D295N Mutant Cathepsin D Exerts a Dominant Negative Effect In Vitro by Promoting α‐Synuclein Accumulation

Abstract

Parkinson's disease (PD) is a neurodegenerative movement disorder affecting more than one percent of people over the age of 65. The histopathological hallmark of PD are proteinaceous inclusions termed Lewy bodies, which contain aberrantly accumulated α‐synuclein. Current therapeutic strategies are aimed at reducing α‐synculein burden within the brain, and understanding factors that contribute to early events which cause synuclein accumulation may facilitate this aim. Cathepsin D (CD) is a lysosomal aspartic protease which has been previously demonstrated to cleave α‐synuclein and reduce α‐synuclein‐associated toxicity both in vitro and in vivo. We show here that introduction of a catalytically inactive mutant CD via a lentiviral vector in SH‐SY5Y cells results in inhibition of endogenous wildtype CD activity and accumulation of α‐synuclein. To further explore the mechanism behind mutant CD's effect on α‐synuclein we used PepstatinA (PepA) to pharmacologically inhibit CD activity. We observed that use of PepA also resulted in significantly increased levels of α‐synuclein. Additionally, LC3I levels were significantly increased in both cells expressing mutant CD or cells treated with PepA, suggesting alterations in the autophagy pathway result from inhibition of CD activity. Our results suggests a dominant negative function for the catalytically inactive mutant CD. We demonstrate that decrease of endogenous CD activity in dopaminergic cells gives rise to α‐synuclein accumulation and alterations in the autophagic pathway, suggesting that improving and/or maintaining CD function within the brain may represent a way to prevent α‐synuclein aggregation and corresponding neuropathology from occurring.