ATXN2-CAG42 Sequesters PABPC1 into Insolubility and Induces FBXW8 in Cerebellum of Old Ataxic Knock-In Mice

Abstract

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 tissue-specific effects, which may be partially alleviated by the induction of FBXW8.

Mammalian ataxin-2 modulates translation control at the pre-initiation complex via PI3K/mTOR and is induced by starvation

“…Another integral component of the pre-initiation complex is the ribosomal protein S6, whose phosphorylation downregulates translation initiation and cell proliferation, while augmenting cell size and affecting glucose homeostasis [57]. It has been demonstrated that ataxin-2 interacts directly with PABP in Drosophila melanogaster and mouse brain [12, 33], colocalizes with the rER and cosediments with RPS6 in mouse brain [58]. We report now that ataxin-2 indeed colocalizes with PABP also in rat hippocampal neuron cultures and murine embryonal fibroblasts, but also colocalizes with eIF4A1, eIF3B and eIF4G (Figure 1) and shows more similar cosedimentation with the initiation factors than with PABP and RPS6 (Figure 5).…”

“…SCA2 is a neurodegenerative disease, which usually manifests with a cerebellar syndrome or alternatively with a Parkinsonian midbrain syndrome, while later stages also affect the brainstem, spinal cord and thalamus [5-11]. The unstable expansion of a polyglutamine (polyQ) domain in the disease protein leads to a toxic gain-of-function pathogenesis, through the accumulation and aggregation of ataxin-2 plus the sequestration of crucial interactor molecules such as the poly(A)-binding protein, PABP [12, 13]. The human ATXN2 gene contains a (CAG) repeat of normally 22-23 units within exon 1; the presence of more than 31 triplets usually causes the clinically manifest multi-system neurodegeneration known as SCA2 with autosomal dominant inheritance, while alleles with 27 to 33 triplets have reduced penetrance and contribute to the risk of motor neuron disease [14-18].…”

“…Recent expression profiling efforts in Atxn2 -knockoout mouse tissues demonstrated a selective downregulation of mitochondrial matrix components global proteome profiles and a preferential downregulation of calcium homeostasis factors together with an upregulation of mRNA translation factors in global transcriptomics [29-31]. The structure of ataxin-2 contains a PAM2 motif which mediates association with the poly(A)-binding protein PABP [12, 13, 32], thought to promote ataxin-2 association with polyribosomes [33]. Ataxin-2 structure also contains two globular domains named Lsm (‘Like Sm’ domain) and LsmAD (Lsm-associated domain), that were shown to mediate direct interaction with RNA [33, 34].…”

Ataxin-2 (Atxn2)-Knock-Out Mice Show Branched Chain Amino Acids and Fatty Acids Pathway Alterations

“…It is well established now that polyQ-expansions trigger an aggregation process, which is toxic through oligomer formation, but also through the longer half-life (35) of the disease protein with a gain in physiological functions. This process also sequestrates interaction molecules into insolubility leading to partial losses of physiological functions (8). Thus, polyQ-expanded proteins contribute to pathogenesis in the context of their cellular function (58 -63).…”

“…The structure of the human ATXN2 protein is characterized (1) by the N-terminal polyQ domain (5), (2) by dispersed proline-rich-domains that mediate association with various SH3-motif containing proteins of the tyrosine kinase receptor endocytosis machinery and thus modulate neuro-trophic signaling (6, 7), (3) by a C-terminal PAM2 motif that mediates interaction with the poly(A)-binding protein PABPC1 that is crucial for mRNA translation (8) and (4) by Lsm and Lsm-AD sequences that mediate the association with RNAs (9 -11). ATXN2 is normally localized at the rough endoplasmic reticulum (12), but it relocalizes during periods of low cell energy together with PABPC1 to stress granules where the quality control of RNA occurs (3) and where fasting responses are modulated (13).…”