A Preliminary Study of Chaperone-Mediated Autophagy in ALS Lymphomonocytes

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective degeneration of both upper and lower motor neurons associated to TDP-43 accumulation. Recent evidences showed that TDP-43 is degraded, at least in part, by the chaperone-mediated autophagy (CMA). In this work, we assessed the expression of the two principal parameters of CMA in peripheral blood mononuclear cells (PBMC) obtained from 30 sporadic ALS patients (sALS), 9 ALS patients carrying mutations in SOD1 gene (SOD1+) and 30 healthy controls (CTRL). Our results showed a significant reduction of hsc70 mRNA and protein levels in sALS and SOD1+ patients with respect to controls, while no changes were observed for lamp2A, the rate limiting protein of CMA. Moreover, to estimate the degradative activity of this pathway, we evaluated the levels of specific and nonspecific substrates: although myocyte enhancer factor 2D (MEF2D) levels were unchanged, we showed increased TDP-43 levels in sALS patients, showing also an increased TDP-43/TDP-35 ratio. Immunohistochemistry analysis, performed on 3 sALS patients and 3 healthy controls, revealed that TDP-43 was aggregated in patients cells outside nuclei. 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 and macroautophagy, and we showed decreased levels of BAG1 in both sporadic and familial ALS patients. In conclusion, our results evidenced reduced hsc70 levels with no change in lamp2A, excluding a significant impairment of CMA in PBMC obtained from ALS patients. Anyway, the accumulation of TDP-43 suggests that other degradative pathways could be altered and further investigations are needed in order to explore this hypothesis.