{"doi":"10.7554/elife.18065.023","title":"Decision letter: Control of the structural landscape and neuronal proteotoxicity of mutant Huntingtin by domains flanking the polyQ tract","abstract":null,"journal":null,"year":2016,"id":47083,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Decision letter: Control of the structural landscape and neuronal proteotoxicity of mutant Huntingtin by domains flanking the polyQ tract","abstract":"Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Many neurodegenerative diseases are linked to amyloid aggregation. In Huntington's disease (HD), neurotoxicity correlates with an increased aggregation propensity of a polyglutamine (polyQ) expansion in exon 1 of mutant huntingtin protein (mHtt). Here we establish how the domains flanking the polyQ tract shape the mHtt conformational landscape in vitro and in neurons. In vitro, the flanking domains have opposing effects on the conformation and stabilities of oligomers and amyloid fibrils. The N-terminal N17 promotes amyloid fibril formation, while the C-terminal Proline Rich Domain destabilizes fibrils and enhances oligomer formation. However, in neurons both domains act synergistically to engage protective chaperone and degradation pathways promoting mHtt proteostasis. Surprisingly, when proteotoxicity was assessed in rat corticostriatal brain slices, either flanking region alone sufficed to generate a neurotoxic conformation, while the polyQ tract alone exhibited minimal toxicity. Linking mHtt structural properties to its neuronal proteostasis should inform new strategies for neuroprotection in polyQ-expansion diseases. https://doi.org/10.7554/eLife.18065.001 eLife digest Huntington's disease is a neurodegenerative disorder in which misshapen proteins accumulate in the brain and kill neurons. The misshapen proteins form as a result of specific mutations in the gene that encodes a protein called huntingtin. These mutations result in a region of the protein called the polyQ tract being longer than normal. Other regions of huntingtin that are near to the polyQ tract can dramatically change the behavior of the mutant protein. Shen et al. investigated how these regions control the shape of mutant huntingtin and how this affects the toxicity of the mutant protein in neurons. The experiments found that the two regions on either side of the polyQ tract dramatically change the shape of mutant huntingtin proteins. In the absence of these flanking regions, the extended polyQ region is not very toxic, demonstrating that the flanking sequences play important roles in generating the toxic protein shapes. These flanking regions help mutant huntingtin to form a particular shape that was strongly linked with the death of neurons in rat brain slices. The flanking regions also change the way that the cellular machinery in neurons recognizes mutated huntingtin proteins and acts to prevent them from causing harm. Misshapen forms of other proteins are responsible for causing other neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. Therefore, the findings of Shen et al. may help researchers to develop new drugs for these conditions, as well as for Huntingdon's disease. https://doi.org/10.7554/eLife.18065.002 Introduction Huntington's disease (HD) is an inherited neurodegenerative disease characterized by movement disorders, behavioral abnormalities, and brain atrophy (Orr and Zoghbi, 2007; Ross et al., 2014; Vonsattel and DiFiglia, 1998). HD arises from mutations in Huntingtin (Htt) that expand a polyglutamine (polyQ)-encoding CAG repeat in exon 1 above a threshold length of 35 Qs (MacDonald, 1993). The length of the polyQ tract correlates with both disease severity and propensity to form amyloid aggregates (Scherzinger et al., 1999). A link between neuronal toxicity and amyloid aggregation is further supported by post-mortem analyses of HD brains, which contain amyloid aggregates formed primarily by N-terminal exon 1 truncations of Htt (Difiglia, 1997; Landles et al., 2010; Mangiarini et al., 1996; Ross and Poirier, 2004). Such N-terminal fragments may arise from aberrant splicing at the Htt exon 1 junction or from caspase cleavage (Sathasivam et al., 2013; Wellington et al., 2002). Importantly, mutant Htt exon 1 carrying a polyQ expansion (herein 'mHtt-Ex1') suffices to cause HD-like disease in animal models (Goldberg et al., 1996; Sathasivam et al., 2013; Wellington et al., 2002) and is thus widely used as a relevant model for HD biology and pathology. Despite the link between aggregation and neurodegeneration, the underlying neurotoxic species in HD and other amyloid-linked neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis remains elusive. One model proposes that amyloid aggregates are toxic because they sequester and deplete essential cellular proteins such as transcription factors or molecular chaperones (Kirstein-Miles et al., 2013; Olzscha et al., 2011). However, a number of findings question the causal relationship between mHtt amyloid aggregates and toxicity. For instance, the medium spiny striatal neurons more vulnerable to HD toxicity show few to no aggregates in HD patient brains. In contrast, less affected neuronal cell-types contain many aggregates (Kuemmerle et al., 1999). In addition, while transgenic mHtt-Ex1 is neurotoxic in mice, a longer N-terminal mHtt fragment encompassing exons 1 and 2 forms many aggregates in transgenic mice but exhibits no neuronal dysfunction (Slow et al., 2005). Finally, longitudinal survival studies of primary neurons expressing fluorescently-tagged mHtt-Ex1 demonstrated that the formation of an amyloid inclusion correlated with neuronal survival (Arrasate et al., 2004). Since fluorescence and EM imaging studies have suggested that mHtt can be sequestered in different types of cellular inclusions (Caron et al., 2014; Lu et al., 2013; Nekooki-Machida et al., 2009; Sahl et al., 2016) one possible explanation for these observations is that only some inclusions are toxic, while others are protective. An alternative hypothesis proposes that toxicity resides in soluble oligomeric mHtt conformations (Behrends et al., 2006; Campioni et al., 2010; Kim et al., 2016; Miller et al., 2011; Sun et al., 2015). Biophysical studies indicate that amyloidogenic proteins, including mHtt, not only form amyloid fibrils but also populate an array of ill-defined soluble, oligomeric conformations. Studies with conformationally-sensitive antibodies indicate that these states are conformationally highly heterogeneous (Duim et al., 2014; Kayed and Glabe, 2006; Nucifora et al., 2012; Sontag et al., 2012). The transient and structurally diverse nature of these soluble species has hindered their characterization; accordingly, the nature and determinants involved in the formation of these oligomeric mHtt species remain elusive. It remains imperative to clarify the mHtt conformational landscape and link the various species – aggregates, oligomers, or even aberrant monomers – to proteotoxicity. We also must understand how various mHtt species engage the cellular protein homeostasis (or 'proteostasis') pathways that clear aberrant and aggregation-prone conformations (Martinez-Vicente et al., 2010; Ravikumar et al., 2004; Rubinsztein et al., 2012; Rui et al., 2015; Tam et al., 2006; Tsvetkov et al., 2013). mHtt interactions with the chaperones and degradation pathways that handle misfolded proteins in the cell (Balch et al., 2008; Hartl et al., 2011) are likely key determinants of the cellular balance of toxic and non-toxic conformers. Recent studies indicate that two domains flanking the expanded polyQ tract greatly influence mHtt aggregation and biology. N17, the 17 amino acid N-terminal flanking domain, has been shown to enhance mHtt aggregation (Tam et al., 2009; Thakur et al., 2009). N17 also mediates mHtt interaction with chaperones, hosts many post-translational modifications regulating mHtt toxicity (Gu et al., 2009; Steffan, 2004; Thompson et al., 2009), and harbors a functional nuclear export sequence (Maiuri et al., 2013; Rockabrand et al., 2007; Zheng et al., 2013). The C-terminal proline-rich flanking domain (herein 'PRD') also binds cellular factors and influences mHtt toxicity in yeast (Duennwald et al., 2006; Gao et al., 2014). The PRD has also been shown to slow aggregation (Bhattacharyya et al., 2006; Tam et al., 2009). Importantly, an integrated understanding of the interplay between the biophysical and cellular modulation of mHtt by these flanking regions should provide insights into the nature of proteotoxicity. Here we combine biophysical and cell biological approaches to define how the domains flanking the polyQ tract modulate the ensemble of mHtt conformations in vitro and in vivo. Importantly, we link the mHtt conformational ensemble to mHtt proteostasis and toxicity in cultured neurons and brain slices. Biophysical and structural analyses demonstrate that N17 and PRD have opposing effects on the energetic barriers dictating the formation of aggregates and oligomers by mHtt both in vivo and in vitro. This interplay between N17 and PRD determines the formation of toxic mHtt conformations and their interaction with cellular proteostasis pathways. One corollary of our data is that neuronal mHtt toxicity cannot be explained by a simple model whereby amyloid fibrils or oligomers are toxic, but rather one that points to specific toxic conformational sub-populations. Our work linking the mHtt conformational landscape with neuronal proteostasis and toxicity informs rational avenues to leverage the roles of the polyQ flanking regions for HD therapeutics. Results Opposing effects of N17 and PRD flanking domains on polyQ amyloid propensity and aggregation To evaluate the impact of N17 and PRD on the expanded polyQ tract, we created a set of mHtt-Ex1 deletion variants containing a pathogenic-length polyQ tract (Q51) and lacking the N17 (∆N), PRD (∆P), or both N17 and PRD domains (∆N∆P). These variants were compared to an otherwise identical mHtt-Ex1 with the same pathogenic polyQ tract but containing both flanking domains (Ex1) (Figure 1A). The mHtt-Ex1 variants were recombinantly expressed and purified as soluble N-terminal GST fusion proteins (Figure 1—figure supplement 1); aggregation is initiated by cleavage of the GST moiety as previously described (Scherzinger et al., 1999; Tam et al., 2006). Figure 1 with 1 supplement see all Download asset Open asset Flanking regions impact pathogenic mHtt aggregation propensity in vitro and in striatal neurons. (A) Schematic representation of polyQ expanded mutant Htt-Ex1 variants (mHtt-Ex1) used in this study. mHtt-Ex1 contains N17 (red), an expanded polyQ tract (grey), the proline-rich domain ('PRD', blue), as well as a short, 10-amino acid C terminal tail ('C', white). Variants were generated by deleting the regions flanking the polyQ domain. An additional C-terminal S tag (not shown) was used for immunoblot detection of recombinantly produced mHtt. For recombinant expression of all mHtt variants, a 51-mer polyQ tract was used. (B) Kinetics of formation of SDS-insoluble, heat-stable aggregates for the mHtt-Ex1 variants as measured by the filter trap assay. Aggregation of purified recombinant mHtt-Ex1 variants from (A) was initiated by cleavage of a solubilizing N-terminal GST tag by TEV protease. All aggregation reactions were performed at a concentration of 3 µM. Data is representative of at least three independent experiments. (C) Rate of accumulation of amyloid aggregates for the mHtt variants measured by the ThioflavinT fluorescence assay. All variants were aggregated at a concentration of 3 µM. (D) Normalized curves of ThioflavinT amyloid aggregation kinetics from (C) used to compare kinetic rates. Variants without the N17 region (∆N, ∆N∆P) form amyloids much more slowly than variants with the N17 region (Ex1, ∆P). (E) Fluorescence images of each of the mHtt mutants transfected into the ST14a striatal neuron-derived cell line. mHtt variants were constructed similar as in (A) with a 51-mer polyQ length. Instead of a N-terminal GST and C-terminal S-tag, constructs had only a C-terminal GFP tag. Images were taken 48 hr post transfection. Scale bar is 20 µm. (F) Percentage of transfected cells containing aggregates for each of the Htt mutants as shown in (E). Similar to as seen in vitro deletion of the N17 region leads to overall less aggregation while deletion of the PRD leads to overall more aggregation. Data are mean ± SEM of three independent experiments counting at least 150 cells. *p <0.05, **p<0.005, ***p<0.001. (G) Summary model for how the N17 and PRD regions contribute to mHtt aggregation propensity. https://doi.org/10.7554/eLife.18065.003 We initially characterized how these flanking regions contribute to the aggregation kinetics of purified mHtt through two complementary approaches. The filter trap assay detects large, SDS-insoluble aggregates through filtration through a 20 µm cellulose acetate membrane, (Wanker et al., 1999). Formation of β-sheet rich amyloid structures was detected using the fluorescent dye ThioflavinT (LeVine, 1999). Consistent with previous reports, deletion of N17 reduces the rate and yield of amyloid formation and aggregation, while deletion of the PRD domain enhances formation of amyloids and aggregates (Figure 1B–C, Figure 1—figure supplement 1B–C) (Crick et al., 2013; Tam et al., 2009; Thakur et al., 2009). ∆N∆P exhibited a complex, combined behavior of both N17 and PRD deletions. Similar to ∆P, the ∆N∆P mutant displayed enhanced aggregation rates measured in the filter trap assay (Figure 1B). On the other hand, similar to ∆N, ∆N∆P had very slow kinetics of amyloid formation as measured by the ThioflavinT assay (Figure 1C). Indeed, fitting the normalized ThioflavinT amyloid formation kinetics to the Finke-Watzky kinetic model (Alvarez et al., 2013; Morris et al., 2009) showed that the elongation rate v for amyloid formation and half-time to saturation of amyloid formation t1/2 for mHtt variants lacking N17 (∆N and ∆N∆P) were much slower than those for mHtt variants containing N17 (Ex1 and ∆P) (Figure 1D, Figure 1—figure supplement 1D). We conclude that the presence of a PRD disfavors formation of large, SDS-insoluble aggregates while N17 exerts a dominant effect to promote the ThioflavinT-reactive, amyloid conformation. To relate these biophysical observations to mHtt-Ex1 behavior in a neuronal cellular environment, the equivalent mHtt-Ex1 variants were fused C-terminally to GFP and expressed in striatal neuron-derived ST14a cells (Cattaneo and Conti, 1998). Formation of GFP-inclusions provided a read-out for the aggregation propensity of the mHtt variants in vivo. As observed in vitro, deleting N17 reduced the formation of visible inclusions in vivo, while deleting PRD enhanced the formation of aggregates (Figure 1E–F). Notably, few aggregates were visible in the ∆N∆P expressing cells, despite rapid formation of insoluble aggregates in vitro. Given the slow kinetics of amyloid aggregation by ∆N∆P in vitro, it is possible that in the absence of the N17 and PRD flanking regions, the polyQ tract does not efficiently generate amyloidogenic fibrils but instead forms non-amyloidogenic aggregates that are less stable in vivo (Crick et al., 2013). We conclude that N17 and PRD have opposing effects of on amyloid formation and aggregation in vitro and in vivo (Figure 1G) and further suggest that the cellular environment destabilizes the non-amyloid aggregates generated by the polyQ tract in ∆N∆P. N17 and PRD control the morphology of mHtt amyloid fibrils Next, we used cryo-electron microscopy (cryo-EM) to gain a structural understanding of how N17 and PRD impact the formation of mHtt amyloid fibrils. mHtt-Ex1 fibrils have a characteristic architecture, in which frayed fibril ends branch out from a bundled central core (Figure 2A, Figure 2—figure supplement 1A) (Bugg et al., 2012; Darrow et al., 2015; Shahmoradian et al., 2013). For the ∆N mHtt variant, we observed dramatically fewer fibrils, consistent with its lower amyloid aggregation propensity (Figure 1). In addition, the fibrils formed by ∆N had a strikingly distinct morphology, which lacked the bundled architecture of Ex1 fibrils and were much thinner and straighter (Figure 2—figure supplement 2). Allowing ∆N aggregation to reach saturation by prolonged incubation increased the number of fibrils but did not change their thin morphology (Figure 1C, Figure 2—figure supplement 1B). Thus, the thin fibril structure of ∆N aggregates is intrinsic to the mutation. In contrast, ∆P formed many large, densely packed aggregates with individual fibrils arranged in parallel bundles (Figure 2A, Figure 2—figure supplement 2), consistent with its increased aggregation propensity. As observed for kinetic measurements, the morphology of ∆N∆P aggregates combined properties from both the ∆N and ∆P fibrils. Similar to ∆N fibrils, the ∆N∆P fibrils were shorter, thinner and lacked the frayed fibril ends observed for Ex1 (Figure 2—figure supplement 2); similar to ∆P fibrils, ∆N∆P aggregates consisted of more densely packed fibrils (Figure 2A). Quantification of at least 10 individual micrographs for each fibril variant supported these observations, indicating that ∆N fibrils were only several nanometers in width, whereas Ex1 and ∆P fibrils were on average almost a micron wide and over a micron long (Figure 2—figure supplement 2). We conclude that N17 and PRD have independent and dramatic effects on the amyloid formation propensity of the polyQ tract and its fibrillar structure. N17 promotes amyloid formation but also enhances interfibrillar contacts, thereby driving the bundling of individual fibrils observed for Ex1 and ∆P (Figure 2B). In contrast, PRD appears to destabilize lateral contacts among fibrils and thus prevents their dense packing (Figure 2B). The combined effects of N17 and PRD in Ex1 lead to the characteristic mHtt structure consisting of fibril bundles with frayed ends (Figure 2C). When N17 is absent, PRD leads to thin sparse fibrils observed for ∆N; while when PRD is absent, N17 leads to thick, bundled aggregates as observed for ∆P (Figure 2C). Figure 2 with 2 supplements see all Download asset Open asset Morphology of mHtt aggregation highlight how N17 and PRD contribute to mHtt aggregation. (A) Morphology of fiber aggregates from each of the mHtt mutants by cryo-EM. mHtt fibers were imaged 24 hr post initiation of aggregation. Scale bar is 500 nm. Top row is the cryo-EM micrographs. The second and third rows are traced annotations of the EM micrographs and zoom-ins of individual areas, respectively. Bottom row is the original cryo-EM micrograph of the zoomed-in area. (B) Summary of the N17 and PRD contributions to aggregation morphology and propensity. (C) Summary model for how the N17 and PRD regions direct morphology of mHtt aggregates. Morphology differences between ∆N∆P and Ex1 showcase how the flanking regions impact mHtt aggregation in a combinatorial manner. https://doi.org/10.7554/eLife.18065.005 The PRD domain controls mechanical stability of mHtt amyloid aggregates Our model predicts that PRD destabilizes the interfibrillar contacts within an aggregate. This effect may be of importance for HD in light of the prion hypothesis that postulates that intercellular transmission of aggregate 'seeds' can nucleate aggregation in naïve cells (Pearce et al., 2015). For prions, distinct fibrillar amyloid stability is a hallmark of different prion strains (Tanaka et al., 2006) as their reduced stability (or increased 'frangibility') is directly linked to their propensity for intercellular transmission (Cushman et al., 2010). We thus tested if the polyQ flanking regions change the mechanical stability of the mHtt amyloid aggregates. Amyloid aggregates of all mHtt variants were gently isolated by centrifugation and subjected to different sonication conditions to test their stability to mechanical disruption (Figure 3A, Figure 3—figure supplement 1A). The size and morphology of the resulting species were measured by Dynamic Light Scattering (DLS) and cryoEM, respectively (Figure 3B–C, Figure 3—figure supplement 1B). Figure 3 with 2 supplements see all Download asset Open asset The PRD region mechanically destabilizes mHtt aggregates. (A) mHtt fibers were isolated by centrifugation and sonicated to test their mechanical stability. Sonicated species were analyzed by cryo-EM imaging and Dynamic Light Scattering (DLS). (B) Size comparisons between sonicated mHtt aggregates lacking PRD (∆N∆P, ∆P) and mHtt variants with the PRD (Ex1, ∆N) as measured by DLS. Sonication of mHtt variants containing the PRD results in much smaller fiber fragments than those lacking PRD. Data are mean ± SEM. (C) Cryo-EM images of sonicated fibers for mHtt variants lacking PRD (∆N∆P, ∆P) or mHtt variants with the PRD (Ex1, ∆N). Scale bar is 100 nm. https://doi.org/10.7554/eLife.18065.008 DLS analyses confirmed that PRD significantly reduced the mechanical stability of mHtt aggregates. mHtt fibrils formed by variants containing a PRD, namely Ex1 and ∆N, were readily fragmented into small,    of   in size (Figure  In contrast, aggregates formed by mHtt variants lacking a PRD, namely ∆P and   disruption and  in  and highly bundled fibrillar structures  from  µm even at the  sonication conditions (Figure 3B–C, Figure 3—figure supplement 1). Cryo-EM imaging confirmed and extended these observations (Figure  The sonicated  of Ex1 and ∆N were  structures only one or two fibrils in width, while the sonication  of ∆P and ∆N∆P were large, thick, and  bundles of    the presence or absence of N17 had no  impact on the size of the fragmented  indicating that while N17  to the formation of these fibrils, it does not contribute to their mechanical stability. We conclude that the PRD mechanically destabilizes mHtt aggregates by   contacts (Figure  thus  a molecular  for reduced aggregation propensity of mHtt variants with a PRD (Figure 1). We    the PRD domain also  to the  stability of mHtt fibrils to protein   such as  or   The isolated amyloid aggregates of Ex1 and ∆P were initially  with   which was  to  either fibril variant (Figure 3—figure supplement  We  subjected  fibrils with   of the much    acid (Figure 3—figure supplement   Ex1 and ∆P fibrils were   to   of  acid and only  incubation with    which can  mHtt aggregates formed in vivo  et al.,  did we    of the mHtt fibrils of both Ex1 and ∆P (Figure 3—figure supplement  These experiments suggest that the mechanical stability differences for fibrils with or without the PRD  from their structural architecture rather than intrinsic differences in their  stability. N17 and PRD regions shape the conformational landscape of oligomeric mHtt species We   how the polyQ flanking regions impact the conformational ensemble of soluble, oligomeric mHtt species by  the kinetics of formation and stability of soluble, oligomeric species by mHtt flanking region variants  and  2007; Nucifora et al., 2012; Sontag et al.,  (Figure   different   TEV cleavage of the GST     were used to  the formation of oligomeric species  than   (Figure   et al., 2012;  et al.,  while       were used to  species smaller than   (Figure   species  than   were    (Figure  Figure  supplement 1A) or     conditions (Figure  Figure  supplement 1B).  each  the filter trap assay was used to  formation of  SDS-insoluble, amyloid aggregates (Figure  filter  Figure  with 1 supplement see all Download asset Open asset N17 and PRD direct the formation and  of oligomer  (A) Schematic of oligomer and aggregate  generated  the mHtt aggregation  We characterized the oligomer  using      either  or     conditions or       were isolated by   of the in vitro mHtt aggregation   at the same conditions as in Figure  (B)     oligomers for the mHtt  ∆N generated a   of mHtt oligomeric species that  through the aggregation  whereas ∆P generated very few oligomeric species that   from the   was  for the C-terminal  on the mHtt  (C)       oligomers over    was  for the C-terminal  on the mHtt   SDS-insoluble mHtt aggregates  not  the   as shown by the  filter trap   filter  ∆N  few aggregates because the protein remains  in the oligomeric  (D)       oligomers    Aggregation  were  in a    and  for the   and the  conformational    only recognizes the smaller oligomers of the mHtt variants  ∆N, and  (E)  landscape model of the different conformational species in the mHtt-Ex1 aggregation   Aggregation through the ∆N or ∆P pathways  the energetic barriers between the oligomeric and fibrillar states of mHtt     analyses  that  oligomers of Ex1    aggregation kinetics and   as aggregation  to form the aggregates  in the filter trap (Figure   et al., 2012). In contrast,   oligomers  to  over  in the   (Figure   contrast, ∆P had much fewer oligomers in both  and    the  oligomers  much more   the  of aggregation that those observed for  consistent with the overall  aggregation of  Thus, the absence of PRD  the balance between oligomer and fibril formation,  by    of these oligomeric species to aggregates (Figure   Surprisingly, very  ∆N oligomers  through the aggregation  in both the  and   (Figure      when ∆N amyloid aggregation was  the  of ∆N oligomers   both  and   conditions (Figure  supplement 1B). Consistent with our previous  the increased presence of ∆N oligomers  in few   aggregates (Figure 1B–C,  We  if these   ∆N oligomers  to the small, thin ∆N amyloid fibrils observed by cryo-EM (Figure 2A). However, while  ∆N oligomers were   as  as 3 hr  (Figure  cryo-EM imaging of ∆N at 3 hr of aggregation showed very few fibrils and no additional   or structures (not   the   species formed by ∆N only  significantly   hr of aggregation (Figure 1C).  these data indicate that ∆N    stable soluble oligomers that may be   but are  fibrillar   These ∆N oligomers, despite being very  in   to be  structurally heterogeneous to yield    by  We   the impact of the polyQ flanking regions on the formation of soluble oligomer  smaller than    et al., 2012;  and    (Figure  The aggregation  was initiated by TEV cleavage of the  and  analyzed by   (Figure   all mHtt variants formed several  oligomer species very  in the aggregation  For Ex1 and ∆P, these  oligomers  as aggregation  while the species of ∆N and ∆N∆P were very  similar to that observed for  oligomers in   (Figure  This   the  that N17 promotes the formation of fibrils (Figure  at the  of oligomer formation, whereas the PRD  to the accumulation of   N17 and PRD  to act  in oligomer formation  the oligomers formed by ∆N∆P exhibited a behavior  between the ∆N and ∆P  in the  oligomer   a   of ∆N∆P species  to the insoluble aggregates (Figure  filter  a  of  oligomers  in the   and in the    It thus appears that N17 and PRD have independent and  effects on the conformational landscape of the polyQ (Figure  N17 promotes amyloid formation and fibril bundling and prevents the accumulation of non-amyloidogenic oligomeric  On the other hand, PRD disfavors aggregation by  the  of soluble oligomeric","is_dataset_classified":null,"base_score":0.0,"endowment":0.0,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"18998783","pmcid":null,"openalex_id":"https://openalex.org/W4230171860","authors":[],"funders":[],"total_grants":0,"fwci":null,"citation_percentile":null,"influential_citations":0,"citation_trend":[],"oa_status":"gold","license":"cc-by","oa_locations":[{"url":"https://doi.org/10.7554/elife.18065.023","host_type":""},{"url":"https://doi.org/10.7554/elife.18065.023","host_type":""}],"fields_of_study":["Genetic Neurodegenerative Diseases","Mitochondrial Function and Pathology"],"mesh_terms":[],"keywords":["Huntingtin","Polyglutamine tract","Proteotoxicity","Proteostasis","Huntingtin Protein","Protein aggregation","Mutant","Biology","Amyloid (mycology)","Cell biology","HSF1","Chemistry","Genetics","Gene","Heat shock protein","Hsp70"],"sdg_mappings":[{"sdg_number":0,"sdg_label":"Life in Land"}],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-07-16T04:25:48.892285Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}