Ubiquitin-proteasome system dysfunction in experimental models of Parkinson's disease

The present study investigates cellular mechanisms underlying the pathogenic role of ubiquity proteasome system (UPS) dysfunction in dopaminergic degeneration following exposure to Parkinsonian neurotoxins. Mutations or overproduction of α-synuclein have been shown to be associated with familial Parkinson’s disease (PD), and wild type αsynuclein is the major component of Lewy bodies, the protein inclusion bodies characteristic of PD. The organochlorine pesticide dieldrin has been implicated as an environmental risk factor for PD. The gene-environment interaction between α-synuclein and dieldrin impaired the proteolytic function of UPS leading to profound formation of α-synuclein aggregates, and enhanced apoptotic cell death in dopaminergic neuronal N27 cells. Proteasome inhibition by MG-132 elicited severe dopaminergic neurotoxicity in both an immortalized dopaminergic cell model of Parkinson’s disease (N27 cells) and primary mesencephalic culture. Stereotaxic injection of MG-132 into the substantia nigra resulted in marked nigrostriatal degeneration in vivo, as demonstrated by prominent loss of nigral dopamine neurons and depletion of striatal dopamine. The proteasome inhibitor MG-132 was utilized as a pharmacologic tool to mimic UPS dysfunction in the remaining studies to investigate the molecular and cellular mechanisms underlying UPS impairment-induced dopaminergic degeneration. Proteasome inhibition by MG-132 depolarized mitochondrial membrane potential, caused mitochondrial release of proapoptotic molecules, and triggered apoptotic cell death exclusively proceeding through the mitochondria-dependent pathway in N27 cells. Caspase-3-dependent proteolytic cleavage of PKCδ into catalytic fragment (PKCδ-CF) and regulatory fragment (PKCδ-RF), plays a crucial role in MG-132-induced dopaminergic v apoptosis; ROS generation was not found to be important in MG-132-related cell death. PKCδ proteolytic cleavage resulted in a substantial increase in its kinase activity. The PKCδ-specific inhibitor rottlerin, but not SOD mimetic MnTBAP, significantly alleviated caspase-9 and -3 activation, indicating that proteolytically activated PKCδ amplified mitochondrial apoptosis cascades. In agreement, mitochondrial translocation of PKCδ-CF led to caspase-3 activation and DNA fragmentation. Suppression of PKCδ proteolytic cleavage by caspase-3 cleavage resistant mutant PKCδ effectively inhibited MG-132induced activation of mitochondrial apoptosis. Further study into the mechanisms of proteasome inhibition activating mitochondrial apoptosis yielded some exciting new findings. The mitochondria may be a key sensor of polyubiquitin stress because ubiquitinated proteins preferentially accumulate in mitochondria as compared to cytosol. Additionally, overexpression of ubiquitin mutant effectively rescues cells from MG-132induced mitochondrial apoptosis without altering antioxidant status of cells, whereas ubiquitin mutant augmented the proapoptotic effect of MG-132. Additionally, ubiquitin conferred neuronal resistance to a variety of dopaminergic neurotoxins that impair UPS including dopamine, MPP and dieldrin. These results suggest that UPS impairment consequent to neurotoxin exposure plays a crucial contributory role in dopaminergic degeneration and that exposure to neurotoxic agents and gene-environment interactions could elicit dopaminergic neurotoxicity by converging to impair UPS function. Additionally, the findings of this work not only provide novel insights into cellular mechanisms of ubiquitin stress in dopaminergic neuronal cells but also serve as a foundation for future study ascertaining biochemical and functional links vi between UPS dysfunction and mitochondrial apoptosis in the degenerative process of Parkinson’s disease.