Summary Hexanucleotide expansions in C9ORF72 are the most frequent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Disease mechanisms were evaluated in mice expressing C9ORF72 RNAs with up to 450 GGGGCC repeats or with one or both C9orf72 alleles inactivated. Chronic 50% reduction of C9ORF72 did not provoke disease, while its absence produced splenomegaly, enlarged lymph nodes, and mild social interaction deficits, but no motor dysfunction. Hexanucleotide expansions caused age-, repeat length- and expression level-dependent accumulation of RNA foci and dipeptide-repeat proteins synthesized by AUG-independent translation, accompanied by loss of hippocampal neurons, increased anxiety, and impaired cognitive function. Single dose injection of antisense oligonucleotides (ASOs) that target repeat-containing RNAs but preserve levels of mRNAs encoding C9ORF72 produced sustained reductions in RNA foci and dipeptide-repeat proteins, and ameliorated behavioral deficits. These efforts identify gain-of-toxicity as a central disease mechanism caused by repeat-expanded C9ORF72 and establish the feasibility of ASO-mediated therapy.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease characterized by motor neuron loss, leading to paralysis and death 2–5 years following disease onset1. Nearly all ALS patients contain aggregates of the RNA-binding protein TDP-43 in the brain and spinal cord2, and rare mutations in the gene encoding TDP-43 can cause ALS3. There are no effective TDP-43-directed therapies for ALS or related TDP-43 proteinopathies, such as frontotemporal dementia (FTD). Antisense oligonucleotides (ASOs) and RNA interference approaches are emerging as attractive therapeutic strategies in neurological diseases4. Indeed, treating a rodent model of inherited ALS (caused by a mutation in SOD1) with ASOs to SOD1 significantly slowed disease progression5. But since SOD1 mutations account for only ~2–5% of ALS cases, additional therapeutic strategies are needed. Silencing TDP-43 itself is probably not warranted given its critical cellular functions1,6 Here we present an unexpectedly powerful alternative therapeutic strategy for ALS, by targeting ataxin 2. Lowering ataxin 2 suppresses TDP-43 toxicity in yeast and flies7, and intermediate-length polyglutamine expansions in the ataxin 2 gene increase risk of ALS7,8. We used two independent approaches to test whether reducing ataxin 2 levels could mitigate disease in a mouse model of TDP-43 proteinopathy9. First, we crossed ataxin 2 knockout mice to TDP-43 transgenic mice. Lowering ataxin 2 reduced TDP-43 aggregation, had a dramatic effect on survival and improved motor function. Second, in a more therapeutically applicable approach, we administered ASOs targeting ataxin 2 to the central nervous system of TDP-43 mice. This single treatment markedly extended survival. Because TDP-43 aggregation is a component of nearly all ALS cases6, targeting ataxin 2 could represent a broadly effective therapeutic strategy.

Spinocerebellar ataxia type 1 (SCA1) is one of several neurodegenerative diseases caused by expansion of a polyglutamine tract in the disease protein, in this case, ATAXIN-1 (ATXN1). A key question in the field is whether neurotoxicity is mediated by aberrant, novel interactions with the expanded protein or whether its wild-type functions are augmented to a deleterious degree. We examined soluble protein complexes from mouse cerebellum and found that the majority of wild-type and expanded ATXN1 assembles into large stable complexes containing the transcriptional repressor Capicua. ATXN1 directly binds Capicua and modulates Capicua repressor activity in Drosophila and mammalian cells, and its loss decreases the steady-state level of Capicua. Interestingly, the S776A mutation, which abrogates the neurotoxicity of expanded ATXN1, substantially reduces the association of mutant ATXN1 with Capicua in vivo. These data provide insight into the function of ATXN1 and suggest that SCA1 neuropathology depends on native, not novel, protein interactions.

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease caused by expansion of a glutamine-encoding repeat in SCA1. In all known polyglutamine diseases, the glutamine expansion confers toxic functions onto the protein. The mechanism by which this occurs remains enigmatic, however, in light of the fact that the mutant protein apparently maintains interactions with its usual partners. Here we show that the expanded polyglutamine tract differentially affects the function of the host protein in the context of different endogenous protein complexes. Polyglutamine expansion in Ataxin1 favors the formation of a particular protein complex containing RBM17, contributing to SCA1 neuropathology via a gain-of-function mechanism. Concomitantly, polyglutamine expansion attenuates the formation and function of another protein complex containing Ataxin1/Capicua, contributing to SCA1 via a partial loss-of-function mechanism. This model provides mechanistic insight into the molecular pathogenesis of SCA1 as well as other polyglutamine diseases.Expansion of an unstable translated CAG repeat located in different disease genes so far causes nine dominantly inherited neurodegenerative disorders, the so-called polyglutamine diseases: Huntington's disease (HD), spinobulbar muscular atrophy (SBMA), dentatorubropallidoluysian atrophy (DRPLA), and six autosomal dominant spinocerebellar ataxias (SCAs) 1 . As would be expected for dominant mutations, polyglutamine expansions confer toxic properties on the host proteins 1-3 ; animal models genetically lacking the polyglutamine-containing proteins do not develop neurodegeneration 4-7 . However, expansion of the polyglutamine tract is necessary but not sufficient to cause pathology: in the case of SCA1, for example, expanded Ataxin1 (ATXN1) does not produce cerebellar degeneration if it lacks the nuclear localization signal 8 or the AXH domain 9 , or if a serine to alanine substitution prevents phosphorylation at residue 776 10 . These and other studies in HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptSBMA and HD indicate that protein domains outside of the polyglutamine tract play a significant role in the selective neurotoxicity observed in these diseases 11-18 . Moreover, they suggest that there is a relationship between the normal functions of the wild-type proteins and the toxic functions of their expanded counterparts. Given that mouse and fly models overexpressing wild-type ATXN1 develop a mild version of SCA1 19 begs the question of whether the glutamine expansion enhances some interactions to mediate the gain-of-function.To gain a foothold on this question, we sought to characterize protein partners of ATXN1 that interact with it in a manner dependent on two criteria necessary for toxicity: polyglutamine expansion and phosphorylation at serine 776 (S776). We have identified RBM17 (RNA binding motif protein 17) as a protein that meets these criteria. Here we show that ATXN1 forms at least two distinct, large native complex...

SUMMARY Spinocerebellar ataxia type 1 (SCA1) is a paradigmatic neurodegenerative proteinopathy, in which a mutant protein (in this case, ATAXIN1) accumulates in neurons and exerts toxicity; in SCA1 this process causes progressive deterioration of motor coordination. Seeking to understand how post-translational modification of ATAXIN1 levels influences disease, we discovered that the RNA-binding protein PUMILIO1 (PUM1) not only directly regulates ATAXIN1 but that it also plays an unexpectedly important role in neuronal function. Loss of Pum1 caused progressive motor dysfunction and SCA1-like neurodegeneration with motor impairment, primarily by increasing Ataxin1 levels. Breeding Pum1+/− mice to SCA1 mice (Atxn1154Q/+) exacerbated disease progression, whereas breeding them to Atxn1+/− mice normalized Ataxin1 levels and largely rescued the Pum1+/− phenotype. Thus, both increased wild-type ATAXIN1 levels and PUM1 haploinsufficiency could contribute to human neurodegeneration. These results demonstrate the importance of studying post-transcriptional regulation of disease-driving proteins to reveal factors underlying neurodegenerative disease.