Proteasome Inhibition-Induced Dopamine Neuron Degeneration: Underlying Mechanisms And Neuroprotective Strategies

Objectives: Parkinson's disease (PD) is characterized by the progressive loss of nigral dopamine (DA) neurons. So far, the pathogenesis of PD remains unclear and there is no cure for this disorder. Ubiquitin-proteasome system (UPS) impairment has been proposed to play an important role in the pathogenesis of PD,but the underlying mechanisms and potential therapeutic strategy for the UPS impairment-induced DA neuron degeneration are largely unknown. In the present study, we attempt to determine whether calcium homeostasis disturbance, c-Jun NH2 terminal kinases (JNK) activation and caspase-3 mediated intrinsic apoptotic pathway, as well as endoplasmic reticulum stress (ERS) are involved in the proteasome inhibition-induced DA neuron degeneration, in addition to test the effectiveness and the possible mechanisms of glial derived neurotrophic factor (GDNF) in prevention of the proteasome inhibition-induced DA neuron degeneration.Methods: This study is consisted of three parts of investigations based on the UPS impairment cellular and animal models that were established by administering the proteasome inhibitor lactacystin in the cultured DA neurons, and into rat medial forebrain bundles via stereotaxic injection. In Part I, using primary ventral mesencephalic (VM) cellular model, we examined the role of calcium homeostasis disturbance in the lactacystin-induced DA neuronal injury by determining the intracellular free calcium levels and the DA neuron survival as well as the involvement of L-type voltage dependent calcium channels (L-VDCC) in these events. In part II, using cellular and animal models, we determined the role of JNK in the lactacystin-induced DA neuronal injury by observing JNK activation and caspase-3 mediated intrinsic apoptosis as well as rat nigral DA neural injury. In Part III, using VM cellular model, we tested the effectiveness of GDNF and the possible mechanisms of its neuroprotection by evaluating the effects of GDNF on the DA neuronal injury, ERS related gene and protein level, caspase-3 activation, and the α-synuclein inclusion formation.Results: We have demonstrated (1) Lactacystin exposure induced the expression of the gene related to calcium homeostasis disturbance, lowered intracellular calcium levels, reduced the depolarization-induced calcium entry and DA release, and caused DA neuronal injury. Depolarization reversed the lactacystin-induced calcium homeostasis disturbance and protected DA neuronal injury. Such protection was mimicked by L-VDCC agonists but blocked by L-VDCC antagonists. (2) In vitro, lactacystin treatment caused apoptosis of DA neurons, induced ERS, activated JNK pathway, and elicited caspase-3 mediated intrinsic apoptosis; JNK inhibitor blocked the activation of JNK substrates and intrinsic apoptotic pathway while protecting DA neurons. In vivo, lactacystin caused changes in motor behaviors, striatal DA levels reduction, JNK activation and nigral DA neuron loss which was significantly ameliorated by JNK inhibitor. (3) GDNF pretreatment attenuated the neuron loss and apoptotic cell death in DA neurons while suppressing ERS and caspase-3 activation, but not affectingα-synuclein inclusion body formation.Conclusions: Our study indicates that, under conditions of UPS impairment, (1) a perturbation in calcium homeostasis resulting from decreased L-VDCC activity is involved in DA neuronal injury. (2) JNK is involved in the DA neuron degeneration through a caspase-3 mediated intrinsic apoptotic pathway.(3) GDNF can prevent DA neuron degeneration by suppressing the ERS and caspase-3 activation, but not affecting inclusion formation. These novel findings may provide valuable information to understand the pathogenesis of DA neuron degeneration and to facilitate the discovery of therapeutic targets for preventing and curing PD.