Limone, Adriana (2024) Modulation of mitochondrial quality control pathway through inhibition of 37/67 kDa Laminin Receptor in human skin fibroblasts from genetic Alzheimer’s disease. [Tesi di dottorato]

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Abstract

Autophagy is a highly conserved cellular catabolic process by which macromolecules or damaged organelles are targeted to lysosomes for degradation, thus representing an essential pathway for the maintenance of protein’s homeostasis. Accordingly, growing evidence implicates autophagic dysfunctions in several neurodegenerative disorders, including Alzheimer’s disease (AD). Hence, autophagy modulation might represent a valuable tool in such pathologies. Searching for a compound that stimulates autophagic pathway, led us to identify 37/67 kDa laminin receptor (LR) inhibitor as modulator of Akt-mTOR pathway, the master regulator of autophagy, highlighting a pivotal role for 37/67 kDa LR in this cellular pathway. Accordingly, we found that 37/67 kDa LR inhibitor, NSC47924 inactivates Akt – mTOR axis in fibroblasts from genetic AD-affected patients, suggesting a downstream activation of autophagy. Indeed, NSC47924 influenced the conversion of the cytosolic microtubule-associated LC3-I into the lipidated LC3-II associated to autophagosome. Moreover, qRT-PCR analysis revealed that NSC47924 treatment influenced the expression of genes linked to autophagy initiation. Interestingly, upon inhibitor treatment, we found a modulation of the turnover of autophagic receptors, e.g. p62, NDP52 and OPTN, as well as the mitophagy regulators PINK1 and Parkin, consistent with an efficient elimination of dysfunctional mitochondria which are known to be abnormally accumulated in AD. In fAD fibroblasts, the inhibitor significantly improved mitochondrial morphology, restoring tubular structures and a highly interconnected network. Furthermore, the morphological improvement correlated with a functional recovery of mitochondrial network, as assessed by the measurement of oxygen consumption rate by Seahorse analysis and mitochondrial ROS production. Collectively, our findings suggest that 37/67 kDa LR inhibitor stimulates an autophagic pathway that allows the efficient removal of damaged organelles counteracting crucial AD pathogenic mechanisms, contributing to the recovery of cellular homeostasis.

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