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SLITRK2 knockdown in human motor neurons rescues axonal and functional neuromuscular deficits in FUS-ALS

ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron (MN) loss caused by early axonal and synaptic changes. The mechanisms underlying these changes are studied intensely but remain poorly understood. Here, we exploited FUS-ALS to interrogate cell compartment-specific changes in RNA expression and regulation. To achieve this, we established a pipeline combining human induced pluripotent stem cell (iPSC)-derived MNs with microfluidic compartmentalization. We first validated this platform and characterized the human axonal transcriptome by performing 3'-tag RNA sequencing on paired soma and axon samples from three healthy control (HC) lines. Subsequently, we applied this pipeline to specifically investigate gene expression and alternative polyadenylation (APA) alterations in the axons of FUS-ALS MNs.

ORGANISM(S): Homo sapiens

PROVIDER: GSE308345 | GEO | 2026/09/07

REPOSITORIES: GEO

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Project description:Through the generation of humanized FUS mice expressing full length human FUS, we identify that when expressed at near endogenous murine FUS levels both wild-type or ALS- and frontotemporal dementia (FTD)-causing mutations complement the essential function(s) of murine FUS. Replacement of murine FUS with mutant, but not wild-type, human FUS causes stress-mediated induction of chaperones, decreased expression of ion channels/transporters essential for synaptic function, and reduced synaptic activity, without loss of nuclear FUS or its cytoplasmic aggregation. Most strikingly, accumulation of mutant human FUS is shown to activate an integrated stress response and inhibit local, intra-axonal protein synthesis in hippocampal neurons and sciatic nerves. Collectively, our evidence demonstrates that human ALS/FTD-linked mutations in FUS induce a gain-of-toxicity that includes stress-mediated suppression in intra-axonal translation, synaptic dysfunction, and progressive, age-dependent motor and cognitive disease without cytoplasmic aggregation, altered nuclear localization, or aberrant splicing of FUS-bound pre-mRNAs. Methods: RNA from mouse spinal cords of 18-month-old mFUS-/-/hgFUS (WT, R521C or R521H) and their Non-Tg control littermates was extracted with TRIzol. RNA quality was measured using the Agilent Bioanalyzer system and processed using the Illumina TruSeq Stranded mRNA Sample Preparation Kit according to manufacturer’s protocols. mRNA profiles were generated by deep sequencing, with n≥3 biological replicates per group. Results: We mapped on average 15 million non-redundant reads per sample. Fastq files were aligned to mouse reference genome (mm9 UCSC Genome Browser) using TopHat workfow and the transcript abundance for each annotated protein-coding gene [as fragments per kilobase of transcript per million mapped reads (FPKM)] was estimated by Cufflinks. 13,468 genes which expressed FPKM>=1 were kept for downstream analyses. RNA profiles from normal (Non-Tg) and humanized hgFUSWT mice were almost undistinguishable. Both humanized mutant FUS lines had highly distinct RNA profiles [determined with unsupervised hierarchical clustering and principal component analysis (PCA)], with 709 down and 348 up-regulated genes relative to age-matched Non-Tg or humanized hgFUSWT littermates (P<0.05). These changes uncovered FUS mutant dependent altered pathways that may contribute to ALS/FTD-linked mutant FUS-mediated toxicity. The validation by RT-QPCR of altered expression of 20 genes is shown in Figure 5.

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SLITRK2 knockdown in human motor neurons rescues axonal and functional neuromuscular deficits in FUS-ALS

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