Lentiviral vector-mediated overexpression of mutant ataxin-7 recapitulates SCA7 pathology and promotes accumulation of the FUS/TLS and MBNL1 RNA-binding proteins

Background

We used lentiviral vectors (LVs) to generate a new SCA7 animal model overexpressing a truncated mutant ataxin-7 (MUT ATXN7) fragment in the mouse cerebellum, in order to characterize the specific neuropathological and behavioral consequences of the genetic defect in this brain structure.

Results

LV-mediated overexpression of MUT ATXN7 into the cerebellum of C57/BL6 adult mice induced neuropathological features similar to that observed in patients, such as intranuclear aggregates in Purkinje cells (PC), loss of synaptic markers, neuroinflammation, and neuronal death. No neuropathological changes were observed when truncated wild-type ataxin-7 (WT ATXN7) was injected. Interestingly, the local delivery of LV-expressing mutant ataxin-7 (LV-MUT-ATXN7) into the cerebellum of wild-type mice also mediated the development of an ataxic phenotype at 8 to 12 weeks post-injection. Importantly, our data revealed abnormal levels of the FUS/TLS, MBNL1, and TDP-43 RNA-binding proteins in the cerebellum of the LV-MUT-ATXN7 injected mice. MUT ATXN7 overexpression induced an increase in the levels of the pathological phosphorylated TDP-43, and a decrease in the levels of soluble FUS/TLS, with both proteins accumulating within ATXN7-positive intranuclear inclusions. MBNL1 also co-aggregated with MUT ATXN7 in most PC nuclear inclusions. Interestingly, no MBNL2 aggregation was observed in cerebellar MUT ATXN7 aggregates. Immunohistochemical studies in postmortem tissue from SCA7 patients and SCA7 knock-in mice confirmed SCA7-induced nuclear accumulation of FUS/TLS and MBNL1, strongly suggesting that these proteins play a physiopathological role in SCA7.

Conclusions

This study validates a novel SCA7 mouse model based on lentiviral vectors, in which strong and sustained expression of MUT ATXN7 in the cerebellum was found sufficient to generate motor defects.

     

Structural basis of the phosphorylation dependent complex formation of neurodegenerative disease protein Ataxin-1 and RBM17

Spinocerebellar Ataxia Type1 (SCA1) is a dominantly inherited neurodegenerative disease and belongs to polyglutamine expansion disorders. The polyglutamine expansion in Ataxin-1 (ATXN1) is responsible for SCA1 pathology. ATXN1 forms at least two distinct complexes with Capicua (CIC) or RNA-binding motif protein 17 (RBM17). The wild-type ATXN1 dominantly forms a complex with CIC and the polyglutamine expanded form of ATXN1 favors to form a complex with RBM17. The phosphorylation of Ser776 in ATXN1 is critical for SCA1 pathology and serves as a binding platform for RBM17. However, the molecular basis of the phospho-specific binging of ATXN1 to RBM17 is not delineated. Here, we present the modeled structure of RBM17 bound to the phosphorylated ATXN1 peptide. The structure reveals the phosphorylation specific interaction between ATXN1 and RBM17 through a salt-bridge network. Furthermore, the modeled structure and the interactions between RBM17 and ATXN1 were validated through mutagenesis study followed by Surface Plasmon Resonance binding experiments. This work delineates the molecular basis of the interaction between RBM17 and the phosphorylated form of ATXN1, which is critical for SCA1 pathology. Furthermore, the structure of RBM17 and pATXN1 peptide might be utilized to target RBM17–ATXN1 interaction to modulate SCA1 pathogenesis.     

Beyond the Glutamine Expansion: Influence of Posttranslational Modifications of Ataxin-1 in the Pathogenesis of Spinocerebellar Ataxia Type 1

Posttranslational modifications are crucial mechanisms that modulate various cellular signaling pathways, and their dysregulation is associated with many human diseases. Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease characterized by progressive ataxia, mild cognitive impairments, difficulty with speaking and swallowing, and respiratory failure. It is caused by the expansion of an unstable CAG trinucleotide repeat encoding a glutamine tract in Ataxin-1 (ATXN1). Although the expansion of the polyglutamine tract is the key determinant of the disease, protein domains outside of the polyglutamine tract and posttranslational modifications of ATXN1 significantly alter the neurotoxicity of SCA1. ATXN1 undergoes several posttranslational modifications, including phosphorylation, ubiquitination, sumoylation, and transglutamination. Such modifications can alter the stability of ATXN1 or its activity in the regulation of target gene expression and therefore contribute to SCA1 toxicity. This review outlines different types of posttranslational modifications in ATXN1 and discusses their potential regulatory mechanisms and effects on SCA1 pathogenesis. Finally, the manipulation of posttranslational modifications as a potential therapeutic approach will be discussed.     

Two Novel SNPs in ATXN3 3’ UTR May Decrease Age at Onset of SCA3/MJD in Chinese Patients

Spinocerebellar ataxia type 3 (SCA3), or Machado—Joseph disease (MJD), is an autosomal dominantly-inherited disease that produces progressive problems with movement. It is caused by the expansion of an area of CAG repeats in a coding region of ATXN3. The number of repeats is inversely associated with age at disease onset (AO) and is significantly associated with disease severity; however, the degree of CAG expansion only explains 50 to 70% of variance in AO. We tested two SNPs, rs709930 and rs910369, in the 3’ UTR of ATXN3 gene for association with SCA3/MJD risk and with SCA3/MJD AO in an independent cohort of 170 patients with SCA3/MJD and 200 healthy controls from mainland China. rs709930 genotype frequencies were statistically significantly different between patients and controls (p = 0.001, α = 0.05). SCA3/MJD patients carrying the rs709930 A allele and rs910369 T allele experienced an earlier onset, with a decrease in AO of approximately 2 to 4 years. The two novel SNPs found in this study might be genetic modifiers for AO in SCA3/MJD.     

Parkinsonian phenotype in Machado-Joseph disease (MJD/SCA3): a two-case report

Background  

Machado-Joseph disease (MJD), or spinocerebellar ataxia type 3 (SCA3), is an autosomal dominant neurodegenerative disorder of late onset, which is caused by a CAG repeat expansion in the coding region of the ATXN3 gene. This disease presents clinical heterogeneity, which cannot be completely explained by the size of the repeat tract. MJD presents extrapyramidal motor signs, namely Parkinsonism, more frequently than the other subtypes of autosomal dominant cerebellar ataxias. Although Parkinsonism seems to segregate within MJD families, only a few MJD patients develop parkinsonian features and, therefore, the clinical and genetic aspects of these rare presentations remain poorly investigated. The main goal of this work was to describe two MJD patients displaying the parkinsonian triad (tremor, bradykinesia and rigidity), namely on what concerns genetic variation in Parkinson's disease (PD) associated loci (PARK2, LRRK2, PINK1, DJ-1, SNCA, MAPT, APOE, and mtDNA tRNA ^Gln T4336C).     

Population Genetics and New Insight into Range of CAG Repeats of Spinocerebellar Ataxia Type 3 in the Han Chinese Population

Spinocerebellar ataxia type 3 (SCA3), also called Machado-Joseph disease (MJD), is one of the most common SCAs worldwide and caused by a CAG repeat expansion located in ATXN3 gene. Based on the CAG repeat numbers, alleles of ATXN3 can be divided into normal alleles (ANs), intermediate alleles (AIs) and expanded alleles (AEs). It was controversial whether the frequency of large normal alleles (large ANs) is related to the prevalence of SCA3 or not. And there were huge chaos in the comprehension of the specific numbers of the range of CAG repeats which is fundamental for genetic analysis of SCA3. To illustrate these issues, we made a novel CAG repeat ladder to detect CAG repeats of ATXN3 in 1003 unrelated Chinese normal individuals and studied haplotypes defined by three single nucleotide polymorphisms (SNPs) closed to ATXN3. We found that the number of CAG repeats ranged from 13 to 49, among them, 14 was the most common number. Positive skew, the highest frequency of large ANs and 4 AIs which had never been reported before were found. Also, AEs and large ANs shared the same haplotypes defined by the SNPs. Based on these data and other related studies, we presumed that de novo mutations of ATXN3 emerging from large ANs are at least one survival mechanisms of mutational ATXN3 and we can redefine the range of CAG repeats as: ANs≤44, 45 ≤AIs ≤49 and AEs≥50.     

Poly(Q) Expansions in ATXN7 Affect Solubility but Not Activity of the SAGA Deubiquitinating Module

Spinocerebellar ataxia type 7 (SCA7) is a debilitating neurodegenerative disease caused by expansion of a polyglutamine [poly(Q)] tract in ATXN7, a subunit of the deubiquitinase (DUB) module (DUBm) in the SAGA complex. The effects of ATXN7-poly(Q) on DUB activity are not known. To address this important question, we reconstituted the DUBm in vitro with either wild-type ATXN7 or a pathogenic form, ATXN7-92Q NT, with 92 Q residues at the N terminus (NT). We found that both forms of ATXN7 greatly enhance DUB activity but that ATXN7-92Q NT is largely insoluble unless it is incorporated into the DUBm. Cooverexpression of DUBm components in human astrocytes also promoted the solubility of ATXN7-92Q, inhibiting its aggregation into nuclear inclusions that sequester DUBm components, leading to global increases in ubiquitinated H2B (H2Bub) levels. Global H2Bub levels were also increased in the cerebellums of mice in a SCA7 mouse model. Our findings indicate that although ATXN7 poly(Q) expansions do not change the enzymatic activity of the DUBm, they likely contribute to SCA7 by initiating aggregates that sequester the DUBm away from its substrates.     

Genetic Variance in the Spinocerebellar Ataxia Type 2 (ATXN2) Gene in Children with Severe Early Onset Obesity

Background

Expansion of a CAG repeat in the coding region of exon 1 in the ATXN2 gene located in human chromosome 12q24.1 causes the neurodegenerative disease spinocerebellar ataxia type 2 (SCA2). In contrast to other polyglutamine (polyQ) disorders, the SCA2 repeat is not highly polymorphic in central European (CEU) controls with Q22 representing 90% of alleles, and Q23 contributing between 5–7% of alleles. Recently, the ATXN2 CAG repeat has been identified as a target of adaptive selection in the CEU population. Mouse lines deficient for atxn2 develop marked hyperphagia and obesity raising the possibility that loss-of-function mutations in the ATXN2 gene may be related to energy balance in humans. Some linkage studies of obesity related phenotypes such as antipsychotic induced weight gain have reported significant lod scores on chromosome 12q24. We tested the hypothesis that rare loss-of-function ATXN2 variants cause obesity analogous to rare mutations in the leptin, leptin receptor and MC4R genes.

Methodology/Principal Findings

We sequenced the coding region of ATXN2 including intron-exon boundaries in 92 severely obese children with a body mass index (BMI) >3.2 standard deviations above age- and gender-adjusted means. We confirmed five previously identified single nucleotide polymorphisms (SNPs) and three new SNPs resulting in two synonymous substitutions and one intronic polymorphism. Alleles encoding >Q22 were overrepresented in our sample of obese children and contributed 15% of alleles in children identified by their parents as white. SNP rs695872 closely flanking the CAG repeat showed a greatly increased frequency of C/C homozygotes and G/C heterozygotes compared with reported frequencies in the CEU population.

Conclusions/Significance

Although we did not identify variants leading to novel amino acid substitutions, nonsense or frameshift mutations, this study warrants further examination of variation in the ATXN2 gene in obesity and related phenotypes in a larger case-control study with emphasis on rs695872 and CAG repeat structure.     

De Novo Mutations in Ataxin-2 Gene and ALS Risk

Pathogenic CAG repeat expansion in the ataxin-2 gene (ATXN2) is the genetic cause of spinocerebellar ataxia type 2 (SCA2). Recently, it has been associated with Parkinsonism and increased genetic risk for amyotrophic lateral sclerosis (ALS). Here we report the association of de novo mutations in ATXN2 with autosomal dominant ALS. These findings support our previous conjectures based on population studies on the role of large normal ATXN2 alleles as the source for new mutations being involved in neurodegenerative pathologies associated with CAG expansions. The de novo mutations expanded from ALS/SCA2 non-risk alleles as proven by meta-analysis method. The ALS risk was associated with SCA2 alleles as well as with intermediate CAG lengths in the ATXN2. Higher risk for ALS was associated with pathogenic CAG repeat as revealed by meta-analysis.     

14-3-3 Binding to ataxin-1(ATXN1) regulates its dephosphorylation at Ser-776 and transport to the nucleus

Spinocerebellar ataxia type 1 (SCA1) is a lethal neurodegenerative disorder caused by expansion of a polyglutamine tract in ATXN1. A prominent site of pathology in SCA1 is cerebellar Purkinje neurons where mutant ATXN1 must enter the nucleus to cause disease. In SCA1, phosphorylation of ATXN1 at Ser-776 modulates disease. Interestingly, Ser-776 is located within a region of ATXN1 that harbors several functional motifs including binding sites for 14-3-3, and splicing factors RBM17 and U2AF65. The interaction of ATXN1 with these proteins is thought to be regulated by the phosphorylation status of Ser-776. In addition, Ser-776 is adjacent to the NLS in ATXN1. Although pS776-ATXN1 is enriched in nuclear extracts of cerebellar cells, the vast majority of 14-3-3 is in the cytoplasmic fraction. We found that dephosphorylation of cytoplasmic pS776-ATXN1 is blocked by virtue of it being in a complex with 14-3-3. In addition, data suggest that binding of 14-3-3 to cytoplasmic ATXN1 impeded its transport to the nucleus, suggesting that 14-3-3 must disassociate from ATXN1 for transport of ATXN1 to the nucleus. Consistent with this hypothesis is the observation that once in the nucleus pS776 is able to be dephosphorylated. Evidence is presented that PP2A is the pS776-ATXN1 phosphatase in the mammalian cerebellum. In the nucleus, we propose that dephosphorylation of pS776-ATXN1 by PP2A regulates the interaction of ATXN1 with the splicing factors RBM17 and U2AF65.     

The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3’-P and 5’-OH, are processed by mammalian polynucleotide kinase 3’-phosphatase (PNKP), a bifunctional enzyme with 3’-phosphatase and 5’-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14–41 to 55–82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP’s 3’ phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3’-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients’ brain. Finally, long amplicon quantitative PCR analysis of human MJD patients’ brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.     

Repeat Associated Non-AUG Translation (RAN Translation) Dependent on Sequence Downstream of the ATXN2 CAG Repeat

Spinocerebellar ataxia type 2 (SCA2) is a progressive autosomal dominant disorder caused by the expansion of a CAG tract in the ATXN2 gene. The SCA2 disease phenotype is characterized by cerebellar atrophy, gait ataxia, and slow saccades. ATXN2 mutation causes gains of toxic and normal functions of the ATXN2 gene product, ataxin-2, and abnormally slow Purkinje cell firing frequency. Previously we investigated features of ATXN2 controlling expression and noted expression differences for ATXN2 constructs with varying CAG lengths, suggestive of repeat associated non-AUG translation (RAN translation). To determine whether RAN translation occurs for ATXN2 we assembled various ATXN2 constructs with ATXN2 tagged by luciferase, HA or FLAG tags, driven by the CMV promoter or the ATXN2 promoter. Luciferase expression from ATXN2-luciferase constructs lacking the ATXN2 start codon was weak vs AUG translation, regardless of promoter type, and did not increase with longer CAG repeat lengths. RAN translation was detected on western blots by the anti-polyglutamine antibody 1C2 for constructs driven by the CMV promoter but not the ATXN2 promoter, and was weaker than AUG translation. Strong RAN translation was also observed when driving the ATXN2 sequence with the CMV promoter with ATXN2 sequence downstream of the CAG repeat truncated to 18 bp in the polyglutamine frame but not in the polyserine or polyalanine frames. Our data demonstrate that ATXN2 RAN translation is weak compared to AUG translation and is dependent on ATXN2 sequences flanking the CAG repeat.