Gene co-expression network analysis for identifying modules and functionally enriched pathways in SCA2

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant neurodegenerative disease caused by CAG repeat expansion in the ATXN2 gene. The repeat resides in an encoded region of the gene resulting in polyglutamine (polyQ) expansion which has been assumed to result in gain of function, predominantly, for the ATXN2 protein. We evaluated temporal cerebellar expression profiles by RNA sequencing of ATXN2Q127 mice versus wild-type (WT) littermates. ATXN2Q127 mice are characterized by a progressive motor phenotype onset, and have progressive cerebellar molecular and neurophysiological (Purkinje cell firing frequency) phenotypes. Our analysis revealed previously uncharacterized early and progressive abnormal patterning of cerebellar gene expression. Weighted Gene Coexpression Network Analysis revealed four gene modules that were significantly correlated with disease status, composed primarily of genes associated with GTPase signaling, calcium signaling and cell death. Of these genes, few overlapped with differentially expressed cerebellar genes that we identified in Atxn2−/− knockout mice versus WT littermates, suggesting that loss-of-function is not a significant component of disease pathology. We conclude that SCA2 is a disease characterized by gain of function for ATXN2

Antisense oligonucleotide therapy for spinocerebellar ataxia type 2

There are no disease-modifying treatments for adult human neurodegenerative diseases. Here we test RNA-targeted therapies1 in two mouse models of spinocerebellar ataxia type 2 (SCA2), an autosomal dominant polyglutamine disease2. Both models recreate the progressive adult-onset dysfunction and degeneration of a neuronal network that are seen in patients, including decreased firing frequency of cerebellar Purkinje cells and a decline in motor function3,4. We developed a potential therapy directed at the ATXN2 gene by screening 152 antisense oligonucleotides (ASOs). The most promising oligonucleotide, ASO7, downregulated ATXN2 mRNA and protein, which resulted in delayed onset of the SCA2 phenotype. After delivery by intracerebroventricular injection to ATXN2-Q127 mice, ASO7 localized to Purkinje cells, reduced cerebellar ATXN2 expression below 75% for more than 10 weeks without microglial activation, and reduced the levels of cerebellar ATXN2. Treatment of symptomatic mice with ASO7 improved motor function compared to saline-treated mice. ASO7 had a similar effect in the BAC-Q72 SCA2 mouse model, and in both mouse models it normalized protein levels of several SCA2-related proteins expressed in Purkinje cells, including Rgs8, Pcp2, Pcp4, Homer3, Cep76 and Fam107b. Notably, the firing frequency of Purkinje cells returned to normal even when treatment was initiated more than 12 weeks after the onset of the motor phenotype in BAC-Q72 mice. These findings support ASOs as a promising approach for treating some human neurodegenerative diseases

Cytoplasmic CUG RNA Foci Are Insufficient to Elicit Key DM1 Features

The genetic basis of myotonic dystrophy type I (DM1) is the expansion of a CTG tract located in the 3′ untranslated region of DMPK. Expression of mutant RNAs encoding expanded CUG repeats plays a central role in the development of cardiac disease in DM1. Expanded CUG tracts form both nuclear and cytoplasmic aggregates, yet the relative significance of such aggregates in eliciting DM1 pathology is unclear. To test the pathophysiology of CUG repeat encoding RNAs, we developed and analyzed mice with cardiac-specific expression of a beta-galactosidase cassette in which a (CTG)400 repeat tract was positioned 3′ of the termination codon and 5′ of the bovine growth hormone polyadenylation signal. In these animals CUG aggregates form exclusively in the cytoplasm of cardiac cells. A key pathological consequence of expanded CUG repeat RNA expression in DM1 is aberrant RNA splicing. Abnormal splicing results from the functional inactivation of MBNL1, which is hypothesized to occur due to MBNL1 sequestration in CUG foci or from elevated levels of CUG-BP1. We therefore tested the ability of cytoplasmic CUG foci to elicit these changes. Aggregation of CUG RNAs within the cytoplasm results both in Mbnl1 sequestration and in approximately a two fold increase in both nuclear and cytoplasmic Cug-bp1 levels. Significantly, despite these changes RNA splice defects were not observed and functional analysis revealed only subtle cardiac dysfunction, characterized by conduction defects that primarily manifest under anesthesia. Using a human myoblast culture system we show that this transgene, when expressed at similar levels to a second transgene, which encodes expanded CTG tracts and facilitates both nuclear focus formation and aberrant splicing, does not elicit aberrant splicing. Thus the lack of toxicity of cytoplasmic CUG foci does not appear to be a consequence of low expression levels. Our results therefore demonstrate that the cellular location of CUG RNA aggregates is an important variable that influences toxicity and support the hypothesis that small molecules that increase the rate of transport of the mutant DMPK RNA from the nucleus into the cytoplasm may significantly improve DM1 pathology

Genetic and Chemical Modifiers of a CUG Toxicity Model in Drosophila

Non-coding CUG repeat expansions interfere with the activity of human Muscleblind-like (MBNL) proteins contributing to myotonic dystrophy 1 (DM1). To understand this toxic RNA gain-of-function mechanism we developed a Drosophila model expressing 60 pure and 480 interrupted CUG repeats in the context of a non-translatable RNA. These flies reproduced aspects of the DM1 pathology, most notably nuclear accumulation of CUG transcripts, muscle degeneration, splicing misregulation, and diminished Muscleblind function in vivo. Reduced Muscleblind activity was evident from the sensitivity of CUG-induced phenotypes to a decrease in muscleblind genetic dosage and rescue by MBNL1 expression, and further supported by the co-localization of Muscleblind and CUG repeat RNA in ribonuclear foci. Targeted expression of CUG repeats to the developing eye and brain mushroom bodies was toxic leading to rough eyes and semilethality, respectively. These phenotypes were utilized to identify genetic and chemical modifiers of the CUG-induced toxicity. 15 genetic modifiers of the rough eye phenotype were isolated. These genes identify putative cellular processes unknown to be altered by CUG repeat RNA, and they include mRNA export factor Aly, apoptosis inhibitor Thread, chromatin remodelling factor Nurf-38, and extracellular matrix structural component Viking. Ten chemical compounds suppressed the semilethal phenotype. These compounds significantly improved viability of CUG expressing flies and included non-steroidal anti-inflammatory agents (ketoprofen), muscarinic, cholinergic and histamine receptor inhibitors (orphenadrine), and drugs that can affect sodium and calcium metabolism such as clenbuterol and spironolactone. These findings provide new insights into the DM1 phenotype, and suggest novel candidates for DM1 treatments

Interaction of musleblind, CUG-BP1 and hnRNP H proteins in DM1-associated aberrant IR splicing

In myotonic dystrophy (DM1), both inactivation of muscleblind proteins and increased levels of CUG-BP1 are reported. These events have been shown to contribute independently to aberrant splicing of a subset RNAs. We demonstrate that steady-state levels of the splice regulator, hnRNP H, are elevated in DM1 myoblasts and that increased hnRNP H levels in normal myoblasts results in the inhibition of insulin receptor (IR) exon 11 splicing in a manner similar to that observed in DM1. In normal myoblasts, overexpression of either hnRNP H or CUG-BP1 results in the formation of an RNA-dependent suppressor complex consisting of both hnRNP H and CUG-BP1, which is required to maximally inhibit IR exon 11 inclusion. Elevated levels of MBNL1 show RNA-independent interaction with hnRNP H and dampen the inhibitory activity of increased hnRNP H levels on IR splicing in normal myoblasts. In DM1 myoblasts, overexpression of MBNL1 in conjunction with si-RNA mediated depletion of hnRNP H contributes to partial rescue of the IR splicing defect. These data demonstrate that coordinated physical and functional interactions between hnRNP H, CUG-BP1 and MBNL1 dictate IR splicing in normal and DM1 myoblasts

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

<i>ATXN2-luc</i> expression driven by the native <i>ATXN2</i> promoter, dependent upon CAG length and the presence of a start codon.

<p>(A) Plasmid constructs used in luciferase assays. (B) Luciferase assays to evaluate <i>ATXN2</i> expression driven by 1062 bp of its native upstream sequence, demonstrated increasing expression with increasing CAG length (ATG constructs). When the start codon was mutated, expression significantly higher than the control was observed only for <i>ATXN2</i>s with CAG repeat lengths of 57 or 102 (CTG constructs). For the longest repeat expression was 25-fold reduced when the start codon was substituted with CTG. Values are mean±SD of three independent experiments. All constructs were cotransfected with SV40-Renilla luciferase and values are represented as mean FLuc / RLuc, the ratio of firefly luciferase to Renilla luciferase. (C) RAN translation products were not observed by western blotting using anti-luciferase (luc) or 1C2 antibodies. Note that polyglutamine proteins detected with the 1C2 anti-polyglutamine antibody are more easily seen as the length of the polyglutamine is increased. Loading was controlled by detecting actin. The mobilities of the smaller ataxin-2-luciferase bands are not consistent with RAN translation bands. (D) Analysis of the luciferase assay results for only the CTG-<i>ATXN2-luc</i> constructs in B revealed significantly increased expression for constructs with 22 or greater CAG repeats but no increasing luciferase expression with increasing CAG repeat length. P<0.001 (**), Bonferroni post-hoc probability of significance. Assays utilized HEK293T cells with assays made 24 hrs after transfection.</p

<i>ATXN2-luc</i> expression driven by the native <i>ATXN2</i> promoter, dependent upon CAG length and the presence of a start codon.

<p>(A) Plasmid constructs used in luciferase assays. (B) Luciferase assays to evaluate <i>ATXN2</i> expression driven by 1062 bp of its native upstream sequence, demonstrated increasing expression with increasing CAG length (ATG constructs). When the start codon was mutated, expression significantly higher than the control was observed only for <i>ATXN2</i>s with CAG repeat lengths of 57 or 102 (CTG constructs). For the longest repeat expression was 25-fold reduced when the start codon was substituted with CTG. Values are mean±SD of three independent experiments. All constructs were cotransfected with SV40-Renilla luciferase and values are represented as mean FLuc / RLuc, the ratio of firefly luciferase to Renilla luciferase. (C) RAN translation products were not observed by western blotting using anti-luciferase (luc) or 1C2 antibodies. Note that polyglutamine proteins detected with the 1C2 anti-polyglutamine antibody are more easily seen as the length of the polyglutamine is increased. Loading was controlled by detecting actin. The mobilities of the smaller ataxin-2-luciferase bands are not consistent with RAN translation bands. (D) Analysis of the luciferase assay results for only the CTG-<i>ATXN2-luc</i> constructs in B revealed significantly increased expression for constructs with 22 or greater CAG repeats but no increasing luciferase expression with increasing CAG repeat length. P<0.001 (**), Bonferroni post-hoc probability of significance. Assays utilized HEK293T cells with assays made 24 hrs after transfection.</p

<i>ATXN2</i> RAN translation was observed for <i>ATXN2</i> sequences with 91 or 102 CAG repeats with C-terminal epitopes driven by the <i>CMV</i> promoter but not the native <i>ATXN2</i> promoter.

<p>(A) <i>CMV</i> promoter driven <i>ATXN2</i> constructs including the <i>ATXN2</i> ATG start codon expressed proteins as expected, detected on western blots by anti-HA (lanes 1 and 3) and anti-FLAG when the epitope was included (lane 3), and anti-1C2 antibodies. When the <i>ATXN2</i> start codon was changed to CTG, <i>ATXN2</i> RAN translation bands were detected by anti-1C2 (lanes 2 and 4), but RAN translation products were not by anti-FLAG and for anti-HA the faintest RAN translation band is present for construct #4 but not #2. (B) <i>ATXN2</i> promoter (<i>ATXN2p</i>) driven <i>ATXN2</i> constructs including the <i>ATXN2</i> ATG start codon expressed proteins as expected, detected on western blots by anti-HA (lanes 5 and 7) and anti-FLAG when the epitope was included (lane 7), and anti-1C2 antibodies. When the <i>ATXN2</i> start codon was changed to CTG, <i>ATXN2</i> RAN translation bands were not observable by anti-1C2, anti-HA, or anti-FLAG antibodies (lanes 6 and 8). All constructs include the hygromycin phosphotransferase (HYG) gene, and uniformity of plasmid transfection and loading was ensured in A and B by detecting blots with anti-HYG and anti-Actin. Note that the intensity of upper bands detected by the anti-HA antibody follow actin band intensity but not HYG indicating that these are non-specific bands. Arrows indicate the specific bands detected by the anti-HA antibody. (C) Anti-HYG detected a doublet of bands in lysates from transfected cells that was absent in untransfected cells (UTC). For each of A, B, and C, we utilized HEK293T cells and 48 hr transfections.</p