The Role And Mechanism Of α-synuclein-miR-7 Pathway In Parkinson’s Disease

Parkinson’s disease（PD）is a disabling and complex neurodegenerative disease,The term parkinsonism is a symptom complex used to describe the motor features of PD,which include resting tremor,muscular rigidity,slow motion,postural and gait disorders.The pathological features of PD are the abnormal accumulation ofα-Synuclein（α-Syn）,the deposition of Lewy body（LB）and Lewy neuritis（LN）,and the progressive loss of dopamine-containing neurons of the substantia nigra pars compacta（SNpc）.PD is a complex neurodegenerative condition,the etiology of PD currently remains a complex puzzle of genes,the environment,and an aging brain,for which the etiology and pathogenic mechanisms remain incompletely understood.It has been more than 100 years since LBs and LNs were discovered as major pathological hallmarks of PD.And the identification ofα-Syn as one of the major constituents of LB.α-Syn is a presynaptic neuronal protein that is abundant in the brain.Under physiological conditions,α-Syn is an unfolded and disordered soluble monomer,whenα-Syn is higher than the critical concentration,there is accumulating and aggregating evidence that misfoldedα-Syn species spread between cells.α-Syn tends to generate a diverse form of aggregates showing toxicity to neuronal cells,which induce neuronal dysfunction and death in the nervous system.Therefore,misfoldedα-Syn pathological aggregation are closely related to neurodegeneration in PD.Researchers Braak found that the distribution of abnormal aggregation ofα-Syn in the brain is closely related to the clinical symptoms of patients.LB and LN begin in the olfactory bulb or the dorsal motor nucleus of the glossopharyngeal nerve and the vagus nerve,and then appear in the raphe nucleus,locus coeruleus,peripheral parasympathetic and sympathetic nerves,The following symptoms were assessed:olfactory disorders,constipation,sleep disorders and so on.LB and LN spread to the dense substantia nigra of the brain,and induce a large number of dopaminergic neuron deaths,and the patient begin to develop motor symptoms such as paralysis and tremor.Therefore,formation,aggregation and diffusion ofα-Syn are the keys to mediate the progression of PD.Studying the molecular mechanism of a vicious cycle ofα-Syn aggregates forming and blocking the continuous effect of this cycle are key strategies to treat PD.Regulating the post-transcriptional level ofα-Syn by miRNAs is one of the important strategies to inhibit the production ofα-Syn.Our laboratory has found that miRNA-7（miR-7）can target the gene SNCA which encodedα-Syn,and could inhibitα-syn protein level to alleviate nerve injury in PD.However,we found that the expression of miR-7 in A53Tα-Syn overexpression mice was reduced in the basal state,and the expression of miR-7 inα-syn knockout mice was increased.This phenomenon suggests that the expression ofα-Syn is not only negatively regulated by miR-7,but may also directly inhibit the expression of miR-7,which forms a vicious circle.Therefore,it is critically important to understand the production,aggregation and diffusion ofα-Syn.To verify the above hypothesis,we recombinantly expressedα-Syn monomer protein in vitro,and obtainedα-Syn pre-formed fibrils by shaking.The exogenousα-Syn PFF short fibers after ultrasonic treatment can mimic the aggregate form ofα-Syn,which is taken up by neighboring neurons after intracerebral injection and then spreads further.The PFF model is prepared by injection into the striatum to simulate the pathological process and clinical symptoms of clinical PD.We can study the expression changes of miR-7 in the production,aggregation and diffusion ofα-Syn and its relationship with nerve damage.First,we proved that exogenousα-Syn PFF can induce the production,aggregation and diffusion ofα-Syn in the brain of mice,thereby inducing pathological PD-like behavioral disorders and pathological damage in mice.At the same time,it was found that miR-7 decreased with the increase ofα-Syn expression.Next,we cultured primary neurons and gave exogenousα-Syn PFF to induce the production ofα-Syn.By studying the pathways ofα-Syn production and degradation,we found that exogenousα-Syn PFF did not affectα-Syn autophagy-lysosome and ubiquitinated proteasome related degradation pathways,but promote the production ofα-Syn by inhibiting the transcription of miR-7.Finally,we reveal that exogenousα-Syn PFF mainly inhibits the expression of miR-7 by acting on the transcription factor c-Myc.In summary,our research found that:exogenousα-Syn PFF inhibit c-Myc to reduce the expression of miR-7,which reduces the post-transcriptional inhibition ofα-Syn,thus forming a vicious circle ofα-Syn accumulation to lead PD nerve damage.PartⅠExogenousα-Syn PFF induces the production and diffusion ofα-Syn in the mice brainAIM:Clarify exogenousα-Syn PFF promotes the production and spread ofα-Syn in mice brain.METHOD:We prepared PFFs from recombinant full-length humanα-syn.3-month-old male mice received stereotactic injections of sterile phosphate buffered saline and 5μgα-Syn PFF into the unilateral striatum.we re-examined mice from the1-,3-,and 6-month-old groups from surgery and detected the behavioral of mouse.Immunohistochemistry was used to detect p-α-Syn expression in olfactory bulb,cortex,striatum,hippocampus and midbrain.Immunohistochemistry was used to detect the resistance of p-α-Syn to PK enzyme in PFF model mice.High performance liquid chromatography（HPLC）was used to detect levels of monoamine transmitters and amino acid neurotransmitters in the striatum.Nissl staining and immunohistochemical staining to detect midbrain neurons,Tyrosine hydroxylase（TH）and glial-derived fibers Glial fibrillary acid protein（GFAP）expression;use tissue immunofluorescence was used to detect the co-localization of p-α-Syn in the SNc area with TH-positive neuronal cells.Western Blot and q RT-PCR were used to detect TH,α-Syn protein level and SNCA m RNA level in midbrain tissue.q RT-PCR was used to detect the expression of miR-7 in striatum tissues of the PFF model,A53T^tg/tgand A30P^tg/tg mice.RESULTS:1)Exogenousα-Syn PFF induced the production,aggregation and spread of p-α-Syn in the olfactory bulb,cortex,striatum,hippocampus and midbrain of mice;2)Exogenousα-Syn PFF induced motor impairment in mice;3)Exogenousα-Syn PFF decreased the level of monoamine neurotransmitters DA and 5-HIAA in the mice striatum;4)Exogenousα-Syn PFF induced TH-positive neuronal loss and activation of GFAP⁺glial;5)Exogenousα-Syn PFF induced an increase in the fluorescence intensity of p-α-Syn in TH-positive neurons;6)Exogenousα-Syn PFF inhibited the expression of TH protein in the midbrain tissue of mice,and increased the level ofα-Syn protein andα-Syn m RNA;7)Exogenousα-Syn PFF inhibited the expression of miR-7 in the midbrain and striatum of mice.CONCLUTION:Exogenousα-Syn PFF induced the formation,aggregation and diffusion ofα-Syn in the brain of mice by inhibiting miR-7,which induced pathological PD-like behavioral disorders and pathological damage in mice.PartⅡExogenousα-Syn PFF promotesα-Syn production by inhibiting the expression of miR-7AIM:To study and clarify the reasons for the accumulation ofα-Syn in primary neurons induced by exogenousα-Syn PFF and its influence on neuron survival.METHOD:Primary cultured neurons were exposed toα-Syn PFF（1μg/ml）.Western Blot was used to detectα-Syn,p-α-Syn and apoptosis-related pathway proteins and synaptic damage-related proteins.Immunofluorescence was used to detect p-α-Syn level and the co-localization of MAP2 and p-α-Syn.In addition,primary neurons were treated with different concentrations ofα-Syn PFF（0.05,0.1,0.5,1 and 5μg/ml）for 7 days.Western Blot was used to detect the expression ofα-Syn.Immunofluorescence was used to detect the expression of p-α-Syn.The primary neurons were treated with 1μg/mlα-Syn monomer andα-Syn PFF respectively for 7days.Western Blot was used to detectα-Syn,synaptic function-related proteins,apoptosis-related pathway proteins,autophagy lysosomes Pathways and ubiquitin protein.QRT-PCR was used to detect the levels of miR-7,miR-34b,miR-34c and SNCA,and immunofluorescence was used to detect the expression of p-α-Syn and the co-localization of MAP2 and p-α-Syn.QRT-PCR was used to detect miR-7 and SNCA m RNA levels of primary neurons that were treated with various concentrations and different time points ofα-Syn monomer andα-Syn PFF.RESULTS:1)Exogenousα-Syn PFF promoted the generation and aggregation of endogenousα-Syn of primary neurons in a time-dependent manner,and induced neuronal damage;2)Exogenousα-Syn PFF promoted the generation of endogenousα-Syn protein and aggregation in primary neurons;3)α-Syn monomer has no effect on the generation and aggregation of endogenousα-Syn of primary neurons,and does not cause neuronal cell damage;4)Exogenousα-Syn PFF does not affect the correlation of intracellular clearing ofα-Syn pathway Protein Lamp1,Atg5,Atg7,P62,LC3B,Ubiquitin expression;5)Exogenousα-Syn PFF inhibited the post-transcriptional regulatory pathway ofα-Syn and inhibited the expression of miR-7;6)Exogenousα-Syn PFF decreased the expression of miR-7 and SNCA m RNA in primary neuronal cells in a concentration-dependent and time-dependent manner.CONCLUTION:Exogenousα-Syn PFF decreased the expression of miR-7 in a concentration-dependent and time-dependent manner,induced the generation and aggregation of endogenousα-Syn and neuronal damage.PartⅢThe mechanism of exogenousα-Syn PFF inhibiting the expression of miR-7AIM:Clarify the molecular mechanism of exogenousα-Syn PFF regulating miR-7 expression in primary neurons.METHOD:Primary cultured neurons were exposed to exogenousα-Syn PFF（1μg/ml）for 1,7,and 14 days.q RT-PCR was used to detect the levels of Pri-miR-7-1,Pri-miR-7-2,Pre-miR-7-1 and Pre-miR-7-2.Exogenousα-Syn PFF treated SH-SY5Y cells for 7 days to detect the expression of miR-7,Pri-miR-7-1 and Pre-miR-7-1.Cultivate 293Ta cells,cytoplasmic nucleus extraction kit was used to extract nuclear proteins,designed biotin-modified DNA probes in the miR-7 promoter sequence,obtained probe-protein complexes through DNA pull down,and perform qualitative identification and screening by mass spectrometry Promoter bound protein.luciferase reporter gene experiment,CHIP,Western Blot were used to identify the miR-7 promoter and Binding of transcription factors.QRT-PCR was used to detect the m RNA levels of transcription factors in primary neuronal cells.Si RNA was used to interfere the expression of transcription factors in SH-SY5Y cells and detected the expression of miR-7 and its precursors.In addition,the website predicted the binding sites of transcription factors and mutated the binding sites respectively,constructed a mutant plasmid of Hnrnpk,transfected into 293Ta cells,and used the luciferase reporter gene experiment to further evaluate the binding sites of transcription factors and promoters.RESULTS:1)Exogenousα-Syn PFF inhibited the expression of Pri-miR-7-1,Pre-miR-7-1,Pri-miR-7-2,and Pre-miR-7-2 in primary neurons;2)α-Syn PFF inhibited the expression of miR-7,Pri-miR-7-1,and Pre-miR-7-1 in SH-SY5Y cells;3)Protein mass spectrometry found the transcription factor c-Myc that binded to the miR-7 promoter;4)The transcription factor c-Myc binded to the miR-7 promote;5)Exogenousα-Syn PFF inhibited the expression of the transcription factor c-Myc in primary neurons;6)Interfering c-Myc inhibited the expression of miR-7,Pri-miR-7-1and Pre-miR-7-1 in SH-SY5Y cells;7)The transcription factor c-Myc binded to both sites of the miR-7 promoter.CONCLUTION:α-Syn PFF inhibited the transcription of miR-7 by interfering with the expression of the transcription factor c-Myc.The major contributions of the present study lie in:The mechanism ofα-Syn PFF as a"seed"transmitted in cell-to-cell and caused pathological damage,which clarify another important way forα-Syn to participate in PD pathology.The expression ofα-Syn is not only negatively regulated by miR-7,but may also directly inhibit the expression of miR-7.Inhibiting miR-7will form a vicious pathological cycle and exacerbateα-Syn generation,aggregation and diffusion,which provide for the exploration of the development of PD therapeutic drugs targeting the process ofα-Syn generation experimental and theoretical basis.