In vitro study of autophagy dysfunctions associated with Parkinson’s disease

Aberrant and misfolded protein accumulation, in particular alpha-synuclein (asyn), is one of the pivotal pathogenic mechanism involved in Parkinson’s disease (PD), the second most common neurodegenerative disorder. As a matter of fact, the abnormal and uncontrolled intracellular overload of undigested proteins relentlessly results in extreme cellular stressor conditions. This is particularly relevant for post-mitotic cells such as neurons. Autophagy is the prominent intracellular clearance mechanism and recent studies have demonstrated dysfunctions of this pathway in PD patients, supporting the hypothesis that these defects can contribute to PD pathogenesis. To investigate autophagic pathways (macroautophagy and chaperone-mediated autophagy, CMA) human neuroblastoma SH-SY5Y cells (basal and differentiated) and mouse cortical neurons were exposed to the PD-related toxin rotenone, a mitochondrial complex I inhibitor, for 24 and 48 hours at increasing concentrations. mRNA and protein levels of autophagy effectors (LC3II, beclin 1, lamp2A, hsc70) and substrates (asyn, MEF2D) were evaluated. Furthermore, the effect of genetic alterations (A53T mutant or wild-type asyn overexpression) and neuroprotective agents on autophagy was also assessed. Results from this study will allow a better understanding of PD pathogenic mechanisms and the identification of new possible therapeutic targets.