Arfaptin 2 regulates the aggregation of mutant huntingtin protein

Huntington's disease (HD) is an inherited neurodegenerative disorder. Here we demonstrate that expression of arfaptin 2/POR1 (partner of Rac1) in cultured cells induces the formation of pericentriolar and nuclear aggregates, which morphologically resemble mutant huntingtin aggregates characteristic of HD. Endogenous arfaptin 2 localizes to aggregates induced by expression of an abnormal amino-terminal fragment of huntingtin that contains polyglutamine (polyQ) expansions. A dominant inhibitory mutant of arfaptin 2 inhibits aggregation of mutant huntingtin, but not in the presence of proteasome inhibitors. Using cell-free biochemical assays, we show that arfaptin 2 inhibits proteasome activity. Finally, we show that expression of arfaptin 2 is increased at sites of neurodegeneration and the protein localizes to huntingtin aggregates in HD transgenic mouse brains. Our data suggest that arfaptin 2 is involved in regulating huntingtin protein aggregation, possibly by impairing proteasome function.     

Nuclear aggregation of huntingtin is not prevented by deletion of chaperone Hsp104

Polyglutamine expansion causes the disease proteins to aggregate, resulting in stable insoluble aggregates in the nucleus. The in vitro aggregation and cellular toxicity of polyglutamine proteins are reduced by chaperone heat shock proteins (Hsp). In polyglutamine disease animal models, however, polyglutamine inclusions remain in the nucleus despite the suppression of neurodegeneration by Hsp. Studies using yeast genetic approach revealed that the balance of Hsp is important for regulating protein aggregation in the cytoplasm of yeast cells. Here we report that N-terminal fragments of huntingtin with an expanded polyglutamine tract form aggregates only in the cytoplasm of yeast cells and, when tagged with nuclear localization sequences (NLS), are able to aggregate in the nucleus. Deletion of the Hsp104 gene prevents the aggregation of huntingtin in the cytoplasm but is unable to eliminate the aggregation of NLS-tagged huntingtin in the nucleus. The inhibitory effect of Hsp104 deletion on the cytoplasmic aggregation of huntingtin only occurs in viable yeast cells, as aggregates can be formed in Hsp104 deletion cells that have been frozen for 72 h. Fresh cytosolic extracts of the Hsp104 deletion strain inhibit the aggregation of huntingtin in vitro, suggesting that the deletion of Hsp104 may alter the activities of other cytoplasmic factors to inhibit polyglutamine aggregation in the cytoplasm. We propose that the regulatory effects of chaperones may mainly be restricted to the cytoplasm and have much less influence on polyglutamine-containing aggregates in the nucleus.     

Tissue transglutaminase does not contribute to the formation of mutant huntingtin aggregates

The cause of Huntington's disease (HD) is a pathological expansion of the polyglutamine domain within the NH(2)-terminal region of huntingtin. Neuronal intranuclear inclusions and cytoplasmic aggregates composed of the mutant huntingtin within certain neuronal populations are a characteristic hallmark of HD. Because in vitro expanded polyglutamine repeats are glutaminyl-donor substrates of tissue transglutaminase (tTG), it has been hypothesized that tTG may contribute to the formation of these aggregates in HD. Therefore, it is of fundamental importance to establish whether tTG plays a significant role in the formation of mutant huntingtin aggregates in the cell. Human neuroblastoma SH-SY5Y cells were stably transfected with truncated NH(2)-terminal huntingtin constructs containing 18 (wild type) or 82 (mutant) glutamines. In the cells expressing the mutant truncated huntingtin construct, numerous SDS-resistant aggregates were present in the cytoplasm and nucleus. Even though numerous aggregates were present in the mutant huntingtin-expressing cells, tTG did not coprecipitate with mutant truncated huntingtin. Further, tTG was totally excluded from the aggregates, and significantly increasing tTG expression had no effect on the number of aggregates or their intracellular localization (cytoplasm or nucleus). When a YFP-tagged mutant truncated huntingtin construct was transiently transfected into cells that express no detectable tTG due to stable transfection with a tTG antisense construct, there was extensive aggregate formation. These findings clearly demonstrate that tTG is not required for aggregate formation, and does not facilitate the process of aggregate formation. Therefore, in HD, as well as in other polyglutamine diseases, tTG is unlikely to play a role in the formation of aggregates.     

Herp Promotes Degradation of Mutant Huntingtin: Involvement of the Proteasome and Molecular Chaperones

In neurodegenerative diseases, pathogenic proteins tend to misfold and form aggregates that are difficult to remove and able to induce excessive endoplasmic reticulum (ER) stress, leading to neuronal injury and apoptosis. Homocysteine-induced endoplasmic reticulum protein (Herp), an E3 ubiquitin ligase, is an important early marker of ER stress and is involved in the ubiquitination and degradation of many neurodegenerative proteins. However, in Huntington’s disease (HD), a typical polyglutamine disease, whether Herp is also involved in the metabolism and degradation of the pathogenic protein, mutant huntingtin, has not been reported. Therefore, we studied the relationship between Herp and N-terminal fragments of huntingtin (HttN-20Q and HttN-160Q). We found that Herp was able to bind to the overexpressed Htt N-terminal, and this interaction was enhanced by expansion of the polyQ fragment. Confocal microscopy demonstrated that Herp was co-localized with the HttN-160Q aggregates in the cytoplasm and tightly surrounded the aggregates. Overexpression of Herp significantly decreased the amount of soluble and insoluble HttN-160Q, promoted its ubiquitination, and inhibited its cytotoxicity. In contrast, knockdown of Herp resulted in more HttN-160Q protein, less ubiquitination, and stronger cytotoxicity. Inhibition of the autophagy-lysosomal pathway (ALP) had no effect on the function of Herp. However, blocking the ubiquitin-proteasome pathway (UPP) inhibited the reduction in soluble HttN-160Q caused by Herp. Interestingly, blocking the UPP did not weaken the ability of Herp to reduce HttN-160Q aggregates. Deletions of the N-terminal of Herp weakened its ability to inhibit HttN-160Q aggregation but did not result in a significant increase in its soluble form. However, loss of the C-terminal led to a significant increase in soluble HttN-160Q, but Herp still maintained the ability to inhibit aggregate formation. We further found that the expression level of Herp was significantly increased in HD animal and cell models. Our findings suggest that Herp is a newly identified huntingtin-interacting protein that is able to reduce the cytotoxicity of mutant huntingtin by inhibiting its aggregation and promoting its degradation. The N-terminal of Herp serves as the molecular chaperone to inhibit protein aggregation, while its C-terminal functions as an E3 ubiquitin ligase to promote the degradation of misfolded proteins through the UPP. Increased expression of Herp in HD models may be a pro-survival mechanism under stress.     

Ubiquilin-1 Overexpression Increases the Lifespan and Delays Accumulation of Huntingtin Aggregates in the R6/2 Mouse Model of Huntington's Disease

Huntington''s Disease (HD) is a neurodegenerative disorder that is caused by abnormal expansion of a polyglutamine tract in huntingtin (htt) protein. The expansion leads to increased htt aggregation and toxicity. Factors that aid in the clearance of mutant huntingtin proteins should relieve the toxicity. We previously demonstrated that overexpression of ubiqulin-1, which facilitates protein clearance through the proteasome and autophagy pathways, reduces huntingtin aggregates and toxicity in mammalian cell and invertebrate models of HD. Here we tested whether overexpression of ubiquilin-1 delays or prevents neurodegeneration in R6/2 mice, a well-established model of HD. We generated transgenic mice overexpressing human ubiquilin-1 driven by the neuron-specific Thy1.2 promoter. Immunoblotting and immunohistochemistry revealed robust and widespread overexpression of ubiquilin-1 in the brains of the transgenic mice. Similar analysis of R6/2 animals revealed that ubiquilin is localized in huntingtin aggregates and that ubiquilin levels decrease progressively to 30% during the end-stage of disease. We crossed our ubiquilin-1 transgenic line with R6/2 mice to assess whether restoration of ubiquilin levels would delay HD symptoms and pathology. In the double transgenic progeny, ubiquilin levels were fully restored, and this correlated with a 20% increase in lifespan and a reduction in htt inclusions in the hippocampus and cortex. Furthermore, immunoblots indicated that endoplasmic reticulum stress response that is elevated in the hippocampus of R6/2 animals was attenuated by ubiquilin-1 overexpression. However, ubiquilin-1 overexpression neither altered the load of htt aggregates in the striatum nor improved motor impairments in the mice.     

Oxidative stress promotes mutant huntingtin aggregation and mutant huntingtin-dependent cell death by mimicking proteasomal malfunction

Huntington's disease (HD) is a familial neurodegenerative disorder caused by an abnormal expansion of CAG repeats in the coding region of huntingtin gene. A major hallmark of HD is the proteolytic production of N-terminal fragments of huntingtin containing polyglutamine repeats that form ubiquitinated aggregates in the nucleus and cytoplasm of the affected neurons. However, the mechanism by which the mutant huntingtin causes neurodegeneration is not well understood. Here, we found that oxidative stimuli enhance the polyglutamine-expanded truncated N-terminal huntingtin (mutant huntingtin) aggregation and mutant huntingtin-induced cell death. Oxidative stimuli also lead to rapid proteasomal dysfunction in the mutant huntingtin expressing cells as compared to normal glutamine repeat expressing cells. Overexpression of Cu/Zn superoxide dismutase (SOD1), Hsp40 or Hsp70 reverses the oxidative stress-induced proteasomal malfunction, mutant huntingtin aggregation, and death of the mutant huntingtin expressing cells. Finally, we show the higher levels of expression of SOD1 and DJ-1 in the mutant huntingtin expressing cells. Our result suggests that oxidative stress-induced proteasomal malfunction might be linked with mutant huntingtin-induced cell death.     

Temporal Separation of Aggregation and Ubiquitination during Early Inclusion Formation in Transgenic Mice Carrying the Huntington's Disease Mutation

Abnormal insoluble ubiqitinated protein aggregates are found in the brains of Huntington's disease (HD) patients and in mice transgenic for the HTT mutation. Here, we describe the earliest stages of visible NII formation in brains of R6/2 mice killed between 2 and 6 weeks of age. We found that huntingtin-positive aggregates formed rapidly (within 24-48 hours) in a spatiotemporal manner similar to that we described previously for ubiquitinated inclusions. However, in most neurons, aggregates are not ubiquitinated when they first form. It has always been assumed that mutant huntingtin is recognised as 'foreign' and consequently ubiquitinated and targeted for degradation by the ubiquitin-proteasome system pathway. Our data, however, suggest that aggregation and ubiquitination are separate processes, and that mutant huntingtin fragment is not recognized as 'abnormal' by the ubiquitin-proteasome system before aggregation. Rather, mutant Htt appears to aggregate before it is ubiquitinated, and then either aggregated huntingtin is ubiquitinated or ubiquitinated proteins are recruited into aggregates. Our findings have significant implications for the role of the ubiquitin-proteasome system in the formation of aggregates, as they suggest that this system is not involved until after the first aggregates form.     

Sir2 is induced by oxidative stress in a yeast model of Huntington disease and its activation reduces protein aggregation

Huntington disease (HD) is a neurodegenerative disorder caused by expansion of CAG trinucleotide repeats, leading to an elongated polyglutamine sequence (polyQ) in the huntingtin protein. Misfolding of mutant polyQ proteins with expanded tracts results in aggregation, causing cytotoxicity. Oxidative stress in HD has been documented in humans as important to disease progression. Using yeast cells as a model of HD, we report that when grown at high glucose concentration, cells expressing mutant polyQ do not show apparent oxidative stress. At higher cell densities, when glucose becomes limiting and cells are metabolically shifting from fermentation to respiration, protein oxidation and catalase activity increases in relation to the length of the polyQ tract. Oxidative stress, either endogenous as a result of mutant polyQ expression or exogenously generated, increases Sir2 levels. Δ sir2 cells expressing expanded polyQ lengths show signs of oxidative stress even at the early exponential phase. In a wild-type background, isonicotinamide, a Sir2 activator, decreases mutant polyQ aggregation and the stress generated by expanded polyQ. Taken together, these results describe mutant polyQ proteins as being more toxic in respiring cells, causing oxidative stress and an increase in Sir2 levels. Activation of Sir2 would play a protective role against this toxicity.     

Chaperone suppression of cellular toxicity of huntingtin is independent of polyglutamine aggregation

Polyglutamine protein aggregation is associated with eight inherited neurodegenerative disorders. In Huntington's disease, N-terminal fragments of mutant huntingtin form intracellular aggregates and mediate cellular toxicity. Recent studies have shown that chaperones inhibit polyglutamine-mediated aggregation and cellular toxicity. Because chaperones also inhibit caspase activation to protect cells from death, it remains unclear whether the protective effect of chaperones on polyglutamine-mediated cellular toxicity is dependent on their inhibition of protein aggregation. In this study, we show that several chaperones including HSP 40, HSP 70, and N-ethylmaleimide-sensitive factor can inhibit cellular toxicity caused by N-terminal mutant huntingtin fragments. However, only HSP 40 is able to inhibit huntingtin aggregation. Furthermore, time-course study suggests that the protection of chaperones against huntingtin toxicity is not the result of their suppression of huntingtin aggregation. Chaperones inhibit caspase-3 and caspase-9 activation mediated by mutant huntingtin, and this inhibition is independent of huntingtin aggregation. We propose that the inhibition of caspase activity by chaperones is involved in their suppression of polyglutamine toxicity.     

Dysfunction of the ubiquitin ligase ube3a may be associated with synaptic pathophysiology in a mouse model of huntington disease

Huntington disease (HD) is a hereditary neurodegenerative disorder characterized by progressive cognitive, psychiatric, and motor symptoms. The disease is caused by abnormal expansion of CAG repeats in the gene encoding huntingtin, but how mutant huntingtin leads to early cognitive deficits in HD is poorly understood. Here, we demonstrate that the ubiquitin ligase Ube3a, which is implicated in synaptic plasticity and involved in the clearance of misfolded polyglutamine protein, is strongly recruited to the mutant huntingtin nuclear aggregates, resulting in significant loss of its functional pool in different regions of HD mouse brain. Interestingly, Arc, one of the substrates of Ube3a linked with synaptic plasticity, is also associated with nuclear aggregates, although its synaptic level is increased in the hippocampus and cortex of HD mouse brain. Different regions of HD mouse brain also exhibit decreased levels of AMPA receptors and various pre- and postsynaptic proteins, which could be due to the partial loss of function of Ube3a. Transient expression of mutant huntingtin in mouse primary cortical neurons further demonstrates recruitment of Ube3a into mutant huntingtin aggregates, increased accumulation of Arc, and decreased numbers of GluR1 puncta in the neuronal processes. Altogether, our results suggest that the loss of function of Ube3a might be associated with the synaptic abnormalities observed in HD.     

Allele-specific silencing of mutant Huntington's disease gene

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by a poly-glutamine expansion in huntingtin, the protein encoded by the HD gene. PolyQ-expanded huntingtin is toxic to neurons, especially the medium spiny neurons of the striatum. At the same time, wild-type huntingtin has important – indeed essential – protective functions. Any effective molecular therapy must preserve the expression of wild-type huntingtin, while silencing the mutant allele. We hypothesized that an appropriate siRNA molecule would display the requisite specificity and efficacy. As RNA interference is incapable of distinguishing among alleles with varying numbers of CAG (glutamine) codons, another strategy is needed. We used HD fibroblasts in which the pathogenic mutation is linked to a polymorphic site: the Δ2642 deletion of one of four tandem GAG triplets. We silenced expression of the harmful Δ2642-marked polyQ-expanded huntingtin without compromising synthesis of its wild-type counterpart. Following this success in HD fibroblasts, we obtained similar results with neuroblastoma cells expressing both wild-type and mutant HD genes. As opposed to the effect of depleting wild-type huntingtin, specifically silencing the mutant species actually lowered caspase-3 activation and protected HD cells under stress conditions. These findings have therapeutic implications not only for HD, but also for other autosomal dominant diseases. This approach has great promise: it may lead to personalized genetic therapy, a holy grail in contemporary medicine.     

Mutant huntingtin promotes the fibrillogenesis of wild-type huntingtin: a potential mechanism for loss of huntingtin function in Huntington's disease

Aggregation of huntingtin (htt) in neuronal inclusions is associated with the development of Huntington's disease (HD). Previously, we have shown that mutant htt fragments with polyglutamine (polyQ) tracts in the pathological range (>37 glutamines) form SDS-resistant aggregates with a fibrillar morphology, whereas wild-type htt fragments with normal polyQ domains do not aggregate. In this study we have investigated the co-aggregation of mutant and wild-type htt fragments. We found that mutant htt promotes the aggregation of wild-type htt, causing the formation of SDS-resistant co-aggregates with a fibrillar morphology. Conversely, mutant htt does not promote the fibrillogenesis of the polyQ-containing protein NOCT3 or the polyQ-binding protein PQBP1, although these proteins are recruited into inclusions containing mutant htt aggregates in mammalian cells. The formation of mixed htt fibrils is a highly selective process that not only depends on polyQ tract length but also on the surrounding amino acid sequence. Our data suggest that mutant and wild-type htt fragments may also co-aggregate in neurons of HD patients and that a loss of wild-type htt function may contribute to HD pathogenesis.     

Roles of Hsp104 and trehalose in solubilisation of mutant huntingtin in heat shocked Saccharomyces cerevisiae cells

Inhibition of huntingtin aggregation, either in the nucleus and/or in the cytosol, has been identified as a major strategy to ameliorate the symptoms of Huntington's disease. Chaperones and other protein stabilisers would thus be key players in ensuring the correct folding of the amyloidogenic protein and its expression in the soluble form. By transient activation of the global heat stress response in Saccharomyces cerevisiaeBY4742, we show that heterologous expression of mutant huntingtin (103Q-htt) could be modulated so that the protein was partitioned off in the soluble fraction of the cytosol. This led to lower levels of reactive oxygen species and improved cell viability. Previous reports had speculated on the relationship between trehalose and the heat shock response in ensuring enhanced cell survival but no direct evidence of such an interaction was available. Using mutants of an isogenic strain which do not express the major trehalose synthetic or metabolising enzymes or the chaperone, heat shock protein 104 (Hsp104), we were able to identify the functions of Hsp104 and the osmoprotectant trehalose in solubilising mutant huntingtin. We propose that the beneficial effect of the protein refolding machinery in solubilising the aggregation-prone protein is exerted by maintaining a tight balance between the trehalose synthetic enzyme, trehalose-6-phosphate synthase 1 and Hsp104. This ensures that the level of the osmoprotectant, trehalose, does not exceed the limit beyond which it is reported to inhibit protein refolding.     

Inhibition of endoplasmic reticulum stress counteracts neuronal cell death and protein aggregation caused by N-terminal mutant huntingtin proteins

Accumulation of abnormal proteins occurs in many neurodegenerative diseases including Huntington's disease (HD). However, the precise role of protein aggregation in neuronal cell death remains unclear. We show here that the expression of N-terminal huntingtin proteins with expanded polyglutamine (polyQ) repeats causes cell death in neuronal PC6.3 cell that involves endoplasmic reticulum (ER) stress. These mutant huntingtin fragment proteins elevated Bip, an ER chaperone, and increased Chop and the phosphorylation of c-Jun-N-terminal kinase (JNK) that are involved in cell death regulation. Caspase-12, residing in the ER, was cleaved in mutant huntingtin expressing cells, as was caspase-3 mediating cell death. In contrast, cytochrome-c or apoptosis inducing factor (AIF) was not released from mitochondria after the expression of these proteins. Treatment with salubrinal that inhibits ER stress counteracted cell death and reduced protein aggregations in the PC6.3 cells caused by the mutant huntingtin fragment proteins. Salubrinal upregulated Bip, reduced cleavage of caspase-12 and increased the phosphorylation of eukaryotic translation initiation factor-2 subunit-alpha (eIF2alpha) that are neuroprotective. These results show that N-terminal mutant huntingtin proteins activate cellular pathways linked to ER stress, and that inhibition of ER stress by salubrinal increases cell survival. The data suggests that compounds targeting ER stress may be considered in designing novel approaches for treatment of HD and possibly other polyQ diseases.     

Effect of Pesticides on the Aggregation of Mutant Huntingtin Protein

The classical reports on neurodegeneration concentrate on studying disruption of signalling cascades. Although it is now well recognized that misfolding and aggregation of specific proteins are associated with a majority of these diseases, their role in aggravating the symptoms is not so well understood. Huntington's disease (HD) is a neurodegenerative disorder that results from damage to complex II of mitochondria. In this work, we have studied the effect of mitochondrial complex I inhibitors, viz. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and rotenone, and complex II inhibitor, viz. 3-nitropropionic acid, on the aggregation of mutant huntingtin (mthtt) protein, whose misfolding and aggregation results in cellular abnormalities characteristic of HD. All three inhibitors were found to accelerate the aggregation of mthtt in vitro, although the amounts of aggregates formed were different in all cases. Thus, apart from their effect on mitochondrial viability, these neurotoxins are capable of interfering with the protein aggregation process and thus, hastening the onset of the disease.     

Alterations in protein synthesis and levels of heat shock 70 proteins in response to salt stress of the halotolerant yeast Rhodotorula mucilaginosa

Responses of the halotolerant yeast Rhodotorula mucilaginosa YRH2 to salt stress was studied. Strain YRH2 was isolated from chemical industry park wastewater evaporation ponds that are characterized by large fluctuations in salinity and pH. Upon shift to high salt medium there is a shutdown of protein synthesis. Radiolabeling and separation of proteins from salt stressed and non-stressed cells identified down-regulated heat shock 70 proteins Ssb1/2p, by N-terminal sequencing and Western blotting. Ssb's role in salt stress in both R. mucilaginosa and S. cerevisiae was examined and we show that its response to salt stress and amino acid limitation is similar. Other proteins such as the heat shock 70 protein Kar2p/BiP and Protein Disulfide Isomerase were strongly induced in response to a shift to high salt in R. mucilaginosa and reacted in a manner similar to the effect of tunicamycin, a known unfolded protein response inducer. Also, assaying carboxypeptidase Y, we showed that high salt medium reduces the specific activity of the enzyme in R. mucilaginosa. It is suggested that the changes in the expression of the heat shock 70 proteins is a part of a mechanism which alleviates the damaging effects of high salt on protein folding in the yeast Rhodotorula mucilaginosa.     

A Highlights from MBoC Selection: Praja1 ubiquitin ligase facilitates degradation of polyglutamine proteins and suppresses polyglutamine-mediated toxicity

A network of chaperones and ubiquitin ligases sustain intracellular proteostasis and is integral in preventing aggregation of misfolded proteins associated with various neurodegenerative diseases. Using cell-based studies of polyglutamine (polyQ) diseases, spinocerebellar ataxia type 3 (SCA3) and Huntington’s disease (HD), we aimed to identify crucial ubiquitin ligases that protect against polyQ aggregation. We report here that Praja1 (PJA1), a Ring-H2 ubiquitin ligase abundantly expressed in the brain, is diminished when polyQ repeat proteins (ataxin-3/huntingtin) are expressed in cells. PJA1 interacts with polyQ proteins and enhances their degradation, resulting in reduced aggregate formation. Down-regulation of PJA1 in neuronal cells increases polyQ protein levels vis-a-vis their aggregates, rendering the cells vulnerable to cytotoxic stress. Finally, PJA1 suppresses polyQ toxicity in yeast and rescues eye degeneration in a transgenic Drosophila model of SCA3. Thus, our findings establish PJA1 as a robust ubiquitin ligase of polyQ proteins and induction of which might serve as an alternative therapeutic strategy in handling cytotoxic polyQ aggregates.     

Identification of novel potentially toxic oligomers formed in vitro from mammalian-derived expanded huntingtin exon-1 protein

Huntington disease is a genetic neurodegenerative disorder that arises from an expanded polyglutamine region in the N terminus of the HD gene product, huntingtin. Protein inclusions comprised of N-terminal fragments of mutant huntingtin are a characteristic feature of disease, though are likely to play a protective role rather than a causative one in neurodegeneration. Soluble oligomeric assemblies of huntingtin formed early in the aggregation process are candidate toxic species in HD. In the present study, we established an in vitro system to generate recombinant huntingtin in mammalian cells. Using both denaturing and native gel analysis, we have identified novel oligomeric forms of mammalian-derived expanded huntingtin exon-1 N-terminal fragment. These species are transient and were not previously detected using bacterially expressed exon-1 protein. Importantly, these species are recognized by 3B5H10, an antibody that recognizes a two-stranded hairpin conformation of expanded polyglutamine believed to be associated with a toxic form of huntingtin. Interestingly, comparable oligomeric species were not observed for expanded huntingtin shortstop, a 117-amino acid fragment of huntingtin shown previously in mammalian cell lines and transgenic mice, and here in primary cortical neurons, to be non-toxic. Further, we demonstrate that expanded huntingtin shortstop has a reduced ability to form amyloid-like fibrils characteristic of the aggregation pathway for toxic expanded polyglutamine proteins. Taken together, these data provide a possible candidate toxic species in HD. In addition, these studies demonstrate the fundamental differences in early aggregation events between mutant huntingtin exon-1 and shortstop proteins that may underlie the differences in toxicity.