Study Of Chaperone Mediated Autophagy in Lymphomonocytes of sporadic and familial als patients evidenced Hsc70 reduction and TDP-43 aggregation

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective degeneration of both upper and lower motor neurons. Causes of disease remain still unknown, although protein aggregate formation in motor neurons is a neuropathological hallmark. These aggregates contain several different proteins, but transactivation response (TAR) DNA-binding protein (TDP-43) is considered the major component. Recent evidences showed that TDP-43 is degraded not only by the ubiquitin proteasome system (UPS) and macroautophagy, but also by the chaperone-mediated autophagy (CMA) through an interaction between the cytosolic chaperone heat shock cognate 70 (Hsc70, also known as HSPA8) and ubiquitylated TDP-43. We assessed the two principal parameters of CMA in easily accessible cells like peripheral blood mononuclear cells (PBMCs) obtained from 30 sporadic ALS patients (sALS), 9 ALS patients carrying mutations in SOD1 gene (SOD1+) and 30 healthy controls. Our results showed a significant reduction of hsc70 in sALS and SOD1+ patients with respect to controls, while no changes were observed for the lysososme-associated membrane protein 2A (lamp2A), the rate limiting protein of CMA. Moreover, to estimate the degradative activity of this pathway we evaluated the levels of specific and nonspecific CMA substrates: although myocyte enhancer factor 2D (MEF2D) levels were unchanged, we showed increased TDP-43 in sALS patients. TDP-43 is physiologically degraded through a caspase-3-mediated cleavage to generate two fragments (35 and 25 kDa, respectively). We here reported an increased TDP-43/TDP-35 ratio in sporadic ALS patients with respect to controls, suggesting a possible reduction of efficiency of the normal proteolytic cleavage. Immunohistochemistry analysis, performed in 3 sALS and 3 healthy controls, revealed that TDP-43 was aggregated in patients cells outside nuclei, confirming that PBMCs are able to recapitulate this pathological feature previously identified in affected tissues of ALS patients. Furthermore, we also investigated parameters related to other degradative mechanisms, reporting a significant reduction of mRNA levels of two macroautophagy-related parameters, SQSTM1/p62 and LC3, in both sALS and SOD1+ patients with respect to controls. Lastly, we assessed mRNA expression of two co-chaperones BAG1 and BAG3, which regulate trafficking of substrates to ubiquitin-proteasome system (UPS) and macroautophagy respectively, and we showed decreased levels of BAG1 in both sporadic and familial ALS patients. In conclusion, our results evidenced reduced hsc70 levels without alteration of lamp2A and accumulation of CMA substrates, excluding an impairment of this catabolic pathway in ALS patients. Anyway, the accumulation of TDP-43 suggests that other degradative pathways could be altered in ALS, further investigations are needed to investigate UPS and macroautophagy in patients cells to explore this hypothesis