GAIN OF TOXIC FUNCTION OF CHAPERONE ASSISTED SELECTIVE AUTOPHAGY MEMBERS IN NEUROMUSCULAR DISEASES: A CHARACTERIZATION OF DISEASE-RELATED MUTANTS

Proteostasis alteration characterizes several diseases affecting muscle and neuronal cells and can be caused by protein misfolding and aggregation and/or mutations in Protein Quality Control system members. For instance, mutations in the Chaperone Assisted Selective Autophagy (CASA) members are causative of neuropathies/myopathies. The CASA complex mainly acts in muscles and neurons, by facilitating the disposal of misfolding and aggregating proteins. This occurs through substrate recognition by the chaperone HSPB8, which interacts with the HSP70 co-chaperone BAG3. If substrates cannot be refolded by HSP70, they are ubiquitinated by the E3 ubiquitin ligase CHIP. Substrates are then routed and compartmentalized into perinuclear deposits of aggregates, or aggresomes, for subsequent autophagic-lysosomal disposal. In the first part of my studies, I showed results on neuropathies/myopathies related P209S/L/Q BAG3 mutants. By overexpressing BAG3 mutants in cells, I observed that all BAG3-P209 mutants are characterized by decreased solubility. The decreased solubility determines BAG3-P209 mutants aggregation and relocation with the other CASA members at the aggresome, resulting in an impairment of CASA activity against misfolded clients. In addition, I demonstrated that BAG3-P209 mutants are preferentially degraded through autophagy. Thus, I showed that boosting autophagy using trehalose, proven to favour the clearance of aggregating proteins related to neurodegenerative diseases, determined BAG3-P209 aggregates disposal, representing a valuable therapeutic approach in BAG3-P209 diseases. In the second part, I firstly showed results on HSPB8 variants S9P, P41S and S181C found in Amyotrophic Lateral Sclerosis (ALS) patients. Using a motoneuron-like cell model overexpressing HSPB8 variants, I observed no differences in HSPB8 variants biochemical behaviour with respect to HSPB8 wildtype (WT), except for the S181C variant, characterized by a structural alteration. However, no differences in HSPB8 variants activity were observed. Instead, I obtained promising results on myopathies/neuropathies-related frameshift mutants pPro173Serfs*43, pGln170Glyfs*45, and pThr194Serfs*23. I found that these mutants present the same C-terminal modification and/or an identical elongated C-terminal tail, predicted to affect protein solubility. Indeed, I observed insolubility and aggregation of these mutants, when overexpressed in cells. Similar to BAG3-P209 mutants, HSPB8 mutants co-segregate with CASA members and associate to an increase in ubiquitinated proteins, suggesting CASA impairment. Again, trehalose-mediated autophagic enhancement favoured the clearance of these aggregating HSPB8 mutants. In the third part, I characterized isogenic iPSCs-derived motoneurons as new cell models to study TDP-43-related proteinopathies. These cell lines were previously gene-edited, to express WT or the ALS-A315T mutated TDP-43, tagged with the DENDRA2 reporter. Using a small molecules-based protocol, I differentiated these iPSCs obtaining a mixed population of neurons and motoneurons. Model validation showed that under untreated conditions all WT and A315T-mutated cell lines are not characterized by TDP-43 misbehaviour, since no hallmark of pathogenic TDP-43 was observed; instead, upon proteasome inhibition, all cell lines showed TDP-43 cleavage, phosphorylation, and aggregation, correlating with activated cleaved caspase-3, in line with the current literature. With my work, I determined shared biochemical and functional alterations between BAG3 and HSPB8 mutants, suggesting that a common therapeutic strategy might be beneficial in the related neuromuscular diseases. In addition, I defined TDP-43-DENDRA2-derived motoneurons as a valuable tool to study TDP-43-related proteinopathies.