Clk1Participates In Neuroglial Activation And Neurotoxicity In MPTP Mouse Model Of Parkinson’s Disease

Object: Investigate the effect of clk1gene in MPTP mouse model to identify thefunction of clk1gene in Parkinson’s disease.Method: Subchronic MPTP mouse model (MPTP,30mg/kg, for5consecutivedays) was employed in male Mclk1+/-mutant mice and wide type C57BL/J mice (WT).Behavioral tests were performed7days after MPTP injection, immunologicalhistochemistry(IHC) was carried on after behavioral tests. Primary astrocyte of WT andMclk1+/-mutant was used to investigate the role of clk1in inflammatory response. Theexpression of inflammatory cytokines and proinflammatory factors were assessed byreal-time PCR, ELISA and NO assay. The role of clk1in the intracellular level ofreactive oxygen species (ROS) generation and mitochondrial membrane potential werealso investigated by using fluorescent probe DCFH-DA and JC-1, respectively. Inaddition, MTT assay were used to detect the neuronal viability in glial conditionedmedium/neuron co-culture system. The expression of HIF-1α, TH and GFAP in braintissue and astrocyte were detected by western blotting.Results: At the basal level, Mclk1+/-mutant appeared no obvious differences inbehavioral appearance, the number of tyrosine hydroxylase (TH)–positive neurons andneuroglial activation in the substantia nigra pars compacta (SNpc) compared with WT.However, in MPTP mouse model, the behavioral deficit of Mclk1+/-mutant mice weremore evident than WT; The IHC showed that the number of TH-positive neurons waslower in Mclk1+/-mutant than WT; GFAP, TH and Iba1immunostaining also showedthat the activation of glial cells in Mclk1+/-mutant around dopaminergic neurons waselevated than that of WT in MPTP mouse model; Moreover, astroglial activation of Mclk1+/-mutant was enhanced than WT after LPS/IFN-γ stimulation. Activatedastrocyte of Mclk1+/-mutant exhibited elevated expression of proinflammatory factorsTNF-α, IL-1β and NO compared with WT. In addition, activated astrocyte of Mclk1+/-mutant also produced higher intracellular ROS and had lower mitochondrial membranepotential than that of WT. In the conditioned medium (CM) from astrocyte/neuronco-culture system, CM from LPS/IFN-γ-treated astrocyte of Mclk1+/-mutant inducedsignificant neuronal cell death, whereas CM from astrocyte with WT had much lesseffect on the death of neurons. The expression of HIF-1α was elevated in substantianigra in MPTP mouse model and LPS/IFN-γ-treated astrocyte of Mclk1+/-mutant.Conclusion:1. These results suggested that Mclk1+/-mutant mice werevulnerable to MPTP-induced dopaminergic neurotoxicity and subsequent reactiveastrogliosis.2. Hyper-inflammatory responses of activated astrocytes might contributeto the MPTP-induced dopaminergic neurotoxicity in Mclk1+/-mutant mice.3. Elevatedsensitivity to MPTP and enhanced glial cells activation affects the immune response viaHIF-1α in Mclk1+/-mutant.