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

Parkinson Phenotype in Aged PINK1-Deficient Mice Is Accompanied by Progressive Mitochondrial Dysfunction in Absence of Neurodegeneration

Background Parkinson's disease (PD) is an adult-onset movement disorder of largely unknown etiology. We have previously shown that loss-of-function mutations of the mitochondrial protein kinase PINK1 (PTEN induced putative kinase 1) cause the recessive PARK6 variant of PD. Methodology/Principal Findings Now we generated a PINK1 deficient mouse and observed several novel phenotypes: A progressive reduction of weight and of locomotor activity selectively for spontaneous movements occurred at old age. As in PD, abnormal dopamine levels in the aged nigrostriatal projection accompanied the reduced movements. Possibly in line with the PARK6 syndrome but in contrast to sporadic PD, a reduced lifespan, dysfunction of brainstem and sympathetic nerves, visible aggregates of alpha-synuclein within Lewy bodies or nigrostriatal neurodegeneration were not present in aged PINK1-deficient mice. However, we demonstrate PINK1 mutant mice to exhibit a progressive reduction in mitochondrial preprotein import correlating with defects of core mitochondrial functions like ATP-generation and respiration. In contrast to the strong effect of PINK1 on mitochondrial dynamics in Drosophila melanogaster and in spite of reduced expression of fission factor Mtp18, we show reduced fission and increased aggregation of mitochondria only under stress in PINK1-deficient mouse neurons. Conclusion Thus, aging Pink1 -/- mice show increasing mitochondrial dysfunction resulting in impaired neural activity similar to PD, in absence of overt neuronal death

Dysregulated expression of lipid storage and membrane dynamics factors in Tia1 knockout mouse nervous tissue

During cell stress, the transcription and translation of immediate early genes are prioritized, while most other messenger RNAs (mRNAs) are stored away in stress granules or degraded in processing bodies (P-bodies). TIA-1 is an mRNA-binding protein that needs to translocate from the nucleus to seed the formation of stress granules in the cytoplasm. Because other stress granule components such as TDP-43, FUS, ATXN2, SMN, MAPT, HNRNPA2B1, and HNRNPA1 are crucial for the motor neuron diseases amyotrophic lateral sclerosis (ALS)/spinal muscular atrophy (SMA) and for the frontotemporal dementia (FTD), here we studied mouse nervous tissue to identify mRNAs with selective dependence on Tia1 deletion. Transcriptome profiling with oligonucleotide microarrays in comparison of spinal cord and cerebellum, together with independent validation in quantitative reverse transcriptase PCR and immunoblots demonstrated several strong and consistent dysregulations. In agreement with previously reported TIA1 knock down effects, cell cycle and apoptosis regulators were affected markedly with expression changes up to +2-fold, exhibiting increased levels for Cdkn1a, Ccnf, and Tprkb vs. decreased levels for Bid and Inca1 transcripts. Novel and surprisingly strong expression alterations were detected for fat storage and membrane trafficking factors, with prominent +3-fold upregulations of Plin4, Wdfy1, Tbc1d24, and Pnpla2 vs. a −2.4-fold downregulation of Cntn4 transcript, encoding an axonal membrane adhesion factor with established haploinsufficiency. In comparison, subtle effects on the RNA processing machinery included up to 1.2-fold upregulations of Dcp1b and Tial1. The effect on lipid dynamics factors is noteworthy, since also the gene deletion of Tardbp (encoding TDP-43) and Atxn2 led to fat metabolism phenotypes in mouse. In conclusion, genetic ablation of the stress granule nucleator TIA-1 has a novel major effect on mRNAs encoding lipid homeostasis factors in the brain, similar to the fasting effect

Genetic ablation of ataxin-2 increases several global translation factors in their transcript abundance but decreases translation rate

Spinocerebellar ataxia type 2 (SCA2) and amyotrophic lateral sclerosis (ALS) are neurodegenerative disorders, caused or modified by an unstable CAG-repeat expansion in the SCA2 gene, which encodes a polyglutamine (polyQ) domain expansion in ataxin-2 (ATXN2). ATXN2 is an RNA-binding protein and interacts with the poly(A)-binding protein PABPC1, localizing to ribosomes at the rough endoplasmic reticulum. Under cell stress, ATXN2, PABPC1 and small ribosomal subunits are relocated to stress granules, where mRNAs are protected from translation and from degradation. It is unknown whether ATXN2 associates preferentially with specific mRNAs or how it modulates RNA processing. Here, we investigated the RNA profile of the liver and cerebellum from Atxn2 knockout (Atxn2−/−) mice at two adult ages, employing oligonucleotide microarrays. Prominent increases were observed for Lsm12/Paip1 (>2-fold), translation modulators known as protein interactor/competitor of ATXN2 and for Plin3/Mttp (>1.3-fold), known as apolipoprotein modulators in agreement with the hepatosteatosis phenotype of the Atxn2−/− mice. Consistent modest upregulations were also observed for many factors in the ribosome and the translation/secretion apparatus. Quantitative reverse transcriptase PCR in liver tissue validated >1.2-fold upregulations for the ribosomal biogenesis modulator Nop10, the ribosomal components Rps10, Rps18, Rpl14, Rpl18, Gnb2l1, the translation initiation factors Eif2s2, Eif3s6, Eif4b, Pabpc1 and the rER translocase factors Srp14, Ssr1, Sec61b. Quantitative immunoblots substantiated the increased abundance of NOP10, RPS3, RPS6, RPS10, RPS18, GNB2L1 in SDS protein fractions, and of PABPC1. In mouse embryonal fibroblasts, ATXN2 absence also enhanced phosphorylation of the ribosomal protein S6 during growth stimulation, while impairing the rate of overall protein synthesis rates, suggesting a block between the enhanced translation drive and the impaired execution. Thus, the physiological role of ATXN2 subtly modifies the abundance of cellular translation factors as well as global translation

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

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

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

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