Proteomic analysis of the TDP-43-associated insoluble fraction from NEFH-TDP-43 mouse brain suggests sustained stress granule formation, CLUH granule recruitment and impaired mitochondrial metabolism

Abstract

AbstractCytoplasmic accumulation and aggregation of TDP-43 is a hallmark of ∼97% of ALS cases. Formation of TDP-43 insoluble aggregates is suggested to either directly or indirectly cause motor neuron loss and progressive neuromuscular degeneration, although how this occurs is not precisely understood. Cytoplasmic TDP-43 is observed in stress granules (SG). SGs are ribonucleoprotein (RNP) complexes formed during stress conditions, consisting of mRNAs and RNA-binding proteins (RNPs). Chronic TDP-43/SG formation may play a role in neuromuscular degeneration in ALS. The composition ofin vivoTDP-43-asscociated SGs in ALS not known. This knowledge may provide insights into the molecular pathways impaired by TDP-43-associated SGs and suggest disease modifying mechanisms. The aim of this study was to isolate and analyse the proteome of the insoluble TDP-43-associated SG fraction from brain tissue of end-stage TDP-43ΔNLS mice. Proteomic analysis identified 134 enriched and 17 depleted proteins in the TDP-43ΔNLS mice, when compared to the control mice. Bioinformatics analyses of the impacted proteins from the SG preparation suggested that brain tissue from end-stage NEFH-TDP-43ΔNLS mice have sustained SG formation, CLUH granule recruitment and impaired mitochondrial metabolism. This is the first time that CLUH granule recruitment has been demonstrated in ALS and the known role of CLUH suggests that cell starvation is a potential mechanism of motor neuron loss that could be targeted in ALS.HighlightsWe present a detailed a protocol for the extraction of cross-linked TDP containing stress granules from brain tissue.We present proteomics data from the insoluble fraction from brain tissue of an ALS mouse model.We identify the mitochondrial mRNA transport protein CLUH and CLUH targets trapped in insoluble SG fraction of brain.Reanalysing proteomics data from axonal soluble fraction supports a link between proteins trapped in the brain and depleted from the axons.Propose a model where metabolic mitochondrial enzymes trapped in the insoluble fraction from the brain via a CLUH dependent mechanism results in motor neuron death by starvation in ALS.