ATXN2-CAG42 sequesters PABPC1 into insolubility and induces FBXW8 in cerebellum of old ataxic knock-in mice

Spinocerebellar Ataxia Type 2 (SCA2) is caused by expansion of a polyglutamine encoding triplet repeat in the human ATXN2 gene beyond (CAG)31. This is thought to mediate toxic gain-of-function by protein aggregation and to affect RNA processing, resulting in degenerative processes affecting preferentially cerebellar neurons. As a faithful animal model, we generated a knock-in mouse replacing the single CAG of murine Atxn2 with CAG42, a frequent patient genotype. This expansion size was inherited stably. The mice showed phenotypes with reduced weight and later motor incoordination. Although brain Atxn2 mRNA became elevated, soluble ATXN2 protein levels diminished over time, which might explain partial loss-of-function effects. Deficits in soluble ATXN2 protein correlated with the appearance of insoluble ATXN2, a progressive feature in cerebellum possibly reflecting toxic gains-of-function. Since in vitro ATXN2 overexpression was known to reduce levels of its protein interactor PABPC1, we studied expansion effects on PABPC1. In cortex, PABPC1 transcript and soluble and insoluble protein levels were increased. In the more vulnerable cerebellum, the progressive insolubility of PABPC1 was accompanied by decreased soluble protein levels, with PABPC1 mRNA showing no compensatory increase. The sequestration of PABPC1 into insolubility by ATXN2 function gains was validated in human cell culture. To understand consequences on mRNA processing, transcriptome profiles at medium and old age in three different tissues were studied and demonstrated a selective induction of Fbxw8 in the old cerebellum. Fbxw8 is encoded next to the Atxn2 locus and was shown in vitro to decrease the level of expanded insoluble ATXN2 protein. In conclusion, our data support the concept that expanded ATXN2 undergoes progressive insolubility and affects PABPC1 by a toxic gain-of-function mechanism with tissuespecific effects, which may be partially alleviated by the induction of FBXW8

Soluble ATXN2 protein levels are reduced and PABPC1 levels change.

<p>In cerebellar tissue, a trend in ATXN2 reduction was observed at 6 weeks and 6 months age, with significance in ATXN2 reduction being reached at 18 months of age. Also, a significant PABPC1 reduction was apparent in 6 weeks and 18 months old CAG42 mice in the cerebellum. In the cortex, a reduction of ATXN2 levels was observed in 6 weeks old animals as a trend and with significance from 6 months onwards. An elevation of PABPC1 levels became significant at 18 months of age (n = 10–12 mice/genotype/tissue).</p

Normal and expanded insoluble ATXN2 drives PABPC1 into insolubility.

<p>(A) Overexpression of CAG22-ATXN2 and CAG74-ATXN2 led to significantly decreased <i>PABPC1</i> transcript levels. (B) Overexpressed Q22-ATXN2 or Q74-ATXN2 reduced endogenous soluble PABPC1 levels, whereas endogenous insoluble PABPC1 levels were significantly increased in the presence of insoluble ATXN2 (n = 3).</p

The paternal and maternal transmission of the 42 CAG repeat is stable.

<p>(A) A CAG-repeat flanking PCR with one FAM-labelled primer was performed. As expected, wild-type (WT) animals showed one product at 94 bp, whereas heterozygotes (CAG1/CAG42) showed an additional larger product. In homozygous mutant mice (CAG42) only the larger product was detected. c: negative control. (B) The exact PCR product size was confirmed by fragment length analysis. The peak in the WT sample represents the 94 bp product, while the 217 bp peak appeared in CAG42 animals. Both peaks were detectable in CAG1/CAG42 mice.</p

A constant weight reduction and a late-onset deficit in a cerebellar test paradigm.

<p>(A) Homozygous CAG42 mice had a reduced body weight by birth up to the age of 21 months, while no change was observed in heterozygous CAG1/CAG42 mice in comparison to wild-type littermates (n≥14 per genotype). (B) Motor coordination was tested on an accelerating rotarod apparatus. An initially increased locomotor performance at 6 weeks of age in the CAG42 mice was replaced by normal activity at 3, 6 and 12 months. At 18 and 21 months age, CAG42 mice showed impaired motor coordination (until 18 months: n≥14 per genotype, 21 months: n≥6 per genotype).</p

Increased sequestration of PABPC1 by insoluble Q42-ATXN2 with age.

<p>(A) In the cerebellum, a progressive insolubility of Q42-ATXN2 from 6 to 12 and 24 months was detectable. The solubility of PABPC1 decreased from 6 to 24 months of age. Insoluble Q42-ATXN2 levels did not change in the cortex, however, the insolubility of PABPC1 increased over time in CAG42 mice. Insoluble PABPC1 levels in WT mice remained stably low in both tissues. (n = 3 mice/genotype/tissue). (B) In the soluble fraction, both wild-type and expanded ATXN2 were able to co-immunoprecipitate PABPC1, with slightly more PABPC1 being pulled down in CAG42 mice.</p

Differentially regulated mRNAs in CAG42 mice at the age of 18 months.

<p>Genes with significant dysregulation and consistency between more than one oligonucleotide spot are shown (n = 4 mice/genotype).</p

Elevated FBXW8 diminishes specifically insoluble expanded ATXN2.

<p>(A) The protein levels of FBXW8 were significantly upregulated in the cerebellum of 18 months old CAG42 mice compared to wild-type (n = 9–11 mice/genotype). (B) Soluble levels of Q22-ATXN2 were unchanged after FBXW8 overexpression, while for Q74-ATXN2 a slight reduction was apparent. With respect to insoluble levels, Q22-ATXN2 was again not influenced by FBXW8 overexpression, while the Q74-ATXN2 reduction became significant. FBXW8 levels were significantly increased by its overexpression both in the soluble and insoluble fraction (n = 7).</p

<i>Atxn2</i> and <i>Pabpc1</i> mRNA levels are elevated in specific ages and areas.

<p>In the cerebellum an <i>Atxn2</i> mRNA elevation was not detectable until 6 months, whereas the <i>Pabpc1</i> transcript levels remained unchanged. In the cortex an <i>Atxn2</i> mRNA elevation was already detected by 6 weeks and <i>Pabpc1</i> mRNA was upregulated by 6 months (n = 9–16 animals/genotype/tissue).</p