The Role Of CDK5-mediated Phosphorylation Of RKIP In Parkinson’s Disease

Neurodegenerative diseases catch more and more attentions in these years, accompanied with the aging of population. Loss of neurons is a common pathological event in all neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntingdon’s disease (HD), and the neuronal loss is responsible for many behavioral symptoms of these diseases. There were a lot of reports about the mechanisms involved in the neuronal loss of neurodegenerative diseases, but the details are still under exploration. The significance of neuronal cell cycle re-entry has been noticed in the studies of neuronal death recently. Cell cycle is a crucial event in normal division and differentiation of proliferative cells, but post-mitotic neurons are not allowed to enter cell cycle in physiological circumstances. Once a neuron enters cell cycle in a stimulated situation, it is called "cell cycle re-entry", and the neuron is doomed because the entry of cell cycle would trigger apoptotic signals rather than cell division signals in a post-mitotic neuron.Cell cycle must be delicately regulated by several signaling pathways together, and mitogen-activated protein kinases cascade (ERK/MAPK cascade) plays an improtant role in this process. ERK/MAPK pathway normally mediates the G1/S transition and spindle assembly checkpoint in proliferative cells, but cell death is usually inevitable once a neuron enters the S-phase. So the study of the regulation of ERK cascade is quite valuable for the understanding of cell cycle re-entry and cell death in neurons.Raf kinase inhibitor protein (RKIP) is a physiological inhibitor of ERK/MAPK pathway, interacting with Raf-1and blocking its activity. Downregulation of RKIP has been reported to correlate with many cancers, but the mechanisms that underlie this downregulation is still poorly understood. Plus, the expression of RKIP is high in the brain, but its functions in the nervous system and neurodegenerative diseases are not clear yet.To explore the regulation of RKIP in neurons, we added another protein in the system, cyclin-dependent kinase5(CDK5). The activity of CDK5is highly specific in the nervous system, and CDK5controls many important events in neurons such as differentiation, axon guidance, synaptic plasticity and migration by means of phosphorylating a string of substrates. In this research, we studied the interaction between CDK5and RKIP, discussed the influence of this interaction on the activity of ERK cascade and mortality of dopaminergic neurons in Parkinson’s disease.Firstly, we identified RKIP as a substrate of CDK5, and the phosphorylation of RKIP at T42attenuated its affinity to Raf-1, so the phosphorylation of RKIP mediated the release of Raf-1from RKIP. On one hand, T42phosphorylation promotes the exposure and recognition of the target motif "179KLYEQ183" in C-terminus of RKIP by chaperone Hsc70and the subsequent degradation of RKIP itself via chaperone-mediated autophagy (CMA). On the other hand, the release of Raf-1rescued its kinase activity, and the signals of p-MEK and p-ERK were both increased in neurons in such circumstance, suggesting the activation of downstream signals of ERK cascade.In the next section, we demonstrated that the number of BrdU+neurons increased when ERK cascade was activated, indicating the neuronal cell cycle re-entry. The neuronal mortality also rised in the mean time, while inhibition of CDK5or ERK pathway was helpful in cell survival. So the phenomenon of cell cycle re-entry participated in the neuronal death. Furthermore, in the cellular model of Parkinson’s disease, we also observed the upregulation of phosphorylated RKIP, the cell cycle re-entry related to activation of ERK cascade, and subsequent cell death in dopaminergic neurons. And in the brain of both MPTP-induced and transgenic models of PD, we found an increase in p-RKIP and activation of ERK cascade, plus a downregulation of RKIP in PD mice of old age. Taken together, we demonstrate that phosphorylation of RKIP is involved in the overactivation of the ERK/MAPK cascade, leading to S-phase re-entry and neuronal loss in Parkinson’s disease.The mechanisms that underlie neuronal loss in Parkinson’s disease have attracted attention for years, and in the current study we supplied a system which united RKIP, CDK5, ERK cascade, autophagy, cell cycle re-entry together in the context of PD. We demostrated that after overactivation of CDK5and enhanced phosphorylation of RKIP, the degradation of RKIP was induced and ERK cascade was overactivated, followed by neuronal cell cycle re-entry and neuronal death. These findings provide evidence for the role of the CDK5/RKIP/ERK pathway in PD pathogenesis and suggest that this pathway may be a suitable therapeutic target in PD.