Study Of Cav1.2 And Cav1.3 Calcium Channels On Iron Accumulation And The Dopamine Neuron Degeneration In The Substantia Nigra Of MPTP-induced Parkinson's Disease Model Mice

IV Parkinson's disease(PD)is characterized by a selective deletion of dopamine(DA)neurons in the substantia nigra pars compact(SNpc),and resulting the exhaust of DA in the striatum.Growing evidence suggests that L-type Ca2+ channels(LTCCs)are involved in neuronal degeneration during the process of PD.Blockage of LTCCs by specific antagonists may delay or terminate the process of PD.However,the underlying mechanisms of LTCCs mediated DA neuron degeneration in the pathogenesis of PD remain unclear.Midbrain DA neurons are autonomic pacemaking when there is no excitatory postsynaptic current input,and their autonomic pacemaking rely on L-type Cav1.3 Ca2+ channels.During pacemaking,the continuous opening of Ca2+ channels result in Ca2+ overload in cytoplasmic.This is one of the important reasons for the DA neurons degeneration.Studies in the PD patients in autopsy and PD animal models have been found that iron selective accumulated in the SN,which may increase the oxidative stress and then resulting in DA neurons degeneration.Until now,the reason of iron selective deposition in the SN is still being investigated.It has been reported that L-type Cav1.2 Ca2+ channels may mediate iron entry into the cardiomyocytes.LTCCs also provide an alternative route of iron entry into neuronal cells.To study effects of LTCCs on the pathogenesis of PD and whether LTCCs mediate the selective iron accumulation in the SN of PD.By a combination of rota rod test,molecular biology,immunofluoresence labeling and primary neuron culture,in the present study,we investigated effects of isradipine,an specific antagonist of LTCCs,on the MPTP-induced DA neuron degeneration and iron deposit in the MPTP-treated PD model mice,as well as MPP+-treated MES23.5 cells and primary cultured ventral mesencephalon(VM)neurons.The results are as follows: 1.After MPTP treatment for 2 weeks,the time remained on the rotating rod significantlydecreased in the MPTP-treated mice compared with that of control(P<0.05).Compared with the MPTP treatment group,mice in the isradipine and MPTP co-treatment group spent longer time on the rotating rod(P<0.05).2.The m RNA expressions of L-type Cav1.2 and Cav1.3 Ca2+ channels ?1 subunit in the SN of MPTP-treated mice were significantly increased compared with that of control after MPTP-treated for 2 and 3 weeks(P<0.01).Co-treatment with isradipine may partly reverse this effect(P<0.01,compared with that of MPTP treatment group).3.The protein expressions of L-type Cav1.2 and Cav1.3 Ca2+ channels ?1 subunit in the SN of MPTP-treated mice were significantly increased compared with that of control after MPTP-treated for 2 and 3 weeks(P<0.01).The peak of Cav1.2 channel ?1 subunit expession appeared after MPTP treated for 2 weeks,while the peak of Cav1.3 channel ?1 subunit expression was presented in the third week(P<0.05,compared with that of control).4.In the SN of MPTP-treated mice,the numbers of TH-positive neurons represented a time-dependent decrease.Compared with that of control,the numbers of TH-positive neurons decreased in the 2nd,3rd,and 4th week of MPTP treatment groups(P<0.01).Co-treatment with isradipine may partly reverse this effect(P<0.05).5.Compared with that of control,the DA and its metabolites DOPAC and HVA contents in the striatum of MPTP-treated mice were significantly decreased after MPTP-treated for 2 weeks(P<0.05),Co-treamtent with isradipine for 2 weeks may partly restore the DA content(P<0.05).However,although isradipine partly reversed the decreased of DOPAC and HVA in the MPTP-treated mice,there was no significant difference(P>0.05).6.In the SN of MPTP-treated mice,the numbers of iron-staining cells represented a time-dependent increase.Compared with that of control,the numbers of iron-staining cells increased after MPTP treated for 2,3 and 4 weeks(P<0.01).Co-treatment with isradipine may partly inhibit this effect(P<0.05).7.The MES23.5 cells and primary cultured ventral midbrain neurons were treated with MPP+ at a dosage of 200 ?mol/L and 100 ?mol/L respectively.Intracellular fluorescence were indicated by calcein.During perfusion with 1 mmol/L Fe SO4,the fluoresence intensity in the MPP+-treated cells and neurons was significantly decreased(P<0.05,compared with that of control).The decrease in the fluoresence intensity was partly inhibited by 0.02 mmol/L isradipine(P<0.05,compared with that of MPP+ treatment group).Perfusion with 0.01 mmol/L of Bayk8644 may significantly accelerate the fluorescence quenching(P<0.05,compared with that of MPP+-treatment group).In summary,in the present study,we found that blockage of LTCCs by isradipine not only improves the motor coordination ability of MPTP-treated mice,reduces the number of iron-positive cells in the SN,as well as partly restores the numbers of DA neurons in the SN and DA content in the striatum.The up-regulation of LTCCs was also inhibited by isradipine.Intracellular iron contents were altered by activating or inactivating the LTCCs.This work may provide a useful framework to advance our knowledge of LTCCs-mediated DA neuron degeneration and might permit the development of a novel alternative approach to treat PD.