Mouse Model For Multiple System Atrophy Induced By α-synuclein Fibrils Seeding In External Urethral Sphincter And The Underlying Mechanism

BackgroundThe formation and propagation ofα-synuclein（α-Syn）-positive inclusions in the nervous system is a key pathological feature ofα-synucleinopathy^[1].Theα-Syn-positive inclusions selectively affect specific regions of the nervous system,leading to different disease syndromes^[2].Multiple system atrophy（MSA）is a sporadic,rapidly progressive type ofα-synucleinopathy,which affects multiple areas of the nervous system and lacks effective treatment with poor prognosis.The clinical manifestations of MSA include autonomic dysfunction,levodopa-insensitive Parkinsonism,cerebellar ataxia,and various combinations of the three symptoms.Autonomic dysfunction,especially the genitourinary dysfunction,is the main clinical manifestation in MSA^[3].Establishing animal models of MSA can recapitulate various clinical phenotypes and pathological features of MSA,and provide a better platform for studying the pathogenesis of MSA and exploring the diagnosis and treatment of MSA.To date,almost all established MSA animal models are neurotoxic or transgenic models and fail to mimic MSA-like autonomic dysfunction,incompletely showing some of pathological changes and motor symptoms of MSA^[4].This study constructed a mouse model whereα-Syn-positive inclusions retrogradely transmit to the central nervous system（CNS）from the nerve terminals of external urethral sphincter（EUS）via the micturition reflex pathway.This model recapitulates MSA-like pathological changes,further inducing MSA-like autonomic dysfunction and dyskinesia.This result suggests that the retrograde transmission ofα-Syn-positive inclusions along the micturition reflex pathway to the CNS possibly lead to MSA-like syndromes.MSA is a fatal multi-system neurodegenerative disorder characterized by variable combinations of rapidly progressive autonomic failure,cerebellar ataxia,and Parkinsonism^[5].It has been recognized that the pathological hallmark of MSA is the accumulation of misfoldedα-Syn in glial cytoplasmic inclusions and neuronal inclusions（NIs）in the CNS^[6].Animal experiments have shown that injection of brain homogenate from MSA patients into the brain of TgM83 mice can induce similar pathological changes and motor dysfunction in MSA,revealing the mechanism ofα-Syn propagation in MSA and otherα-synucleinopathies^[7].However,the origin and propagation pathways of misfoldedα-Syn in MSA remains elusive.According to the latest guidelines,autonomic dysfunction including genitourinary dysfunction,orthostatic hypotension,etc.are necessary the symptom for the diagnosis of MSA.Retrospective data suggest that most MSA patients have developed autonomic symptoms several years before the onset of dyskinesia,with urinary symptoms being more common.In addition,genitourinary dysfunction in patients with extrapyramidal symptoms is thought to help distinguishing Parkinson’s disease（PD）from MSA in the early stage of the disease^[8].Consistently,neuropathological studies have shown widespread degeneration and pathologicalα-Syn deposits in the micturition reflex pathway in CNS of MSA patients,including periaqueductal gray（PAG）,locus caeruleus（LC）,Barrington’s nucleus（BN）,intermediolateral columns（IML）,and Onuf’s nucleus in the spinal cord^[9,10],which may account for the early-onset urogenital dysfunction in MSA patients.To date,the pathogenesis ofα-synucleinopathy such as MSA remains unclear.Researchers have constructed various animal models to explore their pathogenesis and search treatments.However,these animal models are limited to systemic toxic models or transgenic animal models.Although these models partially showing the neuropathological changes ofα-synucleinopathy and related dyskinesias,nearly none of them can mimic the complete pathogenesis ofα-synucleinopathy,and almost no animal model mimics MSA-like autonomic dysfunction^[4].Therefore,the development of animal models ofα-synucleinopathy requires new strategies.Here,we used a transgenic mouse model to study the origin and pathogenesis of MSA.We injected preformedα-Syn fibrils（α-Syn PFFs）into the EUS of TgM83^+/-mice,and inducedα-Syn-positive inclusions to retrogradely propagate to the CNS along the micturition reflex pathway,which recapitulate MSA-like motor and urinary dysfunction.This research proposes a new hypothesis thatα-Syn-positive inclusions retrogradely spread from the nerve terminals of EUS to the brain,leading to MSA-like syndromes.This contributes to a more optimized animal model for revealing the pathological mechanism of MSA and seeking specific molecular targets to prevent the progression of MSA.ObjectiveThis study intends to construct a mouse model of MSA whereα-Syn-positive inclusions retrogradely spread along the micturition reflex pathway,recapitulating MSA-like autonomic and motor dysfunction,and to further explore the mechanisms ofα-Syn transmission inα-synucleinopathy.Methods1.Preparation and identification ofα-Syn fibrils were performed.2.Eight-week-old TgM83 heterozygous transgenic mice were divided into the experimental group and the control group,and their EUS were injected withα-Syn PFFs and phosphate buffer saline（PBS）,respectively.3.Urodynamic evaluation,external anal sphincter（EAS）electromyography（EMG）,and behavioral tests were performed before and every 2 weeks after modeling.Separately 6 mice in the experimental group and the control group were sacrificed at 1 month,2 months,3 months,4 months,5 months,6 months,7 months,8 months,9 months,and 10 months after modeling,and their tissues were reserved after perfusion.4.Immunohistochemistry（IHC）,double immunofluorescence and Western blot（WB）were performed on the EUS,pelvic ganglia,and urinary nuclei in spinal cord and brain layers（Onuf’s nucleus,IML,LC,BN,PAG,etc.）of the mice after modeling,to observe the formation and propagation ofα-Syn-positive inclusions in the nervous system.5.Statistical analyses of urodynamic evaluation,EAS EMG,and behavioral data were performed.Results1.α-Syn-positive inclusions were formed afterα-Syn fibrils were injected into the EUS of TgM83^+/-mice.2.α-Syn-positive inclusions retrogradely spread to the CNS along the micturition reflex pathway,andα-Syn deposits were detected in the urinary-associated nuclei to mimic MSA-like pathological changes.3.Mice performed MSA-like movement disorders in the experimental group after modeling.4.Mice performed MSA-like urinary dysfunction and abnormal EAS EMG in the experimental group after modeling.Conclusion1.Peripheral injection ofα-Syn PFFs could induceα-Syn-positive inclusions in the central nervous system of TgM83 mice.2.Theα-Syn-positive inclusions could retrogradely spread from the nerve terminals of external urethral sphincter along the urogenital-related autonomic pathway（EUS,Onuf’s nucleus,IML,LC,BN,PAG,etc.）to the central nervous system.3.The mouse model whereα-Syn-positive inclusions retrogradely spread from the nerve terminals of external urethral sphincter along the urogenital-related autonomic pathway can recapitulate MSA-like pathological changes,autonomic dysfunction,and dyskinesia.