Research On The Mechanism Of Dopaminergic Neurons Death Induced By Salsolinol Synthase And Its Product

Parkinson's disease(PD)is the second most common neurodegenerative disease in human,which has a higher incidence in the elderly.The main clinical symptoms of PD include rest tremor,muscle rigidity,slowness of movement and postural instability.The main pathological features of PD are the presence of Lewy bodies and the absence of dopaminergic neurons in the substantia nigra pars compacta(SNpc).The exact pathogenesis of PD still remain unclear.Etiological studies have suggested that the onset of PD is the consequence of the co-effects of aging,environmental toxins and genetic factors,among which environmental toxins play a dominant role in the acquired development of PD.Since the environmental neurotoxin1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)was found to induce symptoms similar to Parkinson's disease,great attention on the correlation between PD and its structural analogs catechol tetrahydroisoquinolines(CTIQs)has arisen among researchers.CTIQs are the endogenous neurotoxin enzymatically generated by dopamine and the lipid peroxidation-derived active aldehydes due to oxidative stress in the brain.After N-methylation and oxidation,the CTIQs were transformed to neurotoxin ions,which can accumulate in the mitochondria and finally inhibit the activity of mitochondria complex I.Consequently,the mitochondria dysfunctions cause energy metabolism disorder.This situation aggravates the level of oxidative stress,which formed a vicious circle and eventually lead to degeneration and death of dopaminergic neurons.In our research,we focus on the biological enzymes catalyzing the formation of CTIQs.The lower content and difficulty in purification make it very difficult to study the function of these enzymes.The research on the mechanism of endogenous neurotoxin leading to the pathogenesis of PD is standstill at the same time.Sal synthase(Sal syn)is the first key enzyme in the synthesis and metabolism of CTIQs.It catalyzes the formation of Sal from DA and acetaldehyde and accelerates the generation and metabolism of subsequent products,which is closely related to the pathogenesis of PD.Until now,only few researches were reported on this enzyme worldwide.Our group has been committed to Sal syn for a long time.We isolated,purified and identified Sal syn from rat brains previously,and its enzymatic properties have been preliminarily confirmed.However,the exact biological function of the enzyme remains unknown.The biological function of Sal syn depends on its catalytic product Sal.In recent years,there has been controversy about the neurotoxicity of Sal,and the death mechanism of dopaminergic neurons caused by Sal has not been fully clarified.Therefore,this study mainly focuses on Sal syn and its products Sal,and the results were as follows:1.The PD celluar and animal models were constructed based on 6-OHDA to explore the activity,distribution and product of Sal syn.Results showed that Sal syn distributed in SH-SY5Y cells and striatum,midbrain,hippocampus and cortex of rats.The activity of Sal syn increased in both the celluar model and the striatum of rat models.We successfully detected the content of Sal in rats,which was increased in the midbrain of PD.2.To investigate effects of Sal syn on dopaminergic neurons in vivo and in vitro,it was overexpressed in PC12 cells and mouse.The results showed that Sal syn distributed in the cytoplasm in vitro.After overexpressed for 48 h,the activity of Sal syn and the level of neurontoxin of Sal and NM-Sal increased.Flow cytometry revealed the reduction of mitochondrial membrane potential(MMP)and the increase of apoptosis rate.Western Blot results showed that the Sal syn inhibited the level of Bcl-2 and promoted the expression of Bad,and the similar results were obtained in Sal induced cells.In vivo,we bred FVB Sal syn transgenic mice to detect whether overexpression of Sal syn can cause behavioral and pathological features of PD.Traction test,pole test,rotard test and TH immunohistochemistry stain were peformed.The pole test showed Sal syn transgenic mice showed bradykinesia in 13 months.Nevertheless,there was no significant difference in TH immunohistochemistry stain.Therefore,it is speculated that the neurotoxicity caused by Sal syn in vivo takes time to accumulate,and Sal syn overexpressed solely can't directly cause PD in animals,which is consistent with the complex characteristics of PD pathogenesis.3.In the process of cell apoptosis induced by Sal syn,Sal(the product of Sal syn)plays a key role.In order to elucidate the molecular mechanism that Sal induced the death of dopaminergic neurons,RNA sequencing was performed to screen the differential genes and pathways.The results showed that the expression level of 740genes were up-regulated,such as RNA methylation enzyme(FTO/Mettl3/Mettl23),apoptosis protein(Bad)and transcription factors(Foxa3).And 1665 genes were down-regulated,including autophagy related gene(Atp264/Atg1611),DNA methylation related gene(Dnmt3a/Dnmt1)and transcription factor(FoxO1).GO analysis results indicated that the differentially expressed genes were mainly located in mitochondrial inner membrane,lysosomal body cavity,golgi body and other cell components.The major molecular functions changed in ion binding ability and enzymic catalytic activity,and the major biological processes affected included the regulation of miRNA,autophagy,and the regulation of cell polarity.KEGG enrichment results showed that Sal could affect signaling pathways such as Akt,TOR and FoxO.4.Based on the RNA sequenceing,the Akt-mTOR signaling pathway and the autophagy pathway were focused to reveal the molecular mechanism by which Sal induces the apoptosis of dopaminergic neurons.The Western Blot results showed that Sal induced decreased expression of anti-apoptotic protein Bcl-2 and LC3?/?and increased the phosphorylation level of Akt and mTOR.These results indicate that Sal inhibits the level of autophagy by activating the Akt-mTOR signaling pathway,which eventually leads to the apoptosis of dopaminergic neurons.After Sal induction,we also found that the intracellular Ca²⁺concentration,the expression of the calcium channel-related protein NMDAR1 and the dopamine receptor Drd3 were decreased.These results indicated that Sal induction weakened the signal transduction ability,which may indirectly regulate the level of autophagy.Besides,the expression of demethylase FTO was decreased,which resulted in the increased level of m6A(a kind of RNA methylation).In addition,we also found that Sal can partially reverse the increased expression of FTO and NMDAR1 caused by 6-OHDA,which suggested that Sal may be a potential antagonist of demethylase and NMDAR1 receptor.In conclusion,the potential mechanism of Sal syn and Sal leading to the apoptosis of dopaminergic neurons was clarified.The overexpression of Sal syn promoted the neurotoxin production of CTIQs and then damaged the mitochondria,which eventually leads to the apoptosis of dopaminergic neuron cells.In vitro test showed that Sal induced the death of dopaminergic neurons by activating the Akt-mTOR signaling pathway,which inhibited the autophagy.And this process was modulated by the RNA methylated modification and NMDAR1-Ca²⁺signal transmission pathway.In summary,Sal syn and Sal play crucial roles in PD etiology.