Objective: Our aim is to identify the proteolytic pathways responsible for the clearance of TPPP/P25A in oligodendrocytes, which may eventually represent potential therapeutic targets for Multiple System Atrophy (MSA).

Background: MSA is a rare neurological disorder of unknown etiology and rapid progression. The main pathological hallmark of the disease is the presence of Glial Cytoplasmic Inclusions (GCIs) within oligodendrocytes, mainly composed of alpha-synuclein (SNCA) and the oligodendroglial-specific phosphoprotein TPPP/P25A. TPPP/P25A protein is critical for the aggregation of oligodendroglial SNCA in MSA; therefore manipulation of its expression levels will provide a rational approach to combat the accumulation of SNCA within oligodendrocytes.

Method: For the purposes of the current study we utilized a rat immortalized oligodendroglial cell line stably overexpressing human TPPP/P25A (OLN-p25α) and murine primary oligodendrocytes expressing endogenous TPPP/P25A and treated them with either PBS (as control) or human SNCA Pre-Formed Fibrils (PFFs) as pathological seeds. To assess the role of the autophagy-lysosome pathway (ALP) in TPPP/P25A proteolysis, we used pharmacological inhibitors (general lysosomal: NH₄Cl, macroautophagy: 3-MA) and enhancers [Chaperone-mediated autophagy (CMA): AR7 (Atypical retinoid 7,- RARα antagonist); macroautophagy: rapamycin] of the autophagic pathways or we performed siRNA-based gene silencing of autophagy–related genes (CMA: Lamp2a, macroautophagy: Atg5) and assessed the levels of TPPP/P25A by western immunoblotting and immunocytochemistry.

Results: The pharmacological and molecular inhibition of ALP resulted in a significant accumulation of TPPP/P25A in both cellular models, upon their treatment either with PBS or PFFs. Moreover, our data show that TPPP/P25A bears a KFERQ-like motif and is effectively cleared via CMA in an in vitro system of isolated rat brain lysosomes. Interestingly, pharmacological enhancement of CMA or macroautophagy resulted in a decrease of TPPP/P25A levels both under physiological and under pathological conditions of PFF-induced seeding of SNCA, further supporting the role of ALP in its degradation.

Conclusion: The ALP mediates the clearance of TPPP/P25A in oligodendrocytes; therefore manipulation of autophagic pathways may represent a successful approach for the removal of pathologically accumulated proteins in the context of MSA.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/unraveling-the-mechanisms-of-tubulin-polymerization-promoting-protein-tppp-p25a-degradation-in-oligodendrocytes/