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Mass spectrometry identifies insoluble candidate RBPs in amyotrophic lateral sclerosis

ABSTRACT: To discover RBPs with increased insolubility in a human ALS model, we applied a well established dual-SMAD inhibition-based protocol (Fang et al., 2019; Markmiller et al., 2021; Markmiller et al., 2018; Martinez et al., 2016) to generate iPSC-MN from six control iPSC lines, from four iPSC lines originating from two sALS patients, and from two iPSC lines originating from fALS patients with pathogenic variants in the TARDBP gene (Table S1; Figure S1A). No difference in differentiation capacity was observed (Figure S1B-G), resulting in average 40% ISL1+ MN (Figures S1G), comparable to numbers observed in large scale MN differentation studies (Baxi et al., 2022). The susceptibility of ALS MN to sodium arsenite-induced stress was not changed (Figure S1H and I). Next, we asked which proteins exhibit an increased insolubility in our ALS iPSC-MN. We fractioned iPSC- MN by lysis in radio-immunoprecipitation assay (RIPA) buffer, followed by ultracentrifugation and solubilization of RIPA insoluble proteins in urea buffer. The ultracentrifugation-cleared RIPA insoluble protein fraction is widely used to study protein insolubility in the context of neurodegeneration (Nuber et al., 2013; Walker et al., 2015). Label-free mass spectrometry of the insoluble protein fraction was utilized to identify proteins that are insoluble in sALS and fALS, relative to control iPSC-MNs (Figure 1A). Gene ontology (GO) analysis of the 100 proteins (top 2.9% of all detected proteins) with the highest label free quantification (LFQ) intensities in controls (Figure S1J) revealed that ‘unfolded protein binding’ (corrected P = 7.95 x 10-16) and ‘structural constituent of cytoskeleton’ (corrected P = 1.47 x 10-10) were among the 10 most significantly enriched GO terms, indicating enrichment of insoluble proteins (Figure S1K). Principle component analysis of the insoluble fractions did not distinguish ALS from control samples, suggesting that the overall insoluble proteome is not changed (Figure S1L). At threshold P ≤ 0.05 (Welch’s t-test) and fold change ≥ 1.5, we identified 88 proteins enriched in the insoluble fraction in ALS samples relative to control (Figure 1B). When the sample labels were randomly shuffled, we observed an average of 7.5 proteins (~12-fold lower) as differentially enriched at the same statistical thresholds, indicative of an ALS-specific protein insolubility pattern (Figure 1C). The 88 candidate proteins included cytoskeletal components and motor proteins, functional categories associated with prominent ALS in vitro phenotypes (Akiyama et al., 2019; Egawa et al., 2012; Fazal et al., 2021; Guo et al., 2017; Kreiter et al., 2018) (Figure 1D). Notably, 5 RBPs, NOVA1, ELAVL4, FXR2, RBFOX2, and RBFOX3 were also enriched (Figure 1D). The NOVA1 paralog NOVA2 was significantly enriched (P = 0.03) but did not meet our enrichment threshold (fold change = 1.36). Interestingly, insoluble TDP-43 protein was not significantly different in ALS and control (P = 0.98; fold change = 0.97). Western blot analysis confirmed the increase in insolubility of NOVA1, NOVA2, ELAVL4, RBFOX2 and RBFOX3 (Figure 1E and 1F). The soluble protein levels of NOVA1 and NOVA2 were also increased (Figure 1F). In conclusion, we identified 5 RBPs with elevated insoluble protein levels of ALS-iPSC-MNs.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Pluripotent Stem Cell, Stem Cell

DISEASE(S): Amyotrophic Lateral Sclerosis

SUBMITTER: Karsten Boldt

LAB HEAD: Dr. Karsten Boldt

PROVIDER: PXD032140 | Pride | 2022-06-28

REPOSITORIES: Pride

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Project description:We established iPSCs from healthy donors, SOD1-ALS and TDP43-ALS patients. Using our differentiation protocol originally developed by Reinhardt et al.,2013, we diferentiated these iPSCs toward spinal motor neurons (MNs) and reproduce ALS pathology in a dish. To extend our understanding of finding different molecular mechanisms and pathways related to SOD1- and TDP43 mutations in ALS disease, we have performed a comprehensive gene expression profiling study using RNA-Seq of the iPSC-derived MN models from control individuals and carefully compared with those from SOD1-ALS and TDP43-ALS patients. To generate novel hypotheses of putative underlying molecular mechanisms in ALS, we used human induced pluripotent stem cell (hiPSCs)-derived motor neurons (MNs) from SOD1- and TARDBP (TDP-43 protein)-mutant-ALS patients and healthy controls to perform high-throughput RNA-sequencing (RNA-Seq). An integrated bioinformatics approach was employed to identify differentially expressed genes (DEGs) and key pathways underlying these familial forms of the disease (fALS). In TDP43-ALS, we found dysregulation of transcripts encoding components of the transcriptional machinery and transcripts involved in splicing regulation were particularly affected. In contrast, less is known about the role of SOD1 in RNA metabolism in motor neurons. Here we found that many transcripts relevant for mitochondrial function were specifically altered in SOD1-ALS, indicating transcriptional signatures and expression patterns can vary significantly depending on the causal gene that is mutated. Surprisingly, however, we identified a clear downregulation of genes involved in protein translation in SOD1-ALS suggesting that ALS-causing SOD1 mutations shift cellular RNA abundance profiles to cause neural dysfunction. Altogether, we provided here an extensive profiling of mRNA expression in two ALS models at the cellular level, corroborating the major role of RNA metabolism and protein translation as a common pathomechanism in ALS

Project description: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 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, soluble and insoluble protein levels were increased. In 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 selective induction of Fbxw8 in 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. Factorial design comparing Atxn2 knock-in mice with wild type littermates in three different tissues (brainstem, cerebellum, liver)

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Mass spectrometry identifies insoluble candidate RBPs in amyotrophic lateral sclerosis

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