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Ataxin-2 (ATXN2) is implicated mainly in mRNA processing. Some ATXN2 associates with receptor tyrosine kinases (RTK), inhibiting their endocytic internalization through interaction of proline-rich domains (PRD) in ATXN2 with SH3 motifs in Src. Gain of function of ATXN2 leads to neuronal atrophy in the diseases spinocerebellar ataxia type 2 (SCA2) and amyotrophic lateral sclerosis (ALS). Conversely, ATXN2 knockout (KO) mice show hypertrophy and insulin resistance. To elucidate the influence of ATXN2 on trophic regulation, we surveyed interactions of ATXN2 with SH3 motifs from numerous proteins and observed a novel interaction with Grb2. Direct binding in glutathione S-transferase (GST) pull-down assays and coimmunoprecipitation of the endogenous proteins indicated a physiologically relevant association. In SCA2 patient fibroblasts, Grb2 more than Src protein levels were diminished, with an upregulation of both transcripts suggesting enhanced protein turnover. In KO mouse embryonal fibroblasts (MEF), the protein levels of Grb2 and Src were decreased. ATXN2 absence by itself was insufficient to significantly change Grb2-dependent signaling for endogenous Ras levels, Ras-GTP levels, and kinetics as well as MEK1 phosphorylation, suggesting that other factors compensate for proliferation control. In KO tissue with postmitotic neurons, a significant decrease of Src protein levels is prominent rather than Grb2. ATXN2 mutations modulate the levels of several components of the RTK endocytosis complex and may thus contribute to alter cell proliferation as well as translation and growth.
Parkinson’s disease (PD) is characterized by distinct motor and non-motor symptoms. Sleep disorders are the most frequent and challenging non-motor symptoms in PD patients, and there is growing evidence that they are a consequence of disruptions within the circadian system. PD is characterized by a progressive degeneration of the dorsal vagal nucleus and midbrain dopaminergic neurons together with an imbalance of many other neurotransmitters. Mutations in α-synuclein (SNCA), a protein modulating SNARE complex-dependent neurotransmission, trigger dominantly inherited PD variants and sporadic cases of PD. The A53T SNCA missense mutation is associated with an autosomal dominant early-onset familial PD. To test whether this missense mutation affects the circadian system, we analyzed the spontaneous locomotor behavior of non-transgenic wildtype mice and transgenic mice overexpressing mutant human A53T α-synuclein (A53T). The mice were subjected to entrained- and free-running conditions as well as to experimental jet lag. Furthermore, the vesicular glutamate transporter 2 (VGLUT2) in the suprachiasmatic nucleus (SCN) was analyzed by immunohistochemistry. Free-running circadian rhythm and, thus, circadian rhythm generation, were not affected in A53T mice. A53T mice entrained to the light–dark cycle, however, with an advanced phase angle of 2.65 ± 0.5 h before lights off. Moreover, re-entrainment after experimental jet lag was impaired in A53T mice. Finally, VGLUT2 immunoreaction was reduced in the SCN of A53T mice. These data suggest an impaired light entrainment of the circadian system in A53T mice.
Expansions of the polyglutamine (polyQ) domain (≥34) in Ataxin-2 (ATXN2) are the primary cause of spinocerebellar ataxia type 2 (SCA2). Recent studies reported that intermediate-length (27–33) expansions increase the risk of Amyotrophic Lateral Sclerosis (ALS) in 1–4% of cases in diverse populations. This study investigates the Turkish population with respect to ALS risk, genotyping 158 sporadic, 78 familial patients and 420 neurologically healthy controls. We re-assessed the effect of ATXN2 expansions and extended the analysis for the first time to cover the ATXN2 locus with 18 Single Nucleotide Polymorphisms (SNPs) and their haplotypes. In accordance with other studies, our results confirmed that 31–32 polyQ repeats in the ATXN2 gene are associated with risk of developing ALS in 1.7% of the Turkish ALS cohort (p = 0.0172). Additionally, a significant association of a 136 kb haplotype block across the ATXN2 and SH2B3 genes was found in 19.4% of a subset of our ALS cohort and in 10.1% of the controls (p = 0.0057, OR: 2.23). ATXN2 and SH2B3 encode proteins that both interact with growth receptor tyrosine kinases. Our novel observations suggest that genotyping of SNPs at this locus may be useful for the study of ALS risk in a high percentage of individuals and that ATXN2 and SH2B3 variants may interact in modulating the disease pathway.
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.
The presynaptic protein alpha-synuclein has received much attention because its gain-of-function is associated with Parkinson’s disease. However, its physiological function is still poorly understood. We studied brain regions of knock-out mice at different ages with regard to consistent upregulations of the transcriptome and focused on glyoxalase I (GLO1). The microarray data were confirmed in qPCR, immunoblot, enzyme activity, and behavior analyses. GLO1 induction is a known protective cellular response to glucose stress, representing efforts to decrease toxic levels of methylglyoxal (MG), glyoxal and advanced glycation endproducts (AGEs). Mass spectrometry quantification demonstrated a ubiquitous increase in MG and fructosyl-lysine as consequences of glucose toxicity, and consistent enhancement of certain AGEs. Thus, GLO1 induction in KO brain seems insufficient to prevent AGE formation. In conclusion, the data demonstrate GLO1 expression and glycation damage to be induced by alpha-synuclein ablation. We propose that wild-type alpha-synuclein modulates brain glucose metabolism.
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.
Parkinson's disease (PD) is the most frequent neurodegenerative movement disorder and manifests at old age. While many details of its pathogenesis remain to be elucidated, in particular the protein and mitochondrial quality control during stress responses have been implicated in monogenic PD variants. Especially the mitochondrial kinase PINK1 and the ubiquitin ligase PARKIN are known to cooperate in autophagy after mitochondrial damage. As autophagy is also induced by loss of trophic signaling and PINK1 gene expression is modulated after deprivation of cytokines, we analyzed to what extent trophic signals and starvation stress regulate PINK1 and PARKIN expression. Time course experiments with serum deprivation and nutrient starvation of human SH-SY5Y neuroblastoma cells and primary mouse neurons demonstrated phasic induction of PINK1 transcript up to twofold and PARKIN transcript levels up to sixfold. The corresponding threefold starvation induction of PARKIN protein was limited by its translocation to lysosomes. Analysis of primary mouse cells from PINK1-knockout mice indicated that PARKIN induction and lysosomal translocation occurred independent of PINK1. Suppression of the PI3K-Akt-mTOR signaling by pharmacological agents modulated PARKIN expression accordingly. In conclusion, this expression survey demonstrates that PARKIN and PINK1 are coregulated during starvation and suggest a role of both PD genes in response to trophic signals and starvation stress.
Complexin-1 and foxp1 expression changes are novel brain effects of
alpha-synuclein pathology
(2014)
As the second most frequent neurodegenerative disorder of the aging population, Parkinson’s disease (PD) is characterized by progressive deficits in spontaneous movement, atrophy of dopaminergic midbrain neurons and aggregation of the protein alpha-synuclein (SNCA). To elucidate molecular events before irreversible cell death, we studied synucleinopathy-induced expression changes in mouse brain and identified 49 midbrain/brainstem-specific transcriptional dysregulations. In particular complexin-1 (Cplx1), Rabl2a and 14-3-3epsilon (Ywhae) downregulation, as well as upregulation of the midbrain-specific factor forkhead box P1 (Foxp1) and of Rabgef1, were interesting as early mRNA level effects of alpha-synuclein triggered pathology. The protein levels of complexin-1 were elevated in midbrain/brainstem tissue of mice with A53T-SNCA overexpression and of mice with SNCA-knockout. The response of CPLX1 and Foxp1 levels to SNCA deficiency supports the notion that these factors are regulated by altered physiological function of alpha-synuclein. Thus, their analysis might be useful in PD stages before the advent of Lewy pathology. Because both alpha-synuclein and complexin-1 modulate vesicle release, our findings support presynaptic dysfunction as an early event in PD pathology.
Hereditary Parkinson’s disease can be triggered by an autosomal dominant overdose of alpha-Synuclein (SNCA) or the autosomal recessive deficiency of PINK1. We recently showed that the combination of PINK1-knockout with overexpression of A53T-SNCA in double mutant (DM) mice potentiates phenotypes and reduces survival. Now we studied brain hemispheres of DM mice at age of 18 months in a hypothesis-free approach, employing a quantitative label-free global proteomic mass spectrometry scan of posttranslational modifications focusing on methyl-arginine. The strongest effects were documented for the adhesion modulator CMAS, the mRNA decapping/deadenylation factor PATL1, and the synaptic plasticity mediator CRTC1/TORC1. In addition, an intriguing effect was observed for the splicing factor PSF/SFPQ, known to interact with the dopaminergic differentiation factor NURR1 as well as with DJ-1, the protein responsible for the autosomal recessive PARK7 variant of PD. CRTC1, PSF, and DJ-1 are modulators of PGC1alpha and of mitochondrial biogenesis. This pathway was further stressed by dysregulations of oxygen sensor EGLN3 and of nuclear TMPO. PSF and TMPO cooperate with dopaminergic differentiation factors LMX1B and NURR1. Further dysregulations concerned PRR18, TRIO, HNRNPA1, DMWD, WAVE1, ILDR2, DBNDD1, and NFM. Thus, we report selective novel endogenous stress responses in brain, which highlight early dysregulations of mitochondrial homeostasis and midbrain vulnerability.
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.