Neuroprotective Effect Of Compound Acau Derived From Soil Microorganisms In Models Of Parkinson’s Disease

Parkinson’s disease（PD）is clinically characterized by non-motor symptoms such as hyposmia,constipation,and motor symptoms such as resting tremor and myotonia accompanied by progressive degeneration of dopaminergic neurons,even leading to death in the substantia nigra pars compacta（SNc）.As the second most common neurodegenerative disease,PD patients are expected to exceed 7 million by 2025 worldwide.After two hundred years of research,PD still has not found a therapeutic strategy to stop the disease process.Pathophysiological studies of PD have shown that events such as mitochondrial dysfunction are associated with iron metabolism pathway disorders.A large number of studies have shown that iron metabolism dysregulation is closely related to PD,and the most important pathological feature of PD is the presence of dopaminergic neurons in the substantia nigra pars compacta（SNc）with the iron accumulation.Many clinical studies take the iron signal in the SN region as one of the diagnoses of PD.The current treatment strategies for PD mainly include four aspects:drug therapy,adjuvant therapy,multidisciplinary care,and environmental management of patients and their caregivers.Drug therapy is the most commonly used and basic treatment strategy,and dopamine replacement therapy is still used at present as the golden rule.There are mainly two PD clinical drugs that act through dopaminergic mechanisms or nondopaminergic mechanisms.Most clinical drugs are accompanied by various adverse reactions,such as gastrointestinal symptoms,constipation,nausea.The current treatment methods still mainly focus on reducing motor impairment.Finding safer and more efficient treatments to prevent long-term degeneration of the nervous system in PD and slow down or even terminate the progression of PD is our crucial goal.Historically,plants,fungi,and bacteria have been important and abundant sources of bioactive compounds.Natural products and their molecular frameworks play an extremely important role in the discovery of drugs and medicinal chemistry.Natural products are still the best choice for finding new active compounds or structures.The Lab of Professor Tan Renxiang,Nanjing University,devoted to discovering of active structures,provided 132 soil microbial compounds with new structures or unknown biological activities for this study,including linear peptides and macrocyclic peptides with new skeleton structures.This work aims to screen the compounds with dopaminergic neuron-protective activity from natural productderived compounds extracted from soil microbiota with rich chemical structural resources and to study their mechanism to provide new strategies for the treatment of PD.In the first part of this work,132 compounds were screened for compounds that had no apparent damage to SH-SY5Y cell viability and whether they improved cell survival in two models of SH-SY5Y cell MPP+or H₂O₂ induced was tested.Finally,we further observed the effects of compounds on apoptosis,mitochondrial function,and ROS generation in cell models.The second part used the screened compounds to treat PD animal models to investigate whether they still have dopaminergic neuron protective activity.First,the compounds were used to treat the MPP+induced Caenorhabditis elegans（C.elegans）PD model and then compounds Acau and TAG with nano lipid material-encapsulated carrier were constructed,which proved by In vivo mouse imaging could penetrate the BBB（blood-brain barrier）,and the behavioral experiments in mice were used to prove that the encapsulated carrier has no obvious toxicity,and finally in the mouse MPTP subacute model,it was verified that the two compounds still have the effect of reversing the loss of dopaminergic neurons in the SNc region.In the third part,the macrocyclic peptide compound Acau with a new skeleton structure was selected for in-depth research,and the key protein SFXN2（sideroflexin 2,or SLC56A2）.The key protein of the compound exerting dopaminergic neuroprotective activity,was found through transcriptomics and other means.It is verified that the compound Acau exerts a neuroprotective effect against ferroptosis by regulating iron metabolism.This work is the first to describe the physiological activity of the new scaffold compound Acau and its neuroprotective mechanism through SFXN2 to regulate iron metabolism and exert the antiferroptosis effect,which provides a practical direction for the screening of neuroprotective activities and mechanism research of natural product-derived compounds.Part Ⅰ Screening of soil microbial compounds with neuroprotective activityOBJECTIVE:To screen compounds with protection in the SH-SY5Y cell model from 132 soil microbial compounds.METHODS:SH-SY5Y cells were cultured and treated with 10 nM,100 nM,and 1 μM concentration of compounds.The cell viability was detected by the CCK-8 method to pick compounds with no significant toxicity to cells at 1 concentration for 24 h（cell viability>85%）.The SH-SY5Y cells were given MPP+ or H₂O₂ in different concentrations,and the cell viability was detected by the CCK-8 method to determine the SH-SY5Y cell injury models induced by MPP+or H₂O₂.MPP+-or H₂O₂-induced SH-SY5Y cell models were treated with 1 μM compounds to obtain compounds with neuroprotective effects.In the two cell models treated with compounds,Annexin V-FITC/PI flow cytometry and Hoechst 33342 nuclear staining were used to detect apoptosis,and mitochondrial function was evaluated by MitoSOX and JC-1 fluorescent probe.RESULTS:1)66 compounds of 132 compounds showed no significant toxicity to SH-SY5Y cells at a concentration of 1 μM（cell viability>85%）;2)Compounds 79,83,87,88,92,94 had neuroprotective activity in both MPP+-and H₂O₂-induced SH-SY5Y cell models;3)All of compound 79,83,87,88,92,94 had anti-apoptotic and anti-oxidative effects in the two cell models.CONCLUSION:66 compounds had no significant toxicity to SH-SY5Y cells,and compounds 79,83,87,88,92,and 94 had neuroprotective effects in MPP+-and H₂O₂-induced SHSY5Y cell models.Part Ⅱ Candidate compounds reveal dopaminergic neuron protection in animal models of Parkinson’s diseaseOBJECTIVE:To verify that the compounds obtained from the primary screening have a protective effect on dopaminergic neurons in the PD animal model.METHODS:C.elegans BZ555 was cultured,given MPP+stimulation to induce the PD model,and administered by 6 compounds screened in the first part.Fluorescence intensity and the number of GFP-labeled dopaminergic neurons in the head of C.elegans were detected.Compounds 79（Acau）and 87（TAG）were encapsulated in lipid nanomaterials and observed BBB permeability by In vivo imaging technology.Then the acute toxicity of the encapsulated carriers was simply evaluated by mouse behavioral experiments,survival curves,and dirty-brain ratio,etc.The subacute mouse PD model was treated with compound Acau and TAG encapsulated.The number of dopaminergic neurons with TH positive in SNc was detected to investigate the effect of the compounds on neuron loss.RESULTS:1)All 6 compounds had neuroprotective activity in the C.elegans PD model,among which compounds 79,83,87,and 92 had better protective effects than the positive drug amantadine,while compounds 88 and 92 were less effective than amantadine;2)Compound Acau and TAG with nano-encapsulated carrier had no obvious toxicity to mice and could pass through BBB;3)Compounds 79 and 87 had the effect of reversing the loss of dopamine neurons in SNc region of the mouse MPTP subacute PD model.CONCLUSIONS:Compounds 79,83,87,88,92,and 94 showed dopaminergic neuron protection in the MPP+-induced C.elegans model;Compounds 79（Acau）and 87（TAG）have protective effects on dopaminergic neurons in the subacute MPTP PD mouse model,and as compound Acau is a novel macrocyclic peptide compound,so it is picked as a candidate to explore the mechanism in subsequent work.Part Ⅲ The mechanism of compound Acau exerting protective activity of dopaminergic neuronsOBJECTIVE:To elucidate the mechanism of compound 79 exerting the protective activity of dopaminergic neurons.METHODS:Mouse primary neuron cells were cultured,stimulated with MPP+and treated with the compound Acau.The molecular function of the compound was searched by RNA-seq and the same differentially expressed genes were found in human SH-SY5Y cells by RT-PCR.The iron content of SH-SY5Y cells was detected by iron content detection kit,Prussian blue staining,and ferritin immunofluorescence.The levels of MDA and GSH/GSSG were detected by assay kits,and the SH-SY5Y cells with SFXN2 knockdown or overexpression were used to explore the role of SFXN2 in compound Acau regulating iron metabolism and ferroptosis.RESULTS:1)Analysis of transcriptomic results suggested that compound Acau was related to metal ion binding;2)Compound Acau regulated the expression of SFXN2;3)Compound Acau inhibited MPP+-induced ferroptosis in primary neurons and SH-SY5Y cells;4)Compound Acau reversed erastin-induced ferroptosis in SH-SY5Y cells but was ineffective against RSL3-induced ferroptosis;5)Compound Acau mainly regulated the expression of iron metabolism-related genes in the ferroptosis signaling pathway;6)The key protein that compound Acau exerts neuroprotective effect is SFXN2:Overexpression of SFXN2 abolishes the neuroprotective effect of compound Acau in this cell model and aggravates MPP+-induced cell viability damage and cellular iron dysregulation;SFXN2 knockdown enhances SH-SY5Y cell viability,reduced cellular iron content and inhibited MPP+-induced decrease in cell viability.CONCLUSION:Compound 79（Acau）regulates iron metabolism to inhibit ferroptosis induced by MPP+in neurons through key protein SFXN2.The major contributions of the present study lie in:Structurally novel compounds with dopamine neuron protective activity are obtained from microbially derived natural products;The new structural compound Acau regulates iron metabolism through the key protein SFXN2,inhibits ferroptosis of dopaminergic neurons,and thus plays a role in the preservation of dopaminergic neurons.