The A-type Potassium Channel Blocker 4-AP Protects Nigral Dopaminergic Neurons In The MPTP–induced Mice Model Of Parkinson’s Disease

Parkinson’s disease（PD）is the second most common neurodegenerative disease in the elderly.As the life span of human beings increasing,the prevalence and incidence of PD are also gradullay increased.The major pathologic features of PD are the selective loss of dopamine（DA）neurons in the substantia nigra pars compacta（SNc）and the reduction of DA in the Striatum（Str）.These would result in a series of relative symptoms,especially motor symptoms（e.g.static tremor,muscle rigidity,akinesia,and postural reflex disorder）.Many pathologic mechanisms have been proved to be associated with PD,such as oxidative stress,inflammatory response,mitochondrial dysfunction,iron deposition,abnormal protein aggregation,and apoptosis.These pathologic mechanisms make the treatment of PD extremely complex.Therefore,it is of great significance to search for the effective prevention and treatment of PD.A-type potassium channel,also known as transient outward potassium channel,is a voltage-dependent potassium channel widely existing in substantia nigra DA neurons,which plays an important role in regulation of the amplitude,duration and discharge frequency of neuronal action potential.In mammalian,A-type potassium channel is composed of KCNA4（Kv1.4）、KCNC4（Kv3.4）、KCND1（Kv4.1）、KCND2（Kv4.2）、KCND3（Kv4.3）,in which Kv4.3/KChip3.1 is the main component of A-type potassium channel in substantia nigra DA neurons.Previous studies have shown that the level of Kv4.3mRNA in the remaining DA neurons in the substantia nigra of PD patients is significantly increased,and immunohistochemistry shows that the expression of Kv4.3 in SNc of 7-8 months old A53T mice of PD transgenic animals is selectively increased.Therefore we proposed that the increased expression of A-type potassium channel is closely related to the pathogenesis of PD.Studies have shown that 4-AP plays an important regulatory role in a variety of nervous system diseases.For example,4-AP can improve axonal dysfunction for the treatment of multiple sclerosis;increase the excitability of Purkinje cells to treat nystagmus;and increase the excitability of Purkinje cells to treat nystagmus;improve the conduction of action potential in demyelinated axons for the treatment of multiple sclerosis.However,whether 4-AP can protect DA neurons and delay the pathogenesis of PD has not been reported.Therefore,the purpose of this study was to investigate the modulation of A-type potassium channel in the injury of DA neurons in PD.In this study,1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine（MPTP）-induced damage in PD model mice was prepared.Immunohistochemistry and western blotting were used to observe the effects of lateral ventricle administration of 4-AP on number of positive neurons of tyrosine hydroxylase（TH）in SN and Str,as well as TH protein expression;the changes of DA and its metabolites in Str were detected by high-performance liquid chromatography（HPLC）;to further evaluate the effect of 4-AP on the mice’balance ability and motor speed,rotation behavior and open field test were executed,respectively;to observe the effect of 4-AP on oxidative stress in PD mice,the serum Malondialdehyde（MDA）and Superoxide Dismutase（SOD）were determined by spectrophotometry;To observe the effect of 4-AP on the apoptosis level of PD mouse model,the number of bcl-2and caspase-3 neurons in the SN region was detected by immunohistochemistry.The results are as follows:1.In the MPTP-induced PD mice model,the number of TH immune reactive cells in the SN region was significantly reduced than that in the normal control group（P<0.001）.After 5 days of intraperitoneal pretreatment with 4-AP（0.5 mg/kg,1 mg/kg,5 mg/kg）,the1 mg/kg 4-AP+MPTP co-treatment group can antagonize the reduction of TH immune reactive cells caused by MPTP（P<0.01）.2.In the MPTP-induced PD mice model,TH immune positive fibers were significantly reduced in the Str region than that in the normal control group（P<0.001）.After 5 days of intraperitoneal pretreatment with 4-AP（0.5 mg/kg,1 mg/kg,5 mg/kg）,the 0.5 mg/kg and1 mg/kg 4-AP+MPTP co-treatment group can antagonize the reduction of TH immune positive fibers caused by MPTP（P<0.01）.3.In the MPTP-induced PD mice model,the TH protein expression level in the SN region was decreased（P<0.001）.The 1 mg/kg 4-AP+MPTP co-treatment group could antagonize the decrease of protein expression caused by MPTP（P<0.01）.4.In the MPTP-induced PD mouse model,the content of DA in Str and its metabolites dihydroxyphenylacetic acid（DOPAC）and highvanillic acid（HVA）decreased（P<0.001）.The 1 mg/kg 4-AP+MPTP co-treatment group could antagonize the decrease of DA,DOPAC and HVA content induced by MPTP(P_DA<0.01,P_DOPAC<0.05,P_HVA<0.05).5.The dyskinesia of PD mice was greatly improved by 4-AP.In the open field experiment,the average movement speed of mice in the MPTP group was slower than that in the normal control group（P<0.001）.The speed of movement of the mice was recovered in the 1 mg/kg 4-AP+MPTP co-treatment group（P<0.05）.In the rotating rod experiment,the time spent on the rotating rod in MPTP mice was slower than that in the normal control group（P<0.001）.The 1 mg/kg 4-AP+MPTP co-treatment group could increase the time spent on the rotating rod（P<0.05）.6.The level of oxidative stress of PD mice was greatly improved by 4-AP.Compared with normal mice,the MDA level in the serum of MPTP-induced PD mice was significantly increased（P<0.001）.The 1 mg/kg 4-AP+MPTP co-treatment group could antagonize the increase of MDA levels caused by MPTP（P<0.001）;compared with normal mice,the serum SOD levels in MPTP-induced PD mice were significantly lower（P<0.05）,the 1mg/kg 4-AP+MPTP co-treatment group can antagonize the decrease of SOD level caused by MPTP（P<0.01）.7.The level of apoptosis in SN region of PD mice was greatly improved by 4-AP.Compared with normal mice,the number of Bcl-2 immunoreactive cells in MPTP-induced PD mice decreased（P<0.001）,and the 1 mg/kg 4-AP+MPTP co-treatment group could increase Bcl-2 immunoreactive cells（P<0.001）;Compared with normal mice,the number of Caspase-3 immunopositive cells in MPTP-induced PD mice increased（P<0.001）,and the 1 mg/kg 4-AP+MPTP co-treatment group antagonized the increase in the number of Caspase-3 cells caused by MPTP（P<0.001）.The above results indicate that treating with 4-AP could improve the damage of DA neuron in SN region of MPTP-induced PD model mice.4-AP increase the number of TH positive neurons in SN region,and TH neuronal fibers in Str area,increase the expression of TH protein,promote the release of DA and its metabolites content,thereby improving the function of SN-Str system and alleviating PD mices’dyskinesia.This protection may be through anti-oxidative stress and anti-apoptosis.The results of this experiment provide a new theoretical basis for the DA neuron injury induced by the A-type potassium channels to participation the pathogenesis of PD,and offer a new way possible to prevent and treat PD.