Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder. Disease alleles contain a trinucleotide repeat expansion of variable length, which encodes polyglutamine tracts near the amino terminus of the HD protein, huntingtin. Polyglutamine-expanded huntingtin, but not normal huntingtin, forms nuclear inclusions. A Drosophila model for HD is described. Amino-terminal fragments of human huntingtin containing tracts of 2, 75, and 120 glutamine residues were expressed in photoreceptor neurons in the compound eye. As in human neurons, polyglutamine-expanded huntingtin induces neuronal degeneration. The age of onset and severity of neuronal degeneration correlates with repeat length, and nuclear localization of huntingtin presages neuronal degeneration. In contrast to other cell death paradigms in Drosophila, coexpression of the viral antiapoptotic protein, P35, did not rescue the cell death phenotype induced by polyglutamine-expanded huntingtin (Jackson, 1998).
At least eight dominant human neurodegenerative diseases are due to the expansion of a polyglutamine within the disease proteins. This confers toxicity on the proteins and is associated with nuclear inclusion formation. Recent findings indicate that molecular chaperones can modulate polyglutamine pathogenesis, but the basis of polyglutamine toxicity and the mechanism by which chaperones suppress neurodegeneration remains unknown. In a Drosophila disease model, it has been demonstrated that chaperones show substrate specificity for polyglutamine protein, as well as synergy in suppression of neurotoxicity. This analysis also reveals that chaperones alter the solubility properties of the protein, indicating that chaperone modulation of neurodegeneration in vivo is associated with altered biochemical properties of the mutant polyglutamine protein. These findings have implications for these and other human neurodegenerative diseases associated with abnormal protein aggregation (Chan, 2000).
Analysis of chaperone suppression was extended to the Huntingtonís disease protein, Huntingtin. To do this, Hsp70, dHdj1 or dHdj2 were coexpressed with a truncated form of Huntingtin containing an expanded polyglutamine domain of 120 (Htt-Q120). Flies expressing Htt-Q120 show normal eyes at 1 day, but a severely degenerate eye structure by 10 days. On co-expression of chaperones, Hsp70 was found to strongly suppress neuronal degeneration induced by Htt-Q120, restoring a normal photoreceptor rhabdomere structure. However, dHdj1 showed partial and dHdj2 showed no ability to suppress neurodegeneration induced by mutant Huntingtin. These results demonstrate broad effects of Hsp70 on suppression of polyglutamine toxicity and further emphasize substrate specificity among the Hsp40 class of chaperones (Chan, 2000).
Huntingtin is moderately conserved, with 10 HEAT repeats reported in its amino-terminal half. HD orthologues are evident in vertebrates and Drosophila, but not in Saccharomyces cerevisiae, Caenorhabditis elegans or Arabidopsis thaliana, a phylogenetic profile similar to the NF-kB/Rel/dorsal family transcription factors, suggesting a potential functional relationship. The potential for a relationship between huntingtin and Dorsal was tested by overexpression experiments in Drosophila S2 cells. Drosophila Huntingtin complexes with dorsal via its carboxyl-terminal region, and the two enter the nucleus concomitantly, partly in a lipopolysaccharide (LPS)- and Nup88-dependent manner. Similarly, in HeLa cell extracts, human huntingtin co-immunoprecipitates with NF-kB p50 but not with p105. By cross-species comparative analysis, it has been found that the carboxyl-terminal segment of huntingtin that mediates the association with Dorsal possesses numerous HEAT-like sequences related to those in the amino-terminal segment. Thus, Drosophila and vertebrate huntingtins are composed predominantly of 28 to 36 degenerate HEAT-like repeats that span the entire protein. It is concluded that like other HEAT-repeat filled proteins, huntingtin is made up largely of degenerate HEAT-like sequences, suggesting that it may play a scaffolding role in the formation of particular protein-protein complexes. While many proteins have been implicated in complexes with the amino-terminal region of huntingtin, the NF-kB/Rel/dorsal family transcription factors merit further examination as direct or indirect interactors with huntingtin's carboxyl-terminal segment (Takano, 2002).
Huntington disease is caused by the expansion of a polyglutamine repeat in the Huntingtin protein (Htt) that leads to degeneration of neurons in the central nervous system and the appearance of visible aggregates within neurons. Suppressor polypeptides, containing two polyglutamine repeats separated by a spacer, were developed and tested that bind mutant Htt and interfere with the process of aggregation in cell culture. In a Drosophila model, the most potent suppressor inhibits both adult lethality and photoreceptor neuron degeneration. The appearance of aggregates in photoreceptor neurons correlates strongly with the occurrence of pathology, and expression of suppressor polypeptides delays and limits the appearance of aggregates and protects photoreceptor neurons. These results suggest that targeting the protein interactions leading to aggregate formation may be beneficial for the design and development of therapeutic agents for Huntington disease (Kazantsev, 2002).
Pathogenic polyQ proteins cause axonal transport defects and neuronal apoptosis:
To address whether axonal transport defects are selective to the pathogenic Htt protein, or whether they are a feature of polyQ proteins in general, the effects were examined on axonal transport of various proteins containing polyQ tracts of different lengths and in different contexts. 179Y-GAL4 and APPL-GAL4 were crossed to lines encoding proteins with either a 'normal' length, nondisease-causing polyQ repeat region or proteins with an expanded, disease-causing polyQ repeat region. These proteins consisted either entirely of polyQ repeats (20Q, 22Q, 108Q, 127Q; Marsh, 2000; Kazemi-Esfarjani, 2000) or polyQ repeats embedded in the C-terminal region of the polyQ disease protein Machado-Joseph disease (MJD) protein (MJD-27Q, MJD-78Q; Warrick, 1998). Proteins with normal length polyQ regions (22Q, MJD-27Q) were found to be present within axons, based upon the smooth staining seen with either an antibody against polyQ or an antibody against the HA tag, and these proteins were found to accumulate at neuromuscular junctions, suggesting that they are normally transported within larval axons. In contrast, proteins with expanded polyQ repeats (MJD-78Q, 127Q) exhibited prominent polyQ staining within organelle blockages, while reduced staining was observed at the neuromuscular junctions, suggesting impaired transport of pathogenic polyQ proteins (Gunawardena, 2003).
The extent of axonal accumulations induced by polyQ repeats was length dependent, since a correlation was observed between the number of polyQ repeats and the amount of axonal accumulations. Larvae expressing 20Q, 22Q, or MJD-27Q were similar to wild-type in that they exhibited no axonal accumulations. Larvae expressing the pathogenic proteins MJD-78Q, 108Q, or 127Q exhibited a severe sluggish larval movement phenotype, with prominent axonal accumulations observed in all instances. The expression of MJD-78Q and 127Q at 29°C was also observed to be very toxic such that larvae expressing these proteins never survived to adulthood and died at second or third instar larval stage. Western blot analysis ruled out a general expression difference between proteins with normal length polyQ repeats and proteins with expanded polyQ repeats since, if anything, more MJD-27Q expression was observed compared to MJD-78Q. To reduce the level of toxicity, the amount of MJD-78Q and 127Q made was reduced by growing animals at 25°C (GAL4 activity is temperature dependent). Organelle accumulations were still reduced although these larvae now survived much longer, dying at early pupal stages (Gunawardena, 2003).
To confirm the results seen by immunofluorescent staining, EM analysis was conducted on larvae expressing 127Q and on severe genotypes in which expression of MJD-78Q or 127Q was combined with a 50% reduction in KHC gene dose. Prominent axonal blockages were observed characteristic of those observed in homozygous mutations of motor protein genes. Mutant larval nerves also contained enlarged axons, some almost four or five times the diameter of those observed in wild-type. Sometimes 'holes' were observed lacking organelles within the nerve, perhaps indicative of degeneration (Gunawardena, 2003).
To test directly whether pathogenic polyQ proteins block transport by inducing nonmoving blockages in axonal processes, live analysis was performed of vesicular movement within whole-mount larval axons. YFP-tagged human amyloid precursor protein (APP-YFP) was expressed either in the presence or absence of MJD-78Q, using the GAL4 driver pGAL4-62B SG26-1, which is expressed in only a small population of motor neurons. Neurons expressing only APP-YFP contained many actively motile vesicles moving at velocities of approximately 1 microm/s; large bright nonmotile accumulations such as those seen in the presence of MJD-78Q were never observed. Neurons coexpressing MJD-78Q and APP-YFP revealed nonmoving large, bright aggregates of APP-YFP. Thus, polyQ expression can interfere with transport of APP-YFP vesicles (Gunawardena, 2003).
TUNEL analysis was used to test whether proteins with expanded polyQ repeats can induce neuronal apoptosis. A large increase in neuronal apoptosis was observed in lines expressing MJD-78Q and 127Q, but not in lines expressing the nonpathogenic control proteins 22Q or MJD-27Q. Anti-polyQ staining revealed obvious nuclear inclusions within larval brain cells. Many stained nuclei were obviously enlarged and may be undergoing apoptosis. Expression during embryonic cycle 14 revealed smooth cytoplasmic staining for MJD-78Q protein, while staining for 127Q revealed obvious punctate cytoplasmic aggregates with some nuclear aggregates. At later cycles, both MJD-78Q and 127Q were observed as punctate cytoplasmic and nuclear aggregates. In addition, embryos expressing both MJD-78Q and 127Q died soon after they hatched into larvae, indicating substantial polyQ toxicity on normal development. Taken together, these results confirm that proteins with expanded polyQ repeats cause axonal transport defects, perhaps by blocking axonal processes by polyQ accumulations, neuronal cell death, and neurodegeneration (Gunawardena, 2003).
It is possible that polyQ proteins bind and deplete critical components of the molecular motor machinery, perhaps via a Drosophila version of HAP1. This hypothesis makes two predictions: (1) genetic reduction of motor protein dosage should worsen the phenotypes caused by proteins with polyQ expansions by further depleting the motor protein supply, and (2) motor protein depletion should be observable with biochemical methods. To test this hypothesis, proteins with expanded polyQ repeats were expressed and levels of dynein and kinesin were reduced. While a 50% reduction in the dose of KHC has no significant phenotype on its own, when combined with pathogenic polyQ repeats, it dramatically enhances the axonal organelle accumulation phenotype. Similarly, while a 50% reduction in the dose of DLC or components of the dynactin complex (p150Glued, Arp1, and dynamitin) also normally have no significant phenotypes on their own, when combined with pathogenic polyQ proteins, these reductions substantially enhance the organismal phenotype leading to early larval lethality. This finding is consistent with the observation that the neuronal APPL-GAL4 driver turns on during embryonic stage 15 as observed by the expression pattern of UAS-GFP. The enhanced lethality precluded analysis of axonal transport in these genotypes. Interestingly, organelle accumulations now appeared in transgenic lines expressing normally nonpathogenic poly Q repeats (22Q and MJD-27Q) with a 50% reduced dose of dynein, consistent with the hypothesis that all of these proteins may titrate motor proteins, but to varying extents. To test directly for motor protein depletion mediated by expression of proteins with expanded polyQ regions, early embryos expressing httex1-20Q, MJD-27Q, MJD-78Q, httex1-93Q, and 127Q, were examined using the early embryonic GAL4 driver da-GAL4, which turns on at the blastoderm stage based on its UAS-GFP expression pattern (Gunawardena, 2003).
Two considerations led to an evaluation of the effects of polyQ proteins on available motor protein pools by assessing soluble levels of motor proteins: (1) if motor proteins are titrated from normal cargoes by binding to large aggregates, it may be difficult to distinguish motor proteins bound to sedimentable cargoes from sedimentable aggregates; (2) it is not possible to measure the amount of each motor protein associated with normal cargoes owing to the lack of information about such cargoes and how such cargoes fractionate relative to polyQ aggregates. Thus, soluble levels of motor proteins were assessed under the hypothesis that aggregated polyQ proteins may bind motor proteins and deplete both soluble and cargo bound pools in parallel (Gunawardena, 2003).
At 6 hr of development, no significant change in the amount of total motor protein present in these embryos was observed. In contrast to the normal amounts of total motor proteins, an obvious reduction was observed in the amount of soluble motor proteins in embryos expressing MJD-27Q, MJD-78Q, and 127Q compared to wild-type embryos (i.e., da-GAL4 alone and yw). The amount of soluble DHC, DIC, p150Glued, KHC, and KLC were reduced, with no change observed in tubulin, actin, HDAC3, and Rab8. However, for reasons that are not clear, syntaxin was upregulated. Similar observations were evident from 12 and 16 hr embryo collections. The effect of httex1-93Q expression on levels of soluble motor proteins was not obvious at 6 hr of development. However, at 18 hr of development, there is an obvious reduction in soluble p150Glued and KLC in embryos expressing httex1-93Q but not httex1-20Q or wild-type. Expression of polyQ proteins in embryos was obvious as detected by anti-HA antibody and confirmed by anti-polyQ antibody. The level of 127Q was difficult to evaluate, perhaps due to the formation of aggregates, and was convincingly observed only after immunoprecipitation with anti-HA antibody. These observations indicate that expanded polyQ proteins can deplete or sequester available soluble motor proteins, perhaps into polyQ aggregates. The high expression level of MJD-27Q in embryos and the observed depletion of soluble motor proteins in these embryos are consistent with the finding that axonal blockages can be observed in larvae expressing MJD-27Q when motor protein gene dose is reduced by 50%. This finding is also consistent with the proposal that motor titration and aggregation may act in concert to poison axonal transport (Gunawardena, 2003).
Enhanced expression of chaperones restores neuronal transport and suppresses cell death caused by pathogenic polyQ proteins
The neurodegenerative adult eye phenotype caused by polyQ expansion proteins in Drosophila is suppressed by excess chaperone proteins (Warrick, 1999). This suppression has been proposed to occur by modulating soluble properties of pathogenic polyQ proteins, by preventing abnormal interactions with other proteins, or by rescuing chaperone depletion (Bonini, 2002). Whether expression of excess HSC70 protein would suppress axonal blockages and neuronal cell death was tested. Expression of UAS-HSC70 using APPL-GAL4 in the presence of MJD-78Q and 127Q restores axonal transport within larval nerves and suppresses neuronal death. PolyQ accumulations were absent within larval nerves, while cytoplasmic and punctate polyQ staining was present within larval brains. However, while these larvae were now able to pupate (expression of MJD-78Q or 127Q alone causes death at second or third instar larval stages), they still failed to eclose, suggesting that polyQ toxicity was still sufficient to cause lethality. Expression of HSC70 by itself did not cause axonal blockages or neuronal cell death. These results suggest that chaperones could 'clear' larval axons of blockages caused by polyQ proteins and suppress cell death within the larval brain, although organismal toxicity was not completely suppressed (Gunawardena, 2003).
Do axonal defects instigate neuronal dysfunction?
The pathogenic polyQ proteins accumulate in both axonal and nuclear inclusions. To dissect the relative contributions of nuclear and axoplasmic inclusions to the phenotype, transgenes were used that expressed proteins that had different subcellular localizations.
One transgene encoded a protein with an expanded polyQ repeat with a nuclear localization sequence (MJD-65QNLS). While expression of MJD-65QNLS within the larval brain caused neuronal apoptosis as observed by TUNEL staining, organelle accumulations within larval axons were absent. These larvae pupated but failed to eclose. While reduction in dynein dose by 50% with excess MJD-65QNLS had no effect, reduction in kinesin dose by 50% with excess MJD-65QNLS caused a small number of accumulations, perhaps due to continued motor titration by these proteins even when targeted to nuclei (Gunawardena, 2003).
To test further if dying neuronal cells induce axonal transport defects, the axonal transport phenotype was analyzed of the cell death gene reaper, which also induces neuronal apoptosis. Transport following reaper expression in these genotypes appeared to be normal based on immunostaining with synaptic vesicle markers even though high levels of neuronal apoptosis were induced. In addition, a 50% reduction in KHC combined with excess reaper expression had no effect on axonal transport. These findings emphasize that not all neuronal death is associated with axonal accumulations, and that the axonal transport defects induced by pathogenic polyQ proteins may be specific to cytoplasmic aggregations of expanded polyQ proteins (Gunawardena, 2003).
To test for cytoplasmic or axoplasmic toxicity, a protein with an expanded polyQ repeat with a nuclear export sequence (MJD-77QNES) was studied. Expression of MJD-77QNES within larval neurons caused large numbers of synaptotagmin-containing organelle accumulations within larval nerves. Consistent with primarily cytoplasmic localization of this protein, polyQ/HA staining was absent from cell nuclei within the larval brain, with bright anterior staining (just distal to the brain) present within larval nerves. Neuronal apoptosis as determined by TUNEL staining was completely absent and these larvae died at second or third instar, similar to MJD-78Q. Quantitative analysis indicates that the extent of organelle accumulations within MJD-77QNES is comparable to accumulations observed in mutations of motor proteins, suggesting that perhaps the extent of accumulations causes lethality. Similar to MJD78Q, a 50% reduction in the dose of KHC with MJD-77QNES enhanced organelle blockages, while 50% reduction in DLC combined with MJD-77QNES caused early larval lethality. Additionally, expression of MJD-77QNES in the adult eye using GMR-GAL4 caused a severe degenerative eye phenotype, indicating that cytoplasmic polyQ protein can cause degeneration of adult neurons (Gunawardena, 2003).
To directly test if excess MJD-77QNES sequestered motor proteins, embryos expressing MJD-27Q, MJD-78Q, and MJD-77QNES were compared with wild-type (da-GAL4). While embryos expressing both MJD-78Q and MJD-77QNES showed normal levels of total motor proteins, they exhibited a striking reduction in the amount of soluble motor proteins. MJD-77QNES also exhibited high molecular weight aggregates, which could be immunoprecipitated with the HA antibody. These high molecular weight aggregates also contained sequestered DHC. Although phenotypically normal when expressed in larvae, MJD-27Q appears to titrate more DHC into high molecular weight aggregates than do MJD-78Q, MJD-77QNES, or 127Q. It is conceivable that pathogenic MJD-78Q, MJD-77QNES, and 127Q form high molecular weight aggregates that were not possible trap or to solubilize using current protocols. Indeed, dramatic phenotypes are only observed in MJD-78Q, MJD-77QNES, and 127Q. Additionally, similar to embryos expressing MJD-78Q or 127Q, embryos expressing MJD-77QNES died soon after they hatch into larvae, indicating significant polyQ toxicity on normal development. It is possible that the 'soluble' polyQ aggregates observed in embryos expressing MJD-77QNES represent a subclass of misfolded proteins, while the class of insoluble aggregates, which were not possible to observe directly on SDS-PAGE, may be responsible for polyQ toxicity (Gunawardena, 2003).
To distinguish if MJD-78Q blockages and MJD-77QNES blockages are comparable, whether blockages caused by MJD-77QNES expression can be suppressed by excess HSC70 was tested. Expression of MJD-77QNES with excess HSC70 completely suppressed axonal accumulations, polyQ aggregates, and rescued larval lethality to pupae, suggesting that axonal blockages caused by either MJD-78Q or MJD-77QNES were comparable. PolyQ-containing accumulations were also absent in larval nerves. However, excess HSC70 was not sufficient to suppress organismal lethality (Gunawardena, 2003).
Huntington's disease is an autosomal dominant neurodegenerative disorder caused by expansion of a polyglutamine tract in the huntingtin protein that results in intracellular aggregate formation and neurodegeneration. Pathways leading from polyglutamine tract expansion to disease pathogenesis remain obscure. To elucidate how polyglutamine expansion causes neuronal dysfunction, Drosophila transgenic strains were generated expressing human huntingtin cDNAs encoding pathogenic (Htt-Q128) or nonpathogenic proteins (Htt-Q0). Whereas expression of Htt-Q0 has no discernible effect on behavior, lifespan, or neuronal morphology, pan-neuronal expression of Htt-Q128 leads to progressive loss of motor coordination, decreased lifespan, and time-dependent formation of huntingtin aggregates specifically in the cytoplasm and neurites. Huntingtin aggregates sequester other expanded polyglutamine proteins in the cytoplasm and leads to disruption of axonal transport and accumulation of aggregates at synapses. In contrast, Drosophila expressing an expanded polyglutamine tract alone, or an expanded polyglutamine tract in the context of the spinocerebellar ataxia type 3 protein, display only nuclear aggregates and do not disrupt axonal trafficking. These findings indicate that nonnuclear events induced by cytoplasmic huntingtin aggregation play a central role in the progressive neurodegeneration observed in Huntington's disease (Lee, 2004).
To characterize neuronal defects that result from an expanded polyQ tract within the Htt gene, transgenic Drosophila were generated expressing N-terminal fragments of human Htt containing 0 (Htt-Q0) or 128 (Htt-Q128) glutamines. The Htt constructs were engineered to include the first 548 aa of the human Htt protein; this region includes and extends well beyond the 81-aa product encoded by the first exon of the gene. The 548-aa fragment is truncated close to the site of cleavage by caspase-3, thought to be a crucial step in the generation of aggregate-forming Htt fragments (Kim, 2001; Wellington, 2002). This region also encompasses the highest stretch of homology between the Drosophila and human Htt proteins. Htt-Q0 and Htt-Q128 fragments were expressed by using UAS/GAL4 or a heat-shock promoter. To confirm transgene expression, Htt protein was compared between pHS-Htt Drosophila maintained at room temperature and after exposure to a heat-shock paradigm. Western blotting with anti-human Htt antibodies detected no Htt protein in control Canton S or in pHS-Htt lines maintained at room temperature. In contrast, Htt-Q0 and Htt-Q128 lines showed abundant Htt expression after heat shock. Transgene expression was also established in pUAST-Htt strains that were crossed to a neuronal GAL4 driver (Lee, 2004).
To determine the functional consequences of Htt-Q128 expression on neuronal activity and morphology, effects in the visual system were examined. Previous Drosophila models of polyQ diseases have demonstrated that eye-specific expression of expanded polyQ proteins leads to a rough-eye phenotype and photoreceptor degeneration. To determine whether the 548-aa Htt transgene caused similar effects, Htt-Q0 and Htt-Q128 were expressed by using the eye-specific GMR-GAL4 driver, and the resulting eye phenotypes were observed by external morphology and the corneal pseudopupil method. Whereas expression of Htt-Q0 does not perturb external-eye appearance or ommatidial morphology, expression of Htt-Q128 causes a rough-eye phenotype with corresponding photoreceptor degeneration. Thus, polyQ expansion in the context of a larger Htt fragment results in neurodegeneration, as observed in other polyQ disease models (Lee, 2004).
To characterize the physiological effects of mutant Htt expression, electroretinograms were recorded from transgenic animals. A normal electrical response to light was seen in Drosophila expressing the GMR-GAL4 driver alone, Htt-Q0 with GMR-GAL4, or Htt-Q128 without the GMR-GAL4 driver. In contrast, Drosophila expressing Htt-Q128 with the GMR-GAL4 driver showed reduced photoreceptor depolarization and complete abolishment of synaptic transmission in response to light. Similar abnormal electroretinograms were observed in heat-shocked pHS-Htt-Q128 lines after a developmental heat shock paradigm but not with control pHS-Htt-Q0 strains. Synaptic activity was also assayed in the giant fiber flight circuit, a pathway important in escape responses and flight initiation. Wild-type Drosophila display little to no spontaneous activity when the temperature is raised to 38°C. In contrast, robust spontaneous seizure activity was recorded in Htt-Q128 flies at 38°C after a developmental heat-shock paradigm. No seizure activity was recorded in Htt-Q0 flies at 38°C. Together, these results indicate that Htt-Q128 expression results in neurodegeneration, accompanied by widespread defects in membrane excitability and brain activity (Lee, 2004).
To establish whether neuronal Htt-Q128 transgene expression causes defects at earlier stages of Drosophila development, quantitative locomotion assays were performed to examine the function of the motor central pattern generator in third-instar larvae. When Htt transgenes were expressed with the pan-neuronal elav-GAL4 driver C155, Htt-Q128 larvae showed a significant reduction in locomotor speed of >25. Adult transgenic flies also display abnormal motor behavior caused by pan-neuronal expression of the Htt-Q128 protein. Whereas expression of Htt-Q128 with the C155 neuronal GAL4 driver causes pharate adult lethality with no viable adult escapers, Htt-Q128 driven by a weaker second chromosome elav-GAL4 driver results in fully viable adults. Several days after eclosion, flies expressing Htt-Q128, but not Htt-Q0, begin to exhibit uncoordinated movement and abnormal grooming behaviors. The behavioral defects worsen with age, resulting in premature death. To quantify the reduction in viability, lifespan curves were generated for control adults, Htt-Q128 adults without elav-GAL4, or adults expressing Htt-Q0 or Htt-Q128 with elav-GAL4. Compared to controls, Htt-Q128/elav-GAL4 animals showed a dramatic reduction in lifespan, with a decrease in the T50 (age at which 50% of the culture has died) by 70%, indicating a highly significant effect of Htt-Q128 expression on viability in Drosophila (Lee, 2004).
A hallmark of HD is the formation of Htt-immunopositive intracellular aggregates in neurons. To determine whether intracellular aggregates are formed in transgenic Htt Drosophila, both Htt-Q128 and Htt-Q0 strains were crossed to flies containing C155 elav-GAL4 to direct expression of Htt within the nervous system. Htt-immunopositive staining was visualized in both central and peripheral neurons of dissected third-instar larvae. Whereas Htt staining remained diffuse throughout the cytoplasm of neurons in Drosophila expressing Htt-Q0, distinct aggregates were observed in the cytoplasm and processes of neurons in lines expressing Htt-Q128. Contrary to what has been observed in exon 1 HD models, no evidence was found of nuclear aggregate localization. To verify that Htt aggregation is based on the length of the polyQ tract and not on protein concentration, Htt levels were quantitated for several Htt-Q0 and Htt-Q128 transgenic lines crossed to elav-GAL4. Levels of Htt protein were generally higher in Htt-Q128 lines than in Htt-Q0 lines, likely because of sequestration of the mutant protein into stable aggregates. However, low-expressing Htt-Q128 lines that produced transgenic protein at a level comparable with that in Htt-Q0 strains still exhibited aggregates, whereas Htt-Q0 lines did not, indicating that polyQ tract expansion and not protein concentration alone is necessary for formation of aggregates. Aggregate formation was also time-dependent. Although Htt levels were visibly high in the central and peripheral nervous system of Htt-Q128/elav-GAL4 embryos, the protein remained largely diffuse in the cytoplasm with rare occurrence of aggregates. By the third instar larval stage, essentially all Htt was observed in aggregates, with relatively little nonaggregate staining. It is concluded that Htt-Q128 forms cytoplasmic neuronal aggregates in a time-dependent manner (Lee, 2004).
Although the causative proteins for many of the polyQ repeat diseases are expressed widely or ubiquitously, aggregate formation and cell death occur in subsets of neurons that differ between the diseases. To examine the effect of cellular context on aggregate formation, the Htt-Q128 protein was expressed in different cell types by using a tubulin GAL4 driver. Transgenic lines were generated containing both the UAS-Htt-Q128 construct and UAS-GFP fused to a nuclear localization signal, allowing for covisualization of Htt-immunopositive aggregates and GFP-stained cell nuclei in expressing cells. Immunocytochemical analysis has demonstrated the formation of cytoplasmic aggregates in both neuronal and nonneuronal tissues, including CNS neurons, gut, salivary glands, and trachea. Interestingly, Htt aggregates were differentially distributed in polarized cells such as the gut, with transport of Htt aggregates to the basolateral domain and exclusion from the apical surface. Similar aggregate transport was found in neurons, indicating that Htt aggregates undergo a cytoskeletal association that allows for directed transport. The Htt-Q128 protein was found in a more diffuse, nonaggregated state in certain cell types, including muscle and epidermis, suggesting that some tissues may be more resistant to Htt aggregation. In cell types in which aggregate formation occurred, only cytoplasmic aggregates (as opposed to nuclear aggregates) were observed, suggesting differences between the larger Htt fragments used in this study compared with exon 1 HD models (Lee, 2004).
Although the polyQ repeat diseases share a similar CAG repeat expansion in the causative gene, the pattern of neurodegeneration and behavioral dysfunction is distinct for each, indicating that protein context for expanded polyQ tracts is critical to disease manifestation. To examine the importance of protein context in the subcellular localization of polyQ-containing proteins, immunocytochemical analysis was performed on larvae expressing an expanded polyQ tract alone (Q127), the mutant polyQ protein responsible for Machado-Joseph disease (SCA3-Q78), or an expanded polyQ tract (Q108) previously engineered into the nonpathogenic dishevelled gene. In contrast to the cytoplasmic localization of Htt aggregates, both Q127 and SCA3-Q78 aggregates localized exclusively to the nucleus. Very few Dishevelled-immunopositive aggregates were observed, and the protein was present diffusely in the cytoplasm. These results demonstrate that the protein context in which the polyQ tract is found exquisitely controls both aggregate localization and aggregate formation (Lee, 2004).
To test whether Htt-Q128 can interact and coaggregate with other polyQ repeat proteins, double transgenic Htt-Q128; Q127 and Htt-Q128; SCA3-Q78 strains were generated. When the Htt-Q128 and Q127 proteins were coexpressed, both central and peripheral neurons showed localization of Htt-Q128 aggregates to the cytoplasm, whereas Q127 aggregates were restricted to the nucleus. Likewise, in strains expressing Htt-Q128 and SCA3-Q78, aggregates were segregated independently in the cytoplasm and nucleus (respectively) of both neuronal and nonneuronal cells. These results suggest that the trafficking and aggregation of nuclear and cytoplasmic aggregates are independently regulated (Lee, 2004).
To determine whether Htt-Q128 might interact with cytoplasmic proteins containing an expanded polyQ tract, double transgenic strains were made containing Htt-Q128 and Dishevelled-Q108 (Dsh-Q108). Dsh-Q108 formed few aggregates when expressed alone; however, when coexpressed with Htt-Q128, the subcellular distribution of Dsh-Q108 shifted from a diffuse cytoplasmic pattern to a complete sequestration into aggregates that colocalized with Htt-Q128. These findings indicate that Htt aggregates are able to trap and sequester Dsh-Q108 into cytoplasmic aggregates. Similar interactions between Htt aggregates and endogenous cytoplasmic polyQ-containing proteins might be predicted to play an important role in disease pathology (Lee, 2004).
Htt-Q128 forms cytoplasmic aggregates that are associated with cytoskeletal transport systems. When Htt-Q128 was expressed with the eye-specific driver GMR-GAL4, Htt aggregates were abundantly transported along axons entering the CNS of the developing visual system and accumulated in pathfinding photoreceptor growth cones. Similarly, when Htt-Q128 was expressed with the C155 driver, aggregates were transported in larval motor axons and accumulated at presynaptic neuromuscular junction terminals. No aggregates were observed in axons from animals expressing Htt-Q0. Axonal transport of aggregates was not observed in transgenic animals that produce exclusively nuclear aggregates (Q127 and SCA3-Q78), suggesting that axonal and synaptic defects that may occur downstream of cytoplasmic aggregate formation are likely specific to HD. Additionally, no Dsh-Q108 aggregates were observed in axon bundles. However, when Dsh-Q108 was coexpressed with Htt-Q128, Dsh-Q108 protein trapped by Htt-Q128 aggregates was also transported along axons. Similar trapping of endogenous cytoplasmic polyQ proteins may sequester them away from their natural cellular locations and contribute to neuronal dysfunction (Lee, 2004).
In observing axonal aggregates in Htt-Q128-expressing animals, it was noted that the diameter of Htt aggregates often exceeds that of normal larval axons. This suggests that large Htt aggregates might physically block axonal transport, as would be manifested by axonal swellings at the sites of blockage. This hypothesis was tested in Htt-Q128-expressing animals by observing the localization of synaptotagmin I, a synaptic vesicle protein localized to synapses in Drosophila. Normal transport of the synaptotagmin protein along axons is below the threshold for immunocytochemical detection. This pattern of trafficking, as observed in Htt-Q0-expressing animals, is abruptly altered in Htt-Q128-expressing Drosophila. Instead of the normal diffuse localization along axonal tracts, synaptotagmin became concentrated at specific points along axons that corresponded to large areas of Htt-immunopositive aggregate accumulation, suggesting sites of axonal blockage. These synaptotagmin-rich areas of Htt aggregate accumulation were quantified in 100-µm segments along peripheral nerves and were observed at a density of 6.1 +/- 2.6 sites per 100 microm. In contrast, synaptotagmin-immunopositive accumulations alone without Htt aggregate colocalization were only observed at a density of 1.3 +/- 0.9 per 100 microm. The average diameter of Htt-Q128 aggregate accumulations at putative axonal blockage sites was 2.4 +/- 0.6 µm. These findings suggest that axonal segments can be obstructed by Htt aggregates. Over time, the cumulative blockage of axons and synaptic terminals in postmitotic neurons is likely to contribute to the progressive physiological defects and neuronal dysfunction that has been documented in Htt-Q128-expressing Drosophila, as well as to late-onset neurodegeneration in HD patients (Lee, 2004).
Many human neurodegenerative diseases have been successfully modeled in Drosophila with replication of key neuropathological features, including late onset and progressive neurodegeneration. Existing Drosophila models of HD target expression of the first exon of the mutant Htt protein to the fly retina, either by using an eye-specific promoter (Jackson, 1998) or the GAL4/UAS system (Steffan, 2001). In both HD models, expression of Htt with a pathogenic number of glutamine repeats results in nuclear accumulation of aggregates and progressive neurodegeneration of photoreceptor cells, suggesting a role for nuclear aggregates in disease pathology. Indeed, nuclear aggregate-mediated impairment of transcription has become a favored hypothesis to explain polyQ-mediated neurodegeneration. The findings using a larger Htt transgene suggest that nonnuclear pathology associated with cytoplasmic and neuritic aggregates is likely to play an essential role in disease progression as well. Given that Drosophila polyQ disease models with either nuclear-restricted (expanded polyQ alone, mutant SCA-3, or Htt exon 1) or cytoplasm-restricted (Htt-Q128) aggregates both exhibit neurodegeneration, it is likely that multiple pathways for polyQ-mediated dysfunction exist. Indeed, it has been possible to rescue adult lethality in Htt-Q128-expressing Drosophila with any of the previously published genetic suppressors of Drosophila transgenic polyQ models. These negative results likely reflect distinct modes of toxicity between nuclear and cytoplasmic aggregates and suggest that preventing polyQ-mediated Htt toxicity may require more research on the role of nonnuclear aggregates. A potential role has been demonstrated for cytoplasmic and neuritic aggregates in the sequestration of cytoplasmic polyQ proteins and in the blockage of axonal transport. Consistent with the hypothesis that Htt-Q128 expression causes neurodegeneration secondary to impairment of axonal transport, recent studies have found that neurodegeneration is a primary consequence of axonal transport defects in non-polyQ diseases as well, including Alzheimer's disease. During review of this manuscript, two reports have been published that document similar axonal transport defects in HD models. Determining the precise mechanisms by which Htt aggregates physically attach to the axonal cytoskeleton will likely provide important insights into the mechanisms of axonal transport blockage in HD. In summary, these results indicate that cytoplasmic aggregate formation in HD sequesters endogenous polyglutamine proteins and blocks axonal transport, contributing to neurophysiological dysfunction and neurodegeneration (Lee, 2004).
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