Abstract
BACKGROUND Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disease caused by expansion of a CAG repeat in Ataxin-2 (ATXN2) gene. The mutant ATXN2 protein with a polyglutamine tract is known to be toxic and contributes to the SCA2 pathogenesis.
OBJECTIVE Here we tested the hypothesis that the mutant ATXN2 transcript with an expanded CAG repeat (expATXN2) is also toxic and contributes to SCA2 pathogenesis.
METHODS The toxic effect of expATXN2 transcripts on SK-N-MC neuroblastoma cells and primary mouse cortical neurons was evaluated by caspase 3/7 activity and nuclear condensation assay, respectively. RNA immunoprecipitation assay was performed to identify RNA binding proteins (RBPs) that bind to expATXN2 RNA. Quantitative PCR was used to examine if rRNA processing is disrupted in SCA2 and Huntington disease (HD) human brain tissue.
RESULTS expATXN2 RNA induces neuronal cell death, and aberrantly interacts with RBPs involved in RNA metabolism. One of the RBPs, transducin β-like protein 3 (TBL3), involved in rRNA processing, binds to both expATXN2 and expanded huntingtin (expHTT) RNA in vitro. rRNA processing is disrupted in both SCA2 and HD human brain tissue.
CONCLUSION These findings provide the first evidence of a contributory role of expATXN2 transcripts in SCA2 pathogenesis, and further support the role expHTT transcripts in HD pathogenesis. The disruption of rRNA processing, mediated by aberrant interaction of RBPs with expATXN2 and expHTT transcripts, suggest a point of convergence in the pathogeneses of repeat expansion diseases with potential therapeutic implications.
Introduction
Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant disorder caused by a CAG repeat expansion in the first exon of the ATXN2 gene located on chromosome 12q24 1. The repeat is in-frame to encode polyglutamine (polyQ). The signs and symptoms of SCA2 include progressive deterioration in balance and coordination, neuropathies, nystagmus and slow saccadic eye movements, slurred speech and cognitive impairment 2-5. SCA2 is the second most common form of autosomal dominant ataxia, with a prevalence of 1-2 cases/105 inhabitants, varying somewhat by ethnicity and geographic location 2, 3, 6-8. The highest prevalence of the SCA2 mutation occurs in Cuba (6.57 cases/105 inhabitants) 9, and is likely a consequence of a founder effect 10. SCA2 neuropathology is characterized by a significant loss of cerebellar Purkinje neurons, a less prominent loss of cerebellar granule cells 11; marked neuronal loss in the inferior olive, pontocerebellar nuclei, and substantia nigra; degeneration of the thalamus and pons, and thinning of the cerebellar cortex without changes in neuronal density 11-14. The normal ATXN2 allele contains 15 to 32 CAG triplets, while the disease allele typically has 33 to 64 triplets 15. The most common disease allele has 37 triplets, and neonatal onset SCA2 cases with over 200 CAG repeats have been reported 16. Similar to other CAG repeat diseases, the repeat length in SCA2 is inversely correlated to age of onset 17, 18.
Recently, intermediate CAG expansion in ATXN2 has been associated with a higher risk for amyotrophic lateral sclerosis (ALS) 19. Current evidence indicates that neurotoxicity of ATXN2 protein, which is involved in multiple cellular pathways, including mRNA maturation, translation, and endocytosis, is central to SCA2 pathogenesis 20, 21. This is supported by data from several SCA2 cell and mouse models expressing mutant ATXN2 protein 22-24.
However, multiple laboratories, including ours, have demonstrated an important neurotoxic role for mutant RNA transcripts in CAG/CTG repeat expansion diseases, including myotonic dystrophy type 1 (DM1)25, 26, Huntington disease (HD) 27, 28, Huntington disease-like 2 (HDL2) 29, SCA3 30-32, and SCA8 33. RNA-triggered pathogenic processes are thought to be, at least in part, mediated by aberrant interaction between expanded repeat-containing RNA transcripts and RNA-binding proteins (RBPs) 34-36. The basic hypothesis is that expanded CAG/CUG repeats in transcripts form hairpin structures which sequester multiple RBPs and hence prevent the RBPs from performing their normal function in cells 35. To add to the pathomechanistic complexity of CAG/CUG repeat diseases, antisense transcripts that span the CAG/CUG repeat regions are also expressed at the DM1 (CAG direction)37, HDL2 (CAG direction)38, 39, SCA7 (CUG direction)40, SCA8 (CUG direction) 33 and HD (CUG direction)41 loci. We have recently described a transcript expressed antisense to ATXN2 at the SCA2 locus 42, and provided evidence that this antisense ATXN2 (ATXN2-AS) transcript contributes to SCA2 pathogenesis, and potentially to ALS associated with an intermediate repeat expansion at the ATXN2 locus 19. We hypothesized that, in addition to mutant ATXN2 protein and mutant ATXN2-AS transcript 42, mutant sense ATXN2 RNA also contributes to SCA2 pathogenesis.
As predicted by this hypothesis, the data presented here demonstrate that sense expATXN2 transcripts are neurotoxic in cell models in the absence of expression of mutant ATXN2 protein, aberrantly interact with RBPs that are involved in rRNA processing, and lead to disruption of rRNA processing. We demonstrate a similar disruption of rRNA processing in HD patient brain tissue. Similar to findings in other repeat expansion diseases, SCA2 is therefore the fifth neurodegenerative CAG/CTG repeat expansion disease in which pathogenesis is likely a consequence of a combination of expression of mutant protein and bi-directionally expressed mutant RNA.
Materials and Methods
Description of materials and methods is provided in the supplementary data.
Results
The non-translatable expATXN2 transcript is neurotoxic
To confirm the toxicity of expATXN2 transcripts, we cloned FL ATXN2 cDNA with 22, 58, or 104 CAG triplets into the 3’ untranslated (UTR) region of Renilla luciferase (Rluc) cDNA, thereby allowing expression of expATXN2 RNA transcripts, but preventing ATG-initiated translation of the RNA into FL ATXN2 protein (Fig. 1A). No evidence of expression of an expanded polyglutamine tract was detected by Western blotting using the expanded polyglutamine-specific antibody 1C2 43, confirming that the 3’ UTR cloning approach indeed eliminated detectable ATG-initiated translation of the FL ATXN2 (Fig. 1B). Caspase 3/7 activity assay showed that overexpression of Rluc-ATXN2-(CAG)58 or Rluc-ATXN2-(CAG)104 was significantly more toxic than Rluc-ATXN2-(CAG)22 in SK-N-MC cells (Fig. 1C). Comparable expression levels of overexpressed transcripts in SK-N-MC cells were confirmed by qPCR (Fig. 1D). However, hairpin-forming expanded CAG repeats can also be translated in the absence of ATG start codon through the mechanism of repeat-associated non-ATG translation (RAN translation) 44.
To exclude the possibility that RAN translation of protein fragments with expanded amino acid tracts leads to neurotoxicity in our SK-N-MC model system, we cloned an ATXN2 fragment containing a CAG repeat expansion, multiple upstream stop codons, and 150 bp of ATXN2 sequence flanking the repeat (thereby excluding all ATGs) into a vector with tags for each of the three open reading frames (Fig. S1A). There were no detectable protein fragments from any of the three reading frames (Fig. S1B-E), indicating that SK-N-MC cells do not support the RAN translation of expATXN2 transcripts, and confirming that expression of FL expATXN2 transcript is sufficient to trigger neurotoxicity even when the transcripts are not translated into proteins.
Consistent with these observations in neuroblastoma cells, overexpression of Rluc-ATXN2-(CAG)104 triggers neurotoxicity in primary mouse cortical neurons, as measured by nuclear condensation assay (Fig. 1E). Rluc-ATXN2-(CAG)58 was not toxic in this assay, perhaps reflective of the short time frame of the experiment (nuclear condensation is a later stage event, whereas caspase 3/7 activation occurs at an early stage in cell death), differences in the levels of transcript expression in primary neurons compared to neuroblastoma cell lines, or different sensitivity to transcript-induced toxicity in primary neurons and SK-N-MC cells.
It has been suggested that the CAG repeats form stable hairpin structures 35, while CAA interruptions either break hairpin regularity or induce the formation of branched structures 45. To examine whether preventing the formation of hairpin structures in expATXN2 ameliorate its neurotoxicity, we replaced the pure CAG repeat region in the ATXN2-(CAG)104 with a fragment of heavily interrupted CAG/CAA triplets, to obtain the ATXN2-(CAG/CAA)105 construct. Inserting interruptions abolished expATXN2 toxicity in primary mouse cortical neurons (Fig. 1F), suggesting that the secondary hairpin structure adopted by the pure CAG repeat may be critical for neurotoxicity.
Full-length expATXN2 transcripts form RNA foci in SCA2 cell and mouse models, and in one human SCA2 brain
Repeat-containing mutant transcripts form RNA foci in all CUG/CAG diseases in which RNA neurotoxicity has been demonstrated to contribute to pathogenesis 46-48. We therefore sought to detect similar foci in SCA2 models and human brain by fluorescence in situ hybridization (FISH). CAG RNA foci were absent in SK-N-MC neuroblastoma cells that overexpress GFP alone (Fig. 2A) or a FL ATXN2 construct modified to have only one CAG triplet (GFP-ATXN2Q1, Fig. 2B), and were only rarely detected in cells overexpressing FL normal ATXN2 (nATXN2) transcripts with 22 triplets (GFP-ATXN2Q22, Fig. 2C). Foci were much more abundant in cells overexpressing full-length (FL) expanded ATXN2 (expATXN2) transcripts with 58 or 104 CAG triplets (GFP-ATXN2Q58 or GFP-ATXN2Q104, Fig. 2D and 2F), as quantified in Fig. 2E. The foci are resistant to DNase treatment and are degraded by RNase treatment (Fig. 2G and 2H).
This set of experiments demonstrates that expATXN2 transcripts form RNA foci, and that the extent of foci formation may at least partially correlate with repeat length. Furthermore, while not detected in wildtype (WT, Fig. 2I-J) mice, ATXN2 RNA foci are present in cerebellar Purkinje neurons of SCA2 transgenic mice (Fig. 2K-L) which express FL ATXN2 with 127 CAG triplets specifically in Purkinje neurons 22. Finally, out of the five human postmortem brains available for this study, ATXN2 RNA foci were detected in cerebellar Purkinje cells in one brain (H1 case, Table 1) that had 38 triplets for the mutant allele (Fig. 2O-P), but not in the control human brains (Fig. 2M-N). RNA foci may be only a hallmark for RNA toxicity, and whether RNA foci are toxic or not remains to be further determined.
expATXN2 transcripts aberrantly interact with RNA binding proteins (RBPs)
We next examined whether the neurotoxicity of expATXN2 transcript is mediated by aberrant expATXN2 RNA-RBP interactions. We performed an in vitro biotinylated ATXN2 RNA pull-down assay (Fig. 3A) and identified by mass spectrometry (MS) a total of 57 RBPs that preferentially bind to the expATXN2, compared to the nATXN2 transcript. Go analysis of functional annotation49 and STRING analysis50 of the expATXN2 RBPs are shown in Fig. S2. A selective list of expATXN2 RBPs is shown in Table S1. Out of the 57 expATXN2 RBPs, 40 are localized in the nucleus, with 20 of them in the nucleolus, suggesting that aberrant expATXN2-RBP interactions may predominantly occur in the nucleus. Interestingly, among the 20 nucleolar RBPs, 7 of them contain WD40 repeat domains, of which, five (PWP1, TBL3, WDR3, WDR36 and UTP18; Table S1 and Fig. S2B) are components of the small subunit (SSU) processome for ribosomal RNA (rRNA) processing. We therefore became interested in the SSU processome components that were identified as expATXN2 RBPs. Out of the five SSU components 51, 52 that are potential expATXN2 interactors, we selected TBL3 (transducin β-like protein 3) for further analysis, as we were interested in RNA mediated disease mechanisms shared by both SCA2 and HD, and by the same method, TBL3 appeared to interact with the expanded Huntingtin (expHTT) transcript as well (Fig. 3B-C), and has a relatively greater number of peptide hits and percentage of protein coverage, compared with other SSU components identified by MS (Table S1), though the number of peptide hits does not always imply stronger interaction 53.
TBL3 binds to expanded CAG repeats in vitro
We performed additional RNA pull down experiments and western blots to confirm that TBL3 interacts with expATXN2 in vitro (Fig. 3B). To test whether the interaction is disease-specific, we also included expHTT transcripts, associated with HD, the most prevalent and most studied CAG repeat disease 27, 28, 41, 54. Studies from multiple laboratories, including ours, support the idea that RNA neurotoxicity contributes to HD 27-29, 47 We confirmed that TBL3 interacts in vitro with expanded CAG repeats flanked with either ATXN2 or HTT-specific sequence (Fig. 3B), but not with expanded CUG repeats flanked with either antisense ATXN2 (ATXN2-AS; 42), antisense HTT (HTT-AS; expressed on HD locus)41, or junctophilin-3 (JPH3) flanking sequence 29 (Fig. 3B). To further confirm that the interaction between TBL3 and expATXN2 and expHTT was dependent on the CAG repeat, we pre-incubated expATXN2 and expHTT transcripts with (CTG)8C Morpholino (MO), which we have previously established hybridizes to CAG repeat expansions 55. The pretreatment with (CTG)8C prevented TBL3 from binding to either transcript in vitro and provided further evidence that both expATXN2-TBL3 and expHTT-TBL3 interactions are dependent on the presence of an expanded CAG repeat (Fig. 3C). Taken together, these data indicate that TBL3 binds to expanded CAG repeats independent of flanking sequence.
To investigate whether TBL3 binds to expanded CAG repeats independently of other cellular proteins, we purified the TBL3 N-terminal RNA binding domain as a fusion with maltose binding protein (MBP-NTD-TBL3) and measured its binding with expATXN2 transcripts using an in vitro nitrocellulose filter binding assay 56. The isolated TBL3 NTD associated with ATXN2 CAG RNA, with KD =350 nM and 420 nM for ATXN2-(CAG)22 and ATXN2-(CAG)108, respectively (Fig. 3D). Although overall binding was weak, the yeast homolog of TBL3, Utp13, binds pre-rRNA as a tetramer with other UtpB complex proteins. Therefore, the weak affinity of the isolated MBP-NTD-TLB3 for expATXN2 may be due to the absence of its normal binding partners. The in vitro binding reactions saturated approximately 20-30% of refolded ATXN2 RNA, suggesting that a fraction of the ATXN2 RNA is unable to refold into a conformation that is competent to bind TBL3.
Despite its weak affinity for RNA, MBP-NTD-TBL3 bound ATXN2 CAG repeats more strongly than control RNAs, including the ATXN2-AS-(CUG)110 transcript (KD=650 nM), and a CAG repeat containing CAA interruptions, ATXN2-(CAG/CAA)105 (KD=2.1 μM). This preference for continuous CAG repeats raised the possibility that TBL3 recognizes the hairpin structure of CAG repeat RNA. To test this idea, the filter binding assays were also carried out in the presence of a competitor yeast tRNA, which is expected to be structured under our assay conditions. The tRNA competitor abolished the interaction between MBP-NTD-TBL3 and ATXN2 or ATXN2-AS transcripts (Fig. 3D), consistent with the idea that TBL3 binding depends on the structures of ATXN2 CAG repeats.
The effect of TBL3 reduction on 45S pre-rRNA level and processing
Depletion of UTP13, the yeast homolog of TBL3, increases the steady-state level of unprocessed 35S pre-rRNA in yeast 57. We therefore hypothesized that, although the interaction between expATXN2 and TBL3 may not be direct and likely involves other proteins, sequestration of TBL3 in a complex that interacts with expATXN2 may disrupt its normal function and affect rRNA maturation. We therefore predicted that knockdown of TBL3 in cells, mimicking its sequestration, would increase the level of unprocessed 45S pre-rRNA, the human counterpart of yeast 35S pre-rRNA. Three individual siRNAs were used to knock down TBL3 in HEK293T cells in order to minimize the possibility of alternative mechanisms of TBL3 reductions through off-target effects. Each siRNA reduced TBL3 protein level by 50-80% at 72 hours post transfection (Fig. 4A-B). Next, we examined 45S pre-rRNA levels by qPCR using primers against the 5’ external transcribed spacer 58 as indicated in Fig. 4C. Knockdown of TBL3 in HEK293T cells using each siRNA increased steady-state 45S pre-rRNA levels (normalized to ACTB, Fig. 4D), consistent with a previous study using stable shRNA transfection 59. As previously reviewed 60, 61, a complex sequence of cleavage steps is required to release the mature RNAs (18S, 5.8S and 28S) from the precursor 45S pre-rRNA. qPCR using primers against 18S rRNA would detect the mature 18S rRNA, unprocessed 45S pre-rRNA, as well as any intermediate rRNAs containing 18S sequence. Similarly, qPCR using primers against 28S rRNA would detect the mature 28S rRNA, the 45S pre-rRNA precursor, as well as intermediate rRNAs that contain the 28S sequence (Fig. 4C). We therefore used the ratios of 18S rRNA/45S pre-rRNA and 28S rRNA/45S rRNA measured by qPCR, as readouts for 18S rRNA maturation and 28S rRNA maturation, respectively. Depletion of UTP13 in yeast has been previously shown to decrease 18S rRNA maturation 62, 63. Consistently, we found that knock down of TBL3 in HEK293T cells decreased the ratio of 18S rRNA to 45S pre-rRNA (Fig. 4E), indicating that TBL3 may play a role in 18S rRNA maturation. In addition, 28S rRNA maturation was also decreased after TBL3 knock down (Fig. 4F). We attempted to determine if overexpression of TBL3 has the opposite effect, however, forced expression of TBL3 by itself triggered toxicity and mis-localized the protein into nuclear aggregates (data not shown). Similarly, MBNL1, an RBP previously shown to interact with expanded CAG/CUG (expCAG/CUG) transcript also appeared to be toxic when overexpressed or knocked down 28, suggesting that expression of certain RBPs must be tightly controlled to maintain their normal function.
45S pre-rRNA level and processing is altered in SCA2 and HD postmortem tissue
Finally, we examined the expression of 45S pre-rRNA in human postmortem SCA2 and HD cerebella. qPCR amplification suggested that there was a slight, though not statistically significant, increase of 45S pre-rRNA level (normalized to ACTB) in both SCA2 and HD cerebella, compared with control (Fig. 4G). The qPCR results suggested that there was a decrease in both 18S rRNA maturation and 28S rRNA maturation in SCA2 and HD cerebella, compared with the controls (Fig. 4H-I), consistent with the trend observed with the knock down of TBL3 (Fig. 4D-F). Only the decrease in HD samples, but not in SCA2 samples, reached statistical significance, under the caveat that the relatively lower statistical power in SCA2 samples may not allow the detection of small changes. Taken together, the data supports the idea that aberrant RNA-RBP interactions may affect the steady state level and the maturation of 45S pre-rRNA in both SCA2 and HD.
Discussion
We have previously shown that antisense ATXN2-AS transcripts contribute to SCA2 pathogenesis42. Here we provide the first evidence that sense expATXN2 transcripts is involved in SCA2 pathogenesis. First, in establishing its potential pathogenicity, we show that untranslatable FL ATXN2 transcript is neurotoxic (Fig. 1). This model is not suitable to test the contribution of sense ATXN2 transcript relative to the toxicity of ATXN2 protein, or antisense ATXN2-AS RNA 42, because neither ATXN2 protein nor ATXN2-AS RNA is present in this model. In the future, genome editing approaches can be used to establish SCA2 iPS cell models that specifically model protein-versus RNA-triggered mechanism of pathogenesis. SCA2 iPSCs can also be subjected to transcriptome, proteome, and RNA interactome analysis to identify additional pathways that are involved in RNA-mediated aspects of SCA2 pathogenesis. Differentiation into neuronal types of greater and lesser selective vulnerability in SCA2 (e.g., Purkinje cells, cortical excitatory neurons, etc.) could be used to determine cell type vulnerability to RNA and protein mediated neurotoxicity. Next, we show that the expATXN2 transcripts aggregate into nuclear RNA foci in SCA2 cell and transgenic mouse models, as well as in human SCA2 postmortem brain tissue. However, out of five human SCA2 postmortem brains available for this study, expATXN2 RNA foci were only detected in case H1 that had the 22/38 ATXN2 CAG repeat lengths and the latest disease on-set (Table 1). Interestingly, we have recently characterized a transcript that is expressed in the direction antisense to ATXN2 (ATXN2-AS) and contains an expanded CUG repeat 42. CUG RNA foci containing this transcript were detected in SCA2 cases K3 and M1 (Table 1; 42). Given that SCA2 is associated with a relatively short repeat expansion, detection of foci may require a more sensitive assay. There are a number of alternative explanations for the absence of foci in the other SCA2 brains: (1) CAG RNA foci are highly toxic, or appear in cells marked for early death, such that Purkinje cells with foci may not have been present by the time of death; (2) CAG RNA foci are protective and associated with late onset disease and perhaps slower disease progression; (3) detectable foci were lost consequent to the process of brain collection or storage; (4) RNA foci are a byproduct of neurotoxic processes and have a neutral role in neurotoxicity; 5) RNA foci are an epiphenomenon, present in some SCA2 brains because of an unknown genetic or environmental factor and with no relevance to disease. Recent work describing a transgenic BAC mouse model expressing expanded C9orf72 (expC9orf72) and exhibiting widespread RNA foci, but lacking behavioral abnormalities and neurodegeneration, even at advanced ages, suggests that RNA foci are not sufficient to trigger toxicity in ALS 64. A transgenic mouse model expressing non-translatable FL ATXN2, which could be tracked in live cells in real time 65 might help determine the relevance of CAG RNA foci to disease pathogenesis.
Our data strongly suggests that the neurotoxicity of expATXN2 transcript involves aberrant expATXN2-RBP interactions that perturb rRNA maturation. We initially focused on Transducin beta like protein 3 (TBL3), a component of the SSU processome required for rRNA processing. While our RNA pull-down assay indicates that TBL3 interacts with expATXN2 RNA, this assay cannot be used to prove a direct interaction. Filter-binding assays showed that recombinant TBL3 NTD can weakly interact with expATXN2 RNA, and preferentially binds the structures of the CAG repeats. (Fig. 3D). Other components of the SSU processome likely stabilize the interaction of TBL3 with the expATXN2 RNA in the cell. The yeast homolog of TBL3, Utp13, recognizes double-stranded regions of the pre-rRNA as a heterotetramer with other Utp proteins. Indeed, mass spectrometry analysis of expATXN2 interactors did identify other proteins from the SSU processome in our isolated complexes (Table S1). One interesting possibility is that the multi-dentate recognition of structured RNA by TBL3 and its binding partners, which is a normal feature of their function in pre-rRNA processing, also contributes to the toxic aggregation of CAG repeat RNAs. Future experiments will be needed to determine which proteins are most important for neuronal toxicity.
Our results indicate that a subset of RBPs bind to both expanded ATXN2 and HTT transcripts. This is not surprising, as it is well established that transcripts containing expanded CAG repeats form similar secondary structures in vitro 35, 66, 67 and, hence, at least some of the downstream effects are likely to be shared between different CAG repeat diseases. While SCA2 primarily affects cerebellum, HD is primarily characterized by atrophy of striatum and cerebral cortex 68. However, recent evidence indicates that cerebellum is also affected in HD 69, 70 and, in fact, appears to degenerate early 71 and independently from the striatal atrophy 71. This suggests that similar mechanism of pathogenesis may contribute to cerebellar pathology in both SCA2 and HD. Whether and to which degree mutant RNA-triggered mechanisms contribute to this pathology, remain to be further determined.
Interestingly, it was previously reported that expanded ataxin-3 (ATXN3) transcripts, involved in spinocerebellar ataxia type 3 (SCA3) interact with nucleolin. In SCA3, this aberrant nucleolin-ATXN3 interaction decreases 45S pre-rRNA levels in cell and Drosophila models of SCA3 31. On the other hand, aberrant interaction between the expC9orf72 transcripts and nucleolin, may contribute to the decreased maturation of 28S, 18S and 5.8S rRNAs from the precursor 45S pre-rRNA in ALS patients associated with CCCCGG hexamer expansion in C9orf72 gene 72. It is quite possible that a therapeutic agent that prevents aberrant RNA-RBP interactions between toxic hairpin-forming transcripts and RBPs may be at least partially effective across multiple diseases. Alternatively, similar therapies may target shared pathogenic pathways downstream of the toxic transcripts.
In summary, we provide the first evidence that the ATXN2 transcript with an expanded repeat may contribute to SCA2 pathogenesis, with similar properties to transcript-mediated toxicity in HD. The ATXN2 transcript with an expanded CAG repeat itself, or its protein interactors, may provide valuable therapeutic targets in the future.
Author contributions
D.D.R. and P.P.L conceived the study, oversaw the project, and designed the experiments; P.P.L., R.M., H.F., X.S., N.A., J.J., L.M., E.H., and D.D.R. carried out the experiments and analyzed data; H.Y.E.C., C.A.R., S.M.P., R.L.M., and S.W. provided fundamental reagents and intellectual contribution; P.P.L., R.M., R.L.M., S.W., and D.D.R. wrote the manuscript. D.D.R. contributed to this work prior to her current position. All the authors had final approval of the submitted version.
Acknowledgments
We would like to acknowledge support for the statistical analysis from the National Center for Research Resources and the National Center for Advancing Translational Sciences (NCATS) of the National Institutes of Health through grant 1UL1TR001079. We thank Dr. Laura Ranum for the kind gift of the A8(*KKQEXP)-3Tf1 construct. We thank Dr. Arnulf Koeppen, for providing frozen brain samples of three SCA2 patients. We thank Dr. Olga Pletinikova for providing a frozen brain sample of one SCA2 patient. We thank Dr. Shanshan Zhu for technical assistance regarding confocal imaging. We thank Kathryn A. Carson, Sc.M. for the advice on statistical analysis.
Footnotes
Relevant conflict of interest/financial disclosure: Nothing to report.
Funding agencies: This work was supported by the National Institutes of Health grants NS064138 (to D.D.R.), NS112796 (to P.P.L.), NS112687 (to P.P.L.), NS099397 (to S.W.), and NS033123 (to S.M.P.).