Neuroprotective Effects Of A Novel Synthesized Pyridine Compound On The Dopaminergic Neurons In The Substantia Nigra

Parkinson’s Disease(PD)is a clinically common neurodegenerative disorder characterized by degeneration and deletion of dopamine(DA)ergic neurons in the substantia nigra(SNc),and accumulation of alpha-synuclein(a-syn).Aggregation of a-syn induces neuronal excitatory toxicity.Autophagy is one of the major pathways that clear disordered a-syn.Therefore,autophagy enhancers,which could promote the clearance of aggregated a-syn,are suggested to be a new therapeutic measure for PD.Aminopyridine compounds have been proven to play protective roles in a variety of neurological diseases,including PD,reducing α-syn aggregation,regulating neuronal electrical activity,anti-inflammatory,antioxidant stress,etc.Based on the above,we cooperated with Chengdu University of Traditional Chinese Medicine to screen potential compounds that can be used for PD prevention and treatment from 19 newly synthesized pyridine compounds,and preliminarily explored their mechanism of action.Objective:1.To test the effects of newly synthesized compounds on autophagy induction and a-syn clearance.2.To test the effect of the newly synthesized compounds on the electrical activity of DA ergic neurons in substantia nigra.Method:1.The toxicity of the compounds on PC12 cell lines overexpressing A53T-α-syn were evaluated by lactate dehydrogenase(LDH)kit.2.The expressions of α-syn and autophagy related proteins LC3 was detected by western blotting.3.The electrical activity of DA ergic neurons was recorded by whole cell patch clamp.Results:1.The expression of LC3-II in N2 a cells treated with compounds for 8 h was detected,the results showed that positive control Torin1 and compound 12 could induce the increase of LC3-II protein level(P<0.01).The expression of LC3-II protein was significantly increased in N2 a cells treated with 30 μM autophagy inhibitor chloroquine and 10 μM Compound 12 for 8 h(P<0.05).2.The PC12 cells line overexpressing A53T-α-syn were treated with Compound 12 of 0.5 μM,5 μM and 10 μM for 24 h.The results showed that compound 12 of 0.5 μM,5μM and 10 μM did not affect the release of LDH compared with the control group.3.The PC12 cells line overexpressing A53T-α-syn were treated with 0.5 μM,5 μM and 10 μM compound 12 for 24 h,the results showed that compared with the control group,the protein level of α-syn in the 0.5 μM compound 12 treatment group decreased(P<0.05).4.The PC12 cells line overexpressing A53T-α-syn were treated with 0.1 μM and0.25 μM compound 12 for 48 h,the results showed that compared with the control group,the protein level of α-syn in the 0.1 μM compound 12 treatment group decreased(P<0.01).5.Compound 12(0.25,0.5,5,50 μM)inhibit the spontaneous firing frequency of DA neurons.There was no significant dose dependence among the concentrations.5 μM was selected as the concentration for subsequent experiments.6.Compound 12 inhibited the evoked firing activity of DA ergic neurons.At 40 p A,the number of action potentials decreased,the time between peak potentials,the start time of action potentials and the duration of action potentials increased,and the duration of post-hyperpolarization potential shortened(P<0.05).7.Compound 12 promoted the hyperpolarization of DA ergic neuron membrane potential.The hyperpolarization from-52.01 ± 1.92 m V to-58.32 ± 1.88 m V was statistically significant(P<0.05).8.Compound 12 significantly inhibited Sag amplitude of DA neurons in substantia nigra.After a series of current stimulation(-200-0 p A,2 s),the Sag amplitude decreased compared with that of the basic Sag.The Sag amplitude of-200 to-100 p A was statistically significant before and after perfusion(P<0.05).Conclusion:In conclusion,in PC12 cell lines overexpressing A53T-α-syn,compound 12 does not affect the cell viability,reduces the protein level of α-syn,promote hyperpolarization of membrane potential,and inhibits discharge activities and sag amplitude of DA neurons in substantia nigra.It is speculated that the compound has a certain neuroprotective effect at low concentration and reduces the electrical activity of neurons.In this study,we discussed the regulatory effect and mechanism of a novel compound on DAergic neurons in the substantia nigra,which provided a new experimental idea for further elucidating the neuroprotective effect of pyridine compounds in PD.