derivatives resulting from overexpression of human PS1 occurs in a manner independent of endoproteolytic cleavage. These results are consistent with a model in which the abundance of PS1 and PS2 fragments is regulated coordinately by competition for limiting cellular factor(s) (Thinakaran, 1997).

Mutations in presenilin (PS) genes cause early-onset familial Alzheimer's disease by increasing production of the amyloidogenic form of amyloid beta peptides ending at residue 42 (Abeta42). PS is an evolutionarily conserved multipass transmembrane protein, and all known PS proteins contain a proline-alanine-leucine-proline (PALP) motif starting at proline (P) 414 (amino acid numbering based on human PS2) at the C terminus. Furthermore, missense mutations that replace the first proline of PALP with leucine (P414L) lead to a loss-of-function of PS in Drosophila melanogaster and Caenorhabditis elegans. To elucidate the roles of the PALP motif in PS structure and function, neuro2a as well as PS1/2 null fibroblast cell lines were examined, transfected with human PS harboring mutations at the PALP motif. P414L mutation in PS2 (and its equivalent in PS1) abrogates stabilization, high molecular weight complex formation, and entry to Golgi/trans-Golgi network of PS proteins; this results in failure of Abeta42 overproduction, as is the case in familial Alzheimer's disease mutation, as well as failure of site-3 cleavage of Notch. These data suggest that the first proline of the PALP motif plays a crucial role in the stabilization and formation of the high molecular weight complex of PS, the latter being the active form with intramembrane proteolytic activities (Tomita, 2001).

Proteolytic processing and stabilization of presenilin

Presenilin 1 (PS1), mutated in pedigrees of early-onset familial Alzheimer's disease, is a polytopic integral membrane protein that is endoproteolytically cleaved into 27-kDa N-terminal and 17-kDa C-terminal fragments. Although these fragments are the principal PS1 species found in normal mammalian brain, the role of endoproteolysis in the maturation of PS1 has been unclear. The present study, which uses stably transfected mouse neuroblastoma N2a cells, demonstrates that full-length polypeptides, derived from either wild-type or A246E FAD-mutant human (hu) PS1, are relatively short-lived (t1/2 1.5 h) proteins that give rise to the N- and C-terminal PS1 fragments, which are more stable (t1/2 approximately 24 h). N-terminal fragments, generated artificially by engineering a stop codon at amino acid 306 (PS1-306) of wild-type huPS1, were short-lived, whereas an FAD-linked variant that lacked exon 9 (DeltaE9) and was not endoproteolytically cleaved exhibited a long half-life. These observations suggest that endoproteolytic cleavage and stability are not linked, leading to a model in which wild-type full-length huPS1 molecules are first stabilized then subsequently endoproteolytically cleaved to generate the N- and C-terminal fragments. These fragments appear to represent the mature and functional forms of wild-type huPS1 (Ratovitski, 1997).

The gamma-secretase protein complex

Mutations in the presenilin (PS) genes are linked to early onset familial Alzheimer's disease (FAD). PS-1 proteins are proteolytically processed by an unknown protease to two stable fragments of approximately 30 kDa [N-terminal fragment (NTF)] and approximately 20 kDa [C-terminal fragment (CTF)]. The CTF and NTF of PS-1 bind to each other. Fractionating proteins from extracted membrane preparations by velocity sedimentation reveals a high molecular mass SDS and Triton X-100-sensitive complex of approximately 100-150 kDa. To prove if both proteolytic fragments of PS-1 are bound to the same complex, co-immunoprecipitations were performed using multiple antibodies specific to the CTF and NTF of PS-1. These experiments revealed that both fragments of PS-1 occur as a tightly bound non-covalent complex. Upon overexpression, unclipped wild type PS-1 sediments at a lower molecular weight in glycerol velocity gradients than the endogenous fragments. In contrast, the non-cleavable, FAD-associated PS-1 Deltaexon 9 sediments at a molecular weight similar to that observed for the endogenous proteolytic fragments. This result may indicate that the Deltaexon 9 mutation generates a mutant protein that exhibits biophysical properties similar to the naturally occurring PS-1 fragments. This could explain the surprising finding that the Deltaexon 9 mutation is functionally active, although it cannot be proteolytically processed. Formation of a high molecular weight complex of PS-1 composed of both endogenous PS-1 fragments may also explain the recent finding that FAD-associated mutations within the N-terminal portion of PS-1 result in the hyperaccumulation not only of the NTF but also of the CTF. Moreover, these results provide a model to understand the highly regulated expression and processing of PS proteins (Capell, 1998).

Mutations in presenilin 1 (PS1) and PS2 genes contribute to the pathogenesis of early onset familial Alzheimer's disease by increasing secretion of the pathologically relevant Aß42 polypeptides. PS genes are also implicated in Notch signaling through proteolytic processing of the Notch receptor in C. elegans, Drosophila, and mammals. Drosophila PS (Psn) protein undergoes endoproteolytic cleavage and forms a stable high molecular weight (HMW) complex in Drosophila S2 or mouse neuro2a (N2a) cells in a similar manner to mammalian PS. The loss-of-function recessive point mutations located in the C-terminal region of Psn, that cause an early pupal-lethal phenotype resembling Notch mutant in vivo, disrupts the HMW complex formation, and abolishes gamma-secretase activities in cultured cells. The overexpression of Psn in mouse embryonic fibroblasts lacking PS1 and PS2 genes rescues the Notch processing. Moreover, disruption of the expression of Psn by double-stranded RNA-mediated interference completely abolishes the gamma-secretase activity in S2 cells. Surprisingly, gamma-secretase activity dependent on wild-type Psn is associated with a drastic overproduction of Aß1-42 from human ßAPP in N2a cells, but not in S2 cells. These data suggest that the mechanism of gamma-secretase activities through formation of HMW PS complex, as well as its abolition by loss-of-function mutations located in the C terminus, are highly conserved features in Drosophila and mammals (Takasugi, 2002).

The formation of the stabilized HMW complex of mammalian PS, that requires the integrity of the conserved PS C terminus, is essential to the acquisition of gamma-secretase activity, and an aspartate residue within 7th TMD (TMD7) is crucial to the gamma-secretase activity in mammalian PS. To verify the effects of missense mutations in Psn that cause Notch (i.e, loss-of-function) phenotype in Drosophila in vivo, on the metabolism of Psn polypeptides, the two types of amino acid substitutions (i.e., P507L or G516E) were introduced and stably expressed the mutant Psn in N2a NL/N cells. In addition, N2a NL/N cells, stably coexpressing Psn carrying D461A mutation that replaces the highly conserved aspartate residue in the TMD7 with alanine, were established to see if this mutation works as a dominant negative mutant on gamma-cleavage as in mammalian PS. Neither Psn/P507L, Psn/G516E nor Psn/D461A undergo endoproteolysis to give rise to NTF and CTF that normally occurs with wild type Psn. The replacement of endogenous PS1 did not occur in N2a NL/N cells coexpressing Psn/P507L or Psn/G516E. Upon CHX treatment of the N2a cells, the Psn/P507L or Psn/G516E holoproteins were rapidly degraded in a similar manner to wild type Psn holoprotein. In contrast, the overexpression of Psn/D461A results in a complete replacement of endogenous murine PS1 fragments, and a portion of Psn/D461A is stabilized as a holoprotein, as previously described in aspartate mutants of mammalian PS (i.e., PS1/D385A, PS2/D366A). The HMW complex formation of Psn and its derivatives was analyzed. The unstable Psn/P507L or Psn/G516E holoproteins were fractionated exclusively in the LMW range. In contrast, Psn/D461A, which was stabilized but not cleaved, was present as holoproteins broadly within LMW and HMW ranges in a similar manner to that of mammalian PS2/D366A (Takasugi, 2002).

Psn-dependent gamma-secretase activity in Drosophila has been shown to cleave Notch and other transmembrane proteins in vivo. The amino acid sequence of APPL, a Drosophila homolog of ßAPP, is not homologous to that of mammalian ßAPP especially within the TMD, and gamma-cleavage of APPL has not been documented. However, it has been shown that overexpression of the C-terminal 99 amino acid fragment of human ßAPP elicits the cleavage to generate Aß1-40 by a gamma-secretase-like activity in Drosophila SL-2 cells, although Drosophila cells lack ß-secretase activity. To evaluate the gamma-secretase-like activity for proteolytic processing of the TMD sequence of human ßAPP in Drosophila S2 cells, a cDNA encoding SC100, that corresponds to the C-terminal fragment of human ßAPP starting at the 1st residue of Aß preceded by a signal peptide, was transiently transfected and the conditioned media by ELISA was analyzed. Aß secretion was readily detectable in conditioned media of cells expressing SC100; surprisingly, however, percent Aß42 was ~15%, which was in sharp contrast to the robust Aß1-42 overproduction in mouse N2a cells, that is mediated by the same PS species, i.e., wild type Psn. To exclude the possibility that gamma-secretase-like activity in S2 cells is incapable of producing excessive amounts of Aß1-42, a cDNA encoding SC100 was constructed harboring an isoleucine to phenylalanine substitution at residue 716 of ßAPP (SC100/I716F); this substitution has been shown to cause robust increase in Aß1-42 secretion in COS cells. Transfection of SC100/I716F into S2 cells results in a dramatic increase in Aß1-42 secretion and simultaneous decrease in Aß40 secretion, suggesting that the endogenous gamma-secretase-like activity mediated by Psn normally cleaves the TMD sequence of human ßAPP predominantly at Aß40 position, but is capable of cleaving predominantly at position 42 under pathogenic conditions (e.g., ßAPP mutation) in S2 cells. Thus, Psn-dependent gamma-cleavage in S2 cells shows similar characteristics to those in mammalian cells, whereas it may be shifted to position 42 by some unknown mechanism in mouse N2a cells (Takasugi, 2002).

To examine whether Psn plays an essential role in Aß generation by a gamma-secretase-like activity in S2 cells, an S2 cell line was generated stably expressing SC100 (S2-SC100) and the expression of endogenous Psn gene was suppressed by double-stranded RNA (dsRNA)-mediated interference (RNAi). After a 48-h transfection of Psn dsRNA, the expression of Psn polypeptide in the form of fragments was completely and specifically abolished in S2-SC100 cells. After incubation in fresh media for additional 24 h, the cell lysates and conditioned media were analyzed. Immunoblot analysis has revealed an accumulation of SC100 as well as of a ~10-kDa polypeptide comigrating with C83 of mammalian cells. The latter band presumably represents the SC100 derivative cleaved by an alpha-secretase-like activity, that has been reported in Drosophila and SL-2 cells. No Aß secretion was observed in conditioned media, suggesting that the total suppression of the expression of Psn by RNAi results in a complete loss of gamma-secretase activity. Thus, Psn-dependent gamma-secretase activity is required for Aß generation from a human ßAPP derivative (i.e., SC100) in Drosophila S2 cells (Takasugi, 2002).

Amyloid beta-peptide (Abeta) is generated by the consecutive cuts of two membrane-bound proteases. Beta-secretase cuts at the N terminus of the Abeta domain, whereas gamma-secretase mediates the C-terminal cut. Recent evidence suggests that the presenilin (PS) proteins, PS1 and PS2, may be gamma-secretases. Because PSs principally exist as high molecular weight protein complexes, biologically active gamma-secretases likely require other cofactors such as nicastrin (Nct; see Drosophila Nicastrin) for their activities. Preferentially mature Nct forms a stable complex with PSs. Nct levels have been down-regulated by using a highly specific and efficient RNA interference approach. Very similar to a loss of PS function, down-regulation of Nct levels leads to a massive accumulation of the C-terminal fragments of the beta-amyloid precursor protein. In addition, Abeta production is markedly reduced. Strikingly, down-regulation of Nct destabilizes PS and strongly lowers levels of the high molecular weight PS1 complex. Interestingly, absence of the PS1 complex in PS1(-/-) cells is associated with a strong down-regulation of the levels of mature Nct, suggesting that binding to PS is required for trafficking of Nct through the secretory pathway. Based on these findings it is concluded that Nct and PS regulate each other and determine gamma-secretase function via complex formation (Edbauer, 2002).

The gamma-secretase complex catalyzes the final intramembraneous cleavage of the beta-amyloid precursor protein, liberating the neurotoxic amyloid beta-peptide implicated in Alzheimer's disease. Apart from the catalytic subunit presenilin (PS), three additional subunits, nicastrin, APH-1, and PEN-2, have been identified. In mammals, two PS homologues, PS1 and PS2, which are part of distinct gamma-secretase complexes, exist. Likewise, two APH-1 homologues, APH-1a and APH-1b, have been identified. Furthermore, two APH-1a splice forms, APH-1aS and APH-1aL, have been reported. Both APH-1a splice forms and APH-1b are expressed in peripheral and neuronal cells. APH-1aS, APH-1aL, and APH-1b form separate, proteolytically active gamma-secretase complexes containing either one of the two PSs. Deficiency of APH-1a causes a decrease in nicastrin, PS, and PEN-2 levels and an increase in the levels of APH-1b, whereas deficiency of APH-1b did not affect the levels of APH-1a or the other complex components. Consistent with this finding, it was found that deficiency of APH-1a is associated with reduced gamma-secretase activity, whereas deficiency of APH-1b is not. Thus, APH-1b gamma-secretase complexes may fulfill redundant functions. Taken together, these results suggest that, dependent on the tissue expression of the individual subunits, six distinct gamma-secretase complexes composed of the known subunits can exist in human cells (Shirotani, 2004).

Studies conducted in cell culture indicate that the gamma-secretase involved in amyloid beta-formation and Notch signaling is a multisubunit aspartic protease. Little is known, however, of the structure, function, or localization of gamma-secretase in the adult brain, or possible effects of familial Alzheimer's disease (FAD)-causing mutations on the brain protease. Mouse brain contains a complex composed of gamma-secretase subunits presenilin-1 N-terminal fragment, presenilin-1 C-terminal fragment, Nicastrin, Aph-1a and Pen-2. A homozygous FAD-linked Presenilin-1 knock-in mutation does not alter relative subunit levels. Immunocytochemical localization of gamma-secretase subunits revealed overlapping but distinct regional and subcellular distributions. All subunits are expressed throughout the neuraxis predominantly in neurons, and are present in axons. Their distributions and levels of expression are unaffected by mutant presenilin-1. In a presenilin-1/amyloid precursor protein double knock-in mouse, subunits are associated with plaques, but are expressed at similar levels in amyloid-rich and -poor regions. gamma-Secretase subunits are distributed much more extensively than circumscribed amyloid deposits, suggesting the importance of other factors for localized amyloid deposition. These results indicate a widespread neuronal function for gamma-secretase in the adult brain, and suggest the pathogenic mechanism of FAD-linked mutations does not involve alterations in the composition, expression or brain distribution of the protease. The subcellular localization of gamma-secretase subunits is consistent with a nerve terminal source for amyloid aggregates (Siman, 2004).

Presenilin and nicastrin are essential components of the gamma-secretase complex that is required for the intramembrane proteolysis of an increasing number of membrane proteins including the amyloid-beta precursor protein (APP) and Notch. By using co-immunoprecipitation and nickel affinity pull-down approaches, it has been shown that mammalian APH-1 (mAPH-1: see Drosophila anterior pharynx defective 1), a conserved multipass membrane protein, physically associates with nicastrin and the heterodimers of the presenilin amino- and carboxyl-terminal fragments in human cell lines and in rat brain. Similar to the loss of presenilin or nicastrin, the inactivation of endogenous mAPH-1 using small interfering RNAs results in the decrease of presenilin levels, accumulation of gamma-secretase substrates (APP carboxyl-terminal fragments), and reduction of gamma-secretase products (amyloid-beta peptides and the intracellular domains of APP and Notch). These data indicate that mAPH-1 is probably a functional component of the gamma-secretase complex required for the intramembrane proteolysis of APP and Notch (Lee, 2002).

APH-1 and PEN-2 genes modulate the function of nicastrin and the presenilins in Caenorhabditis elegans. Preliminary studies in transfected mammalian cells overexpressing tagged APH-1 proteins suggest that this genetic interaction is mediated by a direct physical interaction. Using the APH-1 protein encoded on human chromosome 1 [APH-1(1)L; also known as APH-1a] as an archetype, it is reported that endogenous forms of APH-1 are predominantly expressed in intracellular membrane compartments, including the endoplasmic reticulum and cis-Golgi. APH-1 proteins directly interact with immature and mature forms of the presenilins and nicastrin within high molecular weight complexes that display gamma- and epsilon-secretase activity. Indeed APH-1 proteins can bind to the nicastrin delta312-369 loss of function mutant, which does not undergo glycosylation maturation and is not trafficking beyond the endoplasmic reticulum. The levels of expression of endogenous APH-1(1)L can be suppressed by overexpression of any other members of the APH-1 family, suggesting that their abundance is coordinately regulated. Finally, although the absence of APH-1 destabilizes the presenilins, in contrast to nicastrin and PEN-2, APH-1 itself is only modestly destabilized in cells lacking functional expression of presenilin 1 or presenilin 2. Taken together, these data suggest that APH-1 proteins, and APH-1(1) in particular, may have a role in the initial assembly and maturation of presenilin.nicastrin complexes (Gu, 2003).

The Alzheimer disease-associated presenilin (PS) proteins apparently provide the active site of gamma-secretase, an unusual intramembrane-cleaving aspartyl protease. PSs principally occur as high molecular weight protein complexes that contain nicastrin (Nct) and additional as yet unidentified components. Recently, PEN-2 has been implicated in gamma-secretase function. PEN-2 is a critical component of PS1/gamma-secretase and PS2/gamma-secretase complexes. Strikingly, in the absence of PS1 and PS1/PS2, PEN-2 levels are strongly reduced. Similarly, PEN-2 levels are reduced upon RNA interference-mediated down-regulation of Nct. In contrast, down-regulation of PEN-2 by RNA interference is associated with reduced PS levels, impairs Nct maturation, and deficient gamma-secretase complex formation. It is concluded that PEN-2 is an integral gamma-secretase complex component and that gamma-secretase complex components are expressed in a coordinated manner (Steiner, 2002).

A combination of genetic factors and early life events is thought to determine the vulnerability of an individual to develop a complex neurodevelopmental disorder like schizophrenia. Pharmacogenetically selected, apomorphine-susceptible Wistar rats (APO-SUS) display a number of behavioral and pathophysiological features reminiscent of such disorders. Microarray analyses reveal in APO-SUS rats, relative to their counterpart APO-UNSUS rats, a reduced expression of Aph-1b, a component of the gamma-secretase enzyme complex that is involved in multiple neural developmental signaling pathways. The reduced expression is due to a duplicon-based genomic rearrangement event resulting in an Aph-1b dosage imbalance. The expression levels of the other gamma-secretase components were not affected. However, gamma-secretase cleavage activity was significantly changed, and the APO-SUS/-UNSUS Aph-1b genotypes segregate with a number of behavioral phenotypes. Thus, a subtle imbalance in the expression of a single, developmentally important protein may be sufficient to cause a complex phenotype (Coolen, 2005).

gamma-Secretase is an intramembrane-cleaving aspartyl protease complex that mediates the final cleavage of beta-amyloid precursor protein to liberate the neurotoxic amyloid-beta peptide implicated in Alzheimer's disease. The four proteins presenilin (PS), nicastrin (NCT), APH-1, and PEN-2 are sufficient to reconstitute gamma-secretase activity in yeast. Although PS seems to contribute the catalytic core of the gamma-secretase complex, no distinct function has been attributed to the other components so far. In Caenorhabditis elegans, mutation of a glycine to an aspartic acid within a conserved GXXXG motif in the fourth transmembrane domain of APH-1 causes a loss of function phenotype. Surprisingly, it was found that the human homologue APH-1a carrying the equivalent mutation G122D is fully active in yeast co-expressing PS1, NCT, and PEN-2. To address this discrepancy, APH-1a was expressed as G122D in HEK293 cells. Overexpressed APH-1a G122D os not incorporated into the gamma-secretase complex. Separate overexpression of PS1, NCT, or PEN-2 together with APH-1a G122D allows the formation of heterodimers lacking the other endogenous components. Only the combined overexpression of PS1 and NCT together with APH-1a G122D facilitates the formation of a fully active gamma-secretase complex. Under these conditions, APH-1a G122D supports the production of normal amounts of Abeta. It is concluded that cooperative effects may stabilize a trimeric complex of APH-1a G122D together with PS1 and NCT. Upon successful complex assembly, the GXXXG motif becomes dispensable for gamma-secretase activity (Edbauer, 2005).

gamma-Secretase is a multimeric membrane protein complex comprised of presenilin (PS), nicastrin (Nct), Aph-1, and Pen-2. It is a member of an atypical class of aspartic proteases that hydrolyzes peptide bonds within the membrane. During the biosynthetic process of the gamma-secretase complex, Nct and Aph-1 form a heterodimeric intermediate complex and bind to the C-terminal region of PS, serving as a stabilizing scaffold for the complex. Pen-2 is then recruited into this trimeric complex and triggers endoproteolysis of PS, conferring gamma-secretase activity. Although the Pen-2 accumulation depends on PS, the binding partner of Pen-2 within the gamma-secretase complex remains unknown. PS1 was reconstituted in Psen1/Psen2 deficient cells by expressing a series of PS1 mutants in which one of the N-terminal six transmembrane domains (TMDs) was swapped with those of CD4 (a type I transmembrane protein) or CLAC-P (a type II transmembrane protein). The proximal two-thirds of TMD4 of PS1, including the conserved Trp-Asn-Phe sequence, is required for its interaction with Pen-2. Using a chimeric CD4 molecule harboring PS1 TMD4, it was further demonstrated that the PS1 TMD4 bears a direct binding motif to Pen-2. Pen-2 may contribute to the activation of the gamma-secretase complex by directly binding to the TMD4 of PS1 (Watanabe, 2005).

gamma-Secretase is a membrane protein complex that cleaves the beta-amyloid precursor protein (APP) within the transmembrane region, after prior processing by beta-secretase, producing amyloid beta-peptides Abeta(40) and Abeta(42). Errant production of Abeta-peptides that substantially increases Abeta(42) production has been associated with the formation of amyloid plaques in Alzheimer's disease patients. Biophysical and genetic studies indicate that presenilin-1, which contains the proteolytic active site, and three other membrane proteins [nicastrin, anterior pharynx defective-1 (APH-1), and presenilin enhancer-2 (PEN-2)] are required to form the core of the active gamma-secretase complex. This study reports the purification of the native gamma-secretase complexes from HeLa cell membranes and the identification of an additional gamma-secretase complex subunit, CD147 (see Drosophila Basigin), a transmembrane glycoprotein with two Ig-like domains. The presence of this subunit as an integral part of the complex itself was confirmed through coimmunoprecipitation studies of the purified protein from HeLa cells and of solubilized complexes from other cell lines such as neural cell HCN-1A and HEK293. Depletion of CD147 by RNA interference was found to increase the production of Abeta peptides without changing the expression level of the other gamma-secretase components or APP substrates whereas CD147 overexpression has no statistically significant effect on Abeta-peptide production, other gamma-secretase components or APP substrates, indicating that the presence of the CD147 subunit within the gamma-secretase complex down-modulates the production of Abeta-peptides (Zhou, 2005; full text of article).

Macromolecular complexes containing presenilins (PS1 and PS2), nicastrin, anterior pharynx defective phenotype 1 (APH-1), and PS enhancer 2 (PEN-2) mediate the intramembranous, gamma-secretase cleavage of beta-amyloid precursor protein (APP), Notch, and a variety of type 1 membrane proteins. PEN-2 is critical for promoting endoproteolysis of PS1 and the proximal two-thirds of transmembrane domain (TMD) 1 of PEN-2 is required for binding with PS1. This study sought to identify the structural domains of PS1 that are necessary for binding with PEN-2. To address this issue, a series of constructs was generated encoding PS1 mutants harboring deletions or replacements of specific TMDs of PS1-NTF, and the effects of encoded molecules were examined on interactions with PEN-2, stabilization and endoproteolysis of PS1, and gamma-secretase activity. PS1 TMDs 1 and 2 and the intervening hydrophilic loop are dispensable for binding to PEN-2. Furthermore, analysis of chimeric PS1 molecules that harbor replacements of each TMD with corresponding transmembrane segments from the sterol regulatory element-binding protein cleavage activating protein (SCAP) revealed that the PS1-SCAP TMD4 mutant failed to coimmunoprecipitate endogenous PEN-2, strongly suggesting that the fourth TMD of PS1 is required for interaction with PEN-2. Further mutational analyses revealed that the "NF" sequence within the TMD4 of PS1 is the minimal motif that is required for binding with PEN-2, promoting PS1 endoproteolysis and gamma-secretase activity (Kim, 2005).

Regulation of Notch processing by presenilins: Studies with C. elegans

sel-12 (sel means suppressor/enhancer of lin-12) is a transmembrane protein that facilitates Notch signaling in C. elegans. It is related to Presenilin-1 (S182), a mammalian gene that when mutated causes aggressive, early-onset Alzheimer's via an unknown mechanism. In C. elegans, Notch signaling is involved in the anchor cell/ventral uterine precursor cell (AC/VU) decision and vulval precursor cell (VPC) specification during gonadogenesis. The AC/VU decision involves an interaction between two initially equivalent cells of the somatic gonad. When lin-12, which codes for a Notch family member, is eliminated, both precursor cells become ACs. When lin-12 is constitutively activated, both precursor cells become VUs. sel-12 was isolated as a suppressor of a lin-12 gain of function mutation. That is, sel-12 mutation acts to reduce lin-12 signaling. Reducing sel-12 activity reduces lin-12 activity in lateral signaling that specifies the secondary fate of VPCs. Cell ablation experiments show that sel-12 functions within a VPC to lower lin-12 activity. The predicted SEL-12 protein contains multiple potential transmembrane domains, consistent with its function as a receptor, ligand, channel or membrane structural protein. SEL-12 might be directly involved in lin-12-mediated reception, functioning for example as a co-receptor or as a downstream effector that activatesupon LIN-12 activation. Alternatively, sel-12 may be involved in a more general cellular process, such as receptor localization or recycling, and hence influence lin-12 activity indirectly (Levitan, 1995).

The data presented in this paper suggest that the effect of SEL-12/presenilin on LIN-12/Notch is analogous to its effect on APP. LIN-12/Notch proteins are transmembrane proteins with hallmark motifs: epidermal growth factor-like; LIN-12/Notch repeat, and cdc10/SWI6 (ankyrin) motifs. Like APP, LIN-12/Notch proteins must be correctly sorted and transported to the cell surface, and undergo proteolytic cleavage events. There appears to be at least one constitutive proteolytic cleavage event that occurs in the extracellular domain during the transport to the plasma membrane; the cleaved form produced by this constitutive cleavage event may be the major species present at the cell surface. In addition, binding of ligand appears to induce a cleavage event in or near the transmembrane domain; this apparent cleavage event enables the intracellular domain to translocate to the nucleus, where it participates directly in regulating downstream gene expression. It is conceivable that SEL-12/presenilin is involved in promoting one or more of these cleavage events, either by activating protease(s) or promoting trafficking of either LIN-12 or proteases to an appropriate compartment. The strong accumulation of SEL-12::GFP in the ER/Golgi is consistent with a role for SEL-12 in a constitutive cleavage event involved in maturation of LIN-12/Notch proteins. The fact that less LIN-12::GFP was observed at the cell surface in a sel-12 mutant background could be explained in the context of this model by proposing that abnormal processing of LIN-12 leads to its failure to be transported to the plasma membrane or to its degradation. The putative ligand-dependent cleavage of activated LIN-12 might occur at the plasma membrane or in internalized vesicles. The failure to observe SEL-12::GFP in the plasma membranes of the VPCs does not preclude a role for SEL-12 in ligand-dependent cleavage. It is possible that SEL-12::GFP is present at low abundance in the plasma membrane; the ligand-induced event appears to affect a very small proportion of receptor molecules, suggesting that the agent that promotes the cleavage may not be very abundant. Although the issue of the biochemical mechanism of presenilin function is not resolved in any system, the parallels between APP and LIN-12/Notch trafficking and processing suggest that a common mechanism is involved. An important challenge for the future will be to identify the primary effect of SEL-12/presenilin on APP and LIN-12/Notch, since proteolytic processing, intracellular trafficking and degradation are intimately linked, and altering one process can affect another (Levitan, 1998).

Mutant presenilins have been found to cause Alzheimer disease. This paper describes the identification and characterization of HOP-1, a Caenorhabditis elegans presenilin that displays much more lower sequence identity with human presenilins than does the other C. elegans presenilin, SEL-12. Despite considerable divergence, HOP-1 appears to be a bona fide presenilin, because HOP-1 can rescue the egg-laying defect caused by mutations in sel-12 when hop-1 is expressed under the control of sel-12 regulatory sequences. HOP-1 also has the essential topological characteristics of the other presenilins. Reducing hop-1 activity in a sel-12 mutant background causes synthetic lethality and terminal phenotypes associated with reducing the function of the C. elegans lin-12 and glp-1 genes. These observations suggest that hop-1 is functionally redundant with sel-12 and underscore the intimate connection between presenilin activity and LIN-12/Notch activity inferred from genetic studies in C. elegans and mammals (X. Li, 1997).

Mutations in the human presenilin genes PS1 and PS2 cause early-onset Alzheimer's disease. Studies in Caenorhabditis elegans and in mice indicate that one function of presenilin genes is to facilitate Notch-pathway signaling. Notably, mutations in the C. elegans presenilin gene sel-12 reduce signaling through an activated version of the Notch receptor LIN-12. To investigate the function of a second C. elegans presenilin gene hop-1 and to examine possible genetic interactions between hop-1 and sel-12, a reverse genetic strategy was used to isolate deletion alleles of both loci. Animals bearing both hop-1 and sel-12 deletions display new phenotypes not observed in animals bearing either single deletion. These new phenotypes (germ-line proliferation defects, maternal-effect embryonic lethality, and somatic gonad defects) resemble those resulting from a reduction in signaling through the C. elegans Notch receptors GLP-1 and LIN-12. Thus SEL-12 and HOP-1 appear to function redundantly in promoting Notch-pathway signaling. Phenotypic analyses of hop-1 and sel-12 single and double mutant animals suggest that sel-12 provides more presenilin function than does hop-1. There is as yet no evidence that Notch-pathway signaling is involved in the pathophysiology of Alzheimer's disease (AD). Thus, the relationship between the roles of presenilins in proteolytic processing of APP and in facilitating Notch receptor function remains unclear. Intriguingly, recent evidence suggests that multiple proteolytic processing events are required for intracellular trafficking and signal transduction of the Notch receptor: two cleavage events are proposed to occur in the extracellular domain and a third proposed cleavage occurs within or just carboxyl-terminal to the transmembrane region. The apparent similarities between the processing of APP and Notch, particularly the prospect that both are cleaved within the transmembrane domain, raise the possibility that presenilins affect proteolytic processing of APP and Notch in analogous ways. Presenilins might regulate proteolytic processing directly or might do so indirectly, for example, by promoting normal intracellular trafficking of APP or Notch. In support of a role for presenilins in the processing or trafficking of Notch, LIN-12::GFP levels at the plasma membrane are seen to be reduced in a sel-12 mutant background. An understanding of how presenilins affect Notch-receptor activity may be relevant to an understanding of the way in which presenilins affect APP cleavage and to the identification of targets for preventing the pathophysiological effects of presenilin dysfunction in AD (Westlund, 1999).

aph-2 (Drosophila homolog: CG7012) encodes a novel extracellular protein required for GLP-1-mediated signaling (Goutte, 2000). Aph-2, termed Nicastrin in this study (see Drosophila nicastrin), is a transmembrane glycoprotein that forms high molecular weight complexes with presenilin 1 and presenilin 2. Suppression of nicastrin expression in C. elegans embryos induces a subset of notch/glp-1 phenotypes similar to those induced by simultaneous null mutations in both presenilin homologs of C. elegans (sel-12 and hop-1). Nicastrin also binds carboxy-terminal derivatives of beta-amyloid precursor protein (betaAPP), and modulates the production of the amyloid beta-peptide (Abeta) from these derivatives. Missense mutations in a conserved hydrophilic domain of nicastrin increase Abeta42 and Abeta40 peptide secretion. Deletions in this domain inhibit Abeta production. Nicastrin and presenilins are therefore likely to be functional components of a multimeric complex necessary for the intramembranous proteolysis of proteins such as Notch/GLP-1 and betaAPP (Yu, 2000).

In the absence of homology to other proteins, sequence databases were screened for orthologous genes in other species. A full-length C. elegans nicastrin ortholog was found in public databases (accession no. Q23316; identity = 22%; similarity = 41%). Full-length murine and Drosophila nicastrin orthologs from appropriate cDNA libraries were cloned and sequenced using partial cDNA sequences from these databases as start points (mouse nicastrin accession no. AF24069, identity = 89%, similarity = 93%; D. melanogaster nicastrin accession no. AF240470, identity = 30%, similarity = 48%). The four animal nicastrins have similar predicted topologies and have three domains with significant sequence conservation near residues 306-360, 419-458, and 625-662 of human nicastrin. Within the first conserved domain, all four proteins contained the motif DYIGS (residues 336-340), which is also partially conserved in an Arabidopsis protein. All four animal nicastrins also contain four cysteines spaced at 16 to 17-residue intervals in the N terminus (Cys 195, Cys 213, Cys 230 and Cys 248) (Yu, 2000).

To explore whether nicastrin, like the presenilins, might have a role in Notch signaling in vivo, RNA interference (RNAi) was used in C. elegans. Wild-type worms injected with C. elegans nicastrin double-stranded (ds) RNA produce dead embryos, many of which lack an anterior pharynx. This phenotype is highly reproducible and specific. Except for embryonic lethality, none of the other phenotypes associated with a lack of C. elegans presenilin (sel-12) activity were observed. However, this phenotype is identical to that induced when the activity of genes in the notch/glp-1 pathway (glp-1, aph-1 or aph-2) are reduced, or when the activities of both C. elegans presenilin homologs (sel-12 and hop-1) are reduced simultaneously. Thus nicastrin contributes to some aspects of notch/glp-1 signaling in C. elegans embryos (Yu, 2000).

Nicastrin is a multi-pass transmembrane protein that has recently been identified as a member of high-molecular weight complexes containing presenilin. The C. elegans homolog of nicastrin, aph-2, is required for GLP-1/Notch signaling in the early embryo. In addition to the maternal-effect embryonic lethal phenotype, aph-2 mutant animals also display an egg-laying defect. This latter defect is related to the SEL-12/presenilin egg-laying defect. aph-2 and sel-12 genetically interact and cooperate to regulate LIN-12/Notch signaling in the development of the somatic gonad. In addition, aph-2 and lin-12/Notch genetically interact. A new role for aph-2 in facilitating lin-12 signaling in the somatic gonad is illustrated, thus providing evidence that APH-2 is involved in both GLP-1/Notch- and LIN-12/Notch-mediated signaling events. Nicastrin can partially substitute for aph-2, suggesting a conservation of function between these proteins (Levitan, 2001).

Regulation of Notch processing by presenilins: Studies in mammals

Signaling through the receptor protein Notch, which is involved in crucial cell-fate decisions during development, requires ligand-induced cleavage of Notch. This cleavage occurs within the predicted transmembrane domain, releasing the Notch intracellular domain (NICD), and is reminiscent of gamma-secretase-mediated cleavage of beta-amyloid precursor protein (APP), a critical event in the pathogenesis of Alzheimer's disease. A deficiency in presenilin-1 (PS1) inhibits processing of APP by gamma-secretase in mammalian cells, and genetic interactions between Notch and PS1 homologs in Caenorhabditis elegans indicate that the presenilins may modulate the Notch signaling pathway. In mammalian cells, PS1 deficiency also reduces the proteolytic release of NICD from a truncated Notch construct, thus identifying the specific biochemical step of the Notch signaling pathway that is affected by PS1. Moreover, several gamma-secretase inhibitors block this same step in Notch processing, indicating that related protease activities are responsible for cleavage within the predicted transmembrane domains of Notch and APP. Thus the targeting of gamma-secretase for the treatment of Alzheimer's disease may risk toxicity caused by reduced Notch signaling (De Strooper, 1999).

Proteolytic release of the Notch-1 intracellular domain (NICD), an essential step in the activation of Notch signaling, is markedly reduced in presenilin-1 (PS1)-deficient cells and is restored by PS1 expression. Nuclear translocation of the NICD is also markedly reduced in PS1-deficient cells, resulting in reduced transcriptional activation. Mutations in PS1 that are associated with familial Alzheimer's disease impair the ability of PS1 to induce proteolytic release of the NICD and nuclear translocation of the cleaved protein. These results suggest that PS1 plays a central role in the proteolytic activation of the Notch-1-signaling pathway and that this function is impaired by pathogenic PS1 mutations. Thus, dysregulation of proteolytic function may underlie the mechanism by which presenilin mutations cause Alzheimer's disease (Song, 1999).

Genetic analyses in Caenorhabditis elegans demonstrate that sel-12 and hop-1, homologs of the Alzheimer's disease-associated presenilin genes, modify signaling through LIN-12 and GLP-1, homologs of the Notch cell surface receptor. To gain insight into the biochemical basis of this genetic interaction, the possibility that presenilin-1 (PS1) physically associates with the Notch1 receptor in mammalian cells was investigated. Notch1 and PS1 coimmunoprecipitate from transiently transfected human embryonic kidney 293 cell lysates in a detergent-sensitive manner, consistent with a noncovalent physical association between the two proteins. The interaction predominantly occurs early in the secretory pathway prior to Notch cleavage in the Golgi, because PS1 immunoprecipitation preferentially recovers the full-length Notch1 precursor. When PS1 is immunoprecipitated from 293 cells that have been metabolically labeled with [35S]methionine and [35S]cysteine, Notch1 is the primary protein detected in PS1 immunoprecipitates, suggesting that this interaction is specific. Furthermore, endogenous Notch and presenilin coimmunoprecipitate from cultured Drosophila cells, indicating that physical interaction can occur at physiological expression levels. These results suggest that the genetic relationship between presenilins and the Notch signaling pathway derives from a direct physical association between these proteins in the secretory pathway (Ray, 1999a).

Mutations in PS genes cause autosomal dominant Alzheimer disease, with age of onset frequently in the 40s. Notch is known as a developmental protein that plays an important role in lateral inhibition and specifying cell fate decisions in proliferating immature cells, and is not known to be present in adult neurons. It was reasoned that, if Notch1/PS-1 interaction is relevant in Alzheimer disease, Notch1 would also need to be expressed in neurons in adult brain and colocalized with PS-1. Notch1, Notch2, and a Notch ligand, Jagged1, are all expressed in adult brain in mouse and in human, with strongest expression in the hippocampal formation and Purkinje cells of the cerebellum. Double immunofluorescent staining demonstrates neuronal colocalization of Notch1 with PS-1. Moreover, Notch1 expression in sporadic Alzheimer disease hippocampus is elevated more than 2-fold in comparison to that in control human hippocampus by both immunohistochemistry and Western blot analysis (p < 0.007). These results support the hypothesis that Notch1 continues to play a role in terminally differentiated neurons, and that Notch1/PS-1 interactions may occur in adult mammalian brain. The alteration in Notch1 expression in sporadic Alzheimer disease raises the possibility that disruption of Notch1/PS-1 functional interactions may occur in Alzheimer disease (Berezovska, 1998).

The normal functional neurobiology of the Alzheimer's disease (AD) related gene presenilin 1 (PS1) is unknown. One clue comes from a genetic screen of Caenorhabditis elegans, which reveals that the presenilin homolog sel-12 facilitates lin-12 function. The mammalian homolog of lin-12, Notch1, is a transmembrane receptor that plays an important role in cell fate decisions during development, including neurogenesis, but does not have a known function in fully differentiated cells. To better understand the potential role of Notch1 in mammalian postmitotic neurons and to test the hypothesis that Notch and PS 1 interact, the effect of Notch1 transfection on neurite outgrowth in primary cultures of hippocampal/cortical neurons was studied. Notch1 inhibits neurite extension, and thus has a function in postmitotic mature neurons in the mammalian CNS. Furthermore, evidence is presented demonstrating that there is a functional interaction between PS1 and Notch1 in mammalian neurons, analogous to the sel-12/lin-12 interaction in vulval development in C. elegans. The inhibitory effect of Notch1 on neurite outgrowth is markedly attenuated in neurons from PS1 knockout mice, and enhanced in neurons from transgenic mice overexpressing wild type PS1, but not mutant PS1. These data suggest that PS1 facilitates Notch1 function in mammalian neurons, and support the hypothesis that a functional interaction exists between PS1 and Notch1 in postmitotic mammalian neurons (Berezovska, 1999).

Presenilin-1 (PS1), a polytopic membrane protein primarily localized to the endoplasmic reticulum, is required for efficient proteolysis of both Notch and beta-amyloid precursor protein (APP) within their trans-membrane domains. The activity that cleaves APP (called gamma-secretase) has properties of an aspartyl protease; mutation of either of the two aspartate residues located in adjacent transmembrane domains of PS1 inhibits gamma-secretase processing of APP. These aspartates are required for Notch processing, since mutation of these residues prevents PS1 from inducing the gamma-secretase-like proteolysis of a Notch1 derivative. Thus PS1 might function in Notch cleavage as an aspartyl protease or di-aspartyl protease cofactor. However, the ER localization of PS1 is inconsistent with that hypothesis, since Notch cleavage occurs near the cell surface. Using pulse-chase and biotinylation assays, evidence is provided that PS1 binds Notch in the ER/Golgi and is then co-transported to the plasma membrane as a complex. PS1 aspartate mutants are indistinguishable from wild-type PS1 in their ability to bind Notch or traffic with it to the cell surface, and do not alter the secretion of Notch. Thus, PS1 appears to function specifically in Notch proteolysis near the plasma membrane as an aspartyl protease or cofactor (Ray, 1999b).

Mouse Notch1, which plays an important role in cell fate determination in development, is proteolytically processed within its transmembrane domain by unidentified gamma-secretase-like activity that depends on presenilin. To study this proteolytic event, a cell-free Notch cleavage assay system was established using the membrane fraction of fibroblast transfectants of various Notch constructs with deletion of the extracellular portion (NotchDeltaE). The cytoplasmic portion of Notch1DeltaE is released from the membrane upon incubation at 37°C; this is inhibited by the specific gamma-secretase inhibitor, MW167, or by overexpression of dominant negative presenilin1. Likewise, other members of mouse Notch family are proteolytically cleaved in a presenilin-dependent, MW167-sensitive manner in vivo as well as in the cell-free Notch DeltaE cleavage assay system. All four members of the mouse Notch family migrate to the nucleus and activate the transcription from the promoter carrying the RBP-J consensus sequences after they are released from the membrane. These results demonstrate the conserved biochemical mechanism of signal transduction among mammalian Notch family members (Mizutani, 2001).

Following ectodomain shedding, Notch-1 undergoes presenilin (PS)-dependent constitutive intramembranous endoproteolysis at site-3. This cleavage is similar to the PS-dependent g-secretase cleavage of the ß-amyloid precursor protein (ßAPP). However, topological differences in cleavage resulting in amyloid ß-peptide (Aß) or the Notch-1 intracellular domain (NICD) indicate independent mechanisms of proteolytic cleavage. The secretion of an N-terminal Notch-1 Aß-like fragment (Nß) is reported in this study. Analysis of Nß by MALDI-TOF MS revealed that Nß is cleaved at a novel site (site-4, S4) near the middle of the transmembrane domain. Like the corresponding cleavage of ßAPP at position 40 and 42 of the Aß domain, S4 cleavage is PS dependent. The precision of this cleavage is affected by familial Alzheimer’s disease-associated PS1 mutations similar to the pathological endoproteolysis of ßAPP. Considering these similarities between intramembranous processing of Notch and ßAPP, it is concluded that these proteins are cleaved by a common mechanism utilizing the same protease, i.e. PS/g-secretase (Okochi, 2002).

Notch receptors undergo a cascade of endoproteolytic cleavages required for Notch signaling. Upon binding of membrane-anchored ligands from the DSL (Delta/Serrate/Lag-2) family, Notch receptors undergo consecutive cleavages at site-2 (S2) and site-3 (S3). Cleavage of mouse Notch-1 at S2 occurs in its ectodomain by TACE [tumor necrosis factor-a (TNF-a-converting enzyme], a member of the ADAM (a disintegrin and metalloprotease domain) family ~12 amino acids distant from the TM. This 'ectodomain shedding' event results in the generation of NEXT (Notch extracellular truncation), that is cleaved subsequently at S3 within the TM close to the cytoplasmic border. Cleavage of Notch at S3 liberates NICD (Notch intracellular domain) that translocates to the nucleus, where it is involved in target gene transcription. S3 cleavage strictly depends on the biological activity of the presenilin (PS) proteins, which may contribute the catalytic site of g-secretase, an unusual intramembrane-cleaving aspartyl protease complex (Okochi, 2002 and references therein).

Beside the Notch-1-4 receptors, several other type I TM proteins have been identified as substrates for PS-dependent endoproteolysis, including the Alzheimer's disease (AD)-associated ß-amyloid protein precursor (ßAPP), ErbB-4, E-cadherin and LRP. These proteins undergo ‘ectodomain shedding’ in their large extracellular domains, prior to the consecutive PS-dependent cleavage within the TM. In the case of ßAPP, these cleavages are mediated by a-secretase and ß-secretase. Cleavage of ßAPP by a- and ß-secretase (BACE) results in the generation of the respective ßAPP C-terminal fragments (CTFs), CTFa and CTFß, which are the direct substrates for g-secretase cleavage. Cleavage of CTFß and CTFa by g-secretase occurs in the middle of the TM and leads to the liberation of Aß and p3 peptides), respectively. Aß is deposited in the brain of AD patients in 'senile plaques', an invariant pathological hallmark of AD. Recently, the elusive C-terminal cleavage product of g-secretase, AICD (ßAPP intracellular domain), has been identified and characterized. Surprisingly, AICD results from PS-dependent g-secretase cleavage of ßAPP-CTFs predominantly after Leu49 (Aß numbering). This cleavage is almost identical to the S3 cleavage of Notch-1 and does not occur after Val40 and Ala42 (Aß numbering) as predicted. Thus, g-secretase cleaves the ßAPP TM at several sites: one in the middle after position 40 (g40) and 42 (g42) (with major g40 and minor g42 cleavage) and one close to the cytoplasmic border after position 49 (g49) of the Aß domain. Interestingly, AICD may translocate to the nucleus where it could have a role in transcriptional regulation similar to NICD (Okochi, 2002 and references therein).

Because of these striking similarities between Notch and ßAPP endoproteolysis, it was hypothesized that an Aß/p3-like species (called Notch ß-peptide, Nß) derived from NEXT intramembranous proteolysis may be secreted into the extracellular space. This study reports the identification and characterization of secreted Nß peptides derived from endoproteolysis of NEXT derivatives. Sequence analysis revealed that Nß is derived from endoproteolytic cleavage near the middle of the Notch-1 TM at site-4 (S4), which is 12 amino acid residues upstream of S3. Like S3 cleavage, S4 cleavage occurs in a PS- and g-secretase-dependent manner. Strikingly, familial AD (FAD)-associated PS mutants known to cause the increased production of C-terminally elongated pathogenic Aß42 also affect the generation of C-terminally elongated Nß variants, supporting a direct role for PS in the proteolytic cleavage of Notch-1 and ßAPP (Okochi, 2002).

The role of Notch signaling in general and presenilin in particular during mouse somitogenesis was analyzed. Cyclical production of activated Notch (NICD) was visualized and it was established that somitogenesis requires less NICD than any other tissue in early mouse embryos. Indeed, formation of cervical somites proceeds in Notch1; Notch2-deficient embryos. This is in contrast to mice lacking all presenilin alleles, that have no somites. Since Nicastrin-, Pen-2-, and APH-1a-deficient embryos have anterior somites without γ-secretase, presenilin may have a γ-secretase-independent role in somitogenesis. Embryos triple homozygous for both presenilin null alleles and a Notch allele that is a poor substrate for presenilin (N1V→G) experience fortuitous cleavage of N1V→G by another protease. This restores NICD, anterior segmentation, and bilateral symmetry but does not rescue rostral/caudal identities. These data clarify multiple roles for Notch signaling during segmentation and suggest that the earliest stages of somitogenesis are regulated by both Notch-dependent and Notch-independent functions of presenilin (Hupper, 2005).

Activation of mammalian Notch receptor by its ligands induces TNFalpha-converting enzyme-dependent ectodomain shedding, followed by intramembrane proteolysis due to presenilin (PS)-dependent gamma-secretase activity. A modification, monoubiquitination, as well as clathrin-dependent endocytosis, is required for gamma-secretase processing of a constitutively active Notch derivative, DeltaE, which mimics the TNFalpha-converting enzyme-processing product. PS interacts with this modified form of DeltaE, DeltaEu. The lysine residue targeted by the monoubiquitination event has been identified, and its importance for activation of Notch receptor by its ligand, Delta-like 1, has been confirmed. A new model is proposed where monoubiquitination and endocytosis of Notch are a prerequisite for its PS-dependent cleavage, and its relevance for other gamma-secretase substrates is discussed (Gupta-Rossi, 2004).

The ubiquitination pathway involves a multiprotein cascade in which the substrate specificity is determined by the E3 component. Multi-ubiquitin chains at least four subunits long are required for efficient recognition and degradation of ubiquitinated proteins by the proteasome, but ubiquitin has more recently been shown to endorse new functions that do not always involve the proteasome (Gupta-Rossi, 2004).

The results show that a monoubiquitination event takes place on the DeltaE molecule, a constitutively active form of the Notch receptor that mimics the intermediate TACE-processing product generated after ligand binding. This modification is a prerequisite for gamma-secretase cleavage and targets one of the subunits of a dimeric membrane-anchored form of Notch DeltaE. The major site of monoubiquitination has been localized to a juxtamembrane, conserved lysine residue K1749 in mNotch1. Access to the monoubiquitinated form DeltaEu was gained by coimmunoprecipitation with endogenous PS1 when gamma-secretase activity was inhibited by a specific drug. Thus, DeltaEu is a labile intermediate appearing before gamma-secretase cleavage. This form could also be detected after coexpression of PS1 or DeltaC4, a PS2-derived construct. These molecules are probably not included in PS-containing high molecular weight complexes; neither are gamma-secretase components when transiently overexpressed. The existence of DeltaEu was varified by extracting and stabilizing it out of the active complexes. It is proposed that this ubiquitination step is required in the context of the full-length receptor activated by ligand binding. Although the modified intermediate species derived from full-length Notch could not be directly accessed, mutating the crucial lysine residue impaired Dll1-mediated Notch signaling, in accordance with the DeltaE results. It remains to be determined which E3 ubiquitin ligase is involved in this modification. Various proteins carrying such an activity have been associated with the Notch cascade and are candidates to be tested, e.g., Deltex, Suppressor of Deltex, and Cbl. Experiments are in progress to answer this question (Gupta-Rossi, 2004).

The results show that endocytosis of Notch DeltaE and of ligand-activated full-length Notch are necessary for gamma-secretase cleavage. The involvement of a clathrin-dependent endocytosis event for Notch activation complies with the mosaic analysis performed in Drosophila, which revealed that shibire function is required in Notch-expressing cells receiving a lateral inhibition signal. It is proposed that monoubiquitination on a juxtamembrane lysine (K1749) and endocytosis occur after ligand-induced cleavage of the Notch extracellular domain by TACE. The data are in apparent contradiction with a previous model, according to which gamma-secretase cleavage occurs at the plasma membrane. However, the previous data can be reinterpreted in light of the new model. The previous study argued that the TACE-processing product of Notch (similar to the DeltaE construct used in this study) remains associated with the apical membrane in Nicastrin or PS mutant cells, and in WT cells only a small amount of this molecule can be found in endocytic vesicles. This result can be explained by the fact that ubiquitination is one of the limiting steps in Notch signaling, or that active PS is needed to direct the final steps of endocytosis of the ubiquitinated forms. Probably for the same reason, DeltaE is very poorly cleaved by gamma-secretase when overexpressed, and the ubiquitination event can hardly be detected. The current results are also in apparent contradiction with those of another study, which postulates that in Drosophila, PS-mediated proteolysis does not appear to require a particular sequence nor the presence of active dynamin. However, the assay used appears unusually sensitive, as it even detects the cleavage of a Notch molecule carrying a G1743V mutation of the gamma-secretase cleavage site, a mutation that prevents activation in most other assays and, when introduced into mice, gives rise to an almost perfect Notch1 null phenotype. Therefore, a leakage due to overexpression might in some cases be responsible for the activity detected. Experiments are in progress to test the effect of mutating the juxtamembrane lysine of Drosophila Notch (Gupta-Rossi, 2004).

Various papers have described monoubiquitination as a signal for internalization of receptors such as EGFR or glycine receptor. The results do not allow one to discriminate between ubiquitination triggering endocytosis or being concomitant with the first steps of endocytosis. However, the observations show a more internal localization of LLFF (the site of gamma-secretase cleavage) compared with the K1749R mutant, and endocytosis of the K1749R DeltaE mutant seems to be blocked at an earlier stage when compared with the WT or LLFF mutant. These data suggest that ubiquitination is necessary for late events driving Notch to compartments where gamma-secretase cleavage can occur (Gupta-Rossi, 2004).

Canonical Notch signaling is thought to control the endocrine/exocrine decision in early pancreatic progenitors. Later, RBP-Jkappa interacts with Ptf1a and E12 to promote acinar differentiation. To examine the involvement of Notch signaling in selecting specific endocrine lineages, this pathway was deregulated by targeted deletion of presenilin1 and presenilin2, the catalytic core of gamma-secretase, in Ngn3- or Pax6-expressing endocrine progenitors. Surprisingly, whereas Pax6(+) progenitors were irreversibly committed to the endocrine fate, it was discovered that Ngn3(+) progenitors were bipotential in vivo and in vitro. When presenilin amounts are limiting, Ngn3(+) progenitors default to an acinar fate; subsequently, they expand rapidly to form the bulk of the exocrine pancreas. gamma-Secretase inhibitors confirmed that enzymatic activity was required to block acinar fate selection by Ngn3 progenitors. Genetic interactions identified Notch2 as the substrate, and suggest that gamma-secretase and Notch2 act in a noncanonical titration mechanism to sequester RBP-Jkappa away from Ptf1a, thus securing selection of the endocrine fate by Ngn3 progenitors. These results revise the current view of pancreatic cell fate hierarchy, establish that Ngn3 is not in itself sufficient to commit cells to the endocrine fate in the presence of Ptf1a, reveal a noncanonical action for Notch2 protein in endocrine cell fate selection, and demonstrate that acquisition of an endocrine fate by Ngn3(+) progenitors is gamma-secretase-dependent until Pax6 expression begins (Cras-Méneur, 2009).

Presenilin processing of Delta and Jagged

The cleavage of Notch by presenilin (PS)/gamma-secretase is a salient example of regulated intramembrane proteolysis, an unusual mechanism of signal transduction. This cleavage is preceded by the binding of protein ligands to the Notch ectodomain, activating its shedding. It was hypothesized that the Notch ligands, Delta and Jagged, themselves undergo PS-mediated regulated intramembrane proteolysis. The ectodomain of mammalian Jagged is shown to be cleaved by an A disintegrin and metalloprotease (ADAM) 17-like activity in cultured cells and in vivo, similar to the known cleavage of Drosophila Delta by Kuzbanian. The ectodomain shedding of ligand can be stimulated by Notch and yields membrane-tethered C-terminal fragments (CTFs) of Jagged and Delta that accumulate in cells expressing a dominant-negative form of PS or treated with gamma-secretase inhibitors. PS forms stable complexes with Delta and Jagged and with their respective CTFs. PS/gamma-secretase then mediates the cleavage of the latter to release the Delta and Jagged intracellular domains, a portion of which can enter the nucleus. The ligand CTFs compete with an activated form of Notch for cleavage by gamma-secretase and can thus inhibit Notch signaling in vitro. The soluble Jagged intracellular domain can activate gene expression via the transcription factor AP1, and this effect is counteracted by the co-expression of the gamma-secretase-cleaved product of Notch, Notch intracellular domain. It is concluded that Delta and Jagged undergo ADAM-mediated ectodomain processing followed by PS-mediated intramembrane proteolysis to release signaling fragments. Thus, Notch and its cognate ligands are processed by the same molecular machinery and may antagonistically regulate each other's signaling (LaVoie, 2003).

The evolutionary conserved Notch signaling pathway is involved in cell fate specification and mediated by molecular interactions between the Notch receptors and the Notch ligands -- Delta, Serrate, and Jagged. Like Notch, Delta1 and Jagged2 are subject to presenilin (PS)-dependent, intramembranous 'gamma-secretase' processing, resulting in the production of soluble intracellular derivatives. Moreover, and paralleling the observation that expression of familial Alzheimer's disease-linked mutant PS1 compromises production of Notch S3/NICD, the PS-dependent production of Delta1 cytoplasmic derivatives are also reduced in cells expressing mutant PS1. These studies led to the conclusion that a similar molecular apparatus is responsible for intramembranous processing of Notch and it's ligands. To assess the potential role of the cytoplasmic derivative on nuclear transcriptional events, a Delta1-Gal4VP16 chimera was expressed and marked transcriptional stimulation of a luciferase-based reporter was demonstrated. These findings suggest that Delta1 and Jagged2 play dual roles as activators of Notch receptor signaling and as receptors that mediate nuclear signaling events via gamma-secretase-generated cytoplasmic domains (Ikeuchi, 2003).

The structure of presenilin

Presenilins have been implicated in the genesis of Alzheimer's disease and in facilitating LIN-12/Notch activity during development. All presenilins have multiple hydrophobic regions that could theoretically span a membrane, and a description of the membrane topology is a crucial step toward deducing the mechanism of presenilin function. An eight-transmembrane-domain model has been proposed for presenilin, based on studies of the Caenorhabditis elegans SEL-12 presenilin. Experiments are described that support the view that two of the hydrophobic regions of SEL-12 function as seventh and eighth transmembrane domains. Human presenilin 1 behaves like SEL-12 presenilin when analyzed by these methods. These results provide additional experimental support for the eight-transmembrane-domain model of presenilin topology (Li, 1998).

The carboxyl terminus of presenilin 1 and 2 (PS1 and PS2) binds to the neuron-specific cell adhesion molecule telencephalin (TLN) in the brain. TLN (or ICAM-5) is a neuron- and region-specific member of the ICAM subfamily of intercellular adhesion molecules. TLN promotes dendritic outgrowth and contributes to long-term potentiation. PS1 deficiency results in the abnormal accumulation of TLN in a yet unidentified intracellular compartment. The first transmembrane domain and carboxyl terminus of PS1 form a binding pocket with the transmembrane domain of TLN. Remarkably, APP binds to the same regions via part of its transmembrane domain encompassing the critical residues mutated in familial Alzheimer's disease. These data surprisingly indicate a spatial dissociation between the binding site and the proposed catalytic site near the critical aspartates in PSs. They provide important experimental evidence to support a ring structure model for PS (Annaert, 2001).

Only a small fraction of TLN binds PS1 under steady-state conditions, indicating that the interaction is functional and not structural. PS1 is mainly restricted to pre-Golgi compartments in cultured neuronal cells, while TLN resides almost exclusively at the somatodendritic plasma membrane. Occasionally, some PS1-positive membranes closely juxtaposed to TLN positive patches were observed, probably at the level of focal contacts. However, PS1 deficiency causes striking alterations in the subcellular distribution of endogenous TLN, which becomes missorted and accumulates in large intracellular structures. TLN, a strongly developmentally regulated protein, is detected significantly earlier in PS1-/- neurons than in their wild-type counterparts. Also, the relative numbers of neurons expressing TLN at the cell surface increases more rapidly in the absence of PS1. Furthermore, because TLN accumulations are only seen in differentiated PS1-/- neurons and not at early stages, they apparently reflect a time-related cumulative effect of PS1 deficiency. This can easily be interpreted in a context of a defective functional PS1-TLN interaction, even if only small quantities of TLN interact with PS1 at any given point in time (Annaert, 2001).

Interestingly, TLN accumulates in intracellular structures and seems to cause a local reorganization of the subcortical actin cytoskeleton. A link is known to exist between the actin cytoskeleton and TLN. This interaction is important for intercellular adhesion believed to control neurite outgrowth in the telencephalon. Since Notch signaling is also implicated in neurite outgrowth and branching, it follows that PS1 apparently controls at least two pathways involved in neuritic arborization. It is therefore surprising that the absence of PS1 has so little effect on the overall morphology of mature neurons in culture. It is concluded that important compensating mechanisms must be at work and more subtle, yet-to-be discovered physiological alterations in neuritic outgrowth and/or synaptogenesis caused by the absence of PS1 are anticipated. In line with this hypothesis are recent observations in PS1 conditionally targeted mice. These mice do not show abnormalities in the Notch signaling pathway, but nevertheless display subtle cognitive deficits. Whether accumulation of TLN can explain this phenotype is an interesting possibility. It is to be noted that mice lacking TLN also display no abnormalities apart from changes in hippocampal LTP (Annaert, 2001).

The interaction of TLN with the C terminus of PS1 requires at least five amino acids (Val-829 toTrp-833) in the N-terminal part of the transmembrane region. This suggests that the hydrophobic PS1 C terminus can intrude into the lipid bilayer to interact with this part of the TLN transmembrane domain. Also for APP, the PS1 binding site is in the transmembrane domain, but located in the 11 amino acids (Thr-639 to Lys-649) situated at its C-terminal end. This extends previous work demonstrating that the cytoplasmic domain of APP and large parts of the ectodomain are not needed for PS1 binding. It is speculated that the binding region is crucial for the presentation of APP to the catalytic domain of gamma-secretase. In support of this conclusion, phenylalanine-scanning mutagenesis of this region significantly influenced gamma-secretase processing of APP. Also peptidomimetics that inhibit the gamma42 cleavage of APP include amino acids of this region. Most importantly, all known FAD-causing missense mutations in APP that shift the gamma-secretase cleavage toward Aß42 production are located within this short sequence. It seems likely that the FAD mutations may affect the binding of APP with PS1 and that, by doing so, they modulate the presentation of APP to gamma-secretase. Therefore, small compounds mimicking the binding sites in APP or in TLN, or binding selectively to the APP and not the TLN sequence, could possibly prevent the processing of APP by gamma-secretase (Annaert, 2001).

TLN was identified via two-hybrid screening using the C-terminal eight amino acids of PS1 as a bait. Residues Leu-460 to Ile-467 in PS1 thus define the minimal binding region with TLN. The interaction is more efficient when the whole C terminus of PS1 (or PS2) is used (Lys-429-Ile-467, and the corresponding sequence in PS2), which is not unexpected since the structure of a peptide in a protein is strongly influenced by neighboring sequences. Importantly, when the PS1 protein was scanned for additional binding sites, it was established that the first transmembrane region of PS1 (Val-82-Ser-102) determines a second important binding site for TLN and APP. Two domains at opposing sites in the PS1 sequence are therefore apparently involved in TLN and APP binding. These results suggest that certain type I membrane proteins bind via their transmembrane domain to a common binding pocket constituting the C-terminal domain and the first integral membrane domain of PS1 (Annaert, 2001).

The fact that the binding domains in PS1 are exceptionally well conserved among different species further corroborates the hypothesis that they are of major functional importance. Consistently, only few disease-linked mutations are found in these regions while some loss-of-function mutations in these domains are found in the PS homologs of C. elegans and Drosophila. If both domains comprise together a functional binding pocket, they should be closely juxtaposed in the lipid bilayer, suggesting a circular or ring-like structure for PS1. Although provocative, such a model supports recent findings that intramolecular associations between different domains of PS1, as well as cooperative interactions between both fragments, are important for the functionality of the PS complex. While other, more complicated models could be envisaged, the fact that the PS1Deltaexon 9, as well as other mutations that prevent endoproteolysis of PS1, maintains gamma-secretase cleavage of APP supports the model that both fragments remain closely associated in a ring-like structure. The model implies the possibility that the hydrophobic C terminus of PS1 can penetrate to different extents into the membrane, and a regulatory function is postulated for this part of PS1. Furthermore, the model suggests that the supposedly catalytic site in PS1 and the APP binding site are remote. This implies that binding and cleavage of substrate are two separate events. If PS1 is indeed the gamma-secretase, it has to be assumed that substrate presentation to the catalytic domain near the aspartate residues involves a refolding of the PS1 binding module into the interior of the ring structure. It is also possible that PSs, after binding substrates like APP(-CTF), Notch, or TLN, tag or transport these proteins for destination to a downstream compartment. The missorting of TLN in PS1-deficient neurons is at least compatible with this possibility as well (Annaert, 2001).

To date, the type I membrane proteins shown to bind PS can be subdivided in three functional classes. APP and Notch are gamma-secretase substrates, and their regulated intramembrane proteolysis depends critically on PSs. Nicastrin binds strongly to PSs, but appears to be a modulator of and not a substrate for gamma-secretase. Finally, cadherin and TLN are both cell adhesion proteins, and PS1 may modulate their correct cell membrane insertion and therefore their adhesive functions at the cell membrane. PS1 also regulates (to a certain extent) Wnt signaling via its interaction with ß-catenin (Annaert, 2001).

Therefore, the PSs are at the crossroads of several important signaling pathways, and the question is now whether PSs are a means of cross-talk between these pathways. By defining precisely the molecular domains involved in the interaction of PS1 with APP and TLN, a structural basis for further investigations of this question is provided. It is also clear that these binding sites provide novel potentially important targets for drug development in the fight against Alzheimer's disease (Annaert, 2001).

The role of presenilin in beta-amyloid precursor protein processing

Point mutations in the presenilin-1 gene (PS1) are a major cause of familial Alzheimer's disease. They result in a selective increase in the production of the amyloidogenic peptide amyloid-beta(1-42) by proteolytic processing of the amyloid precursor protein (APP). An investigation was carried out to see whether PS1 is also involved in normal APP processing in neuronal cultures derived from PS1-deficient mouse embryos. Cleavage by alpha- and beta-secretase of the extracellular domain of APP is not affected by the absence of PS1, whereas cleavage by gamma-secretase of the transmembrane domain of APP is prevented, causing carboxyl-terminal fragments of APP to accumulate and a fivefold drop in the production of amyloid peptide. Pulse-chase experiments indicate that PS1 deficiency specifically decreases the turnover of the membrane-associated fragments of APP. As in the regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor, PS1 appears to facilitate a proteolytic activity that cleaves the integral membrane domain of APP. These results indicate that mutations in PS1 that manifest clinically cause a gain of function and that inhibition of PS1 activity is a potential target for anti-amyloidogenic therapy in Alzheimer's disease (De Strooper, 1998).

Recent reports indicate that missense mutations on presenilin (PS) 1 are likely responsible for the main early-onset familial forms of Alzheimer's disease (FAD). Consensual data obtained through distinct histopathological, cell biology, and molecular biology approaches have led to the conclusion that these PS1 mutations clearly trigger an increased production of the 42-amino-acid-long species of beta-amyloid peptide (A beta). Overexpression of wild-type PS1 in HK293 cells increases A beta40 secretion. By contrast, FAD-linked mutants of PS1 trigger increase secretion of both A beta40 and A beta42 but clearly favor the production of the latter species. Overexpression of the wild-type PS1 augments the alpha-secretase-derived C-terminally truncated fragment of beta-amyloid precursor protein (APP alpha) recovery, whereas transfectants expressing mutated PS1 secrete drastically lower amounts of APP alpha when compared with cells expressing wild-type PS1. This decrease was also observed when comparing double transfectants overexpressing wild-type beta-amyloid precursor protein and either PS1 or its mutated congener M146V-PS1. Altogether, these data indicate that PS mutations linked to FAD not only trigger an increased ratio of A beta42 over total A beta secretion but concomitantly down-regulate the production of APP alpha (Ancolio, 1997).

Progressive cerebral deposition of the amyloid beta-protein (Abeta) is believed to play a pivotal role in the pathogenesis of Alzheimer's disease (AD). The highly amyloidogenic 42-residue form of Abeta (Abeta42) is the first species to be deposited in both sporadic and familial AD. Mutations in two familial AD-linked genes, presenilins 1 (PS1) and 2 (PS2), selectively increase the production of Abeta42 in cultured cells and the brains of transgenic mice, and gene deletion of PS1 shows that it is required for normal gamma-secretase cleavage of the beta-amyloid precursor protein (APP) to generate Abeta. To establish the subcellular localization of the PS1 regulation of APP processing to Abeta, fibroblasts from PS1 wild-type (wt) or knockout (KO) embryos as well as Chinese hamster ovary (CHO) cells stably transfected with wt or mutant PS1 were subjected to subcellular fractionation on discontinuous Iodixanol gradients. APP C-terminal fragments (CTF) are markedly increased in both endoplasmic reticulum- (ER-) and Golgi-rich fractions of fibroblasts from KO mice; moreover, similar increases have been documented directly in KO brain tissue. No change in the subcellular distribution of full-length APP is detectable in fibroblasts lacking PS1. In CHO cells, a small portion of APP, principally the N-glycosylated isoform, forms complexes with PS1 in both ER- and Golgi-rich fractions, as detected by coimmunoprecipitation. Mutant PS1 significantly increased Abeta42 levels in the Golgi fraction. These results indicate PS1 and APP can interact in the ER and Golgi, where PS1 is required for proper gamma-secretase processing of APP CTFs, and that PS1 mutations augment Abeta42 levels principally in Golgi-like vesicles (Xia, 1998).

Most of early onset familial forms of Alzheimer's disease (FAD) are due to inherited mutations located on two homologous proteins, presenilins 1 and 2 (PS1 and PS2) encoded by chromosomes 14 and 1, respectively. The expression of wild type (wt)-PS2 in human HEK293 cells increases the production of the physiological alpha-secretase-derived product, APPalpha. By contrast, APPalpha secretion is drastically reduced in cells expressing the FAD-linked N141I-PS2. Wild-type-PS2, N141I-PS2 and their C-terminal maturation fragment are degraded by the enzymatic multicatalytic complex, proteasome. Interestingly, two selective proteasome inhibitors, Z-IE(Ot-Bu)A-Leucinal and lactacystin potentiate the APPalpha secretion observed in wtPS2-expressing cells and further amplify the N141I-PS2-induced decrease in APPalpha production. By contrast, a series of pharmacological agents unable to affect the proteasome do not modify PS2 immunoreactivities and APPalpha recoveries. Altogether, these data indicate that: (1) wtPS2 positively modulates the alpha-secretase physiological pathway of betaAPP maturation in human cells; (2) N141I mutation on PS2 drastically lowers the secretion of APPalpha; (3) Proteasome inhibitors prevent the degradation of wtPS2, N141I-PS2 and their C-terminal maturation product. This protection against proteasomal degradation directly modulates the APPalpha secretion response elicited by wt- and FAD-linked PS2 expression in human HEK293 cells (Marambaud, 1998)

Mutations in the presenilin genes PS1 and PS2 cause the most common form of early-onset familial Alzheimer's disease. The influence of PS1 mutations on the generation of endogenous intracellular amyloid beta-protein (A beta) species was assessed using a highly sensitive immunoblotting technique with inducible mouse neuroblastoma (Neuro 2a) cell lines expressing the human wild-type (wt) or mutated PS1 (M146L or delta exon 10). The induction of mutated PS1 increases the intracellular levels of two distinct A beta species ending at residue 42 that are likely to be A beta1-42 and its N-terminally truncated variant(s) A beta x-42. The induction of mutated PS1 results in a higher level of intracellular A beta1-42 than of intracellular A beta x-42, whereas extracellular levels of A beta1-42 and A beta x-42 are increased proportionally. In addition, the intracellular generation of these A beta42 species in wt and mutated PS1-induced cells is completely blocked by brefeldin A, whereas it exhibita differential sensitivities to monensin: the increased accumulation of intracellular A beta x-42 versus inhibition of intracellular A beta1-42 generation. These data strongly suggest that A beta x-42 is generated in a proximal Golgi, whereas A beta1-42 is generated in a distal Golgi and/or a post-Golgi compartment. Thus, it appears that PS1 mutations enhance the degree of 42-specific gamma-secretase cleavage that occurs in the normal beta-amyloid precursor protein processing pathway (a) in the endoplasmic reticulum or the early Golgi apparatus prior to beta-secretase cleavage or (b) in the distinct sites where A beta x-42 and A beta1-42 are generated (Sudoh, 1998).

Mutations in the presenilin-1 (PS-1) gene account for approximately 50% of the cases of autosomal dominant, early onset, inherited forms of Alzheimer's disease (AD). PS-1 is an integral membrane protein expressed in neurons and is localized primarily in the endoplasmic reticulum (ER). PS-1 mutations may promote neuronal degeneration by altering the processing of the beta-amyloid precursor protein (APP) and/or by engaging apoptotic pathways. Alternative processing of APP in AD may increase production of neurotoxic amyloid beta-peptide (Abeta) and reduce production of the neuroprotective alpha-secretase-derived form of APP (sAPPalpha). In differentiated PC12 cells expressing an AD-linked PS-1 mutation (L286V), sAPPalpha activates the transcription factor NF-kappaB and preventes apoptosis induced by Abeta. Treatment of cells with kappaB decoy DNA blocka the antiapoptotic action of sAPPalpha, demonstrating the requirement for NF-kappaB activation in the cytoprotective action of sAPPalpha. Cells expressing mutant PS-1 exhibit an aberrant pattern of NF-kappaB activity following exposure to Abeta, which is characterized by enhanced early activation of NF-kappaB followed by a prolonged depression of activity. Blockade of NF-kappaB activity in cells expressing mutant PS-1 by kappaB decoy DNA is associated with enhanced Abeta-induced increases of [Ca2+]i and mitochondrial dysfunction. Treatment of cells with sAPPalpha stabilizes [Ca2+]i and mitochondrial function and suppresses oxidative stress by a mechanism involving activation of NF-kappaB. Blockade of ER calcium release prevents (and stimulation of ER calcium release by thapsigargin induces) apoptosis in cells expressing mutant PS-1, suggesting a pivotal role for ER calcium release in the proapoptotic action of mutant PS-1. Finally, a role for NF-kappaB in preventing apoptosis induced by ER calcium release has been demonstrated by data showing that sAPPalpha prevents thapsigargin-induced apoptosis, an effect blocked by kappaB decoy DNA. It is concluded that sAPPalpha stabilizes cellular calcium homeostasis and protects neural cells against the proapoptotic action of mutant PS-1 by a mechanism involving activation of NF-kappaB. The data further suggest that PS-1 mutations result in aberrant NF-kappaB regulation that may render neurons vulnerable to apoptosis (Guo, 1998).

Progressive cerebral deposition of the amyloid beta-protein (Abeta) is believed to play a pivotal role in the pathogenesis of Alzheimer's disease (AD). The highly amyloidogenic 42-residue form of Abeta (Abeta42) is the first species to be deposited in both sporadic and familial AD. Mutations in two familial AD-linked genes, presenilins 1 (PS1) and 2 (PS2), selectively increase the production of Abeta42 in cultured cells and the brains of transgenic mice, and gene deletion of PS1 shows that it is required for normal gamma-secretase cleavage of the beta-amyloid precursor protein (APP) to generate Abeta. To establish the subcellular localization of the PS1 regulation of APP processing to Abeta, fibroblasts from PS1 wild-type (wt) or knockout (KO) embryos as well as Chinese hamster ovary (CHO) cells stably transfected with wt or mutant PS1 were subjected to subcellular fractionation on discontinuous Iodixanol gradients. APP C-terminal fragments (CTF) are markedly increased in both endoplasmic reticulum- (ER-) and Golgi-rich fractions of fibroblasts from KO mice; moreover, similar increases have been documented directly in KO brain tissue. No change in the subcellular distribution of full-length APP is detectable in fibroblasts lacking PS1. In CHO cells, a small portion of APP, principally the N-glycosylated isoform, forms complexes with PS1 in both ER- and Golgi-rich fractions, as detected by coimmunoprecipitation. Mutant PS1 significantly increased Abeta42 levels in the Golgi fraction. These results indicate PS1 and APP can interact in the ER and Golgi, where PS1 is required for proper gamma-secretase processing of APP CTFs, and that PS1 mutations augment Abeta42 levels principally in Golgi-like vesicles (Xia, 1998).

Accumulation of the amyloid-beta protein (Abeta) in the cerebral cortex is an early and invariant event in the pathogenesis of Alzheimer's disease. The final step in the generation of Abeta from the beta-amyloid precursor protein is an apparently intramembranous proteolysis by the elusive gamma-secretase(s). The most common cause of familial Alzheimer's disease is mutation of the genes encoding presenilins 1 and 2, which alters gamma-secretase activity to increase the production of the highly amyloidogenic Abeta42 isoform. Moreover, deletion of presenilin-1 in mice greatly reduces gamma-secretase activity, indicating that presenilin-1 mediates most of this proteolytic event. Mutation of either of two conserved transmembrane (TM) aspartate residues in presenilin-1, Asp 257 (in TM6) and Asp 385 (in TM7), substantially reduces Abeta production and increases the amounts of the carboxy-terminal fragments of beta-amyloid precursor protein that are the substrates of gamma-secretase. These effects have been observed in three different cell lines as well as in cell-free microsomes. Either of the Asp to Ala mutations also prevented the normal endoproteolysis of presenilin-1 in the TM6 to TM7 cytoplasmic loop. In a functional presenilin-1 variant (carrying a deletion in exon 9) that is associated with familial Alzheimer's disease and which does not require this cleavage, the Asp 385 to Ala mutation still inhibits gamma-secretase activity. These results indicate that the two transmembrane aspartate residues are critical for both presenilin-1 endoproteolysis and gamma-secretase activity, and suggest that presenilin 1 is either a unique diaspartyl cofactor for gamma-secretase or is itself gamma-secretase, an autoactivated intramembranous aspartyl protease (Wolfe, 1999).

The discovery that a deficiency of presenilin 1 (PS1) decreases the production of amyloid beta-protein (Abeta) has identified the presenilins as important mediators of the gamma-secretase cleavage of beta-amyloid precursor protein (APP). Two conserved transmembrane (TM) aspartates in PS1 are critical for Abeta production, providing evidence that PS1 either functions as a required diaspartyl cofactor for gamma-secretase or is itself gamma-secretase. Presenilin 2 (PS2) shares substantial sequence and possibly functional homology with PS1. The two TM aspartates in PS2 are also critical for gamma-secretase activity, providing further evidence that PS2 is functionally homologous to PS1. Cells stably co-expressing TM Asp to Ala mutations in both PS1 and PS2 show further accumulation of the APP-derived gamma-secretase substrates, C83 and C99. The production of Abeta is reduced to undetectable levels in the conditioned media of these cells. Furthermore, endoproteolysis of the exogenous Asp mutant PS2 is absent, and endogenous PS1 C-terminal fragments are diminished to undetectable levels. Therefore, the co-expression of PS1 and PS2 TM Asp --> Ala mutants suppresses the formation of any detectable PS1 or PS2 heterodimeric fragments and essentially abolishes the production of Abeta. These results explain the residual Abeta production seen in PS1-deficient cells and demonstrate the absolute requirement of functional presenilins for Abeta generation. It is concluded that presenilins, and their TM aspartates in particular, are attractive targets for lowering Abeta therapeutically to prevent Alzheimer's disease (Kimberly, 2000).

A presenilin-1 (PS1) conditional knockout mouse (cKO) has been generated in which PS1 inactivation is restricted to the postnatal forebrain. The PS1 cKO mouse is viable and exhibits no gross abnormalities in contrast to the pleiotropic phenotypes associated with PS1 deficiency in the embryonic brain. The carboxy-terminal fragments of the amyloid precursor protein differentially accumulate in the cerebral cortex of cKO mice, while generation of ß-amyloid peptides is reduced. Expression of Notch downstream effector genes, Hes1, Hes5, and Dll1, is unaffected in the cKO cortex. Although basal synaptic transmission, long-term potentiation, and long-term depression at hippocampal area CA1 synapses are normal, the PS1 cKO mice exhibit subtle but significant deficits in long-term spatial memory. These results demonstrate that inactivation of PS1 function in the adult cerebral cortex leads to reduced Aß generation and subtle cognitive deficits without affecting expression of Notch downstream genes (Yu, 2001).

The most striking feature of the disrupted APP processing is the 30-fold accumulation of the APP C-terminal fragments (CTFs) in PS1 cKO mice by the age of 6 months. Interestingly, the APP ß-CTFs (C89 and C99) accumulate differentially in the absence of PS1, with the increase in the level of C89 and C99 cleavage fragments measuring approximately 30- and 3-fold, respectively. The levels of the alpha-CTF (C83) are also elevated by as much as 30-fold. Since the CTFs represent the substrates for gamma-secretase cleavage, these results are consistent with a requirement of PS1 for gamma-secretase activity. The differential accumulation of the CTFs, particularly C89 and C99, which are cleavage products of ß-secretase, is likely due to the differences in their half lives. Alternatively, C99 could be converted to C89 by ß-secretase, resulting in much lower levels of C99 relative to C89 in the cKO mice. Furthermore, all APP CTFs are present in phosphorylated and nonphosphorylated forms in the brain of both control and cKO mice. The cytoplasmic domain of full-length APP has been shown to be phosphorylated in cultured neurons and adult rat brain by cdk5 on Thr668, which resides in the C-terminal region common to all APP CTF species. This phosphorylation event may therefore account for the observed phosphorylation of the CTFs. Although the physiological significance of APP phosphorylation is unclear, there is evidence suggesting that it may be associated with the regulation of Aß generation and neurite extension (Yu, 2001).

Sequential processing of the amyloid precursor protein (APP) by beta- and gamma-secretases generates the Abeta peptide, a major constituent of the senile plaques observed in Alzheimer's disease. The cleavage by gamma-secretase also results in the cytoplasmic release of a 59- or 57-residue-long C-terminal fragment (Cgamma). This processing resembles regulated intramembrane proteolysis of transmembrane proteins such as Notch, where the released cytoplasmic fragments enter the nucleus and modulate gene expression. This study examines whether the analogous Cgamma fragments of APP also exert effects in the nucleus. Ectopically expressed Cgamma is present both in the cytoplasm and in the nucleus. Interestingly, expression of Cgamma59 causes disappearance of PAT1 (a protein that interacts with the APP cytoplasmic domain) from the nucleus and induces its proteosomal degradation. Treatment of cells with lactacystin prevents PAT1 degradation and retains its nuclear localization. By contrast, Cgamma57, a minor product of gamma-cleavage, is only marginally effective in PAT1 degradation. Furthermore, Cgamma59 but not Cgamma57 potently represses retinoic acid-responsive gene expression. Thus, these studies provide the evidence that, as predicted by the regulated intramembrane proteolysis mechanism, Cgamma seems to function in the nucleus (Gao, 2001).

Alzheimer's disease is associated with increased production and aggregation of amyloid-ß (Aß) peptides. Aß peptides are derived from the amyloid precursor protein (APP) by sequential proteolysis, catalysed by the aspartyl protease BACE, followed by presenilin-dependent gamma-secretase cleavage. Presenilin interacts with nicastrin, APH-1 and PEN-2, all of which are required for gamma-secretase function. Presenilins also interact with alpha-catenin, ß-catenin and glycogen synthase kinase-3ß (GSK-3ß), but a functional role for these proteins in gamma-secretase activity has not been established. Therapeutic concentrations of lithium, a GSK-3 inhibitor, block the production of Aß peptides by interfering with APP cleavage at the gamma-secretase step, but do not inhibit Notch processing. Importantly, lithium also blocks the accumulation of Aß peptides in the brains of mice that overproduce APP. The target of lithium in this setting is GSK-3alpha, which is required for maximal processing of APP. Since GSK-3 also phosphorylates tau protein, the principal component of neurofibrillary tangles, inhibition of GSK-3alpha offers a new approach to reduce the formation of both amyloid plaques and neurofibrillary tangles, two pathological hallmarks of Alzheimer's disease (Phiel, 2003).

In summary, this study shows that GSK-3alpha facilitates APP processing and that lithium inhibits the generation of Aß peptides through inhibition of GSK-3alpha. In support of this conclusion: (1) lithium reduces Aß production in cultured cells and in the brains of mice that overproduce Aß peptides; (2) kenpaullone, an alternative GSK-3alpha inhibitor, also inhibits Aß production; (3) RNAi-mediated depletion of GSK-3alpha reduces Aß production, and (4) moderate overexpression of GSK-3alpha increases Aß production. Lithium inhibits the GSK-3-mediated phosphorylation of tau, which, in its hyperphosphorylated state, is the main component of neurofibrillary tangles. Thus, GSK-3alpha offers an attractive target for pharmacological agents aimed at reducing the formation of amyloid plaques and neurofibrillary tangles, the pathological hallmarks of Alzheimer's disease. Lithium also protects neurons from proapoptotic stimuli and could therefore reduce neuronal cell death associated with Alzheimer's disease. Lithium has been used for more than 50 years to treat bipolar disorder, but has a narrow therapeutic window and a higher frequency of side effects in older patients. Thus, although lithium might be considered for the prevention of Alzheimer's disease, especially in younger patients with FAD mutations or Down's syndrome, new agents that specifically target GSK-3alpha may prove to be valuable in the treatment of Alzheimer's disease (Phiel, 2003).

The beta-amyloid precursor protein (APP) and the Notch receptor undergo intramembranous proteolysis by the Presenilin-dependent gamma-secretase. The cleavage of APP by gamma-secretase releases amyloid-beta peptides, which have been implicated in the pathogenesis of Alzheimer's disease, and the APP intracellular domain (AID), for which the function is not yet well understood. A similar gamma-secretase-mediated cleavage of the Notch receptor liberates the Notch intracellular domain (NICD). NICD translocates to the nucleus and activates the transcription of genes that regulate the generation, differentiation, and survival of neuronal cells. Hence, some of the effects of APP signaling and Alzheimer's disease pathology may be mediated by the interaction of APP and Notch. Membrane-tethered APP binds to the cytosolic Notch inhibitors Numb and Numb-like in mouse brain lysates. AID also binds Numb and Numb-like, and represses Notch activity when released by APP. Thus, gamma-secretase may have opposing effects on Notch signaling; positive by cleaving Notch and generating NICD, and negative by processing APP and generating AID, which inhibits the function of NICD (Roncarati, 2002).

Amyloid β-peptide (Aβ), which plays a central role in Alzheimer's disease, is generated by presenilin-dependent γ-secretase cleavage of β-amyloid precursor protein (βAPP). The presenilins (PS1 and PS2) also regulate Aβ degradation. Presenilin-deficient cells fail to degrade Aβ and have drastic reductions in the transcription, expression, and activity of neprilysin (see Drosophila Neprilysin 4), a key Aβ-degrading enzyme. Neprilysin activity and expression are also lowered by γ-secretase inhibitors and by PS1/PS2 deficiency in mouse brain. Neprilysin activity is restored by transient expression of PS1 or PS2 and by expression of the amyloid intracellular domain (AICD), which is cogenerated with Aβ, during γ-secretase cleavage of βAPP. Neprilysin gene promoters are transactivated by AICDs from APP-like proteins (APP, APLP1, and APLP2), but not by Aβ or by the γ-secretase cleavage products of Notch, N- or E- cadherins. The presenilin-dependent regulation of neprilysin, mediated by AICDs, provides a physiological means to modulate Aβ levels with varying levels of γ-secretase activity (Pardossi-Piquard, 2005).

If Aβ production and degradation are tightly linked, this raises the question of why Aβ accumulates in AD. The net accumulation of Aβ in AD pathology likely reflects the cumulative effect of multiple events acting on production, fibrillogenesis, and degradation. In many forms of AD, especially the late-onset sporadic forms, it has not been shown that there is increased β- and γ-secretase activity. In fact, some have suggested that these forms may reflect defective degradation of Aβ. Therefore, AICD levels are likely to be unchanged in these late-onset forms of AD, and as a result, the AICD-mediated ability to upregulate neprilysin activity would not be efficiently brought into play to protect the brain. In contrast, in those cases of AD arising from mutations in APP and PS1, which activate γ-secretase and AICD production, the principal effect is to produce longer Aβ isoforms such as Aβ42. However, although Aβ40 is efficiently degraded by NEP, Aβ42 is degraded by NEP both in vitro and in vivo at a 6-fold lower rate. As a result, the upregulation of AICD (and thus, NEP expression), which would be anticipated in subjects with presenilin mutations, would not completely abolish the accumulation of Aβ42 in these cases. It should be noted that, in agreement with the above hypothesis, neprilysin expression and activity were higher only in brain tissues with familial Alzheimer’s disease linked to various presenilin-1 mutations, while sporadic AD cases displayed neprilysin levels similar to those exhibited by normal brain tissues. Interestingly, PS1 mutations selectively affect neprilysin and do not alter insulin-degrading enzyme expression (Pardossi-Piquard, 2005).

The above observations are also of direct practical interest because they indicate the possibility of new avenues for controlling Aβ levels without directly affecting γ-secretase. This latter concept is important because of the various developmental and postnatal side-effects associated with the inhibition of γ-secretase-mediated cleavage of other signaling molecules, including Notch. This work now suggests that Aβ levels might be modulated by directly increasing neprilysin expression, using AICD or small molecule mimics of AICD. Upregulation of neprilysin by transgenic overexpression, at least to modest levels, appears to be sufficient to reduce brain Aβ levels and to pose few toxic side effects. This strategy would also circumvent the other side effects of γ-secretase inhibitors, including the potentially self-defeating effect of reducing AICD and thus preventing NEP-mediated degradation of Aβ (Pardossi-Piquard, 2005).

Subcellular localization of presenilin

The mechanisms by which mutations in presenilin-1 (PS1) and presenilin-2 (PS2) result in the Alzheimer's disease phenotype are unclear. Full-length PS1 and PS2 are each processed into stable proteolytic fragments after their biosynthesis in transfected cells. PS1 and PS2 have been localized by immunocytochemistry to the endoplasmic reticulum (ER) and Golgi compartments, but previous studies could not differentiate between the full-length presenilin proteins and their fragments. Subcellular fractionation of cells stably transfected with PS1 or PS2 were carried out to determine the localization of full-length presenilins and their fragments. Full-length PS1 and PS2 are principally distributed in ER fractions, whereas the N- and C-terminal fragments are localized predominantly to the Golgi fractions. In cells expressing the PS1 mutant lacking exon 9 (DeltaE9), only full-length molecules are observed that are present in the ER and Golgi fractions. The turnover rate is considerably slower for the DeltaE9 holoprotein, apparently due to decreased degradation within the ER. These results suggest that that full-length presenilin proteins are primarily ER resident molecules and undergo endoproteolysis within the ER. The fragments are subsequently transported to the Golgi compartment, where their turnover rate is much slower than that of the full-length presenilin in the ER (J. Zhang, 1998).

The presenilin (PS) genes associated with Alzheimer disease encode polytopic transmembrane proteins which undergo physiologic endoproteolytic cleavage to generate stable NH2- and COOH-terminal fragments (NTF or CTF) which co-localize in intracellular membranes, but are tightly regulated in their stoichiometry and abundance. Linear glycerol velocity and discontinuous sucrose gradient analysis were to investigate the distribution and native conformation of PS1 and PS2 during this regulated processing in cultured cells and in brain. The PS1 NTF and CTF co-localize in the endoplasmic reticulum (ER) and in the Golgi apparatus, where they are components of a approximately 250-kDa complex. This complex also contains beta-catenin but not beta-amyloid precursor protein. In contrast, the PS1 holoprotein precursor is predominantly localized to the rough ER and smooth ER, where it is a component of a approximately 180-kDa native complex. PS2 forms similar but independent complexes. Restricted incorporation of the presenilin NTF and CTF along with a potentially functional ligand (beta-catenin) into a multimeric complex in the ER and Golgi apparatus may provide an explanation for the regulated accumulation of the NTF and CTF (Yu, 1998).

Interactions of members of the armadillo family with presenilins

Alzheimer's disease-related presenilins are thought to be involved in Notch signaling during embryonic development and/or cellular differentiation. Proteins mediating the cellular functions of the presenilins are still unknown. The yeast two-hybrid system was used to identify an interacting armadillo protein, termed p0071, that binds specifically to the hydrophilic loop of presenilin 1. In vivo, the presenilins constitutively undergo proteolytic processing, forming two stable fragments. The C-terminal fragment of presenilin 1 directly binds to p0071. Nine out of 10 armadillo repeats in p0071 are essential for mediating this interaction. Since armadillo proteins, like beta-catenin and APC, are known to participate in cellular signaling, p0071 may function as a mediator of presenilin 1 in signaling events (Stahl, 1999).

Missense substitutions in the presenilin 1 (PS1) and presenilin 2 (PS2) proteins are associated with early-onset familial Alzheimer's disease. Yeast-two-hybrid and coimmunoprecipitation methods were used to show that the large cytoplasmic loop domains of PS1 and PS2 interact specifically with three members of the armadillo protein family, including beta-catenin, p0071, and a novel neuronal-specific armadillo protein--neural plakophilin-related armadillo protein (NPRAP). The PS1:NPRAP interaction occurs between the arm repeats of NPRAP and residues 372-399 at the C-terminal end of the large cytoplasmic loop of PS1. The latter residues contain a single arm-like domain and are highly conserved in the presenilins, suggesting that they form a functional armadillo protein binding site for the presenilins (Levesque, 1999).

The presenilin proteins are components of high-molecular-weight protein complexes in the endoplasmic reticulum and Golgi apparatus that also contain beta-catenin. Presenilin mutations associated with familial Alzheimer disease (but not the non-pathogenic Glu318Gly polymorphism) alter the intracellular trafficking of beta-catenin after activation of the Wnt/beta-catenin signal transduction pathway. As with their effect on betaAPP processing, the effect of PS1 mutations on trafficking of beta-catenin arises from a dominant 'gain of aberrant function' activity. These results indicate that mistrafficking of selected presenilin ligands is a candidate mechanism for the genesis of Alzheimer disease associated with presenilin mutations, and that dysfunction in the presenilin-beta-catenin protein complexes is central to this process (Nishimura, 1999).

Presenilin-1 can associate with members of the catenin family of signalling proteins, but the significance of this association is unknown. Presenilin-1 forms a complex with beta-catenin in vivo that increases beta-catenin stability. Pathogenic mutations in the presenilin-1 gene reduce the ability of presenilin-1 to stabilize beta-catenin, and lead to increased degradation of beta-catenin in the brains of transgenic mice. Moreover, beta-catenin levels are markedly reduced in the brains of Alzheimer's disease patients with presenilin-1 mutations. Loss of beta-catenin signalling increases neuronal vulnerability to apoptosis induced by amyloid-beta protein. Thus, mutations in presenilin-1 may increase neuronal apoptosis by altering the stability of beta-catenin, predisposing individuals to early-onset Alzheimer's disease (Z. Zhang, 1998).

Families bearing mutations in the presenilin-1 (PSI) gene develop Alzheimer's disease (AD). However, the mechanism through which PS1 causes AD is unclear. The co-immunoprecipitation with PS1 in transfected COS-7 cells indicates that PSI directly interacts with endogenous beta-catenin, and the interaction requires residues 322-450 of PSI and 445-676 of beta-catenin. Both proteins are co-localized in the endoplasmic reticulum. Over-expression of PS1 reduces the level of cytoplasmic beta-catenin, and inhibits beta-catenin-T cell factor-regulated transcription. These results indicate that PSI plays a role as inhibitor of the beta-catenin signal, which may be connected with the AD dysfunction (Murayama, 1998).

A screen was carried out for proteins that interact with presenilin (PS) 1, and the full-length cDNA of human delta-catenin, which encodes 1225 amino acids was cloned. Delta-catenin interactes with a hydrophilic loop region in the endoproteolytic C-terminal fragment of PS1, but not with that of PS-2. These results suggest that PS1 and PS2 partly differ in function. PS1 loop fragment containing the pathogenic mutation retains the binding ability. Another armadillo-protein, p0071 interacts with PS1 (Tanahashi, 1999).

The two hybrid system and confirmatory co-immunoprecipitations were used to identify a novel catenin, termed delta-catenin, which interacts with PS1 and is principally expressed in brain. The catenins are a gene family related to the Armadillo gene in Drosophila, some of which appear to have dual roles-they are components of cell-cell adherens junctions, and may serve as intermediates in the Wingless (Wg) signaling pathway, which, like Notch/lin-12, is also responsible for a variety of inductive signaling events. In the non-neuronal 293 cell line, PS1 interacts with beta-catenin, the family member with the greatest homology to Armadillo. Wg and Notch interactions are mediated by the Dishevelled gene, which may form a signaling complex with PS1 and Wg pathway intermediates to regulate the function of the Notch/lin-12 gene (Zhou, 1997).

The Alzheimer's disease-linked gene presenilin 1 (PS1) is required for intramembrane proteolysis of APP and Notch. In addition, recent observations strongly implicate PS1 as a negative regulator of the Wnt/ß-catenin signaling pathway, although the mechanism underlying this activity is unknown. Presenilin has been shown to function as a scaffold that rapidly couples ß-catenin phosphorylation through two sequential kinase activities independent of the Wnt-regulated Axin/CK1alpha complex. Thus, presenilin deficiency results in increased ß-catenin stability in vitro and in vivo by disconnecting the stepwise phosphorylation of ß-catenin, both in the presence and absence of Wnt stimulation. These findings highlight an aspect of ß-catenin regulation outside of the canonical Wnt-regulated pathway and a function of presenilin separate from intramembrane proteolysis (Kang, 2002).

Presenilin targets E-cadherin

E-cadherin controls a wide array of cellular behaviors including cell-cell adhesion, differentiation and tissue development. Presenilin-1 (PS1), a protein involved in Alzheimer's disease, controls a gamma-secretase-like cleavage of E-cadherin. This cleavage is stimulated by apoptosis or calcium influx and occurs between human E-cadherin residues Leu731 and Arg732 at the membrane-cytoplasm interface. The PS1/gamma-secretase system cleaves both the full-length E-cadherin and a transmembrane C-terminal fragment, derived from a metalloproteinase cleavage after the E-cadherin ectodomain residue Pro700. The PS1/gamma-secretase cleavage dissociates E-cadherins, beta-catenin and alpha-catenin from the cytoskeleton, thus promoting disassembly of the E-cadherin-catenin adhesion complex. Furthermore, this cleavage releases the cytoplasmic E-cadherin to the cytosol and increases the levels of soluble beta- and alpha-catenins. Thus, the PS1/gamma-secretase system stimulates disassembly of the E-cadherin-catenin complex and increases the cytosolic pool of beta-catenin, a key regulator of the Wnt signaling pathway (Marambaud, 2002).

Miscellaneous presenilin interactions

The unfolded protein response (UPR) mediates signaling from the endoplasmic reticulum to the nucleus. Presenilin plays an important role in the proteolysis of a protein involved in the UPR, terned Ire1p. Cells respond to the accumulation of unfolded proteins in the lumen of the ER by activating transcription in the nucleus of a set of genes involved in protein folding, such as the molecular chaperones BiP (or GRP78), GRP94, calreticulin, and protein disulfide isomerase. As such, the UPR adjusts the protein folding capacity of the ER according to need. UPR signaling is initiated by Ire1p, a bifunctional ER transmembrane protein with both serine/threonine kinase and endoribonuclease activities. Ire1p is a single spanning ER membrane protein oriented with its N-terminal half inside the ER lumen, and its C-terminal half (which contains both the kinase and nuclease domains) in the cytosol or nucleus. The ER-lumenal portion of Ire1p is thought to function as a sensor domain that detects changes in the concentration of unfolded proteins or unbound chaperones. Activation of Ire1p leads to its phosphorylation and oligomerization, ultimately resulting in the induction of its endoribonuclease activity by unknown means. The substrate of Ire1p is the mRNA encoding the UPR-specific transcription factor Hac1p that binds to the unfolded protein response element (UPRE) in the promoters of direct target genes of the pathway. Upon induction of the UPR, a 252-nucleotide intron present toward the 3' end of HAC1 mRNA is removed, generating the spliced form (HAC1i mRNA, i = induced). The first catalytic step, cleavage of HAC1 mRNA at both intron-exon junctions, is carried out by Ire1p. The second step, ligation of the two liberated exons, is carried out by tRNA ligase, an enzyme that is shared with the pre-tRNA splicing pathway. Thus, rather than using spliceosomes, the HAC1 intron is removed by a mechanism that resembles pre-tRNA splicing. Both HAC1u mRNA (u = unspliced, uninduced) and HAC1i mRNA are exported to the cytosol and become engaged in polyribosomes, but only the spliced form gives rise to Hac1 protein (Niwa, 1999).

The recent identification of Ire1p homologs suggests that at least some aspects of the UPR are conserved in higher eukaryotic cells. In mammals, two Ire1 isoforms have been identified, Ire1alpha and Ire1beta. Overexpression of either isoform is sufficient to induce the UPR, and overexpression of dominant-negative forms blocks the pathway. Sequence comparisons show strong conservation of the C-terminal kinase and endoribonuclease domains among all known Ire1 homologs. In contrast, the ER-lumenal domains are more divergent, even between the two mammalian isoforms. The conservation of the Ire1p kinase and nuclease domains, together with the fact that human Ire1alpha has been shown to cleave the 5' splice junction of yeast HAC1u mRNA, suggests that a nonconventional splicing event also plays a key role during UPR induction in higher eucaryotes. A basic leucine zipper transcription factor, ATF6, has been identified that is initially synthesized as a transmembrane protein and then becomes proteolyticlly cleaved to release a fragment that participates in transcriptional regulation upon UPR induction. Overexpression of a cytosolic fragment can activate transcription of UPR target genes. In contrast to yeast Hac1p, however, its mRNA is not spliced upon UPR induction (Niwa, 1999).

A remaining enigma in understanding of the UPR concerns the intracellular localization of Ire1p. From its glycosylation pattern, yeast Ire1p is known to reside in the ER membrane and/or inner nuclear membrane, which are continuous around the nuclear pores. The partitioning, if any, of Ire1p between these two membrane domains is unknown, i.e., it has not been determined whether the C-terminal domain (bearing both its kinase and nuclease functions) is cytosolic or nuclear. It is possible, however, that splicing of HAC1 mRNA is a nuclear event because tRNA ligase is localized to the nucleus. Thus, if the C-terminal half of Ire1p is in the cytosol, then activated Ire1p molecules must somehow enter the nucleus to participate in splicing. Ire1p might migrate to the nucleus after its biosynthesis or upon activation of the UPR. There is currently no precedent, however, for a membrane protein with a large cytoplasmic domain to move within the plane of the membrane through nuclear pores. Another possibility is that a fragment of Ire1p is proteolytically severed from the ER membrane upon UPR induction. This fragment could then migrate as a soluble protein into the nucleus to participate in splicing. A precedent for this latter mechanism is found in the pathways controlling sterol biosynthesis and Notch signaling (Niwa, 1999).

Experimental support is provided for the hypothesis that the unusual features of the yeast UPR pathway are conserved. In mammalian cells, the mechanism characterized in yeast is expanded upon to suggest that induction of the UPR involves proteolytic cleavage of Ire1, which allows its cytosolic domains to move into the nucleus, presumably as a prerequisite to participate in RNA splicing. Nuclear localization and induction of the UPR are reduced in cells lacking presenilin-1 (PS1), suggesting PS1 is a new component of the UPR that governs an essential proteolytic step. Yeast HAC1 mRNA is correctly spliced in mammalian cells upon UPR induction and mammalian Ire1 can precisely cleave both splice junctions. Surprisingly, UPR induction leads to proteolytic cleavage of Ire1, releasing fragments containing the kinase and nuclease domains that accumulate in the nucleus. Nuclear localization and UPR induction are reduced in presenilin-1 knockout cells. These results suggest that the salient features of the UPR are conserved among eukaryotic cells and that presenilin-1 controls Ire1 proteolysis in mammalian cells (Niwa, 1999).

These results suggest that misfolding of proteins in the ER leads to increased proteolysis of Ire1 by a PS1-dependent pathway. This raises the question whether other substrates of this proteolytic system, such as APP and/or Notch, are also cleaved at an increased rate when the UPR is induced. Interestingly, it has been suggested that ATF6 is also synthesized as an ER transmembrane protein that becomes cleaved and enters the nucleus upon induction of the UPR. These observations suggest that the processing of ATF6 and Ire1 could be coordinated, possibly both being carried out by gamma-secretase (gamma-secretase is known to cleave in the middle of the transmembrane domain of amyloid precursor protein APP). This putative cross-talk could be explained by either of the two pathways. Ire1-dependent activation of gamma-secretase might directly lead to increased processing of other substrates, including ATF6. Alternatively, activation of Ire1 might lead to the formation of protein complexes in the membrane that, in addition to Ire1, include other proteins which then also become substrates for cleavage by a constitutively active gamma-secretase. Thus, it is possible that APP cleavage is activated following UPR induction; ER stress may therefore lead to an increased production of Abeta42, in turn leading to increased amyloid deposits characteristic of Alzheimer's disease. Environmental exposure to agents that cause ER protein misfolding or inherited mutations that predispose an individual to protein folding defects in the ER could therefore cause or accelerate the rate of onset of Alzheimer's disease. Conversely, cells bearing PS1 mutations are hypersensitive to UPR-inducing agents. In the broadest sense, it is intriguing that Alzheimer's disease involves the extracytosolic deposit of aggregated (misfolded?) Abeta42 and that the UPR regulates the cell's extracytosolic protein folding capacity. Thus, the observation that PS1 plays a role in the UPR suggests a potential link between a disease that results from deposits of aberrant protein and a system that monitors their proper maturation. In this light, it is particularly interesting that levels of the molecular chaperone BiP are reduced in the brain of Alzheimer's disease patients. The potential connection between the UPR and the generation of amyloid deposits in Alzheimer's disease raises new possibilities for understanding and modifying the pathogenesis of this disease (Niwa, 1999).

Missense mutations in the human presenilin-1 (PS1) gene, which is found on chromosome 14, cause early-onset familial Alzheimer's disease (FAD). FAD-linked PS1 variants alter proteolytic processing of the amyloid precursor protein and cause an increase in vulnerability to apoptosis induced by various cell stresses. However, the mechanisms responsible for these phenomena are not clear. Mutations in PS1 affect the unfolded-protein response (UPR), which responds to the increased amount of unfolded proteins that accumulate in the endoplasmic reticulum (ER) under conditions that cause ER stress. PS1 mutations also lead to decreased expression of GRP78/Bip, a molecular chaperone, present in the ER, that can enable protein folding. Interestingly, GRP78 levels are reduced in the brains of Alzheimer's disease patients. The downregulation of UPR signaling by PS1 mutations is caused by disturbed function of IRE1, which is the proximal sensor of conditions in the ER lumen. Overexpression of GRP78 in neuroblastoma cells bearing PS1 mutants almost completely restores resistance to ER stress to the level of cells expressing wild-type PS1. These results show that mutations in PS1 may increase vulnerability to ER stress by altering the UPR signaling pathway (Katayama, 1999).

Knowledge of proteins with which the presenilins interact should lead to a better understanding of presenilin function in normal and disease states. A calcium-binding protein, calmyrin, has been identified that interacts preferentially with presenilin 2 (PS2). Calmyrin is myristoylated, membrane-associated, and colocalizes with PS2 when the two proteins are overexpressed in HeLa cells. Calmyrin accumulates in the nucleus and cytoplasm, but When coexpressed with PS2 these two proteins colocalize at the ER. Yeast two-hybrid liquid assays, affinity chromatography, and coimmunoprecipitation experiments confirm binding between PS2 and calmyrin. Two lines of evidence favor the PS2-loop region as the critical site of calmyrin interaction: reduced in vivo colocalization when calmyrin is coexpressed with a loop-deficient PS2 construct and increased yeast liquid culture binding of calmyrin to the PS2-loop rather than the PS2- COOH-terminal domain. Deletion analysis indicates that calmyrin binding is mediated primarily by the NH2-terminal 31 amino acids of the PS2-loop. Remarkably, despite only a three-amino acid difference, the comparable loop region of PS1 interactes with less than one-tenth the strength in similar yeast two-hybrid assays. The loop region is a site associated with several PS-processing phenomena, including proteolytic cleavage, caspase cleavage, as well as abnormal splicing. Apart from calmyrin, several other proteins have been found to interact with the PS-loop, namely, gamma-catenin, filamin, calselinin, mu-calpain, and armadillo protein p007. The data showing that minor (single amino acid) alterations in the loop sequence can produce dramatic changes in protein binding not only has implications in terms of calmyrin function, but may also have important consequences for the other processing events and binding partners associated with this region. Therefore, it is not surprising that many FAD mutations map to the PS1 loop. Functionally, calmyrin and PS2 increase cell death when cotransfected into HeLa cells. These results allude to several provocative possibilities for a dynamic role of calmyrin in signaling, cell death, and AD (Stabler, 1999).

Most early-onset familial Alzheimer's disease cases are caused by mutations in the highly related genes presenilin 1 (PS1) and presenilin 2 (PS2). Presenilin mutations produce increases in beta-amyloid (Abeta) formation and apoptosis in many experimental systems. A cDNA (ALG-3) encoding the last 103 amino acids of PS2 has been identified as a potent inhibitor of apoptosis. Using this PS2 domain in the yeast two-hybrid system, a neuronal protein has been identified that binds calcium and presenilin, which has been called calsenilin. Calsenilin interacts with both PS1 and PS2 in cultured cells, and can regulate the levels of a proteolytic product of PS2. Thus, calsenilin may mediate the effects of wild-type and mutant presenilins on apoptosis and on Abeta formation. Further characterization of calsenilin may lead to an understanding of the normal role of the presenilins and of the role of the presenilins in Alzheimer's disease (Buxbaum, 1998).

A screened for proteins that interact with PS2 to understand its pathological and physiological functions. Using the PS2 loop domain as the bait, the yeast two-hybrid system was used for screening, and mu-calpain was identified as a PS2 binding protein. In COS-1 cells, the interaction of PS2 with mu-calpain was confirmed by immunoprecipitation. These results suggest that PS2 and mu-calpain interact with each other, and might regulate each other's functions (Shinozaki, 1998).

Although structural features indicate that the presenilins are membrane proteins, their function(s) is unknown. The presenilins have been localized to the nuclear membrane, its associated interphase kinetochores, and the centrosomes-all subcellular structures involved in cell cycle regulation and mitosis. The colocalization of the presenilins with kinetochores on the nucleoplasmic surface of the inner nuclear membrane, together with other results, suggests that they may play a role in chromosome organization and segregation, perhaps as kinetochore binding proteins/receptors. A pathogenic pathway for familial Alzheimer's disease is discussed in which defective presenilin function causes chromosome missegregation during mitosis, resulting in apoptosis and/or trisomy 21 mosaicism (J. Li, 1997).

A novel function of the presenilins (PS1 and PS2) in governing capacitative calcium entry (CCE), a refilling mechanism for depleted intracellular calcium stores, is reported. Abrogation of functional PS1, by either knocking out PS1 or expressing inactive PS1, markedly potentiates CCE, suggesting a role for PS1 in the modulation of CCE. In contrast, familial Alzheimer's disease (FAD)-linked mutant PS1 or PS2 significantly attenuates CCE and store depletion-activated currents. While inhibition of CCE selectively increases the amyloidogenic amyloid ß peptide (Aß42), increased accumulation of the peptide has no effect on CCE. Thus, reduced CCE is most likely an early cellular event leading to increased Aß42 generation associated with FAD mutant presenilins. These data indicate that the CCE pathway is a novel therapeutic target for Alzheimer's disease (Yoo, 2000).

It is suggested that autosomal dominant FAD mutant presenilins exert a gain of function by downregulating CCE while increasing IP3-mediated release from the ER store, leading to diminished luminal Ca2+ concentration ([Ca2+]ER). It is interesting to note that changes in [Ca2+]ER influence a number of cellular functions, including chaperone activities and gene expression. Therefore, it is tempting to speculate that reduced CCE may also be an upstream event leading to other molecular phenotypes associated with FAD mutant presenilins, including altered unfolded protein response. Interestingly, in transgenic mice harboring spinocerebellar ataxia type 1 (SCA1) mutant gene products, TRP3, SERCA2, and IP3-R (all components of CCE), are specifically downregulated. This suggests the potential contribution of CCE dysregulation in other neurodegenerative diseases in addition to AD. CCE involves direct physical interaction between the ER and plasma membrane constituents. According to this conformational coupling mechanism, a conformational change of the IP3 receptor (IP3-R) upon agonist stimulation and subsequent release of Ca2+ leads to the formation of a molecular complex containing IP3-R bound to molecular constituents in the plasma membrane harboring CCE channels. This then allows extracellular Ca2+ to replenish the ER store. It has been postulated that the presenilins modulate the gamma-secretase activity via few possible mechanisms: the presenilins might be the gamma-secretases themselves, and serve as essential cofactors for the gamma-secretase action, or regulate intracellular trafficking of a putative gamma-secretase to the target site where relevant substrates are localized. Given a role for presenilins in governing CCE, the presenilins may also modulate proteolytic processing of APP and Notch at or near the cell surface at sites of ER-plasma membrane coupling. It is conceivable that the presenilins may regulate or directly mediate the cleavage of protein(s) involved in modulating CCE. In any event, a gain in the biological activity of the presenilins, owing to autosomal dominant FAD mutations, may attenuate CCE while increasing gamma-secretase activity. Further experimentation will be necessary to elucidate this connection. Finally, augmentation of CCE, through the identification of agonists of plasma membrane store-operated Ca2+ channels (e.g., TRP or as yet undiscovered CCE channels) that mediate CCE, could potentially be employed to reduce PS-associated gamma-secretase activity, and the generation of Aß as a novel therapeutic means for preventing or treating AD (Yoo, 2000).

Mutations in the highly homologous presenilin genes encoding presenilin-1 and presenilin-2 (PS1 and PS2) are linked to early-onset Alzheimer's disease (AD). However, apart from a role in early development, neither the normal function of the presenilins nor the mechanisms by which mutant proteins cause AD are well understood. The properties are described of a novel human interactor of the presenilins named ubiquilin. Yeast two-hybrid (Y2H) interaction, glutathione S-transferase pull-down experiments, and colocalization of the proteins expressed in vivo, together with coimmunoprecipitation and cell fractionation studies, provide compelling evidence that ubiquilin interacts with both PS1 and PS2. Ubiquilin is noteworthy since it contains multiple ubiquitin-related domains typically thought to be involved in targeting proteins for degradation. However, ubiquilin promotes presenilin protein accumulation. Pulse-labeling experiments indicate that ubiquilin facilitates increased presenilin synthesis without substantially changing presenilin protein half-life. Immunohistochemistry of human brain tissue with ubiquilin-specific antibodies reveal prominent staining of neurons. Moreover, the anti-ubiquilin antibodies robustly stain neurofibrillary tangles and Lewy bodies in AD and Parkinson's disease affected brains, respectively. These results indicate that ubiquilin may be an important modulator of presenilin protein accumulation and that ubiquilin protein is associated with neuropathological neurofibrillary tangles and Lewy body inclusions in diseased brain (Mah, 2000).

It will be interesting to determine the precise mechanism by which ubiquilin induces increased presenilin protein synthesis. Ubiquilin could increase presenilin synthesis by simply increasing presenilin transcription, increasing presenilin translation, or facilitating correct polypeptide folding, maturation, and intracellular targeting of the polytopic transmembrane presenilin protein. The possibility that ubiquilin may act as a molecular chaperone is especially intriguing. Studies of the Xenopus ubiquilin homologue, XDRP1, have suggested that ubiquilin can act posttranscriptionally like a molecular chaperone and prevent degradation of in vitro translated cyclin A protein. Chap1 (ubiquilin 2) has been shown to bind Stch, an Hsp70-like protein. In turn, many heat-shock proteins have been shown to function as molecular chaperones, preventing protein aggregation and protein degradation. Recent evidence has linked ubiquilin proteins to the proteasome. Meanwhile, the 19S regulatory subunit of the 26S proteasome (the degradation complex for ubiquitin-tagged proteins) has been shown to possess protein-unfolding activity. Indirect evidence that ubiquilin may aid in presenilin protein folding or targeting comes from the observation that the presenilin construct PS2(DeltaLC), with deletions of both the loop and COOH-terminal ubiquilin-interaction sites, frequently accumulates into large cytoplasmic aggregates. In contrast, presenilin molecules containing ubiquilin interaction sites, rarely form large protein aggregates. Finally, the presenilins have themselves been linked to molecular chaperones of the ER, that are involved in the unfolded-protein response. Another mechanism by which ubiquilin might increase presenilin accumulation is to alter presenilin degradation rates, especially those of the ubiquitinated forms of presenilins. In fact, evidence has been found that overexpression of human ubiquilin proteins, hPLIC-1 (ubiquilin 1) and hPLIC-2 (ubiquilin 2), interfers with ubiquitin-dependent degradation of p53 and IkBalpha. Although no significant change in the turnover rate of the major 54-kD PS2 polypeptide species (corresponding to full-length PS2) has been found, the possibility that certain ubiquitinated forms of presenilins may have altered turnover rates cannot be excluded. It will be important in future studies to determine if ubiquilin is involved in ubiquitin-dependent degradation of presenilins (Mah, 2000 and references therein).

The influence of presenilins on the genetic cascades that control neuronal differentiation have been examined in Xenopus embryos. Resembling sonic hedgehog (shh) overexpression, presenilin mRNA injection reduces the number of N-tubulin plus primary neurons and modulates Gli3 and Zic2 according to their roles in activating and repressing primary neurogenesis, respectively. Presenilin increases shh expression within its normal domain, mainly in the floor plate, whereas an antisense X-presenilin-alpha morpholino oligonucleotide reduces shh expression. Both shh and presenilin promote cell proliferation and apoptosis, but the effects of shh are widely distributed, while those resulting from presenilin injection coincide with the range of shh signaling. It is suggested that presenilin may modulate primary neurogenesis, proliferation, and apoptosis in the neural plate, through the enhancement of shh signaling (Paganelli, 2001).

Presenilin (PS) genes linked to early-onset familial Alzheimer's disease encode polytopic membrane proteins that are presumed to constitute the catalytic subunit of gamma-secretase, forming a high molecular weight complex with other proteins. During attempts to identify binding partners of PS2, CALP (calsenilin-like protein)/KChIP4, a novel member of calsenilin/KChIP protein family that interacts with the C-terminal region of PS, was cloned. Upon co-expression in cultured cells, CALP directly binds to and co-localizes with PS2 in endoplasmic reticulum. Overexpression of CALP does not affect the metabolism or stability of PS complex, and gamma-cleavage of betaAPP or Notch site 3 cleavage was not altered. However, co-expression of CALP and a voltage-gated potassium channel subunit Kv4.2 reconstitutes the features of A-type K(+) currents and CALP directly binds Kv4.2, indicating that CALP functions as KChIPs that are known as components of native Kv4 channel complex. Taken together, CALP/KChIP4 is a novel EF-hand protein interacting with PS as well as with Kv4 that may modulate functions of a subset of membrane proteins in brain (Morohashi, 2002).

Mutations in presenilins (PS) are the major cause of familial Alzheimer's disease (FAD) and have been associated with calcium (Ca2+) signaling abnormalities. FAD mutant PS1 (M146L)and PS2 (N141I) interact with the inositol 1,4,5-trisphosphate receptor (InsP3R) Ca2+ release channel and exert profound stimulatory effects on its gating activity in response to saturating and suboptimal levels of InsP3. These interactions result in exaggerated cellular Ca2+ signaling in response to agonist stimulation as well as enhanced low-level Ca2+ signaling in unstimulated cells. Parallel studies in InsP3R-expressing and -deficient cells revealed that enhanced Ca2+ release from the endoplasmic reticulum as a result of the specific interaction of PS1-M146L with the InsP3R stimulates amyloid beta processing, an important feature of AD pathology. These observations provide molecular insights into the 'Ca2+ dysregulation' hypothesis of AD pathogenesis and suggest novel targets for therapeutic intervention (Cheung, 2008).

Mutation of mammalian Presenilin

Genetic studies in worms, flies, and humans have implicated the presenilins in the regulation of the Notch signaling pathway and in the pathogenesis of Alzheimer's disease. There are two highly homologous presenilin genes in mammals: presenilin 1 (PS1) and presenilin 2 (PS2). In mice, inactivation of PS1 leads to developmental defects that culminate in a perinatal lethality. To test the possibility that the late lethality of PS1-null mice reflects genetic redundancy of the presenilins, PS2-null mice have been generated by gene targeting, and subsequently, PS1/PS2 double-null mice were generated. Mice homozygous for a targeted null mutation in PS2 exhibit no obvious defects; however, loss of PS2 on a PS1-null background leads to embryonic lethality at embryonic day 9.5. Embryos lacking both presenilins, and surprisingly, those carrying only a single copy of PS2 on a PS1-null background, exhibit multiple early patterning defects, including lack of somite segmentation, disorganization of the trunk ventral neural tube, midbrain mesenchyme cell loss, anterior neuropore closure delays, and abnormal heart and second branchial arch development. In addition, Delta like-1 (Dll1) and Hes-5, two genes that lie downstream in the Notch pathway, are misexpressed in presenilin double-null embryos: Hes-5 expression is undetectable in these mice, whereas Dll1 is expressed ectopically in the neural tube and brain of double-null embryos. It is concluded that the presenilins play a widespread role in embryogenesis; that there is a functional redundancy between PS1 and PS2, and that both vertebrate presenilins, like their invertebrate homologs, are essential for Notch signaling (Donoviel, 1999).

Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes cause the most common and aggressive form of early onset familial Alzheimer's disease. To elucidate their pathogenic mechanism, wild-type (wt) or mutant (M146L, C410Y) PS1 and wt or mutant (M239V) PS2 genes were stably transfected into Chinese hamster ovary cells that overexpress the beta-amyloid precursor protein (APP). The identity of the 43-45-kDa PS1 holoproteins was confirmed by N-terminal radiosequencing. PS1 is rapidly processed (t1/2 = 40 min) in the endoplasmic reticulum into stable fragments. Wild-type and mutant PS2 holoproteins exhibit similar half lives (1.5 h); however, their endoproteolytic fragments show both mutation-specific and cell type-specific differences. Mutant PS1 or PS2 consistently induce a 1.4-2.5-fold increase in the relative production of the highly amyloidogenic 42-residue form of amyloid beta-protein (Abeta42) as determined by quantitative immunoprecipitation and by enzyme-linked immunosorbent assay. In mutant PS1 and PS2 cell lines with high increases in Abeta42/Abeta total ratios, spontaneous formation of low molecular weight oligomers of Abeta42 is observed in media, suggesting enhanced Abeta aggregation from the elevation of Abeta42. It is concluded that mutant PS1 and PS2 proteins enhance the proteolysis of beta-amyloid precursor protein by the gamma-secretase cleaving at Abeta residue 42, thereby promoting amyloidogenesis (Xia, 1997).

PS function is required for normal Notch signaling in Drosophila. Mutations in mammalian Presenilin-1 (PS1) are a major cause of familial Alzheimer’s disease. PS1 is required for murine neural development. Lack of PS1 leads to premature differentiation of neural progenitor cells, indicating a role for PS1 in a cell fate decision between postmitotic neurons and neural progenitor cells. Neural proliferation and apoptotic cell death during neurogenesis are unaltered in PS1-/- mice, suggesting that the reduction in the neural progenitor cells observed in the PS1-/- brain is due to premature differentiation of progenitor cells, rather than to increased apoptotic cell death or decreased cell proliferation. In addition, the premature neuronal differentiation in the PS1-/- brain is associated with aberrant neuronal migration and disorganization of the laminar architecture of the developing cerebral hemisphere. In the ventricular zone of PS1-/- mice, expression of the Notch1 downstream effector gene Hes5 is reduced and expression of the Notch1 ligand Dll1 is elevated, whereas expression of Notch1 is unchanged. The level of Dll1 transcripts is also increased in the presomitic mesoderm of PS1-/- embryos, while the level of Notch1 transcripts is unchanged, in contrast to a previous report. These results provide direct evidence that PS1 controls neuronal differentiation in association with the downregulation of Notch signaling during neurogenesis (Handler, 2000).

These results indicate an important difference in the consequences of reduced Notch signaling during neurogenesis in Drosophila and mice. In Drosophila neurogenesis, Notch controls a cell-fate decision between two cell types produced from a multipotent common precursor, promoting epidermal production at the expense of neuronal production. Loss of function mutations in Notch thereby lead to excessive neuronal production. Findings in mice suggest that Notch1 regulates a cell-fate choice between neural progenitor cells and differentiated neurons early in neurogenesis, promoting regeneration of neuronal precursor cells at the expense of differentiation of postmitotic neurons. Therefore, although Notch functions to suppress the production of postmitotic neurons in both mice and Drosophila, downregulation of Notch activity in mice and Drosophila results in a reduction in neuronal population and a neurogenic phenotype, respectively (Handler, 2000).

The Notch signaling pathway plays essential roles during the specification of the rostral and caudal somite halves and subsequent segmentation of the paraxial mesoderm. The role of presenilin 1 (Ps1; encoded by Psen1) during segmentation has been investigated using newly generated alleles of the Psen1 mutation. In Psen1-deficient mice, proteolytic activation of Notch1 is significantly affected and the expressions of several genes involved in the Notch signaling pathway are altered, including Delta-like3, Hes5, lunatic fringe (Lfng) and Mesp2, which encodes a bHLH transcriptional regulator expressed in the rostral region of the presomitic mesoderm. Thus, Ps1-dependent activation of the Notch pathway is essential for caudal half somite development. Defects were observed in Notch signaling in both the caudal and rostral region of the presomitic mesoderm. In the caudal presomitic mesoderm, Ps1 is involved in maintaining the amplitude of cyclic activation of the Notch pathway, as represented by significant reduction of Lfng expression in Psen1-deficient mice. In the rostral presomitic mesoderm, rapid downregulation of the Mesp2 expression in the presumptive caudal half somite depends on Ps1 and is a prerequisite for caudal somite half specification. Chimaera analysis between Psen1-deficient and wild-type cells reveals that condensation of the wild-type cells in the caudal half somite is concordant with the formation of segment boundaries, while mutant and wild-type cells intermingle in the presomitic mesoderm. This implies that periodic activation of the Notch pathway in the presomitic mesoderm is still latent to segregate the presumptive rostral and caudal somite. A transient episode of Mesp2 expression might be needed for Notch activation by Ps1 to confer rostral or caudal properties. In summary, it is proposed that Ps1 is involved in the functional manifestation of the segmentation clock in the presomitic mesoderm.

To examine the in vivo function of presenilin-1 (PS1), the PS1 gene was selectively deleted in excitatory neurons of the adult mouse forebrain. These conditional knockout mice were viable and grew normally, but they exhibited a pronounced deficiency in enrichment-induced neurogenesis in the dentate gyrus. This reduction in neurogenesis did not result in appreciable learning deficits, indicating that addition of new neurons is not required for memory formation. However, postlearning enrichment experiments lead to a postulate that adult dentate neurogenesis may play a role in the periodic clearance of outdated hippocampal memory traces after cortical memory consolidation, thereby ensuring that the hippocampus is continuously available to process new memories. A chronic, abnormal clearance process in the hippocampus may conceivably lead to memory disorders in the mammalian brain (Feng, 2001).

Why should memory traces in the hippocampus be destabilized periodically by adult dentate neurogenesis? The hippocampus is crucial for converting short-term memories into long-term memories and can process and temporarily store new memories during this transition period before transferring those labile memories to the cortex for permanent storage. In rodents this transition period is often about 3 weeks, which coincides with the turnover rate (3 weeks) of adult generated neurons in the dentate gyrus. Because the hippocampus has limited storage capacity, such a closely correlated time course makes dentate neurogenesis an attractive mechanism to degrade those temporarily stored memory traces in the hippocampus once the consolidation of cortical memories has taken place, thus preventing the hippocampus from overload and making room for a new round of memory acquisition and processing (Feng, 2001).

This neurogenesis-memory clearance hypothesis has three major predictive features. (1) The neurogenesis-based memory clearance should be a time-dependent process, because continuous production and periodic turnover of newborn neurons predict that neurogenesis-based destabilization of memory traces is a gradual and accumulative process. (2) Such a clearance mechanism should be preserved in many mammalian species and should be available throughout the entire adult life. Recent findings that neurogenesis occurs in the dentate gyrus of monkeys and humans, even at old ages, appear to be consistent with this hypothesis. (3) This clearance process is also use dependent, and levels of neurogenesis should be positively correlated with the amount of experience or memory acquisition. As more memories are formed and processed in the hippocampus, more active neurogenesis is required to meet the demand for removing more old memory traces. Indeed, a series of experiments reports that hippocampal learning, enrichment, or even running exercise (which certainly produces episodic memories) increases neurogenesis in the dentate gyrus (Feng, 2001).

What, then, is the advantage of choosing dentate gyrus neurogenesis to perform memory clearance in the hippocampus? The dentate gyrus is known to be the first input station within the trisynaptic hippocampal circuits. Adult-generated neurons in the dentate gyrus are known to insert themselves into the granule layers and extend axons into CA3 even during migration, rapidly making new synapses long before they become fully mature. It has been estimated that one granule cell can contact a dozen CA3 pyramidal cells, and each CA3 cell then, in turn, contacts at least 40-60 other nearby CA3 pyramidal cells and 20-30 nearby inhibitory cells. Therefore, such an upstream location for the addition of 'transient new neurons' in the dentate gyrus makes it ideal for amplifying the destabilization effect within the entire hippocampus. This may perhaps explain why the dentate gyrus of the hippocampus has continuous, and the most robust, adult neurogenesis in the entire mammalian brain (Feng, 2001).

Neural stem cells, which exhibit self-renewal and multipotentiality, are generated in early embryonic brains and maintained throughout the lifespan. The mechanisms of their generation and maintenance are largely unknown. This study shows, by using RBP-Jkappa-/- embryonic stem cells in an embryonic stem cell-derived neurosphere assay, that neural stem cells are generated independent of RBP-Jkappa, a key molecule in Notch signaling. However, Notch pathway molecules are essential for the maintenance of neural stem cells; stem cells are depleted in the early embryonic brains of RBP-Jkappa-/- or Notch1-/- mice. Neural stem cells also are depleted in embryonic brains deficient for the presenilin1 (PS1) gene, a key regulator in Notch signaling, and are reduced in PS1+/- adult brains. Both neuronal and glial differentiation in vitro are enhanced by attenuation of Notch signaling and suppressed by expressing an active form of Notch1. These data are consistent with a role for Notch signaling in the maintenance of the neural stem cell, and inconsistent with a role in a neuronal/glial fate switch (Hitoshi, 2002).

Historically, Notch signaling in Drosophila was thought to maintain cells in an undifferentiated state. More recently, gain-of-function evidence in mammals has suggested that Notch signaling directly and instructively induces glial differentiation. Some Notch-signaling loss-of-function studies in mammals seem consistent with this neuronal/glial fate switch idea, in that there is a premature appearance and increased number of postmitotic neurons expressing MAP2 or ßIII tubulin between E10.5 and E13.5 in the PS1-/- brain. Similarly, mice with mutations in other Notch-signaling molecules such as Notch1, RBP-Jkappa, or Hes1/5 have revealed premature neuronal differentiation. However, it is worth noting that such mice with null mutations in Notch-signaling genes die in mid-to-late embryogenesis, when neurogenesis predominates over gliogenesis in vivo. A clonal analysis of E10 cortical cells in vitro shows that neuronal differentiation from single neural stem cells precedes gliogenesis in clonal cell colonies. Thus, the in vivo analyses of Notch mutants may not allow sufficient time to assess whether gliogenesis is increased or decreased (Hitoshi, 2002).

The present study of the loss-of-function and gain-of-function in Notch pathway molecules in vitro reveals that the PS1 homozygous mutation drives E14.5 neural stem cells to differentiate both into more neurons and more astroglia, and that the expression of the active form of Notch1 suppresses the differentiation of postnatal neural stem-cell progeny both into neurons and into astroglia. These findings are therefore inconsistent with the idea that Notch signaling controls a neuronal/glial fate switch of neural stem cells in the central nervous system, although it remains possible that the different times of the introduction of active Notch in neural stem cells (and thus the different in vivo progenitor cell environments) result in the apparently contradictory findings. These data are more consistent with the idea that Notch signaling keeps cells in an undifferentiated state. PS1-/- neural stem cells have a greater probability of dividing asymmetrically to produce neuronal progenitors early in vivo (and neuronal and glial progenitors in vitro), rather than of dividing symmetrically to produce two daughter neural stem cells as wild-type neural stem cells often do during early embryogenic development. Hence, neuronal progenitor cells in the PS1-/- brain may differentiate prematurely from early asymmetric neural stem-cell divisions. Note that this hypothesis of premature neuronal division as a by product of the failure of symmetric divisions of forebrain neural stem cells with deficits in Notch signaling can be seen as an alternative to the idea that Notch signaling is directly and instructively involved in the fate choice between neuronal and glial differentiation in the mammalian central nervous system. These findings, therefore, are consistent with the idea of a primary defect in symmetric stem-cell self-renewal within the central nervous system (Hitoshi, 2002).

The gain-of-function study in vivo shows that enhanced Notch signaling (by transducing an active form of Notch1 via retroviral infection) increases the number of postnatal neural stem cells in the subependyma of the forebrain lateral ventricle. The cells expressing the active form of Notch1 shows self-renewal and multipotentiality, and, thus, they are neural stem cells. These data suggest that Notch signaling encourages neural stem cells to divide symmetrically to increase the size of the neural stem-cell population, rather than to divide asymmetrically to produce progenitor cells in the embryonic brain, consistent with other gain-of-function studies showing that constitutively active Notch signaling inhibits the differentiation of neural progenitor cells in mammals (Hitoshi, 2002).

Presenilin-1 (PS1) is a gene responsible for the development of early-onset familial Alzheimer's disease. To explore the potential roles of PS1 in vascular development, the vascular system was examined of mouse embryos lacking PS1. PS1-deficient embryos exhibit cerebral hemorrhages and subcutaneous edema by mid gestation. Immunohistochemical analysis reveals vascular remodeling failure in the stomach and trunk dorsal median region of the skin and insufficient formation of the perineural plexus around the spinal cord of the PS1 mutant embryos. The number of capillary sprouting sites are reduced and the capillary diameter are increased in the mutant brains, especially at the amygdaloid and striatal regions. Endothelial cells in the sprouting capillaries of the mutant mice show abnormal morphologies such as multiplication, apoptotic and necrotic images, in contrast to pericytes showing a normal appearance. An in vitro assay using para-aortic splanchnopleural mesoderm (P-Sp) reveals aberrant angiogenesis in the explant culture from the mutant. These findings suggest the essential roles of PS1 in angiogenesis (Nakajima, 2003).

Morphogenesis of the central nervous system relies in large part upon the correct migration of neuronal cells from birthplace to final position. Two general modes of migration govern CNS morphogenesis: radial, which is mostly glia-guided and topologically relatively simple; and tangential, which often involves complex movement of neurons in more than one direction. The consequences of loss of function of presenilin 1 on these fundamental processes is described. Previous studies of the central nervous system in presenilin 1 homozygote mutant embryos identified a premature neuronal differentiation that is transient and localized, with cortical dysplasia at later stages. Widespread effects are documented on CNS morphogenesis that appear strongly linked to defective neuronal migration. Loss of presenilin 1 function perturbs both radial and tangential migration in cerebral cortex, and several tangential migratory pathways in the brainstem. The inability of cells to execute their migratory trajectories affects cortical lamination, formation of the facial branchiomotor nucleus, the spread of cerebellar granule cell precursors to form the external granule layer and development of the pontine nuclei. Finally, overall morphogenesis of the mid-hindbrain region is abnormal, resulting in incomplete midline fusion of the cerebellum and overgrowth of the caudal midbrain. These observations indicate that in the absence of presenilin 1 function, the ability of a cell to move can be severely impaired regardless of its mode of migration, and, at a grosser level, brain morphogenesis is perturbed. These results demonstrate that presenilin 1 plays a much more important role in brain development than has been assumed, consistent with a pleiotropic involvement of this molecule in cellular signaling (Louvi, 2004).

Since radial and tangential modes of migration are affected in the Psen1 mutants, it seems plausible that fundamental cellular mechanisms required for cell movement might be perturbed in the absence of functional Psen1 protein. In preparation to move, cells extend a leading process sensing the immediate environment, followed by translocation of the nucleus into the leading process and subsequent retraction of the trailing process. The first step heavily depends on polymerization and reorganization of actin microfilaments and is controlled by Rho family GTPases, while the second step relies on microtubules. Evidence suggests that Psen1 may indeed interact with cytoskeletal elements. (1) In hippocampal cultures, Psen1 associates with microtubules and microfilaments in a developmentally regulated manner and is localized in lamellipodia and filopodia of neuronal growth cones. (2) The microtubule-associated protein Tau can associate with Psen1 in cultured cells and to a lesser extent in brain extracts. (3) Presenilins can interact in vivo and in vitro with at least two actin-binding family members, filamin A and filamin homolog 1. In Drosophila, filamin interacts genetically and physically with presenilin. In humans, mutations in filamin A prevent migration of cerebral cortical neurons causing periventricular heterotopia. (4) In Drosophila, Psn (presenilin) mutations disrupt the spectrin cytoskeleton, whereas Psen1-dependent gamma-secretase cleavage of E-cadherin leads to its dissociation from the cytoskeleton. Interestingly enough, alpha-spectrin accumulates in cytoplasmic inclusions in the brains of individuals with Alzheimer’s disease. Thus, evidence, albeit circumstantial, exists to suggest a link between presenilins and the cytoskeleton that could provide a mechanistic explanation for some of the migration defects seen in the Psen1 mutants (Louvi, 2004).

The role of Notch signaling during skin development was analyzed using Msx2-Cre to create mosaic loss-of-function of multiple Notch alleles individually or in combination, as well loss of Notch signaling by removal of γ-secretase (i.e., both presenilin genes, PS1 and PS2, with precise temporal and spatial resolution. γ-secretase is not involved in skin patterning or cell fate acquisition within the hair follicle. In its absence, however, inner root sheath cells fail to maintain their fates and by the end of the first growth phase, the epidermal differentiation program is activated in outer root sheath cells. This results in complete conversion of hair follicles to epidermal cysts that bears a striking resemblance to Nevus Comedonicus. Sebaceous glands also fail to form in γ-secretase-deficient mice. Importantly, mice with compound loss of Notch genes in their skin phenocopy loss of γ-secretase in all three lineages, demonstrating that Notch proteolysis accounts for the major signaling function of this enzyme in this organ and that both autonomous and nonautonomous Notch-dependent signals are involved (Pan, 2004).

Presenilin-1 (PS1), the major causative gene of familial Alzheimer disease, regulates neuronal differentiation and Notch signaling during early neural development. To investigate the role of PS1 in neuronal migration and cortical lamination of the postnatal brain, the perinatal lethality of PS1-null mice was circumvented by generating a conditional knockout (cKO) mouse in which PS1 inactivation is restricted to neural progenitor cells (NPCs) and NPC-derived neurons and glia. BrdU birthdating analysis revealed that many late-born neurons fail to migrate beyond the early-born neurons to arrive at their appropriate positions in the superficial layer, while the migration of the early-born neurons is largely normal. The migration defect of late-born neurons coincides with the progressive reduction of radial glia in PS1 cKO mice. In contrast to the premature loss of Cajal-Retzius (CR) neurons in PS1-null mice, generation and survival of CR neurons are unaffected in PS1 cKO mice. Furthermore, the number of proliferating meningeal cells, which have been shown to be important for the survival of CR neurons, is increased in PS1-null mice but not in PS1 cKO mice. These findings show a cell-autonomous role for PS1 in cortical lamination and radial glial development, and a non-cell-autonomous role for PS1 in CR neuron survival (Wines-Samuelson, 2005).

Mice with a null mutation of the presenilin 1 gene (Psen1–/–) die during late intrauterine life or shortly after birth and exhibit multiple CNS and non-CNS abnormalities, including cerebral hemorrhages and altered cortical development. The cellular and molecular basis for the developmental effects of Psen1 remain incompletely understood. Psen1 is expressed in neural progenitors in developing brain, as well as in postmitotic neurons. Transgenic mice were crossed with either neuron-specific or neural progenitor-specific expression of Psen1 onto the Psen1–/– background. Neither neuron-specific nor neural progenitor-specific expression of Psen1 can rescue the embryonic lethality of the Psen1–/– embryo. Indeed neuron-specific expression rescues none of the abnormalities in Psen1–/– mice. However, Psen1 expression in neural progenitors rescues the cortical lamination defects, as well as the cerebral hemorrhages, and restores a normal vascular pattern in Psen1–/– embryos. Collectively, these studies demonstrate that Psen1 expression in neural progenitor cells is crucial for cortical development and reveal a novel role for neuroectodermal expression of Psen1 in development of the brain vasculature (Wen, 2005 ).

Presenilin 1 (PS1) regulates environmental enrichment (EE)-mediated neural progenitor cell (NPC) proliferation and neurogenesis in the adult hippocampus. Transgenic mice that ubiquitously express human PS1 variants linked to early-onset familial Alzheimer's disease (FAD) neither exhibit EE-induced proliferation, nor neuronal lineage commitment of NPCs. Remarkably, the proliferation and differentiation of cultured NPCs from standard-housed mice expressing wild-type PS1 or PS1 variants are indistinguishable. In contrast, wild-type NPCs cocultured with primary microglia from mice expressing PS1 variants exhibit impaired proliferation and neuronal lineage commitment, phenotypes that are recapitulated with mutant microglia conditioned media in which altered levels of selected soluble signaling factors were detected. These findings lead to the conclusion that factors secreted from microglia play a central role in modulating hippocampal neurogenesis, and argue for non-cell-autonomous mechanisms that govern FAD-linked PS1-mediated impairments in adult hippocampal neurogenesis (Choi, 2008).

Signaling downstream of Presenilin

Presenilin 1 (PS1) plays a pivotal role in Notch signaling and the intracellular metabolism of the amyloid ß-protein. To understand intracellular signaling events downstream of PS1, the action of PS1 on mitogen-activated protein kinase pathways has been investigated. Overexpressed PS1 suppresses the stress-induced stimulation of stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) in human embryonic kidney 293 cells. Interestingly, two functionally inactive PS1 mutants, PS1(D257A) and PS1(D385A), fail to inhibit UV-stimulated SAPK/JNK. Furthermore, H2O2- or UV-stimulated SAPK activity is higher in mouse embryonic fibroblast (MEF) cells from PS1-null mice than in MEF cells from PS+/+ mice. MEFPS1(-/-) cells were more sensitive to the H2O2-induced apoptosis than MEFPS1(+/+) cells. Ectopic expression of PS1 in MEFPS1(-/-) cells suppressesH2O2-stimulated SAPK/JNK activity and apoptotic cell death. Together, these data suggest that PS1 inhibits the stress-activated signaling by suppressing the SAPK/JNK pathway (Kim, 2001).

Thus, PS1 inhibits the SAPK/JNK pathway. Ectopically expressed PS1 blocks the stress-induced stimulation of SAPK/JNK and its upstream kinases, including SEK1 and MEKK1. FAD-linked PS1 mutants, M146V, C410Y, and L286V, are also able to inhibit the SAPK stimulation. Interestingly, biologically inactive PS1 mutants D257A and D385A, both of which have been shown to lack gamma-secretase activation and PS1 endoproteolysis, fail to inhibit SAPK stimulation. Furthermore, PS1DeltaEx9, which lacks the endoproteolysis site but is competent for activation of gamma-secretase activity, retains the inhibitory effect on the SAPK/JNK pathway. These data suggest that the gamma-secretase activation, rather than the PS1 endoproteolysis, is required for the PS1-induced inhibition of the SAPK/JNK pathway. gamma-Secretase has two major substrates, APP and Notch. The cleavage of APP or Notch by gamma-secretase produces Aß or the intracellular domain of Notch, Notch-IC, respectively. Aß1-42 does not inhibit the SAPK/JNK activity. In comparison, preliminary data showed that overexpression of the Notch intracellular domain, which is the active form of intracellular Notch, results in suppression of SAPK/JNK activation. These findings imply that PS1-mediated cleavage of Notch might be involved in the mechanism of PS1-induced suppression of the SAPK pathway. In this regard, Notch has been previously proposed to play a role in the regulation of the SAPK/JNK pathway (Kim, 2001 and references therein).

Several lines of evidence suggest that presenilins are involved in apoptosis. Overexpression of PS2 has been shown to potentiate apoptosis of PC12 cells induced by NGF withdrawal or neurotoxic Aß1-42. ALG3, a truncated form of murine PS2, reduces T cell receptor-induced or Fas-induced apoptosis in a mouse T cell hybridoma. Studies using PS1-null mice have demonstrated that PS1 is involved in neuronal survival. Ectopic PS1 suppresses the H2O2-induced apoptosis in B103 neuroblastoma cells. Moreover, deficiency of PS1 causes an elevation in the H2O2-induced apoptosis in MEF cells from PS1-null mice, as compared with MEF cells from PS1+/+ wild-type mice. The H2O2-induced apoptosis is blocked by overexpression of SEK1(K129R), suggesting that the SAPK/JNK pathway is involved in the mechanism of the H2O2-induced apoptosis. These findings suggest that PS1, by inhibiting the SAPK pathway, can protect cells from stress-induced apoptotic cell death. However, further study is needed to determine the precise mechanism by which PS1 inhibits the SAPK/JNK pathway (Kim, 2001).

Elaborate metamerism in vertebrate somitogenesis is based on segmental gene expression in the anterior presomitic mesoderm (PSM). Notch signal pathways with Notch ligands Dll1 and Dll3, and the bHLH transcription factor Mesp2 (Mesoderm posterior 2) are implicated in the rostrocaudal patterning of the somite. Changes in the Mesp2 expression domain from a presumptive one somite into a rostral half somite results in differential activation of two types of Notch pathways, dependent or independent of presenilin 1 (Psen1), which is a Notch signal mediator. To further refine this hypothesis, genetic interactions between Dll1, Dll3, Mesp2 and Psen1 have been analyzed, and the roles of Dll1- and Dll3-Notch pathways, with or without Psen1, in rostrocaudal patterning have been elucidated. Dll1 and Dll3 are co-expressed in the PSM and so far are considered to have partially redundant functions. Positive and negative feedback loops comprising Dll1 and Mesp2 appear to be crucial for this patterning; Dll3 may be required for the coordination of the Dll1-Mesp2 loop. Additionally, epistatic analysis reveals that Mesp2 affects rostrocaudal properties more directly than Dll1 or Dll3. Finally, Psen1 is found to be involved differently in the regulation of rostral and caudal genes. Psen1 is required for Dll1-Notch signaling for activation of Dll1, while the Psen1-independent Dll3-Notch pathway may counteract the Psen1-dependent Dll1-Notch pathway. These observations suggest that Dll1 and Dll3 may have non-redundant, even counteracting functions. It is concluded that Mesp2 functions as a central mediator of such Notch pathways and regulates the gene expression required for rostrocaudal patterning of somites (Takahashi, 2003).

Mutations in presenilins are the major cause of familial Alzheimer's disease, but the pathogenic mechanism by which presenilin mutations cause memory loss and neurodegeneration remains unclear. Conditional double knockout mice lacking both presenilins in the postnatal forebrain exhibit impairments in hippocampal memory and synaptic plasticity. These deficits are associated with specific reductions in NMDA receptor-mediated responses and synaptic levels of NMDA receptors and alphaCaMKII. Furthermore, loss of presenilins causes reduced expression of CBP and CREB/CBP target genes, such as c-fos and BDNF. With increasing age, mutant mice develop striking neurodegeneration of the cerebral cortex and worsening impairments of memory and synaptic function. Neurodegeneration is accompanied by increased levels of the Cdk5 activator p25 and hyperphosphorylated tau. These results define essential roles and molecular targets of presenilins in synaptic plasticity, learning and memory, and neuronal survival in the adult cerebral cortex (Saura, 2004).

gamma-Secretase activity and organogenesis

Notch signaling is involved in pronephros development in Xenopus and in glomerulogenesis in mice. However, owing to early lethality in mice deficient for some Notch pathway genes and functional redundancy for others, a role for Notch signaling during early stages of metanephric development has not been defined. Using an antibody specific to the N-terminal end of gamma-secretase-cleaved Notch1, evidence was found for Notch1 activation in the comma and S-shaped bodies of the mouse metanephros. Mouse metanephroi were therefore cultured in the presence of a gamma-secretase inhibitor, N-S-phenyl-glycine-t-butyl ester (DAPT), to block Notch signaling. Slightly reduced ureteric bud branching was observed but normal mesenchymal condensation and expression of markers was observed, indicating that mesenchyme induction had occurred. However, fewer renal epithelial structures were observed, with a severe deficiency in proximal tubules and glomerular podocytes, which are derived from cells in which activated Notch1 is normally present. Distal tubules were present but in reduced numbers, and this was accompanied by an increase in intervening, non-epithelial cells. After a transient 3-day exposure to DAPT, proximal tubules expanded, but podocyte differentiation failed to recover after removal of DAPT. These observations suggest that gamma-secretase activity, probably through activation of Notch, is required for maintaining a competent progenitor pool as well as for determining the proximal tubule and podocyte fates (Cheng, 2003).


Search PubMed for articles about Drosophila Presenilin

Ancolio, K., et al. (1997). Alpha-secretase-derived product of beta-amyloid precursor protein is decreased by presenilin 1 mutations linked to familial Alzheimer's disease. J. Neurochem. 69(6): 2494-9. PubMed Citation: 9375682

Annaert, W. G., et al. (2001). Interaction with Telencephalin and the Amyloid precursor protein predicts a ring structure for Presenilins. Neuron 32: 579-589. 11719200

Berezovska, O., Xia, M. Q. and Hyman, B. T. (1998). Notch is expressed in adult brain, is coexpressed with presenilin-1, and is altered in Alzheimer disease. J. Neuropathol. Exp. Neurol. 57(8): 738-45. PubMed Citation: 9720489

Berezovska, O., et al. (1999). The alzheimer-related gene presenilin 1 facilitates notch 1 in primary mammalian neurons. Brain Res. Mol. Brain Res. 69(2): 273-80. PubMed Citation: 10366748

Boulianne, G. L., et al. (1997). Cloning and characterization of the Drosophila presenilin homologue. Neuroreport 8(4): 1025-9. PubMed Citation: 9141085

Buxbaum, J. D., et al. (1998). Calsenilin: a calcium-binding protein that interacts with the presenilins and regulates the levels of a presenilin fragment. Nat. Med. 4(10): 1177-81. 98442695

Capell, A., et al. (1998). The proteolytic fragments of the Alzheimer's disease-associated presenilin-1 form heterodimers and occur as a 100-150-kDa molecular mass complex. J. Biol. Chem. 273: 3205-3211. 9452432

Casso, D. J. Biehs, B. and Kornberg, T. B. (2011). A novel interaction between hedgehog and Notch promotes proliferation at the anterior-posterior organizer of the Drosophila wing. Genetics 187(2): 485-99. PubMed Citation: 21098717

Chan, Y.-M. and Jan, Y. N. (1999). Presenilins, processing of ß-Amyloid precursor protein, and notch signaling. Neuron 23: 201-204

Chávez-Gutiérrez, L., et al. (2008). Glu(332) in the Nicastrin ectodomain is essential for gamma-secretase complex maturation but not for its activity. J. Biol. Chem. 283: 20096-20105. PubMed Citation: 18502756

Chen, F., et al. (2001). Nicastrin binds to membrane-tethered Notch. Nat. Cell Biol. 3: 751-755. 11483961

Cheng, H.-T., et al. (2003). gamma-Secretase activity is dispensable for mesenchyme-to-epithelium transition but required for podocyte and proximal tubule formation in developing mouse kidney. Development 130: 5031-5042. 12952904

Cheung, K. H., et al. (2008). Mechanism of Ca2+ disruption in Alzheimer's disease by presenilin regulation of InsP3 receptor channel gating. Neuron 58(6): 871-83. PubMed Citation: 18579078

Choi, S. H., et al. (2008). Non-cell-autonomous effects of presenilin 1 variants on enrichment-mediated hippocampal progenitor cell proliferation and differentiation. Neuron 59(4): 568-80. PubMed Citation: 18760694

Chung, H. M. and Struhl, G. (2001). Nicastrin is required for Presenilin-mediated transmembrane cleavage in Drosophila. Nat. Cell Biol. 3(12): 1129-32. 11781576

Cinar, H. N., et al. (2001). The SEL-12 presenilin mediates induction of the Caenorhabditis elegans uterine pi cell fate. Dev. Bio. 237: 173-182. 11518514

Coolen, M. W., et al. (2005). Gene dosage effect on gamma-secretase component Aph-1b in a rat model for neurodevelopmental disorders. Neuron 45(4): 497-503. 15721236

Cox, R. T., et al. (2000). A screen for mutations that suppress the phenotype of Drosophila armadillo, the ß-catenin homolog. Genetics 155: 1725-1740. PubMed Citation: 10924470

Cras-Méneur, C., Li, L., Kopan, R. and Permutt, M. A. (2009). Presenilins, Notch dose control the fate of pancreatic endocrine progenitors during a narrow developmental window. Genes Dev. 23(17): 2088-101. PubMed Citation: 19723764

De Strooper, B., et al. (1998). Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein. Nature 391(6665): 387-90. PubMed Citation: 9450754

De Strooper, B., et al. (1999). A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain. Nature 398(6727): 518-22. PubMed Citation: 10206645

Donoviel, B. D., et al. (1999). Mice lacking both presenilin genes exhibit early embryonic patterning defects. Genes Dev. 13: 2801-2810. PubMed Citation: 10557208

Edbauer, D., Winkler, E., Haass, C. and Steiner, H. (2002). Presenilin and nicastrin regulate each other and determine amyloid -peptide production via complex formation. Proc. Natl Acad. Sci. 99: 8666-8671. 12048259

Edbauer, D., Kaether, C., Steiner, H. and Haass, C. (2005). Co-expression of nicastrin and presenilin rescues a loss of function mutant of APH-1. J. Biol. Chem. 279(36): 37311-5. 15210705

Eimer, S., Lakowski, B., Donhauser, R. and Baumeister, R. (2002a). Loss of spr-5 bypasses the requirement for the C. elegans presenilin sel-12 by derepressing hop-1. EMBO J. 21: 5787-5796. 12411496

Eimer, S., Donhauser, R. and Baumeister, R. (2002b). The Caenorhabditis elegans Presenilin sel-12 is required for mesodermal patterning and muscle function. Dev. Bio. 251: 178-192. 12413907

Feng, R., et al. (2001). Deficient neurogenesis in forebrain-specific Presenilin-1 knockout mice is associated with reduced clearance of hippocampal memory traces. Neuron 32: 911-926. 11738035

Francis, R., et al. (2002). aph-1 and pen-2 are required for Notch pathway signaling, gamma-Secretase cleavage of ßAPP, and Presenilin protein accumulation. Dev. Cell 3: 85-97. 12110170

Gao, Y. and Pimplikar, S. W. (2001). The gamma-secretase-cleaved C-terminal fragment of amyloid precursor protein mediates signaling to the nucleus. Proc. Natl. Acad. Sci. 98: 14979-14984. 11742091

Goutte, C., et al. (2000). aph-2 encodes a novel extracellular protein required for GLP-1-mediated signaling. Development 127(11): 2481-92. 10804188

Gu, Y., et al. (2003). APH-1 interacts with mature and immature forms of presenilins and nicastrin and may play a role in maturation of presenilin-nicastrin complexes. J. Biol. Chem. 278: 7374-7380. 12471034

Guo, Q., Robinson, N. and Mattson, M. P. (1998). Secreted beta-amyloid precursor protein counteracts the proapoptotic action of mutant presenilin-1 by activation of NF-kappaB and stabilization of calcium homeostasis. J. Biol. Chem. 273(20): 12341-51. PubMed Citation: 9575187

Guo, Y., et al. (1999). Drosophila presenilin is required for neuronal differentiation and affects Notch subcellular localization and signaling. J. Neurosci. 19(19): 8435-8442. PubMed Citation: 10493744

Gupta-Rossi, N., et al. (2004). Monoubiquitination and endocytosis direct gamma-secretase cleavage of activated Notch receptor. J. Cell Biol. 166(1): 73-83. 15240571

Handler, M., Yang, X. and Shen, J. (2000). Presenilin-1 regulates neuronal differentiation during neurogenesis. Development 127: 2593-2606. PubMed Citation: 10821758

Hass, M. R., Sato, C. Kopan, R. and Zhao, G. (2009). Presenilin: RIP and beyond. Semin. Cell Dev. Biol. 20: 201-210. PubMed Citation: 19073272

Herranz, H., Stamataki, E., Feiguin, F. and Milan, M. (2006). Self-refinement of Notch activity through the transmembrane protein Crumbs: modulation of gamma-secretase activity. EMBO Rep. 7(3): 297-302. Medline abstract: 16440003

Hong, C. S. and Koo, E. H. (1997). Isolation and characterization of Drosophila presenilin homolog. Neuroreport 8(3): 665-8. PubMed Citation: 9106743

Hitoshi, S., et al. (2002). Notch pathway molecules are essential for the maintenance, but not the generation, of mammalian neural stem cells. Genes Dev. 16: 846-858. 11937492

Hu, Y., Ye, Y. and Fortini, M. E. (2002). Nicastrin is required for gamma-secretase cleavage of the Drosophila Notch receptor. Dev. Cell 2: 69-78. 11782315

Huppert, S. S., Ilagan, M. X., De Strooper, B. and Kopan, R. (2005). Analysis of Notch function in presomitic mesoderm suggests a gamma-secretase-independent role for presenilins in somite differentiation. Dev. Cell 8(5): 677-88. 15866159

Ikeuchi, T. and Sisodia, S. S. (2003). The Notch ligands, Delta1 and Jagged2, are substrates for presenilin-dependent 'gamma-secretase' cleavage. J. Biol. Chem. 278(10): 7751-4. 12551931

Jarriault, S. and Greenwald, I. (2002). Suppressors of the egg-laying defective phenotype of sel-12 presenilin mutants implicate the CoREST corepressor complex in LIN-12/Notch signaling in C. elegans Genes Dev. 16: 2713-2728. 12381669

Kang, D. E., et al. (2002). Presenilin couples the paired phosphorylation of ß-Catenin independent of Axin: Implications for ß-Catenin activation in tumorigenesis. Cell 110: 751-762. 12297048

Katayama, T., et al. (1999). Presenilin-1 mutations downregulate the signalling pathway of the unfolded-protein response. Nat. Cell Biol. 1: 479-485

Kidd, S., Lieber, T. and Young, M. W. (1998). Ligand-induced cleavage and regulation of nuclear entry of Notch in Drosophila melanogaster embryos. Genes Dev. 23: 3728-3740

Kim, J. W., et al. (2001). Negative Regulation of the SAPK/JNK Signaling Pathway by Presenilin. J. Cell Bio. 153: 457-464. 11331298

Kim, S. H. and Sisodia, S. S. (2005). Evidence that the "NF" motif in transmembrane domain 4 of presenilin 1 is critical for binding with PEN-2. J. Biol. Chem. 280(51): 41953-66. 16234243

Klein, T., et al. (2003). The tumor suppressor gene l(2)giant discs is required to restrict the activity of Notch to the dorsoventral boundary during Drosophila wing development. Dev. Bio. 255: 313-333. 12648493

Koizumi, K-i., et al. (2001). The role of presenilin 1 during somite segmentation Development 128: 1391-1402. 11262239

Lakowski, B., et al. (2003). Two suppressors of sel-12 encode C2H2 zinc-finger proteins that regulate presenilin transcription in Caenorhabditis elegans. Development 130: 2117-2128. 12668626

LaVoie, M. J. and Selkoe, D. J. (2003). The Notch ligands, Jagged and Delta, are sequentially processed by alpha-secretase and presenilin/gamma-secretase and release signaling fragments. J. Biol. Chem. 278(36): 34427-37. 12826675

Lecourtois, M. and Schweisguth, F. (1998). Indirect evidence for Delta-dependent intracellular processing of Notch in Drosophila embryos. Curr. Biol. 8(13): 771-4

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Biological Overview

date revised: 10 November 2015

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