ß amyloid protein precursor-like
The view that only the production and deposition of Abeta plays a decisive role in Alzheimer's disease has been challenged by recent evidence from different model systems, which attribute numerous functions to the amyloid precursor protein (APP). To investigate the potential cellular functions of APP and its paralogs, transgenic Drosophila was used as a model. Upon overexpression of the APP-family members, transformations of cell fates during the development of the peripheral nervous system were observed. Genetic analysis showed that APP, APLP1 and APLP2 induce Notch gain-of-function phenotypes, identified Numb as a potential target and provided evidence for a direct involvement of Disabled and Neurotactin in the induction of the phenotypes. The severity of the induced phenotypes not only depended on the dosage and the particular APP-family member but also on particular domains of the molecules. Studies with Drosophila APPL confirmed the results obtained with human proteins and the analysis of flies mutant for the appl gene further supports an involvement of APP-family members in neuronal development and a crosstalk between the APP family and Notch (Merdes, 2004).
These studies show that the ectopic expression of human APP-family members induces Notch gain-of-function phenotypes during the development of the adult PNS. The severity of the induced phenotypes not only depends on the dosage and the particular APP-family member, but also on particular domains of the molecules. This led to the identification of the NPTY motif as the only critical motif within the ICD for the interference with PNS development and for the interaction of APP with Numb/Pon and Dab in vitro and in vivo (Merdes, 2004).
An interaction between APP and Numb has been demonstrated by Roncarati (2002). In mouse brain lysates as well as in cell culture, APP or APP.ICD bind to all four isoforms of Numb and to Numb-like. Surprisingly, in this study, the processing of APP and the release of the ICD of APP resulted in an inhibition of Notch signaling. Numb is a negative regulator of Notch signaling and binds directly via its PTB domain to Notch. Therefore, a direct interaction between APP and the PTB domain of Numb should result in an increase rather than in a decrease of Notch activation. From the known crystal structure of PTB-NPTY interactions, a trimeric complex between Notch, APP and Numb seems unlikely. In this study, the induced Notch gain-of-function phenotypes, the strong genetic interaction, the dependence of the asymmetric localization of APPL on Numb and the direct binding between APP and Numb support a crosstalk between Notch signaling and APP-family members. One explanation for the APP induced Notch gain-of-function phenotypes during mechano-sensory organ (MSO) development would indeed be the sequestration and inactivation of Numb by APP-family members. However, several lines of evidence are provided that (if APP competes with Notch for the binding to Numb) suggest this binding and competition must be highly regulated and requires factors which have not previously been known to be involved in MSO development (Merdes, 2004).
(1) Expression of the human APP-family proteins induces cell fate transformations during MSO development in a dosage- and construct-dependent manner, but the potency in phenotype induction of the different proteins does not correlate with their in vitro and in vivo binding affinity to Numb. Nevertheless, the NPTY motif proves to be essential both for binding to Numb and phenotype induction, suggesting that the binding to Numb might be necessary but not sufficient for phenotype induction. This implies that there is at least one additional factor which plays an important role and which must have different affinities to the APP-family members than Numb, for example, strong binding to APLP2 but weak binding to APP.
(2) Deletion of the ECD of APP results in an inactive molecule, which can no longer induce any phenotypes. This stands in contrast to all in vitro binding studies that have been performed between the NPTY motif of APP and PTB-containing proteins in cell culture. In these assays, the affinity of such a molecule to Fe65, Dab-1/2, X11L, Numb and Numb-like did not change significantly.
(3) APP molecules with a deletion of the NPTY motif could suppress the phenotypes induced by wt APP and induce the loss of macrochaete in wt flies. Such a dominant-negative effect can only be explained if APP-family members have a receptor-like function. In this scenario, APP.DeltaNPTY would compete with wt APP or APPL for ligand binding, but could not relay the 'signal', for example, crosstalk to Notch and/or inactivating Numb. Another possibility would be the necessity of homodimer formation. Such a dimer formation has been postulated, but so far no in vivo data are available. Furthermore, structural data do not provide any evidence for a dimerization of APP molecules prior to the binding of PTB-containing proteins.
(4) Overexpression of Drosophila APPL induces only very weak phenotypes, whereas the overexpression of APPL.sd induces very strong phenotypes. The difference in phenotype induction could not be correlated with significant differences in expression levels, metabolism or processing. This was surprising, since APPL.sd had been generated to impair secretion and therefore processing. As a consequence, it is postulated that the 33 aa deletion in APPL.sd changes the conformation of the ECD, confirming again the important role the ECD plays in determining the potency of the APP-family members for interference with PNS development.
(5) Overexpression of APLP2 results in bold patches, suggesting that presumptive SOPs are transformed into epidermal cells by the induction of a Notch gain-of-function phenotype very early during MSO development. This step during PNS development is known to be independent of Numb and functions via the lateral inhibition mechanism, indicating that APP-family members can also interact with Numb-independent Notch signaling processes. During these processes, so far unknown factors might take over the role of Numb as a negative regulator of Notch to add an additional level of control to the system. From the literature, it seems to be clear that endocytosis is important for inhibition and for the promotion of Notch signaling, but almost nothing is known about the factors directly involved in these events.
(6) Ectopically expressed APPL and APPL.sd as well as APP and APP/APLP2 are asymmetrically localized during MSO development and co-localization and co-immunoprecipitation with Pon has been be demonstrated in vivo. This is an interesting result since APPL and APP induce only weak phenotypes, but APPL.sd and APP/APLP2 induce very strong phenotypes. Nevertheless, both types of proteins are recognized with the same efficiency by the Numb-dependent machinery responsible for the asymmetric distribution of factors during MSO development, thus completely uncoupling this event from phenotype induction. This implies that the phenotype induction occurs after completion of the separation of the SOP siblings and that APP, even if it binds to Numb, does not compete with other binding partners of Numb for asymmetric segregation (Merdes, 2004).
During MSO development, the asymmetric distribution of Numb ensures that the siblings arising from one mother cell show a difference in response to the activation of the Notch receptor. Numb is responsible for the asymmetric segregation of α-adaptin and binds both the ICD of Notch and α-adaptin, suggesting that Numb may regulate Notch by controlled endocytosis. The difference in response to Notch signaling is further amplified by the asymmetric localization of the E3 ubiquitin ligase Neuralized, which upregulates the endocytosis of the Notch ligand Delta. However, one has to take into account that it has also been reported that Numb can (1) bind the ICD of Notch after release, (2) inhibit the ability of this ICD to cause nuclear translocation of Su(H) and (3) can inhibit Notch signaling during wing development by ectopic misexpression. Therefore, even if it is very tempting to suggest that Numb solely regulates Notch by endocytotic mechanisms, there might still be other Numb functions (Merdes, 2004).
Nevertheless, more and more evidence is emerging that regulated endocytosis is an important general feature for the modulation of developmental signals. In this respect, it is especially intriguing that Drosophila Dab has been identified as an essential factor for the interaction of APP with Notch signaling. Whereas the overexpression of Dab enhances the phenotype induced by APP, a reduction of the endogenous protein level by RNAi suppresses the phenotype. Notch gain-of-function phenotypes during MSO development can be induced by expression of high levels of Dab alone. This is remarkable since it has been proposed that the mammalian Dab-2 homologs belong to a family of cargo-specific adaptor proteins, which, like Numb and β-Arrestin, regulate cargo selection and pit formation. Accordingly, APP molecules could induce the observed phenotypes during PNS development, influencing endocytosis and processing of Notch with the help of Dab. A function for APP as endocytotic receptor is supported by the finding that full-length APP is internalized via clathrin-coated vesicles. Furthermore, a direct interaction between Drosophila Dab and Notch has been demonstated previously (Giniger, 1998). These binding studies have been reproduced, but the binding of Dab to Notch in vitro was shown to be very weak in comparison to the binding affinity of Su(H) or Numb. However, additional studies suggest not only the presence of a second Notch-binding motif within the C-terminal domain of Dab, but also reveal the presence in vivo of a complex which contains Notch and Dab in Drosophila embryos (LeGall and Giniger, personal communication to Merdes, 2004). The second binding motif could allow a direct interaction between the Notch receptor and APP mediated by Dab, and it will be of great interest to elucidate the role of Dab with respect to Notch and APP signaling in the future. A crosstalk between the APP family and Notch receptors has also been shown to take place in the mammalian system (Merdes, 2004).
Originally, mutations in the dab gene were isolated by genetic interactions with the Drosophila Abl homolog. It has recently been reported that these mutations have been erroneously attributed and that all mutations isolated as dab alleles in fact affect the nrt locus (Liebl, 2003). Nrt is a single-pass type-II transmembrane protein and belongs to the family of neuronal cell adhesion molecules (N-CAMs). Nrt mutants are viable and fertile, but its function in growth cone guidance can be revealed in combination with other N-CAM mutants. Since the originally described dab alleles were used for the first genetic studies, mutations were identified in nrt as dominant suppressors of the APP-induced phenotype and also the overexpression of Nrt itself induces very strong and very specific Notch gain-of-function phenotypes. However, genetic studies ruled out an involvement of Abl in the APP-induced phenotype. Preliminary genetic data suggest a genetic interaction between appl and nrt mutations resulting in lethality of the otherwise viable alleles. Additional experiments will be necessary both in Drosophila and vertebrates to further explore this interaction. Especially, the isolation of new mutants for Drosophila dab and appl generated in a clearly defined genetic background, and their use for genetic interactions with Notch, numb and nrt, should provide insights into the mechanisms underlying the potential functions of APP-family members in endocytosis, Notch signaling and PNS development. However, the identification of appl as a quantitative trait locus already provides evidence for a function of appl during PNS developmen (Merdes, 2004).
Although it has not been established that the binding interactions between APP, Numb and Dab are functionally important in AD, signaling pathways emanating from aberrant APP function, as it occurs in AD, may influence Dab/Numb and thus Notch activity. Also, the use of drugs to lower APP processing and Aβ production could result in altered APP functions and an interference with Notch signaling in the adult brain. As already mentioned, an interaction between APP and Numb and Numb-like in the mouse brain has been demonstrated and there is accumulating evidence for a role of the Notch signaling pathway not only in early events during cell fate specifications but also in stem cells, in already differentiated neuronal cells and in neurodegeneration in the adult vertebrate nervous system. Furthermore, the view that only the production and deposition of Aβ plays a decisive role in AD has been challenged by recent evidence from different model systems that attribute numerous functions to APP and derivatives thereof. These findings together with the current data make it likely that alterations in the processing of APP either during the onset and progression of AD or by the use of therapeutics may result in loss- as well as in gain-of-function phenotypes contributing to the disease or side effects (Merdes, 2004).
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).
A novel protein has been isolated based on its association with Drosophila APP-like protein (APPL), a homolog of the beta-amyloid precursor protein (APP) that is implicated in Alzheimer's disease. This novel APPL-interacting protein 1 (APLIP1) contains a Src homology 3 domain and a phosphotyrosine interaction domain and is expressed abundantly in neural tissues. The phosphotyrosine interaction domain of APLIP1 interacts with a sequence containing GYENPTY in the cytoplasmic domain of APPL. APLIP1 is highly homologous to the carboxyl-terminal halves of mammalian c-Jun NH(2)-terminal kinase (JNK)-interacting protein 1b (JIP1b) and 2 (JIP2), which also contain Src homology 3 and phosphotyrosine interaction domains. The similarity of APLIP1 to JIP1b and JIP2 includes interaction with component(s) of the JNK signaling pathway and with the motor protein kinesin and the formation of homo-oligomers. JIP1b interacts strongly with the cytoplasmic domain of APP (APPcyt), as APLIP1 does with APPL, but the interaction of JIP2 with APPcyt is weak. Overexpression of JIP1b slightly enhances the JNK-dependent threonine phosphorylation of APP in cultured cells, but that of JIP2 suppresses it. These observations suggest that the interactions of APP family proteins with APLIP1, JIP1b, and JIP2 are conserved and play important roles in the metabolism and/or the function of APPs including the regulation of APP phosphorylation by JNK. Analysis of APP family proteins and their associated proteins is expected to contribute to understanding the molecular process of neural degeneration in Alzheimer's disease (Taru, 2002; full text of article).
APPs possess a membrane-associated receptor-like structure, and the amino acid sequence of their short cytoplasmic region is highly conserved among a wide variety of species. Protein interactions between the cytoplasmic domains of APPs and cytoplasmic proteins are thought to be important for regulating the metabolism of APPs and/or for putative physiological function of APP. Drosophila APLIP1 and its putative mammalian homologs JIP1b and JIP2 can interact with the cytoplasmic domain of the APPs. These proteins, APLIP1, JIP1b, and JIP2, resemble each other in their structure, especially in their carboxyl-terminal regions that contain SH3 and PI domains. They also share properties such as interactions with APPs, MAP kinase kinase, and kinesin; an abundant expression in the nervous system; and the formation of homo-oligomers. These similarities suggest that APLIP1, JIP1b, and JIP2 belong to same protein family functionally conserved in various species. In mammals, JIP3/JSAP was reported as another member of the JIP family of proteins, which display scaffold function in the JNK signaling pathway as do JIP1 and JIP2. In Drosophila, a putative homolog of JIP3 has been designed Sunday Driver protein (SYD). However, they differ from APLIP1, JIP1b, and JIP2 in their domain structure; they do not possess the SH3 and PI domains that are important regions for binding several proteins including APPs, and they may have some different roles from APLIP1, JIP1, and JIP2 (Taru, 2002).
In mammal several proteins bind the cytoplasmic domain of APP (APPcyt), whereas the physiological role(s) of these interaction have not been sufficiently revealed. JIP1b and JIP2, mammalian counterparts of APLIP1 interact with APPcyt. Regarding JIP1b, the binding to APPcyt is relatively lower than the binding of other APP-binding proteins, such as mDab1, X11, and Fe65. It was also observed that the binding of JIP1b was slightly lower than that of the other binding protein X11L in vitro but not in the cell. However, the faint differences in the binding activities do not necessarily deny the physiological importance of JIP1b for APP. In fact, a novel function of JIP1 and JIP2 was found to be the modulation of the phosphorylation of APP at Thr-668 residue induced by the activation of JNK. Expression of JIP1b slightly enhances the phosphorylation of APP, whereas the expression of JIP2 or JIP1a suppresses the phosphorylation. From the previous reports that JIP1b, JIP1a, or JIP2 equally facilitate the activation of JNK signaling, it was expected that these proteins similarly regulate the phosphorylation of APP when JNK is activated. Nevertheless, only JIP1b facilitates the phosphorylation, and others decrease the level of the phosphorylation of APP. The interaction of JIP2 and JIP1a with APP is remarkably weaker than that of JIP1b in the cell. Therefore, it is conceivable that the effect of JIP1a or JIP2 to decrease the level of the phosphorylation of APP reflects their weaker binding properties to APP rather than their properties of regulating the JNK signaling cascade. It is assumed that formation of the complex between JIP1a or JIP2 and JNK may suppress the approach of JNK to the phosphorylation site of APP, whereas the complex of JIP1b and JNK can easily approach APPcyt. Indeed ut has been reported that formation of the tripartite complex composed of JIP1b, JNK, and APP can be observed in cultured cells. Phosphorylation of APP at Thr-668 has been implicated in the metabolism and/or putative function(s) of APP, and modulation of the phosphorylation level of APP by JIPs in mammal possibly has physiological importance (Taru, 2002).
In invertebrates only Drosophila APLIP1 and dX11L have been reported to interact with APPs except kinesin interacts genetically with APPL. They may all implicated in evolutionarily conserved roles relative to metabolism and/or function of APPs, besides the role of mammalian JIP in the phosphorylation of APP. In Drosophila, APPL does not have a phosphorylation site corresponding to the Thr-668 residue of mammalian APP695. In addition, there are some differences in the function of APLIP1 on the JNK signaling pathway from that of mammalian JIP1 and JIP2 because APLIP1 could not interact with DJNK, whereas it can interact with Drosophila JNK kinase Hep. Thus APLIP1 cannot form a complex with DJNK and facilitate JNK activation in Drosophila in the same manner as JIPs do in mammals, whereas a possibility of regulating JNK signaling through an interaction with Hep still remains. Therefore, the effect modulating the phosphorylation of APP by JIP1 and JIP2 may be acquired in the evolutionary process. Questions of what the evolutionarily conserved role(s) of the interaction of APLIP1, JIP1b, and JIP2 with APPs are remain to be elucidated. Several physiological roles for the mammalian JIP family proteins have been proposed other than acting as scaffold molecules of JNK cascades: as a transactivator of the GLUT2 gene and as cargo for kinesin to mediate the transportation of several transmembrane proteins. In Drosophila APLIP1 interacts with the kinesin light chain as well as mammalian JIP1 and JIP2 do, but interaction with the molecules of the JNK cascades is only partly conserved. The metabolic scheme of APPs is basically conserved between Drosophila and mammals. Kinesin is involved in intracellular transport and metabolism of APP in mammals and is associated with APPL in Drosophila. Accordingly, it is assumed that Drosophila APLIP1 and mammalian JIP share a role in the intracellular metabolism of APPs (Taru, 2002).
In conclusion, Drosophila APLIP1 and mammalian JIP1b and JIP2 are binding proteins of APPs. APLIP1, JIP1b, and JIP2 comprise an evolutionary conserved protein family and share properties in their domain structure, expression pattern, and interaction profiles with proteins such as APPs, kinesin, and JNK kinase, although a few exceptions are observed. It is proposed that a novel function of mammalian JIP1 and JIP2 is to modulate the phosphorylation of APP. Further analysis of conserved or different roles of APLIP1, JIP1b, and JIP2 may contribute to our understanding of the mechanisms of APPs metabolism and the pathogenesis of Alzheimer's disease (Taru, 2002).
Although Abl functions in mature neurons, work to date has not addressed Abl's role on Cdk5 in neurodegeneration. β-amyloid (Aβ42) initiates Abl kinase activity and blockade of Abl kinase rescues both Drosophila and mammalian neuronal cells from cell death. Activated Abl kinase is necessary for the binding, activation, and translocalization of Cdk5 in Drosophila neuronal cells. Conversion of p35 into p25 is not observed in Aβ42-triggered Drosophila neurodegeneration, suggesting that Cdk5 activation and protein translocalization can be p25-independent. These genetic studies also showed that abl mutations repress Aβ42-induced Cdk5 activity and neurodegeneration in Drosophila eyes. Although Aβ42 induces conversion of p35 to p25 in mammalian cells, it does not sufficiently induce Cdk5 activation when c-Abl kinase activity is suppressed. Therefore, it is proposed that Abl and p35/p25 cooperate in promoting Cdk5-pY15, which deregulates Cdk5 activity and subcellular localization in Aβ42-triggered neurodegeneration (Lin, 2007).
Like Cdk5, cellular Abl functions in neural development and its kinase activity and subcellular localization are tightly regulated. This study shows that Abl appears to be essential for Aβ42-triggered Drosophila neurodegeneration both in vivo and in vitro. It is of interest in this regard that Abl may serve as a putative molecular target to stop the progress of neurodegeneration. Interestingly, the anti-leukemic agent Abl kinase inhibitor, STI571, has been shown to rescue the Aβ42-induced neurodegeneration in both Drosophila and mammalian cells. However, STI571 is probably not an ideal reagent for testing this idea in vivo because of its low penetration capability through the blood-brain barrier. Another previous link between Aβ42 and Abl inhibition by STI571 has been reported. Aβ42 production is reduced by STI571 in neuronal cultures and in guinea-pig brain. Therefore, it is reasonable to speculate that Abl kinases might affect amyloid signaling at various points including Aβ42 production (Lin, 2007).
In situ RNA localization has demonstrated that the Appl transcript is found in post-mitotic neurons in all developmental stages in the central and peripheral nervous systems. Within the nervous system, transcripts are observed in neuroblasts, newly generated neurons and at least one class of presumed glial cells. The temporal and spatial specificity of Appl expression suggests that the gene product has a function that is common to most neurons. Appl cDNA predicts an 886-amino acid polypeptide that exhibits strong sequence similarity to the human beta-amyloid protein precursor (APP). It has been suggested that during evolution, a neural-specific function encoded by the APP gene has been selectively maintained (Martin-Morris, 1990).
The recently identified Drosophila gene amyloid protein precursor-like (appl) has a predicted amino acid sequence that shares extensive homology with the beta-amyloid protein precursor (APP) associated with Alzheimer's disease. Characterization of proteins encoded by the appl gene was initiated with the expectation that this simple model system might help elucidate the basic function provided by Appl and APP proteins. Two forms of the Appl protein have been identified in embryonic extracts, primary cultures, and transfected cells. Appl is synthesized as a 145-kDa membrane-associated precursor that is converted to a 130-kDa secreted form that lacks the cytoplasmic domain. Both forms are N-glycosylated. Pulse-chase and subcellular localization studies suggest that the conversion is very rapid. The similarities of biogenesis between APP and Appl provide further evidence that Appl and APP might be functionally homologous, and that the secretion event is of physiological significance. Immunocytochemical studies show that the Appl proteins are first detected in developing neurons concomitant with axonogenesis, and remain associated with differentiated neurons. Appl immunoreactivity is observed in neuronal cell bodies, axonal tracts, and neuropil regions. In the embryo, Appl proteins are expressed exclusively in the CNS and PNS neurons, consistent with the APPL transcript localization. The expression pattern of Appl proteins suggests an ancestral function for this protein in the nervous system (Luo, 1990).
Appl protein is present as a 145 kDa transmembrane protein and a 130 kDa soluble protein (Luo, 1990). The 145 kDa holoprotein is converted to the 130 kDa secreted form by proteolytic cleavage. To gain insights into the function and intracellular trafficking and secretion properties of Appl, the localizations of Appl protein within the different functional regions of the nervous system and at a subcellular level were examined. The insect nervous system can be subdivided operationally into three compartments: the cortical layer where neuronal somata reside; the neuropil region where processes from the overlaying cortex and incoming fibers from other centers ramify and make synaptic contacts, and the fiber pathways that connect cortical areas to the neuropil or connect adjacent neuropil regions. The relative distribution of Appl protein in neuronal cell bodies, neuronal processes, and the extracellular matrix can be revealed by analyzing the presence of the protein in these three compartments (Torroja, 1996).
To reveal Appl proteins, an affinity-purified polyclonal antibody generated against the amino terminal ectodomain common to both Appl forms (Ab952M) was used. In the eye disk only cells posterior to the morphogenetic furrow, where photoreceptors are differentiating, are Appl-immunoreactive. In the CNS, Appl is not detected in regions containing neuroblasts, such as the optic proliferation centers. These observations are consistent with the presence of APPL transcripts in postmitotic neurons (Martin-Morris, 1990). Most neurons in the ventral ganglion (VG) and the brain lobes (BL) show similar levels of Appl immunoreactivity. Under the same conditions, no immunoreactive signal is detected in brains from Appld larvae (Torroja, 1996).
Trafficking and processing of Appl protein is regulated precisely. Differences in trafficking and processing may be dependent on the physiological or developmental stage of a specific neuronal population. To gain insights into the biological significance of the regulation of Appl metabolism, an examination was made of Appl protein distribution during metamorphosis in two regions: the ventral ganglion and the optic lobes. Metamorphosis is a time when the nervous system undergoes dramatic change. For this study both whole-mount preparations and paraffin or cryostat sections stained with anti-APPL antibody Ab952M were used (Torroja, 1996).
The ventral ganglion was chosen as a model to study developmental changes during metamorphosis in Appl distribution because of the simplicity of the pattern of Appl immunoreactivity observed and because the process of remodeling that occurs during metamorphosis has been well characterized in this structure. Appl is observed along the axon tracks in the longitudinal and transverse commissures of the ventral cord in the embryo. In the ventral ganglion of third-instar larval CNS, Appl is no longer discerned along the longitudinal commissures but is concentrated in certain areas of the neuropil. During the first 24 hr of metamorphosis, axons and dendrites of larval neurons prune back, and new arborizations are formed. Axonal growth from new adult-specific neurons and from preexisting larval neurons starts at ~24 hr after pupariation and is completed by 72 hr. Appl immunoreactivity changes during the metamorphosis of the ventral ganglion. Immediately after pupariation, Appl distribution in the ventral ganglion resembles that described for the third-instar larval CNS. The most noticeable changes occur during the early stages of metamorphosis. Six hours after pupariation, the neuropil of the thoracic segments show reduced Appl immunoreactivity compared with the third-instar larval CNS. By 12 hr after pupariation, APPL-immunoreactive signal in the thoracic neuropil is lower and comparable to that in the abdominal segments. By 48 hr, levels of Appl protein in the neuropil are very low, and isolated immunoreactive varicosities and processes are clearly distinguishable. This pattern of Appl immunoreactivity remains unchanged until adult stages (Torroja, 1996).
Most of the neurons that form the optic lobes are new adult-specific neurons. Differentiation of these neurons progresses during late larval and pupal stages and has been well characterized. The first half of metamorphosis is characterized by axonal growth, whereas in the second half, synapses are formed. The process of synaptogenesis seems to continue into the adult. Thus, the optic lobe provides a well-studied structure for correlating Appl metabolism and neuronal differentiation. At 0 hr after pupariation, Appl protein is concentrated in photoreceptor axons within the eye stalk and in the three neuropils of the optic lobe: lamina, medulla, and lobula complex. During development, the optic lobe rotates dorsally, and the relative position of the optic neuropils changes. By 25 hr, Appl signal in the optic neuropils remains intense. Sixty hours after pupariation, Appl immunoreactivity in the medulla is arranged in a modular distribution in three layers that are reminiscent of the synaptic layers described in the adult medulla. Appl staining in the lamina becomes more intense, and axons are distinguished clearly. In the adult, intense Appl immunoreactivity remains in the lamina neuropil. Appl signal in the medulla and lobula neuropils is very low and comparable with the rest of the brain neuropil, except for some isolated axonal processes observed in the medulla (Torroja, 1996).
Thus during metamorphosis, the pattern of Appl immunoreactivity in the neuropil of the nervous system displays dynamic changes. In the ventral ganglion, these changes coincide with the period of axon retraction and outgrowth. During the major period of synaptogenesis in the optic lobes, the Appl immunoreactivity pattern in the neuropil resembles the distribution of the synaptic layers (Torroja, 1996).
The cell bodies of adult neurons display punctate Appl immunoreactivity. Some isolated cells in the brain and thoracic ganglion show higher levels of Appl expression. The lamina is highly stained, and separate processes are detected in the medulla. Interestingly, the highest levels of Appl protein are localized in the neuropil of the mushroom bodies. Mushroom bodies are a principal site of olfactory information processing and are involved in associative olfactory learning and memory in Drosophila. They consist of two complex bilaterally symmetrical groups of neurons (Kenyon cells) in the dorsal-posterior brain that receive input predominantly from the antennal lobes. Kenyon cell dendrites form the calyces, whereas their axons extend through the peduncle to the anterior of the brain. There, these axons form three different neuropils: the alpha lobe, which extends dorsally, and the beta and gamma lobes, which extend medially. All of the mushroom body axonal neuropils stain intensely, including the peduncle, alpha lobe, and beta/gamma lobes. The cell bodies of the Kenyon cells show slightly higher signal than the rest of the cortex. Their dendrites, however, do not appear enriched in Appl protein. Appl protein is also detected in the central complex, the major structure of Drosophila brain controlling locomotor behavior that has been shown to play an important role in learning in Drosophila. In the rest of the brain neuropil, some isolated processes are highly stained (Torroja, 1996).
Appl is found in the neuropil of the larval mushroom bodies, although the relative amount of protein in these structures, differing from what is observed in the adult brain, is comparable to or even lower than the levels of Appl protein detected in other brain and ventral ganglion neuropil areas. Interestingly, the mutant protein secretion-deficient APPLsd concentrates in the mushroom bodies at much higher levels than the endogenous Appl protein, whereas induced wild-type Appl and mutant secreted APPLs protein are detected at very low levels in this structure. The distribution of Appl protein expressed under a heat-shock promoter in Appld;hsp:Appl+/+ adult brains was examined. Four hr after the heat shock, induced wild-type Appl protein is especially concentrated in the axonal neuropil of the mushroom bodies and in the lamina, those areas where endogenous Appl is enriched. As is the case with the endogenous protein, the mushroom body calyces do not show enrichment of induced Appl. Similar to what was observed in larvae, mutant secretion-deficient APPLsd protein induced in adult brains is found enriched in the axonal neuropil of the mushroom bodies and along processes going from the lamina into the medulla (Torroja, 1996).
In the adult, Appl is concentrated in regions known to mediate behavioral plasticity. It is interesting that Appl is found enriched in the axons but not in the dendrites of the Kenyon cells. Moreover, the cell-type specificity of Appl processing and trafficking is involved in generating its differential distribution in the adult neuropil. The requirement of Appl function in the mature nervous system could explain the behavioral deficits displayed by Appl-null mutants. The fact that Appl is concentrated in the axon termini, but not in dendritic fields, suggests that the protein is transported to the presynaptic terminals in the Kenyon cells. Experiments with mutant Appl forms indicate that the membrane-bound Appl holoprotein is the most likely form concentrated in the mushroom bodies. In response to stimulation of the Kenyon cells, Appl transport, cleavage, and secretion in the presynaptic site could be regulated, and this regulation might be involved in modification of synaptic contacts between Kenyon cells and their synaptic targets (Torroja, 1996).
Appl protein associated with neuronal processes might correspond to the membrane-bound holoprotein, whereas Appl that is not associated with processes might be the secreted ectodomain. To analyze how the different forms of Appl protein contribute to Appl immunoreactivity, use was made of transgenic flies that express wild-type Appl or mutant Appl proteins, which mimic the membrane-bound or the secreted forms, in an Appld genetic background. First the heat-shock response of the hsp:Appl+ transgene was studied by heat-shocking adult flies for 30 min and the Appl proteins were examined by immunoblot analysis after specified intervals. Maximum levels of protein are reached 4 hr after the heat shock. Two forms of APPL+ protein are generated that show a precursor-product relationship and correspond to the membrane-bound holoprotein (145 kDa) and the secreted (130 kDa) forms. Therefore, in vivo processing of APPL+ protein produced from the heat-shock transgene is similar to the processing of endogenous Appl protein. On the contrary, mutant Appld flies carrying the secretion-defective hsp:Applsd transgene express a single form of Appl and are unable to generate a secreted protein. The secreted hsp:Appls transgene contains a mutated Appl cDNA, in which codon 789GAA has been changed to generate a translation-stop TAA, so that the encoded protein lacks the transmembrane and cytoplasmic domains. Flies carrying this construct in an Appld background express only a secreted Appl protein. The overall kinetics of expression of APPLsd and APPLs are similar to those of the APPL+ proteins when they are expressed under the influence of a heat-shock promoter (Torroja, 1996).
Appl immunoreactivity was compared in Canton S brains and in brains from Appld larvae carrying hsp:Appl+, hsp:Applsd, or hsp:Appls transgenes. The antibody Ab952M was used, and larval CNSs were fixed and immunoprocessed 2-4 hr after the heat shock. These preparations were analyzed with confocal microscopy, and the Appl immunoreactivity was compared in the cortex, in the thoracic neuropil, and in the optic lobe neuropil (Torroja, 1996).
Because the differential distribution of Appl in the neuropil could be a consequence of cell-specific differences in the levels of Appl transcription, Appl immunoreactivity generated from the hsp:Appl+ transgene was characterized. Under these conditions, Appl protein should be produced at similar levels in all neurons, because the heat-shock response is expected to be the same in nearly all cells. In general, Appl immunoreactivity in heat-shocked transgenic brains is lower than in the wild-type brain. In the cortex, neuronal cell bodies show a punctate Appl immunoreactivity. In the ventral ganglion, Appl signal in the cortex is quite uniform, and although some cells show slightly higher levels of protein, the position of these cells varies from sample to sample and does not correlate with the pattern of immunoreactivity observed in the neuropil. Appl immunoreactivity in the neuropil, however, resembles the pattern observed in wild-type larval CNS, in both the ventral ganglion and brain lobes. As in the wild type, only the neuropil regions of the three thoracic neuromeres and the eighth abdominal neuromere display positive immunoreactivity. Thus, in a situation where all neurons produce similar amounts of Appl protein, Appl immunoreactivity in the neuropil still mimics the normal pattern, suggesting that Appl trafficking varies between different neuronal cell types and results in differential distribution of this protein in the neuropil (Torroja, 1996).
Mutant Appl proteins are detected in cell bodies at levels similar to the induced wild-type protein, and they display the characteristic punctate aspect; however, in the neuropil the two mutant forms behave differently. In the thoracic neuropil, induced APPLs protein shows a distribution similar to the induced wild-type Appl protein. In contrast, secretion-defective APPLsd induction results in a very low signal in the thoracic neuropil, and the residual immunoreactive signal frequently is found along axonal tracts. The situation in the optic lobe is the converse of that in the thoracic and central brain neuropils. Although induced wild-type Appl and secretion-defective APPLsd proteins show enrichment in this structure at levels similar to endogenous APPL, the secreted APPLs form is found at very low levels (Torroja, 1996).
In summary, these results show that those regions where Appl seems to be associated with neuronal processes (optic lobes) are the regions where secretion-defective APPLsd is enriched. Secreted APPLs, however, concentrates in regions that are rich in the Appl form that is not associated with processes (thoracic neuromeres). These data suggest that Appl is secreted in some areas of the neuropil, whereas in others it remains as a transmembrane protein, indicating that the proteolytic processing of Appl is differentially regulated (Torroja, 1996).
To understand the in vivo function of Drosophila Appl protein, flies deleted for the Appl gene were generated. These flies are viable, fertile, and morphologically normal, yet they exhibit subtle behavioral deficits. A fast phototaxis defect in Appl- flies is partially rescued by transgenes expressing the wild-type, but not a mutant, Appl protein. A functional homology between Appl and APP is demonstrated, since transgenes expressing human APP show a similar level of rescue as transgenes expressing fly Appl (Luo, 1992).
The importance of the amyloid precursor protein (APP) in the pathogenesis of Alzheimer's disease (AD) became apparent through the identification of distinct mutations in the APP gene, causing early onset familial AD with the accumulation of a 4-kDa peptide fragment (betaA4) in amyloid plaques and vascular deposits. However, the physiological role of APP is still unclear. In this work, Drosophila was used as a model system to analyze the function of APP by expressing wild-type and various mutant forms of human APP in fly tissue culture cells as well as in transgenic fly lines. After expression of full-length APP forms, secretion of APP but not of betaA4 is observed in both systems. By using SPA4CT, a short APP form in which the signal peptide is fused directly to the betaA4 region, transmembrane domain, and cytoplasmic tail, betaA4 release in flies and fly-tissue culture cells is observed. Consequently, a gamma-secretase activity has been shown to be present in flies. Interestingly, transgenic flies expressing full-length forms of APP have a blistered-wing phenotype. Since the wing is composed of interacting dorsal and ventral epithelial cell layers, this phenotype suggests that human APP expression interferes with cell adhesion/signaling pathways in Drosophila, independent of betaA4 generation (Fossgreen, 1998).
The two pathological hallmarks of Alzheimer's disease, amyloid plaques and neurofibrillary tangles, involve two apparently unrelated proteins, the amyloid precursor protein (APP) and Tau. Although it is known that aberrant processing of APP is associated with Alzheimer's disease, the definitive role of APP in neurons is not yet clear. Tau regulates microtubule stabilization and assembly in axons and is, thus, an essential component of the microtubule-associated organelle transport machinery. Although several groups have reported physical interaction between APP and Tau, and induction of Tau phosphorylation by APP and beta-amyloid peptide, the functional connection between APP and Tau is unclear. To explore the possibility that the functions of these two proteins may somehow converge on the same cellular process, Appl, the Drosophila homolog of APP, was overexpressed along with Tau in Drosophila neurons. Panneural coexpression of Appl and Tau results in adults that, upon eclosion, fail to expand wings and harden the cuticle, which is suggestive of neuroendocrine dysfunction. Axonal transport was analyzed when Tau and Appl were coexpressed and transport of axonal cargo was found to be disrupted, as evidenced by increased retention of synaptic proteins in axons and scarcity of neuropeptide-containing vesicles in the distal processes of peptidergic neurons. Demonstrated in an independent approach were genetic interaction and phenotypic similarity between APPL overexpression and mutations in the Kinesin heavy chain gene, the product of which is a motor for anterograde vesicle trafficking (Torroja, 1999).
The hypothesis was tested that amyloid precursor protein (APP) and its relatives function as vesicular receptor proteins for kinesin-I. Deletion of the Drosophila APP-like gene (Appl) or overexpression of human APP695 (an alternatively spliced version of APP) or APPL constructs causes axonal transport phenotypes similar to kinesin and dynein mutants. Genetic reduction of kinesin-I expression enhances while genetic reduction of dynein expression suppresses these phenotypes. Deletion of the C terminus of APP695 or APPL, including the kinesin binding region, disrupts axonal transport of APP695 and APPL and abolishes the organelle accumulation phenotype. Neuronal apoptosis was induced only by overexpression of constructs containing both the C-terminal and Ab regions of APP695. The possibility is discussed that axonal transport disruption may play a role in the neurodegenerative pathology of Alzheimer's disease (Gunawardena, 2001).
Although reducing the amount of kinesin-I to 50% of normal by deleting one of two copies of either the klc or khc gene ordinarily has no significant phenotype, such a reduction in an animal overexpressing APP proteins that contain the cytoplasmic C terminus is predicted to significantly enhance the axonal blockage phenotype. This behavior is expected, because if kinesin-I becomes limiting by virtue of binding excess APP C termini, then further reduction of kinesin-I by deleting one gene copy should dramatically enhance the axonal transport phenotype. To test this prediction, larvae were generated that overexpressed APPL or APP695 and that were also heterozygous for a null mutation, khc8, so that kinesin-I was reduced to 50% of normal. Although larvae overexpressing APPL or APP by themselves or reduced in KHC dosage alone have no striking organismal phenotype, larvae combining these two features exhibit a dramatic new neuromuscular phenotype. These larvae flip their tail and head upwards during crawling, rocking back and forth as they struggled to crawl. Their neurons also contained an enhanced number of organelle accumulations. The extent of accumulations in larvae expressing wild-type APP695 and APPL in the context of reduced KHC dosage was comparable to homozygotes for kinesin-I or dynein mutants and was similarly lethal. Quantitative analysis has revealed a statistically significant difference between siblings with a normal and reduced dose of KHC (Gunawardena, 2001).
To confirm the specificity of the genetic interactions observed between reduction in KHC and overexpression of APP695 or APPL, larvae heterozygous for a null mutation of klc [Df(3L)8ex94, which removes the entire kinesin light chain gene] were generated in combination with constructs expressing APPL and APP695. Neurons from these larvae contain an increased number of organelle jams relative to larvae with a normal dose of KLC. Quantitative analysis reveals a statistically significant difference between siblings containing a normal and reduced dose of KLC, although the extent of enhancement is not as dramatic as when the dosage of KHC is reduced. Although these larvae do not show a larval neuromuscular phenotype as dramatic as was observed when the dosage of KHC was reduced, these larvae show a clear posterior paralysis phenotype. This result is again consistent with a direct functional interaction of the C-terminal region of APP695 and APPL with kinesin-I (Gunawardena, 2001).
Although reducing the amount of cytoplasmic dynein to 50% of normal by deleting one of two copies of either dynein heavy chain (dhc) or dynein light chain (dlc) genes ordinarily has no significant phenotype, such a reduction in an animal overexpressing APP family members that contain the cytoplasmic C terminus is predicted to suppress significantly the severity of the axonal accumulation phenotype. The basis for this prediction is that dynein drives retrograde axonal transport, which is antagonistic to kinesin-I-mediated anterograde axonal transport. In addition, many vesicles or organelles that exhibit net anterograde movement experience periods of retrograde movement owing to the simultaneous presence of kinesins and dyneins on the same vesicle or organelle. Thus, vesicle stalling and axonal accumulations induced by APP are predicted to be ameliorated by dynein reduction by (1) reducing the rate at which vesicles and organelles moved by dynein are transported into regions that have stalled or accumulated vesicles caused by APP expression; or (2) reducing the contribution of dynein-driven movement to a vesicle experiencing stalling because of reduced kinesin-driven activity; this reduction should attenuate vesicle stalling by restoring the balance of movements toward normal (Gunawardena, 2001).
To test this prediction, larvae overexpressing APP695 or APPL were generated that were also heterozygous for either a deficiency of either dhc [Df(3L)GN24] or dlc (roblk). Reduction of dynein suppresses the extent of organelle accumulations in APP695 or APPL transgenic lines. In addition, no significant larval crawling phenotype was observed in these animals. Surprisingly, reduction in dynein dosage also rescues the inviability of males overexpressing APP695. No effect is seen in the lines expressing a C-terminal deletion of APP695 or APPL. Thus, reduction in dosage of a retrograde motor protein appears to be sufficient to decrease organelle accumulations induced by APP or APPL expression (Gunawardena, 2001).
Whether larvae bearing heterozygous deletions of dynein components and the Appl gene in combination would exhibit abnormal axonal transport was investigated. In contrast to the situation with kinesin and APPL, female larvae with one copy (50% dose) of Appl and one copy of dhc or dlc do not have typical axonal accumulations. Intriguingly, male larvae bearing a deletion of the Appl gene, and hence lacking all APPL function, in combination with one copy of dhc or dlc also lack axonal accumulations. Thus, reduction in dynein levels appears to suppress axonal accumulations induced by loss of APPL (Gunawardena, 2001).
Genetic analysis in Drosophila strongly supports the hypothesis that mammalian APP and its homolog, APPL, have kinesin-I receptor functions in vivo. The genetic data and tests complement the in vitro biochemical evidence for a kinesin-I receptor function for APP. In these experiments, APP has been shown to form a complex with conventional kinesin by directly binding to KLC. Transport of APP depends upon kinesin-I and KLC in particular. In addition, the finding that, in Drosophila, APP695 can enter and be transported down axons to neuromuscular junctions and that this transport depends upon the cytoplasmic C terminus containing the proposed KLC binding region supports this view. In toto, these data strongly support the hypothesis that APP functions as a kinesin-I receptor. Perhaps APP bound to kinesin-I may be required for the axonal transport of a subset of cargoes, such as vesicles containing signaling or other molecules used at the synapse. Identifying these vesicles and their cargoes is an important next step (Gunawardena, 2001).
A surprising finding is the suppression of APP and APPL-induced organelle accumulations by genetic reduction of cytoplasmic dynein. Although further work is needed to define the mechanism of this suppression, one simple explanation comes from previous observations about the functionally antagonistic relationship of dynein and kinesin. A general observation is that kinesin and dynein are both present on many of the same axonal vesicles and organelles. Such vesicles and organelles often exhibit alternating anterograde (kinesin) and retrograde (dynein) movements, with net anterograde or retrograde movement resulting from a regulated bias in the balance of opposing movements along the microtubule. Thus, reduction of kinesin-I on non-APP vesicles caused by binding of kinesin-I to excess APP might cause vesicle stalling and organelle accumulations. Stalling of these vesicles and subsequent phenotypes might be rescued by reducing the antagonistic component of movement produced by dynein (Gunawardena, 2001).
An important related issue is whether APP and APPL have functions in addition to their likely roles as kinesin-I receptors on vesicles. Thus, one extreme possibility is that all phenotypic effects caused by genetic manipulation of APPL or APP result from either titration of available kinesin-I or failure to deliver other components of vesicles whose movement depends upon APP or APPL. However, a number of observations support the view that APP and APPL may have additional functions, perhaps mediated by the extracellular domain of the protein. For example, the secreted form of APP has been implicated in the regulation of hemostasis and neuroprotection, while the intact molecule may be involved in cell-extracellular matrix adhesion and in the sequestration of potentially toxic transition metals. There is also evidence that APPL may have a role in synapse differentiation. Thus, APPL and APP may have additional roles at the nerve terminal following their role in the axon as receptors for kinesin-I-dependent transport (Gunawardena, 2001).
It is also striking that several other proteins thought to play a role in Alzheimer's disease have recently been linked to the axonal transport machinery. For example, axonal transport defects are observed in transgenic mice expressing human ApoE4, a gene whose allelic state is associated with increased risk of AD. In the axonal blockages found in these animals, accumulations of synaptophysin, neurofilaments, mitochrondria, and vesicles are seen. Similarly, overexpression of tau protein, a major component of neurofibillary tangles, has been proposed to inhibit kinesin-I-dependent trafficking of vesicles, mitochondria, and endoplasmic reticulum in cultured cells and the PNS of transgenic mice. It is also striking that JIP-1/2 proteins, which are scaffolds for components of JNK signaling pathways, may link kinesin-I to ApoER2 and LRP, which are receptor proteins for ApoE. The allelic state of LRP itself has also been reported to be a predisposing factor for AD. Thus, it is conceivable that not only motor proteins but their cargoes and receptors, such as APP, ApoE, and perhaps secretases and other signaling molecules, coaccumulate when axonal transport is impaired in any way. Accumulating these proteins together at the same site, at the same time, may by itself be neurotoxic, may cause induction of cellular suicide signals, may block neurotrophic and other signaling needed for neuronal viability, or may lead to biochemical changes causing excess production of Aß, any or all of which may lead to cell death. Whether these correlations are unrelated or truly indicative of a causative link remains to be tested (Gunawardena, 2001).
The novel Drosophila mutant löchrig (loe) shows progressive neurodegeneration and neuronal cell death, in addition to a low level of cholesterol ester. loe affects a specific isoform of the gamma-subunit of AMP-activated protein kinase (AMPK: see SNF4/AMP-activated protein kinase gamma subunit), a negative regulator of hydroxymethylglutaryl (HMG)-CoA reductase and cholesterol synthesis in vertebrates. Although Drosophila cannot synthesize cholesterol de novo, the regulatory role of fly AMPK on HMG-CoA reductase is conserved. The loe phenotype is modified by the level of HMG-CoA reductase and suppressed by the statin-induced inhibition of this enzyme; statin has been used for the treatment of Alzheimer patients. In addition, the degenerative phenotype of loe is enhanced by a mutation in amyloid precursor protein-like (APPL), the fly homolog of the human amyloid precursor protein involved in Alzheimer's disease. Western analysis has revealed that the loe mutation reduces APPL processing, whereas overexpression of Loe increases it. These results describe a novel function of AMPK in neurodegeneration and APPL/APP processing that could be mediated through HMG-CoA reductase and cholesterol ester (Tschäpe1, 2002).
Cholesterol metabolism has been investigated for a long time in peripheral cells, yet relatively little is known about it in brain cells. This is all the more surprising as the brain is the organ richest in cholesterol. Most cells in the body take up the required amount of cholesterol via the LDL or VLDL (low- and very low-density lipoprotein) receptor pathway. After uptake, the lipoproteins are degraded and the cholesterol released within the cell where it can be either used as free cholesterol or stored in the form of cholesterol ester. This transport mechanism is highly conserved in vertebrates and invertebrates. In addition, vertebrate cells can produce cholesterol by de novo synthesis in the endoplasmic reticulum. Due to the blood-brain barrier, brain cells are unable to receive their supply of lipoproteins from the plasma and it has been suggested that only very little is supplied by uptake. At least oligodendrocytes seem to meet their demand for cholesterol by de novo synthesis. Nevertheless, the cerebrospinal fluid contains special lipoproteins, the apolipoproteins apoE and apoAI, and most probably these brain lipoproteins are not involved in the transport of cholesterol to and from the brain but rather in the redistribution of cholesterol within the brain (Tschäpe1, 2002 and references therein).
Cholesterol regulates the physical properties of the cell membrane, and its level is therefore tightly controlled. Recent work has shown that cholesterol plays a role in membrane compartmentalization and in the formation of lipid rafts. This important function might be the reason for the connection between cholesterol and neurodegeneration. Studies have shown that the cholesterol level influences the production of the pathogenic Aß peptide, which is produced from the amyloid precursor protein (APP) by cleavage through ß- and gamma-secretase. It has been suggested that Aß processing occurs within rafts, whereas the non-amyloidogenic alpha-processing occurs outside. Cholesterol synthesis in neurons is regulated by hydroxymethylglutaryl-CoA (HMG-CoA) reductase, which again has been connected to Alzheimer's disease. Inhibition of this enzyme by statins not only reduces cholesterol synthesis but also inhibits ß-secretase cleavage of APP. In addition, clinical studies indicate that patients treated with statins have a decreased prevalence of Alzheimer's disease. HMG-CoA reductase activity is negatively regulated via phosphorylation through the AMP-activated protein kinase (AMPK), a heterotrimeric complex, consisting of the catalytic alpha-subunit and ß- and gamma-subunits, found in all eukaryotes (Tschäpe1, 2002).
The Drosophila mutant löchrig (loe) disrupts a specific isoform of the AMPK gamma-subunit, which leads to a low level of cholesterol ester together with a strong neurodegenerative phenotype. loe interacts genetically with HMG-CoA reductase and influences processing of the ß-amyloid protein precursor-like (APPL) gene. Although the regulation and most downstream targets of HMG-CoA reductase are conserved, this enzyme is not involved in cholesterol synthesis in insects, because they cannot synthesize cholesterol de novo. The loe mutant now shows that HMG-CoA reductase and its regulator AMPK are also involved in neurodegeneration in insects. The low level of cholesterol ester suggests that the mediator could be cholesterol ester rather than cholesterol, which might be important in the context of Alzheimer's disease because the level of cholesterol ester has been directly correlated with Aß production in cell culture experiments (Tschäpe1, 2002).
loe was isolated from a collection of P-element insertion lines. About 800 lines that have a shortened adult life span were aged and screened histologically for signs of neurodegeneration. Two of these lines showed severe vacuolization of the central nervous system (CNS) which increased with aging, and one of them was named löchrig (the German term for 'full of holes'). The vacuolar pathology is most prominent around the central complex and in the central parts of the brain, while the optic lobes are less affected. Developmental studies have suggested that the vacuolization and degeneration in loe are confined to differentiated, probably synaptically active neurons, whereas neuroblasts and developing neurons are unaffected (Tschäpe1, 2002).
cDNAs of the loe gene represent at least six alternatively spliced transcripts for the Drosophila gamma-subunit of AMPK. The different mRNAs encode at least three different protein isoforms, all sharing the same C-terminus while varying in their N-terminal part. The C-terminus includes the so-called CBS (cystathionine-ß-synthase) domains that are highly conserved between yeast, mammals and Drosophila. Interestingly, a region in the unique N-terminus of the LoeI isoform shows homology to the X11alpha protein which can bind to the APP protein (Borg, 1998); LoeI and X11 are 28% identical and 41% similar over a stretch of 80 amino acids. The P-element is inserted in the seventh intron of this transcript and 38 bp upstream of the transcription start site of LoeII, suggesting that one or two transcripts are affected by the insertion (all other transcripts are >10 kb downstream of the insertion site and therefore most probably are not affected by the P-element). A small deletion of 1.3 kb was created around the insertion site, removing exon 1 of the LoeII transcript, and these flies do not show a degeneration phenotype. This indicates that LoeII is not required for CNS integrity (Tschäpe1, 2002).
To assess whether the loe mutation influences cholesterol metabolism, a role described for AMPK, the lipid composition of fly heads was measured. The analysis of phospholipids, triglycerides and free cholesterol did not reveal any significant differences between 1- to 5-day-old wild-type and mutant flies. The amount of cholesterol ester, however, was reduced by ~40%. Expressing LoeI in neurons restored the wild-type level of cholesterol ester in the mutant, confirming the role of Loe/AMPK in cholesterol homeostasis. The expression of LoeI restores the cholesterol ester level as well as the neurodegenerative phenotype, directly connecting cholesterol ester and neurodegeneration in the loe mutant. These results reveal an involvement of AMPK in cholesterol ester levels in the brain independent of de novo cholesterol synthesis. In peripheral tissues, vertebrate AMPK inhibits the activation of a hormone-sensitive lipase, an enzyme involved in the breakdown of cholesterol ester. A conserved regulatory pathway in the brain could account for the decreased amount of cholesterol ester (Tschäpe1, 2002 and references therein).
A functional homology to the mammalian AMPK is supported further by the accumulation of fatty acids in the mutant, another pathway regulated by AMPK (Tschäpe1, 2002).
AMPK negatively regulates HMG-CoA reductase, a key enzyme in cholesterol synthesis in vertebrates. In Drosophila, this protein is encoded by the columbus (clb) gene (Van Doren, 1998). To assess whether loe interacts with the clb mutation, flies were created homozygous for loe and heterozygous for two strong, embryonic lethal alleles of clb (which both had the same effect). clb/+; loe mutants show a weak but significant suppression of vacuolization compared with loe mutant flies. To confirm an interaction, lines expressing Clb were used in the loe background. In contrast to the clb mutation, Clb overexpression enhances the phenotype. Control flies, containing only the UAS-Clb construct but no neuronal promoter construct, did not differ from the original loe mutants. The interaction was quantified by counting holes in the different genotypes and measuring their total volume. The enhancement by Clb overexpression and suppression by the clb/+ mutant suggests that HMG-CoA reductase is negatively regulated by AMPK as in other organisms. In addition, an influence on the cholesterol ester level of loe was investigated. Overexpression of Clb slightly reduced, and introduction of one mutant copy of clb slightly increased, the cholesterol ester level in loe; however, the differences are not significant. Nevertheless, they are in agreement with the results on the neurodegenerative phenotype because the clb mutation suppresses and additional Clb enhances the phenotype. Interestingly, the function of HMG-CoA reductase in cholesterol synthesis is not conserved because insects cannot synthesize cholesterol de novo. However, many other downstream genes and regulatory feedback mechanisms are conserved, and one of them might connect HMG-CoA reductase and cholesterol ester (Tschäpe1, 2002).
HMG-CoA reductase can be inhibited pharmacologically by a class of drugs called statins, which have also been shown to decrease the prevalence of Alzheimer's disease. To assess whether treatment with statins influences the neurodegeneration in loe, flies fed on glucose were compared with or without the drug lovastatin. Flies kept on lovastatin showed a suppression of the vacuolization compared with control animals. Treatment of wild-type flies with lovastatin revealed no adverse effects. These results show that the progressive neurodegeneration in loe can be slowed successfully by treatment with statins. The level of cholesterol ester was tested in loe flies treated with statins, but no significant difference could be found (Tschäpe1, 2002).
Cholesterol homeostasis has been implicated in the processing of Aß from APP, as has statin treatment, which can dramatically decrease Aß production. Therefore, whether loe influences APPL, the fly homolog of human APP, was investigated. Appld mutants, which carry a deletion in the Appl gene, do not reveal any signs of neurodegeneration. However, crossing Appld with loe flies shows an enhancement of the loe vacuolization. The effect is weaker in loe flies carrying one copy of Appld (loe/loe; Appld/+) compared with homozygous double mutants (loe/loe; Appld/Appld) and can be detected in the central brain as well as the optic system (Tschäpe1, 2002).
To determine whether loe might influence the APPL protein, Western blot analysis of brain extracts was performed. Using an anti-APPL polyclonal antibody, two bands were detected in w1118 flies, representing the genetic background used to induce the loe mutation. The bands correspond to the membrane-associated 145 kDa precursor and the 130 kDa secreted form, which are absent in Appld. In the loe mutant, similar amounts of APPL precursor protein are found; however, the level of the processed secreted form is reduced. Conversely, more of the secreted form is found when additional LoeI is expressed in neurons. This reveals a role for loe in APPL processing or stabilization of the processed form. To assess whether this effect is specific for APPL, the processing of Notch, which is cleaved by a mechanism similar to that of APP, was investigated. No differences were detected in the processing of Notch, suggesting a specific function of loe in APPL processing, possibly mediated by the X11alpha similarity domain. In addition, whether Columbus or statin treatment influences APPL processing in loe was investigated. Additional expression of Clb, which enhanced the neurodegenerative phenotype of loe, also enhances the processing effect, causing a slight further reduction of APPL processing. On the contrary, one copy of mutant clb or statin treatment slightly increased processing. This suggests that the neurodegenerative phenotype is correlated with the processing of APPL (Tschäpe1, 2002).
Thus, a mutation in the AMPK gamma-subunit causes progressive neurodegeneration in Drosophila. AMPK is a central component of a protein kinase cascade conserved in eukaryotes that acts as a metabolic sensor to monitor the cellular AMP and ATP levels. In cases of ATP depletion, the major ATP function described to date is to activate energy-providing mechanisms while inactivating energy-consuming processes. AMPK is a heterotrimer, consisting of the catalytic alpha-subunit and the ß- and gamma-subunits, which are required for stabilization of the complex and kinase activity. The activity of the complex is regulated by phosphorylation through an upstream kinase, and both phosphorylation and dephosphorylation are sensitive to AMP levels. For all three subunits, different isoforms have been identified that assemble into specific AMPK complexes with distinguishable tissue distribution in peripheral tissues in vertebrates. Whereas most tissues predominantly express one gamma isoform, the human brain expresses three different isoforms. Interestingly, two of them have extended N-termini with no significant homology to each other, LoeI or any other protein. The loe mutation shows, for the first time, that such a brain-specific isoform has a unique function in brain maintenance, which cannot be substituted by other isoforms. This function probably goes beyond the basic role in energy regulation because all isoforms share the C-terminus, which is sufficient for a functional gamma-subunit and, therefore, a functional AMPK complex. It will be interesting to discover whether one of the human isoforms is also required specifically for neuronal survival (Tschäpe1, 2002).
AMPK has a central role in cholesterol metabolism by regulating HMG-CoA reductase and hormone-sensitive lipase, which is involved in the breakdown of cholesterol ester in vertebrates. Although hormone-sensitive lipase has not been found in the brain, a cholesterol ester hydrolase activity is described for the brain; however, nothing is known about the potential regulation of this enzyme by AMPK. An inhibitory function of AMPK in the brain would lead to an overactivity of this hydrolase and, therefore, to a reduced level of cholesterol ester. A Drosophila protein with homology to hormone-sensitive lipase can be found in the Drosophila Sequencing Project, but unfortunately no mutant has been described so far. However, a deficiency deleting this enzyme was tested for genetic interactions with loe. Because this deficiency had no influence on the loe phenotype, it is assumed that it is not involved in the neurodegenerative phenotype. In contrast, this study shows a genetic as well as a pharmacologically induced interaction of loe with HMG-CoA reductase (clb). The interaction reveals that, as in vertebrates, AMPK acts upstream of HMG-CoA reductase. Because a mutation in clb suppresses and overexpression enhances the neurodegenerative loe phenotype, the inhibitory function of AMPK on HMG-CoA reductase seems to be conserved. Interestingly, the function of HMG-CoA reductase is not completely conserved between vertebrates and insects, because arthropods cannot synthesize cholesterol de novo. Rather, HMG-CoA reductase is involved in the production of non-sterol isoprenoids from mevalonate. The effect of HMG-CoA reductase on neurodegeneration cannot, therefore, be mediated through cholesterol synthesis and, as measurements show, the cholesterol level is unaltered in loe. However, the amount of cholesterol ester is lowered in loe and adding or removing Clb has a slight influence on it, and APPL processing in loe is influenced by Clb. In this context, it is worth mentioning that statins dramatically decrease Aß production before a reduction in cholesterol can be detected. This suggests that other members of the cholesterol pathway might regulate APP processing, possibly cholesterol ester (Tschäpe1, 2002).
The loe mutation reveals a connection between cholesterol ester and progressive neurodegeneration in the model system Drosophila. In vertebrates, such a link has been established by the finding that accumulation of Aß can decrease cholesterol esterification in neurons (Koudinova, 1996; Liu, 1998). found that The level of cholesterol ester directly correlates with Aß production (Puglielli. 2001), and that elevated concentrations of cholesterol ester but not free cholesterol increase the generation of Aß. In contrast, it has been shown that lowering the cholesterol concentration inhibits APP cleavage by secretases and interferes with the localization of APP in membrane rafts (Simons, 1998; Frears, 1999). These are membrane microdomains consisting of lipids, proteins and cholesterol, and their correct composition seems to be required for normal APP processing. These results strengthen the likelihood of a role for cholesterol ester because the loe mutant links a reduced level of cholesterol ester, leaving free cholesterol unaltered, with decreased processing of APPL (Tschäpe1, 2002).
These results clearly reveal a function of AMPK in APPL processing. However, the Appl mutant enhances the neurodegenerative phenotype of loe. Like knock-outs of APP in mice, the Appld null mutation of Drosophila displays only subtle neurological deficits. In the loe mutant background, however, Appl can be connected to progressive neurodegeneration, which might help to understand the function of APP proteins. Because the lack of APPL enhances the phenotype, this hints at a neuroprotective function, perhaps specifically of the soluble form of APPL -- this was also suggested by cell culture studies of APP. In this model, neurons would be more vulnerable to the effect of the loe mutation when APPL and its soluble form are missing. The Appl mutant itself might not show degeneration because the damaging event is absent (Tschäpe1, 2002).
With the isolation of the loe mutant, a connection has been made between AMPK, a second enzyme (in addition to HMG-CoA reductase involved in cholesterol homeostasis) and neurodegeneration and APPL processing. This underlines the importance of the cholesterol biosynthesis pathway for the maintenance of the nervous system and for understanding of neurodegenerative diseases such as Alzheimer's. With the Drosophila loe mutant available, the role of this pathway in neurodegeneration can now be studied in an easily accessible model organism (Tschäpe1, 2002).
Nucleokinesis has recently been suggested as a critical regulator of neuronal migration. Disabled 1, which is required for neuronal positioning in mammals, regulates the nuclear position of postmitotic neurons in a phosphorylation-site dependent manner. Dab1 expression in the Drosophila visual system partially rescues nuclear position defects caused by a mutation in the Dynactin subunit Glued. Furthermore, a loss-of-function allele of amyloid precursor protein (APP)-like, a kinesin cargo receptor, enhances the severity of a Dab1 overexpression phenotype characterized by misplaced nuclei in the adult retina. In mammalian neurons, overexpression of APP reduces the ability of Reelin to induce Dab1 tyrosine phosphorylation, suggesting an antagonistic relationship between APP family members and Dab1 function. This is the first evidence that signaling that regulates Dab1 tyrosine phosphorylation determines nuclear positioning through Dab1-mediated influences on microtubule motor proteins in a subset of neurons (Pramatarova, 2006).
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