Cdk5Phosphorylates CLIC4and Promotes Neuronal Apoptosis

Plaques consisting of β-amyloid (Aβ) peptide, neurofibrillary tangles consisting largely of hyper-phosphorylated microtubule-associated tau protein and neuron loss in the hippocampus and cortex regions are the major pathological hallmarks of Alzheimer’s disease(AD). And the neuronal death directly gives rise to pathogenesis of AD. So it is important to study the mechanism of neuronal apoptosis in AD to the prevention and therapy of AD.Cyclin-dependent kinase5(Cdk5) is a unique member of the Cdks family. Unlike the other Cdks, Cdk5was activated by specific brain protein protein:P35. In AD pathogenic progress, excessive Aβ induces P35cleaved to P25,and P25can consistently actives Cdk5then results in neuronal apoptosis. But the mechanism of Cdk5over activation remains not clearly. After selection and identification of Cdk5substrates, we infer that CLIC4may involve in the regulation of the process of Cdk5induced neuronal apoptosis.Chloride Intracellular Channel4(CLIC4) is the best characterized protein of the CLIC family, and its major function is the maintenance of the mitochondria function and structure. Cytoplasmic CLIC4can translocate to the nucleus in response to cellular stress conditions,then participates in the apoptosis.But the mechanism of CLIC4nuclear translocation still not clear, what’s more, the research of CLIC4function in nervous system is short of. We via stable isotope labeling with amino acids in cell culture(SILAC) identify that CLIC4can phosphorylated by Cdk5at point S108. We deduced that CLIC4may a new phosphorylated substrate of Cdk5.We initially verify that overexpressed P25/Cdk5can interact with CLIC4by CO-IP technology, and activated Cdk5can induce CLIC4transport to the nucleus. In vitro cultural primary neurons, we find that oxidative stress and Aβ activated Cdk5then CLIC4also translocate to nucleus. In order to test whether the phosphorylation of CLIC4-S108mediates the localization of CLIC4in intracellular. We mutate the point S108to D108to simulate the phosphorylation. In neurons and cell lines, CLIC4-S108D obviously resides in nucleus. All above results confirm that activated Cdk5phosphorylates CLIC4at position108which gives rise to the nuclear translocation. We further study find that nuclear CLIC4accumulation can trigger caspase3activity thus induces neuronal apoptosis.In this study, we first identify that CLIC4is a Cdk5phosphorylated substrate, and confirm that the phosphorylation site is S108. In the pathogenesis process of AD, Cdk5excess activated can induce the phosphorylation of CLIC4at S108site which gives rise to nuclear CLIC4accumulation and then triggers occurrence of neuronal apoptosis. Our findings provide mechanistic insights into the molecular basis for neuronal apoptosis in AD and other neurodegenerative diseases.