Effects of Mutation and Overexpression (part 2/2)

String and eye development

The onset of pattern formation in the developing Drosophila eye is marked by the simultaneous synchronization of all cells in the G1 phase of the cell cycle. Cells will either commit to another round of division or differentiate into neurons. Although cell cycle synchronization occurs in roughex (rux) mutants, these cells circumvent G1 and all cells enter S phase, including cells that would normally differentiate. rux is suppressed by mutations in genes that promote cell cycle progression (i.e., Cyclin A and string) and enhanced by mutations in genes that promote differentiation (i.e., Ras1 and Star). rux encodes a novel protein of 335 amino acids. Rux appears to function as a negative regulator of G1 progression in the developing eye (Thomas, 1994).

During Drosophila eye development, the posterior to anterior movement of the morphogenetic furrow coordinates cell cycle progression with the early events of pattern formation. The cdc25 phosphatase string (stg) has been proposed to contribute to the synchronization of retinal precursors anterior to the furrow by driving cells in G2 through mitosis and into a subsequent G1. Genetic and molecular analysis of Drop (Dr) mutations suggests that they represent novel cis-regulatory alleles of stg that inactivate expression in eye. Dr mutations are associated with restriction fragment length polymorphisms (RFLPs) mapping upstream of the transcription start site. Retinal precursors anterior to the furrow that lack stg arrest in G2 and fail to enter mitosis, while cells within the furrow accumulate high levels of cyclins A and B. Although G2-arrested cells initiate normal pattern formation, the absence of stg results in retinal patterning defects due to the recruitment of extra photoreceptor cells. G1 arrest in the furrow is not essential for the onset of pattern formation. These results demonstrate a requirement for stg in cell cycle regulation and cell fate determination during eye development (Mozer, 1999).

Dominant alleles of the Drop (Dr) locus have a small eye phenotype, are homozygous lethal, and exhibit a semi-lethal genetic interaction with mutations in stg. An additional recessive phenotype in the eye was revealed in somatic mosaics in which retinal clones homozygous for a lethal revertant of Dr1 contain supernumerary photoreceptor cells (Tearle, 1994). A homozygous viable mutant highway (hwy), whose phenotype and cytogenetic map position suggested it was a recessive viable Dr allele was identified by Hugo Stocker and Ernst Hafen (cited in Mozer, 1999). Adult hwy mutant flies have mildly rough eyes that are slightly smaller than wildtype and are missing macrochaetes on the notum. In apical cross sections of the wildtype retina, ommatidia containing the rhabdomeres of seven of the eight photoreceptor cells appear as a precise repeating array. In sections of the hwy mutant eye, ommatidia with one or more additional photoreceptor cells are also readily detected. The extra photoreceptor cells present in the mutant ommatidia consist of both large and small rhabdomeres suggesting no obvious bias in cell type (Mozer, 1999).

The bristle and eye phenotypes of transheterozygous mutant combinations were scored following inter se crosses between the hwy mutant and Dr and stg alleles. Recessive Dr alleles and all stg alleles fail to complement the bristle phenotype of hwy. In the eye, the supernumerary photoreceptor cell phenotype of hwy was not complemented by revertants of DrMio (Drmr) and a recessive lethal Dr allele (Drfa30). Sectioning of retinas from transheterozygous mutants revealed extra photoreceptor cells at a frequency identical to hwy homozygotes. In complementation tests using stg mutants, allele-specific effects in the eye were observed in trans to hwy. Transcriptional null alleles of stg or cytologically visible chromosome deficiencies that remove the stg gene fail to complement the supernumerary photoreceptor cell phenotype of the hwy mutant. In contrast, ems induced alleles of stg complement the retinal phenotype of hwy, since sectioning of eyes of transheterozygous hwy;stg flies shows the wildtype number of photoreceptor cells. These genetic data show that three independently isolated Dr alleles as well as certain stg alleles share a common recessive phenotype resulting in the formation of extra photoreceptor cells in the eye. Results of genetic complementation and cytogenetic mapping are consistent with the hypothesis that mutations in the stg gene contribute in part to some of the phenotypes associated with Dr alleles. Likewise, the fact that a defect in the expression of stg anterior to the furrow is also observed in eye discs from Dr mutants suggests that these lethal Dr alleles are defective in their ability to express stg in the developing eye. It is suggested that Dr mutations are alleles of stg affecting cis-regulatory elements required for the expression in eye (Mozer, 1999).

In order to determine the function of stg during eye formation cell cycle progression was examined in third instar larval eye discs from viable Dr mutants or in eye disc clones using cell cycle markers. In the Drhwy mutant, the pattern and number of S-phase cells in the eye disc is unaffected, suggesting that stg is not required for G1-S progression. In the wildtype eye disc, CyclinA and Cyclin B protein is uniformly expressed in anterior retinal precursors and abruptly down-regulated within the stg expression domain and the furrow, coincident with cell cycle synchronization and G1 arrest (Thomas, 1994). In anterior retinal precursors in the Drhwy mutant eye disc or within anterior Drmr21 eye disc clones spanning the furrow, CyclinB protein is expressed ubiquitously. Similar effects on CyclinA accumulation within the furrow are also observed in Drhwy;stgX1 mutant eye disc. Thus, the absence of stg ahead of the furrow results in the failure of Cyclin A and B protein down-regulation. The uniform expression of CyclinB within the furrow of Dr eye discs suggests that mutant precursor cells arrest their cell cycle in G2. Consistent with this hypothesis, a decrease in the number of mitotic cells anterior to the furrow is observed in Drhwy eye discs. In the wildtype, equivalent numbers of mitotic cells are observed in the anterior and posterior regions of the eye disc but are absent within the furrow. In contrast, few or no mitotic cells are detected within the stg expression domain of the Drhwy eye disc, while mitosis in more anterior cells or behind the furrow is unaffected. Overall, a two-fold reduction has been found in the number of mitotic cells in the anterior eye disc of the mutant, when compared to wildtype (Mozer, 1999).

Retinal precursor cells in the furrow of Dr- eye discs fail to undergo G1 arrest but instead are blocked in G2. In order to investigate the effect of the lack of G1 arrest on early patterning in the eye, the expression of Atonal, the proneural gene for photoreceptor cell neurons was examined. In wildtype, Atonal is expressed within clusters of cells within the furrow and then becomes restricted to the R8 precursor in the two posterior adjacent rows of cells. In the Drhwy mutant the pattern of Atonal staining is indistinguishable from wildtype, suggesting that stg (and G1 arrest) is not required for the onset of pattern formation. The expression pattern of the neuron-specific marker Elav, in wildtype and recessive Dr mutant eye discs, was examined in order to investigate the events of photoreceptor cell differentiation behind the furrow. In the wildtype eye disc, Elav expression is restricted to the developing photoreceptor cells, commencing behind the furrow initially in a pair of cells in each precluster, and then subsequently in all eight photoreceptor cells. In the Drhwy mutant, Elav expression in the developing photoreceptor cells is normal, however in the basal region of the eye disc and within the optic stalk, ectopic expression of Elav is observed in the nuclei of glial cells. Using Elav staining it was difficult to identify the supernumerary photoreceptor cells in third instar larval eye discs from the Dr mutants, however in 24-30 hr. pupal eye discs they were readily apparent. In order to determine the effects of the Dr mutants on later patterning events, 48-54 hr pupal eyes were stained with cobalt sulfide. In the wildtype pupal eye, the outlines of four cone cells, and surrounding pigment cells were highlighted. In the Drhwy mutant most of the ommatidia contain one or more additional cone cells. In summary, it was found that stg (and G1 arrest) is not required for the onset of pattern formation but that it does play a role in later patterning events behind the furrow (Mozer, 1999).

It is proposed that stg is required in a subset of retinal precursor cells in the eye disc to antagonize signaling pathways specifying the neuronal cell fate. In the absence of stg, these cells are inappropriately recruited to become photoreceptor neurons. Thus, ectopic expression of a neuronal marker (Elav) in retinal glial cells of Dr mutants is the result of the failure to down-regulate neuronal cell signaling pathways in the eye disc. Cell type specification during Drosophila eye development requires a number of ubiquitously expressed molecules that comprise the Ras/Map kinase signaling cascade. These observations suggests the possibility that stg may contribute cell type specificity through the negative regulation of the Ras/Map kinase pathway (Mozer, 1999).

Cell cycle regulation and cell fate specification is coordinately regulated during retinal development by the movement of the morphogenetic furrow. The activation of stg gene expression ahead of the furrow is thought to be the result of hedgehog signaling, however, eye disc clones lacking smoothened (smo) a down-stream component of the hedgehog signaling pathway have no effect on retinal patterning. Given the observation that viable Dr mutants or lethal Dr- adult eye clones have patterning defects, it has been suggested that in the eye the regulation of stg by hedghog is not direct. In the embryo, activation of stg expression is dependent on a number of genes involved in embryonic patterning as well as cis-regulatory sequences upstream of the transcription start site. Although the genes that mediate activation of stg gene expression in the imaginal discs are unknown, the Achaete and Scute genes have been implicated in the negative regulation of stg expression in the wing disc. Molecular genetic mapping of RFLPs associated with Dr alleles to a large region (greater than 80kb) upstream of the stg coding region should lead to the identification of the cis-regulatory elements in the stg promoter that specify expression in the eye. In addition, genetic screens utilizing the eye phenotypes of Dr mutants should prove fruitful as a means to identify the trans-acting factors that bind to these sites as well as additional molecules that coordinate cell cycle progression and pattern formation during development (Mozer, 1999).

A Drosophila cyclin E hypomorphic mutation, DmcycEJP, has been generated and characterized that is homozygous viable and fertile, but results in adults with rough eyes. The mutation arose from an internal deletion of an existing P[w+lacZ] element inserted 14 kb upstream of the transcription start site of the DmcycE zygotic mRNA. The presence of this deleted P element, but not the P[w+lacZ] element from which it was derived, leads to a decreased level of DmcycE expression during eye imaginal disc development. Eye imaginal discs from DmcycEJP larvae contain fewer S phase cells, both anterior and posterior to the morphogenetic furrow. This results in adults with small rough eyes, largely due to insufficient numbers of pigment cells. Altering the dosage of the Drosophila cdk2 homolog cdc2c, retinoblastoma, or p21(CIP1) homolog dacapo, all of which encode proteins known to physically interact with Cyclin E, modifies the DmcycEJP rough eye phenotype as expected. Decreasing the dosage of the S phase transcription factor gene, dE2F, enhances the DmcycEJP rough eye phenotype. Surprisingly, mutations in G2/M phase regulators cyclin A and string (cdc25), but not cyclin B1, B3, or cdc2, enhance the DmcycEJP phenotype without affecting the number of cells entering S phase; instead, the mutations decrease the number of cells entering mitosis. This analysis establishes the DmcycEJP allele as an excellent resource for searching for novel cyclin E genetic interactors. In addition, this analysis has identified cyclin A and string as DmcycEJP interactors, suggesting a novel role for cyclin E in the regulation of Cyclin A and String function during eye development. Existing mechanisms do not explain the genetic interaction between DmcycE and string that was observed or between roughex and string observed in another study. In Drosophila embryos at least, phosphorylation of the tyr 15 and thr 14 residues of Cdc2 in Cyclin A/Cdc2 complexes inhibits Cdk activity. String (Cdc25) acts to dephosphorylate these residues and activate Cdk activity. In mammalian cells Cyclin E/Cdk2 phosphorylates and activates the Cdc25A phosphatase in the G1 to S phase transition. One possible explanation, therefore, is that DmcycE acts to phosphorylate and activate String phosphatase activity, leading to the activation of Cyclin A/Cdc2 activity (Secombe, 1998).

The receptor tyrosine kinase (RTK) signaling pathway is used reiteratively during the development of all multicellular organisms. While the core RTK/Ras/MAPK signaling cassette has been studied extensively, little is known about the nature of the downstream targets of the pathway or how these effectors regulate the specificity of cellular responses. Drosophila yan is one of a few downstream components identified to date, functioning as an antagonist of the RTK/Ras/MAPK pathway. Ectopic expression of a constitutively active protein (yanACT) inhibits the differentiation of multiple cell types. In an effort to identify new genes functioning downstream in the Ras/MAPK/yan pathway, a genetic screen was performed to isolate dominant modifiers of the rough eye phenotype associated with eye-specific expression of yanACT. Approximately 190,000 mutagenized flies were screened, and 260 enhancers and 90 suppressors were obtained. Among the previously known genes recovered are four RTK pathway components [rolled (MAPK), son-of-sevenless, Star, and pointed], and two genes (eyes absent and string) that have not been implicated previously in RTK signaling events. Mutations in five previously uncharacterized genes were also recovered. One of these, split ends, has been characterized molecularly and is shown to encode a member of the RRM family of RNA-binding proteins (Rebay, 2000).

yan itself has been implicated in cell cycle control. Whereas hypomorphic yan mutations are semi-viable and have an extra photoreceptor phenotype, null mutations in yan are embryonic lethal, with the embryos dying as a result of overproliferation of cells in the dorsal neuroectoderm. Thus, depending on the developmental context, yan regulates not only the transition between undifferentiated and differentiated cell types, but also the choice between differentiation and cell division. Recovery of string alleles in this screen could reflect cross-talk between cell cycle and differentiation pathways that occurs in part at the level of transcriptional regulation. Thus, it is possible that the downstream targets of yan will include cell cycle regulators, such as string, or that yan expression and stability may be linked to cell cycle controls. Alternatively, String could have postmitotic functions essential to differentiation (Rebay, 2000).

string has not been isolated in any other Ras pathway screens; however, it was isolated, again as a suppressor, in a screen for modifiers of activated Notch. The direction of interaction suggests string acts as a positive regulator of Notch signaling. Expression of NACT and yanACT have similar developmental consequences since both inhibit or delay differentiation of the cell types in which they are expressed. Isolation of string in both screens could indicate a point of cross-talk between the Notch and the RTK/Ras pathway. Alternatively, isolation of string as a suppressor of both NotchACT and yanACT could have more to do with the similar terminal phenotype of these two backgrounds rather than reflecting direct interactions with the two pathways. Supporting the first hypothesis, cross-talk between the Notch and RTK pathways has been reported by numerous labs. Despite all the genetic interaction data, the mechanisms whereby the Notch and RTK pathways intersect remain to be determined. Experiments designed to study signaling by both pathways in vivo have suggested an antagonistic relationship, which would be consistent with string acting as a negative regulator of Ras signal transduction and a positive regulator of Notch signal transduction (Rebay, 2000 and references therein).

Wee1 kinases catalyze inhibitory phosphorylation of the mitotic regulator Cdk1, preventing mitosis during S phase and delaying it in response to DNA damage or developmental signals during G2. Unlike yeast, metazoans have two distinct Wee1-like kinases, a nuclear protein (Wee1) and a cytoplasmic protein (Myt1). The genes encoding Drosophila Wee1 and Myt1 have been isolated and genetic approaches are being used to dissect their functions during normal development. Overexpression of Dwee1 or Dmyt1 during eye development generates a rough adult eye phenotype. The phenotype can be modified by altering the gene dosage of known regulators of the G2/M transition, suggesting that these transgenic strains can be used in modifier screens to identify potential regulators of Wee1 and Myt1. To confirm this idea, a collection of deletions for loci that can modify the eye overexpression phenotypes was tested and several loci were identified as dominant modifiers. Mutations affecting the Delta/Notch signaling pathway strongly enhance a GMR-Dmyt1 eye phenotype but do not affect a GMR-Dwee1 eye phenotype, suggesting that Myt1 is potentially a downstream target for Notch activity during eye development. Interactions with p53 were observed, suggesting that Wee1 and Myt1 activity can block apoptosis (Price, 2002).

Dwee1 and Dmyt1 overexpression eye phenotypes are sensitive to modification by mutations in known cell cycle regulatory genes, illustrating the feasibility of screening for mutations of genes that are potential regulators of either Wee1 or Myt1. Mutations in genes that promote mitosis, such as cdc2 and cdc25string, should dominantly enhance these overexpression phenotypes and this expectation was confirmed for both of these genes with Dmyt1. Although a GMR-Dwee1 eye phenotype is also enhanced by mutations in cdc2, it is not enhanced by mutations in cdc25string, providing evidence that Wee1 and Myt1 kinases have distinct Cdk1 regulatory effects in this developmental context. This result could be explained by a requirement for higher levels of cdc25string activity to overcome GMR-Dmyt1 inhibition of Cdk1 relative to GMR-Dwee1, perhaps because it is inherently more difficult to dephosphorylate Cdk1 inhibited on both T14 and Y15 by Myt1 activity, compared with Cdk1 inhibited on Y15 alone by Wee1 (Price, 2002).

String and glial development

Neurons and glia are produced in stereotyped patterns after neuroblast cell division during development of the Drosophila central nervous system. The first cell division of thoracic neuroblast 6-4 (NB6-4T) is asymmetric, giving rise to a glial precursor cell and a neuronal precursor cell. In contrast, abdominal NB6-4 (NB6-4A) divides symmetrically to produce two glial cells. To understand the relationship between cell division and glia-neuron cell fate determination, the effects of known cell division mutations on the NB6-4T and NB6-4A lineages were examined and compared. Based on observation of expression of glial fate determination and early glial differentiation genes, the onset of glial differentiation occurs in NB6-4A but not in NB6-4T when both cell cycle progression and cytokinesis are genetically arrested. In contrast, glial differentiation starts in both lineages when cytokinesis is blocked with intact cell cycle progression. These results show that NB6-4T, but not NB6-4A, requires cell cycle progression for acquisition of glial fate, suggesting that distinct mechanisms trigger glial differentiation in the different lineages (Akiyama-Oda, 2000).

Cell division mutants stg, cycA, and pbl were used to investigate the relationship between cell division and glia- neuron cell fate determination in NB6-4. To determine the effects of cell cycle arrest on cell fate of NB6-4, expression of a glial fate determination protein, Gcm, and an early glial marker protein, Repo, were examined in stg mutant embryos. In normal development of NB6-4T, these proteins are detectable in the medial daughter cell after the first cell division and in its progeny cells, while they are expressed in both daughter cells of NB6-4A. In stg mutant embryos, neither Gcm nor Repo is detectable in NB6-4T, whereas both proteins are expressed in NB6-4A at levels comparable to those of wild-type embryos. This indicates that stg activity is required for the onset of glial differentiation in neuroglioblast NB6- 4T, but not in glioblast NB6-4A. GCM mRNA was examined to determine whether the lack of Gcm expression in stg mutant NB6-4T resulted from loss of transcription or failure of posttranscriptional regulation. In contrast to wild-type embryos, in which GCM mRNA is detected in both the NB6-4T and the NB6-4A lineages, GCM mRNA is detected in NB6-4A, but not in NB6-4T of stg mutant embryos. This indicates that transcription of gcm in NB6-4T does not occur or occurs at only a very low level in the mutant embryos (Akiyama-Oda, 2000).

There are at least two possibilities that explain how stg mutation affects the onset of glial differentiation in NB6-4T: (1) that phosphatase activity of Stg protein is directly required for Gcm expression and (2) that stg-mediated cell cycle progression is needed for function and/or distribution of some factors that are necessary for Gcm expression. The latter possibility is favored, since not all the stg-induced cells expressed the glial proteins in the rescue experiments. The normal expression of glial markers in cycA mutant embryos, in which the first cell division is normal, is consistent with the notion that the first cell division is a critical point for the onset of glial differentiation in the NB6-4T lineage (Akiyama-Oda, 2000).

In pbl mutant embryos, which lack cytokinesis, all the nuclei of NB6-4T express the glial proteins, suggesting that cytokinesis is not required for the onset of glial differentiation in the NB6-4T lineage. It has been suggested that cytokinesis may be required for negative regulation of glial differentiation, since more than three nuclei in the pbl mutant NB6-4T, in contrast to three glial cells in the wild-type, express Repo. In wild-type embryos, expression of Gcm protein becomes prominent in one of the daughter cells shortly after the first cell division of NB6-4T. Gcm protein is not detected in NB6-4T of the stg mutant, while beta-gal is detected, although rather weakly, in the cell of stg mutants bearing gcmp-lacZ (gcm promoter regulating lacZ expression). This indicates that the gcm promoter may be initially activated even when cell cycle progression is arrested by stg mutation (Akiyama-Oda, 2000).

Cell cycle progression of NB6-4T appears to be more closely related to up-regulation of Gcm expression. This regulatory mechanism may lead to a sufficient level of Gcm expression, which enables transcription of downstream glia-specific genes including repo. GCM mRNA is present from before the first cell division of NB6-4T in wild-type embryos. The level of Gcm protein in the NB6-4T lineage is possibly controlled by several steps of regulation, including transcription, stability of mRNA, and translation. The findings of this study suggest that such regulatory mechanisms involve stg-mediated cell cycle progression (Akiyama-Oda, 2000).

NB6-4T and NB6-4A are the corresponding cells in thoracic and abdominal segments that share expression of some marker genes, including en and eg. However, these cells show distinct patterns of proliferation and cell fate. NB6-4T produces three glial cells and four to six neuronal cells, while NB6-4A produces only two glial cells. Despite this difference, glial fate arises from both lineages. The analyses using stg mutant embryos reveals that the effects of the mutation on glial differentiation in the NB6-4T and NB6-4A lineages are distinct. In the mutant, expression of Gcm and Repo is detected in NB6-4A but not in NB6-4T. This indicates that the start of glial differentiation in NB6-4T is dependent on stg-mediated cell cycle progression but that in NB6-4A this is not the case (Akiyama-Oda, 2000).

This raises the question of whether the different regulatory mechanisms for glial differentiation in these cells are reflected by their distinct cell types: neuroglioblast and glioblast. In the other glioblast GP, Gcm and Repo expression are detected in the stg mutant, indicating that glial differentiation in the glioblasts is independent of cell cycle progression. In addition to these glioblasts, a few cells expressing the glial proteins are observed in the mutant. These cells might have been neuroglioblasts, suggesting that there may be another mechanism to control the onset of glial differentiation in neuroglioblast lineages (Akiyama-Oda, 2000).

In the NB6-4T lineage, the first cell division is a critical step for triggering glial differentiation. Coincident with the onset of glial differentiation is the occurence of cell fate bifurcation. In the cell division rescue experiments using eg-GAL4, Gcm-positive and Gcm-negative cells appear after the first cell division, although surrounding cells are still stg mutant. The cell fate bifurcation is probably regulated cell intrinsically and coupled to cell division. In contrast, all the nuclei of NB6-4T in the pbl mutant express Gcm and Repo. This may be because these proteins contain the nuclear localization signal that enables them to enter the nuclei within the single cell after translation, even if asymmetry might initially appear within the cell (Akiyama-Oda, 2000).

During cell division of NBs, the transcription factor Prospero is asymmetrically segregated to ganglion mother cells, in which this protein has a role in specification of cell identity. The first cell division of NB6-4T shows some similarity to such NB division, since the transcription factor Gcm is expressed preferentially in one daughter cell after cell division to start sublineage-specific differentiation. There may be a repressor and/or an activator of Gcm expression, which should be segregated to or expressed only in the neuronal daughter cell and the glial daughter cell (Akiyama-Oda, 2000).

Mutations in String/CDC25 inhibit cell cycle re-entry and neurodegeneration in a Drosophila model of Ataxia telangiectasia

Mutations in ATM (Ataxia telangiectasia mutated) result in Ataxia telangiectasia (A-T), a disorder characterized by progressive neurodegeneration. Despite advances in understanding how ATM signals cell cycle arrest, DNA repair, and apoptosis in response to DNA damage, it remains unclear why loss of ATM causes degeneration of post-mitotic neurons and why the neurological phenotype of ATM-null individuals varies in severity. To address these issues, a Drosophila model of A-T was generated. RNAi knockdown of ATM in the eye caused progressive degeneration of adult neurons in the absence of exogenously induced DNA damage. Heterozygous mutations in select genes modified the neurodegeneration phenotype, suggesting that genetic background underlies variable neurodegeneration in A-T. The neuroprotective activity of ATM may be negatively regulated by deacetylation since mutations in a protein deacetylase gene, RPD3, suppressed neurodegeneration, and a human homolog of RPD3, histone deacetylase 2, bound ATM and abrogated ATM activation in cell culture. Moreover, knockdown of ATM in post-mitotic neurons caused cell cycle re-entry, and heterozygous mutations in the cell cycle activator gene String/CDC25 inhibited cell cycle re-entry and neurodegeneration. Thus, it is hypothesized that ATM performs a cell cycle checkpoint function to protect post-mitotic neurons from degeneration and that cell cycle re-entry causes neurodegeneration in A-T (Rimkus, 2008).

The data indicate that ATM knockdown by RNAi causes degeneration of Drosophila post-mitotic photoreceptor neurons. The neurodegeneration phenotype of ATM knockdown flies is similar to that observed in A-T patients. Neurodegeneration in the fly model occurred in the absence of exogenously induced DNA damage, it occurred independently of developmental defects, and it was progressive, increasing in severity as flies aged. Thus, ATM knockdown flies appear to be an appropriate model to study the cellular mechanisms underlying neurodegeneration in A-T (Rimkus, 2008).

A-T is a monogenic disease resulting from mutation of the ATM gene; however, the genetic screen identified second site genes that affect an A-T phenotype, neurodegeneration. Remarkably, independent lines of evidence, from the literature and from the current studies, support the relevance of each of the modifier genes to the mechanism underlying neurodegeneration in A-T (Rimkus, 2008).

ATM is recruited to DSBs by the trimeric MRE11-RAD50-NBS1 (MRN) DNA repair complex, which possesses ATP- dependent nuclease (MRE11) and DNA-tethering (RAD50) activities (Abraham, 2005). Three of the six genes identified in the screen (Stg, RAD50, and PP2A-B') are known components of the ATM signaling pathway that responds to DNA damage in mammals. A role for RAD50 in promoting neurodegeneration is not specific to the eye, since mutation of RAD50 suppress the lethality of Elav-ATMi flies. In addition, mutation of the gene encoding the NBS1 subunit of MRN suppresses the GMR-ATMi rough eye phenotype, suggesting that suppression by RAD50 and NBS1 mutants is due to reduced activity of the MRN complex. Nevertheless, the mechanism underlying suppression of neurodegeneration is unclear since reduced levels of the MRN complex would intuitively be expected to enhance GMR-ATMi phenotypes. One possibility is that the MRN complex is deregulated in the absence of ATM and carries out activities that are lethal to neurons. Finally, PP2A has been shown to dephosphorylate several ATM signaling pathways substrates, including ATM. Mutation of PP2A-B', which encodes a regulatory subunit of the PP2A complex, may enhance neurodegeneration in ATM knockdown flies by affecting the phosphorylation state of ATM substrates (Rimkus, 2008).

Two of the identified genes, MEKK4 and Delta, have potential links to ATM. Mutation of MEKK4 may enhance neurodegeneration in GMR-ATMi flies by allowing cell cycle progression. Published studies suggest a model whereby ATM and MEKK4 pathways collaborate to prevent cell cycle re-entry of post-mitotic neurons by maintaining the latency of CDC25 proteins. Delta encodes a ligand for the Notch receptor, which regulates cell cycle progression and differentiation in many tissues, including the eye. Thus, there may be cross-talk between the ATM and Notch signaling pathways in neurons (Rimkus, 2008).

Finally, studies in cultured cells revealed a direct link between HDAC2, the human homolog of Drosophila RPD3, and ATM. HDAC2 was found to directly associate with ATM and regulate its kinase activity in the absence of exogenously induced DNA damage. Thus, HDAC2 is likely the TSA-sensitive deacetylase that negatively regulates ATM kinase activity. HDAC2 may function by counteracting acetylation of ATM or downstream components of the ATM signaling pathway. It is important to note that although deacetylation of ATM by HDAC2 may regulate ATM activity, HDAC2 is not necessarily an important factor in A-T since the majority of mutations in A-T patients are nonsense or frameshift mutations that result in complete loss or truncation of ATM protein. Nevertheless, the demonstrated physical and functional interactions between HDAC2 and ATM indicate that HDAC2 is an important component of the ATM signaling paradigm and that information garnered from studies of ATM knockdown flies can advance understanding of ATM function in humans (Rimkus, 2008).

Results from the genetic screen predict that A-T patients with mild neurodegeneration will carry heterozygous mutations in suppressor genes, such as CDC25 family members, whereas A-T patients with severe neurodegeneration will carry heterozygous mutations in enhancer genes, such as MEKK4. It will be interesting to see if genes that enhance neurodegeneration in ATM knockdown flies also enhance neurodegeneration in mice. For example, do ATM-/- MEKK4+/- mice exhibit progressive degeneration of cerebellar neurons? If so, this would make mice a practical model for studying the neurodegenerative aspects of A-T (Rimkus, 2008).

The data indicate a causal relationship between cell cycle re-entry and neurodegeneration in the Drosophila model of A-T presented in this study. (1) ATM knockdown in photoreceptor neurons resulted in cell cycle re-entry and neurodegeneration, implicating ATM in both processes. (2) Heterozygous mutation of cell cycle regulatory genes Stg/CDC25, Cdk2, dE2F1, and dE2F2 modified the neurodegeneration phenotype of ATM knockdown flies, highlighting the importance of cell cycle regulation in neurodegeneration. (3) Inhibition of cell cycle re-entry by mutation of Stg/CDC25 also inhibited degeneration of ATM knockdown neurons. In contrast, inhibition of neurodegeneration by expression of P35 did not inhibit cell cycle re-entry. (4) Inhibition of neurodegeneration by expression of P35 caused the accumulation of neurons in S/G2/M phases of the cell cycle, indicating that the neurons that re-entered the cell cycle are the ones that degenerated (Rimkus, 2008).

These findings add to a growing literature linking cell cycle re-entry and neurodegeneration. The observations that terminally differentiated neurons are resistant to oncogenic transformation and that brain tumors of neuronal origin rarely occur suggest that cell cycle re-entry of post-mitotic neurons results in death rather than proliferation. In fact, it has been shown in a variety of systems, including flies and humans, that when neurons re-enter the cell cycle, the result is degeneration rather than proliferation and that ectopic cell cycle activation in neurons is sufficient to trigger degeneration. Furthermore, up-regulation of cell cycle genes, such as proliferating cell nuclear antigen, cyclin A, and cyclin B, has been shown to occur in post-mitotic Purkinje and granule cells of A-T patients; and neurons of ATM-/- mice have been found to undergo DNA replication. Similarly, studies in both fly and mammalian models of Alzheimer’s disease support a causative link between cell cycle re-entry and neurodegeneration. Thus, failure of cell cycle regulation may be a common cause of neurodegenerative disorders, including A-T (Rimkus, 2008).

It is important to keep in mind that equally plausible and nonexclusive models have been put forth for why neurodegeneration occurs in A-T. The oxidative stress model proposes that neurodegeneration occurs as a consequence of increased oxidative stress, and the DNA damage model proposes that neurodegeneration occurs as a consequence of the accumulation of DNA damage. While these models are described as functioning independently, it is unlikely that this is the case. For example, oxidative stress could lead to cell cycle re-entry through several different pathways, and in response to DNA damage, neurons may re-enter the cell cycle before undergoing cell death (Rimkus, 2008).

This paper has described a powerful experimental model in Drosophila to study the molecular mechanisms that underlie neurodegeneration in the human disease A-T. ATM knockdown in flies caused post-mitotic neurons to re-enter the cell cycle and die by programmed cell death. This finding suggests that ATM performs a cell cycle checkpoint function in post-mitotic neurons, as it does in response to DNA damage in proliferating nonneuronal cells and neuroblasts. Heterozygous mutation of Stg/CDC25 suppressed neurodegeneration in ATM knockdown flies and inhibited cell cycle re-entry, suggesting that cell cycle re-entry is causative for neurodegeneration in A-T. In the future, further genetic, cell biological, and molecular analysis of the Drosophila A-T model will allow addressing of unresolved issues, such as the extent to which oxidative stress and DNA damage contribute to neurodegeneration in the absence of ATM, what factors trigger cell cycle re-entry in the absence of ATM, and what factors link cell cycle re-entry to programmed cell death as opposed to cell division in the absence of ATM (Rimkus, 2008).

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string: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | References

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