Understanding the contribution of mtHsp70 towards mitochondrial dysfunction in Parkinson's disease: A yeast model

Abstract

Parkinson's disease (PD) is one of the most prevalent neurodegenerative disorders, characterized by loss of dopaminergic neuron function; leading to bradykinesia and motor dysfunction. It is observed that various genes which have been implicated in the familial and sporadic forms of the disease are involved in pathways which focalize at the mitochondria. This organelle is a central regulator of cell death, and mitochondrial dysfunction is a common phenomenon associated with multiple neurodegenerative diseases. Not surprisingly, a clinical screen identified PD-associated mutations in mitochondrial Hsp70 (mtHsp70), which is a critical protein involved in mitochondria biogenesis and maintenance of homeostasis. In order to specifically understand the role of the mtHsp70 protein, we have developed a Saccharomyces cerevisiae model for studying the disease variants in isolation from other players of this multifactorial disease. We generated the analogous mutants R103W and P486S in yeast mtHsp70 which remarkably recapitulated the symptoms of mitochondrial dysfunction in affected neurons including cell death, increased generation of ROS, mtDNA loss and respiratory incompetence. Spectral analysis of a fluorescent carbocyanine dye uptake in isolated mitochondria indicated compromised membrane potential gradients as a cause for dysfunction. At the molecular level, circular dichroism based-thermal melt analyses confirmed the observed in vivo aggregation propensity of R103W, while P486S exhibited futile enhanced interaction with J-protein co-chaperone partners thereby resulting in loss of chaperoning activity by mtHsp70. Remarkably, these altered biochemical properties reflect identical defects found by us in the human mtHsp70 variants, thereby confirming a direct involvement of mtHsp70 in PD progression. Thus, by utilizing the significant structural and functional homology between the yeast and human mtHsp70 proteins, and an integrated in vivo and in vitro approach, we demonstrate the basic underlying mechanisms of mtHsp70 association with PD.