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Reallocation of ribosomes as a disease mechanism of Amyotrophic Lateral Sclerosis

ABSTRACT: The molecular mechanism underlying Amyotrophic Lateral Sclerosis (ALS) is still largely unclear. The RNA binding protein TDP-43 is a major contributor and hallmark of the disease. Here, we show that in multiple TDP-43 models of ALS the alterations of the physiological crosstalk between free RNA/granules and polysomes lead to the global reorganisation of translational resources. We exploited in cellulo and in vivo models of TDP-43 proteinopathy and a unique tag-free miniaturised polysomal profiling coupled with RNA-Seq and biochemical assays with subcellular (i.e. cell body and axon) and compartment (i.e. RNA granules and polysomes) resolution. Using these models, we identified: i) a widespread axonal- and polysome-specific downregulation of TDP-43 target mRNAs, ii) the rewiring of mRNAs in granule and polysome compartments, iii) decrease in ribosome kinetics, iv) a robust ribosome engagement with residual mRNAs and v) downstream alterations in protein synthesis of TDP-43 non-target mRNAs. Irrespective of the ALS model used, we observed a snowball effect in RNA metabolism with redistribution of translational resources. Based on our results we propose a systems-oriented mechanism of disease pathogenesis, leveraging on the conceptual framework of reallocation of cellular resources.

ORGANISM(S): Mus musculus

PROVIDER: GSE239419 | GEO | 2023/12/06

REPOSITORIES: GEO

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Project description:Aims: Loss of nuclear TDP-43 characterises sporadic and most familial forms of amyotrophic lateral sclerosis (ALS). TDP-43 (encoded by TARDBP) has multiple roles in RNA processing. We aimed to determine whether 1) RNA splicing dysregulation is present in lower motor neurons in ALS and in a motor neuron-like cell model, and 2) TARDBP mutations (mtTARDBP) are associated with aberrant RNA splicing using patient-derived fibroblasts. Methods: Affymetrix exon arrays were used to study mRNA expression and splicing in lower motor neurons obtained by laser capture microdissection of autopsy tissue from individuals with sporadic ALS and TDP-43 proteinopathy. Findings were confirmed by qRT-PCR and in NSC34 motor neuronal cells following shRNA-mediated TDP-43 depletion. Exon arrays and immunohistochemistry were used to study mRNA splicing and TDP-43 expression in fibroblasts from patients with mtTARDBP-associated, sporadic and mutant SOD1-associated ALS. Results: We found altered expression of spliceosome components in motor neurons and widespread aberrations of mRNA splicing that specifically affected genes involved in ribonucleotide binding. This was confirmed in TDP-43 depleted NSC34 cells. Fibroblasts with mtTARDBP showed loss of nuclear TDP-43 protein and demonstrated similar changes in splicing and gene expression, that were not present in fibroblasts from patients with sporadic or SOD1-related ALS. Conclusion: Loss of nuclear TDP-43 is associated with RNA processing abnormalities in ALS motor neurons, patient-derived cells with mtTARDBP, and following artificial TDP-43 depletion, suggesting that splicing dysregulation directly contributes to disease pathogenesis. Key functional pathways affected include those central to RNA metabolism. RNA was extracted from fibroblasts grown from neurologically healthy controls (n=6) and 3 groups of patients with ALS: 1) sporadic cases (n=6); 2) cases due to mutations of SOD1 (n=4); 3) cases due to mutations of TARDBP (n=3). The three ALS groups were compared to the controls.

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Reallocation of ribosomes as a disease mechanism of Amyotrophic Lateral Sclerosis

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