Identification Of The Genetic Modifier For An Spinocerebellar Ataxia Type 3

BackgroundSpinocerebellar ataxia type 3(SCA3),also known as Machado-Joseph disease(MJD),is a neurodegenerative disease caused by CAG expansion in the ATXN3 gene,and is the most commonly inherited autosomal-dominant ataxia worldwide.The CAG repeat length is inversely correlated with age-of-onset,and is positively related to the severity of the disease.However,only approximately 55.2%of the age-of-onset variance and clinical heterogeneity of SC A3 can be explained by the CAG expansion length,indicating that genetic modifiers playing an important role in the pathogenesis of disease.Previously,much work has been done to identify genetic modifiers for SCA3 through association analysis.However,the specific clinical contributions of the existing modifiers is still unclear,since the modifiers lack repeatable verification in different populations,and lack functional confirmation.Therefore,identifying family-specific genetic modifiers is invaluable for interpreting the heterogeneous phenotypes of specific SCA3 families and achieve individualized clinical intervention.The polyQ-expanded ATXN3 diseased proteins,translated from CAG repeated expansion in ATXN3 gene,form neuronal pathological aggregates,is the classical pathological change of SCA3.Neuronal nuclear inclusions(NNIs)play the primary role of aggregates,while smaller granular cytoplasmic staining(GCS),diffused nuclear staining(DNA),and distal axonal aggregates also occur.Among them,the toxcity of GCS is the strongest,and the severity of GCS serves as the most reliable predictor of neuronal survival.Degeneration of SCA3 results in progressive neuronal dysfunction,neuronal loss,gliosis and myelin deficiency.Pathology involves certain diseased-brain regions,including the dentate nuclei,pon,red nuclei,globus pallidus,substantia nigra and spinal cord.The involvement of diseased regions is associated with the CAG expansion length,and could involve broader regions,including cerebral cortex,thalamus,striatum and cerebellum.For cells,it’s of great significance to maintain efficient protein quality control and protein homeostasis by promoting the correct generation,folding and elimination.The primary pathways for protein quality control includes ubiquitin proteasome system,chaperone mediated autophagy,and macroautophagy.For many neurodegenerative diseases,redundant diseased proteins overwhelm the protective pathways for the clearance of misfolding proteins,and aggregate from soluble oligomers to higher-form multimers,and then form insoluble filament aggregates or large insoluble inclusion bodies.In the formation process of pathological aggregates,the cytotoxic effect of the soluble oligomers of diseased proteins on neurons,which leads to neuronal dysfunction and neuronal loss,is the predominant pathogenesis of disease.PPP4R3A(Protein Phosphatase 4 Regulatory Subunit 3A),also called SMEK1(Suppressor of MEK null 1),located in 14q32.12 of human chromosome,is 4416bp of full length,and includes 15 exons.The encoding product PPP4R3A is a highly conserved factor through the conservation process,and is broadly expressed in various tissues and cells,mainly located in the nucleus.PPP4R3A has been reported to play an important role in the nervous system,affecting the neurogenesis in the early development stage,and is associated with the pathogenesis of neurodegenerative diseases,neuronal autoimmune diseases,and psychiatric diseases.Notably,A PPP4R3A variant,rs2273647-T,was identified in Alzheimer’s disease as a genetic modifier,indicating the plausible clinical significance in other types of neurodegenerative diseases.Objectives(1)Clinical,molecular and pathological diagnosis of a spinocerebellar ataxia type 3 family.(2)Identifying genetic modifiers of the SCA3 family,and analyzing the biological function of the variant.(3)Generating the SCA3 mouse model carrying the genetic modifier,and analyzing the effect of the variation on the disease phenotype.Methods(1)To collect clinical data of family patients by describing the medical history and clinical examination;to collect EDTA blood samples of SCA3 family members for molecular diagnosis;to analyze the distribution of pathological aggregates and neurodegenerative changes by histopathological analysis with autopsy specimens from a patient.(2)To identify the candidate genetic modifiers by linkage analysis combined with whole-exome sequencing;to predict the function of the variant by bioinformatic analysis,and to analyze the biological function of the variant by expressing the full length of mutant PPP4R3A in cells;to construct the Ppp4r3a p.K585T knock in mouse model by Using an embryonic stem(ES)cell targeting approach,and to analyze the behavioral and neurodegenerative changes of mice,to reveal the pathogenicity of the variant in vivo.(3)To generate the dygenic Ppp4r3aK585T/+MJD84.2 mouse model by mating Ppp4r3aK585T/+mice with MJD84.2/SCA3 mice,and verify the modification effects of the variant on disease genotype by behavioral and pathological analysis.Results(1)Part I:A case report for an SCA3 familyIn this study,we recruited a rare SCA3 family from Shandong.The patients exhibited earlier age-onset(among 20-30 years old),more severe clinical presentation,and shorter life span(about 20 years after the disease onset).In the postmortem study of brain tissues from one patient,we demonstrated a widespread distribution of neuronal pathological aggregates in diseased regions,including the cerebral cortex,hippocampus,cervical cord,and the Purkinje cells in cerebellum.The predominant pattern of aggregates is GCS,while DNS and NNIs also occur.Ubiquitination and autophagy of aggregates have been reported as late events during the disease.In our research,we observed Ub-and P62(Autophagy-associated factor)-positive expression in the neuronal pathological aggregates in the patient,mainly expressing in GCS.Through analyzing the neurodegenerative changes of the patient,we observed massive neuronal loss in the cerebral cortex,hippocampus,medulla oblongata,and the Purkinje cells in cerebellum.And the absence of astrocytes in the white matter of the cerebral cortex was observed the patient.Therefore,the severe involvement of clinical and pathological phenotypes of patients from this SCA3 family prompted us to study the family-specific genetic modifier,which could be a novel prospective target to achieve precision medicine in this family.(2)Part II:The identification and functional analysis of the genetic modifier for the SCA3 familyWith linkage analysis and whole-exome sequencing,a PPP4R3A c.1715A>C(p.K585T)variant was identified as the prospective genetic modifier for the SCA3 family.Combined with bioinformatics prediction and functional validation,we demonstrated that the variation might be an ubiquitination site.The variant leads to reduced ubiquitination level of proteins,causing slower protein degradation rate and abnormal accumulation of mutant PPP4R3A proteins.With PPP4R3A immunostaining,immunoreactive signals of PPP4R3A were observed in neuronal pathological aggregates,indicating the participation of PPP4R3 A in the formation of aggregates.In addition,to validate the pathogenic effects of the PPP4R3A variant in vivo,we constructed the Ppp4r3a p.K585T knock in mouse model.Through behavioral tests,pathological staining,and analysis of the expression of mutant PPP4R3A and PQC components,we demonstrated the progressive motor dysfunction,widely neurodegenerative changes in the brain,spinal cord and peripheral nerves,abnormal accumulation of mutant PPP4R3A,and the collapse of proteostasis in the brain tissues in the Ppp4r3aK585T/+ mice.Therefore,our results reveal that the PPP4R3A p.K585T variant is pathogenic and is the plausible genetic modifier for the SCA3 family.(3)Part III:The identification and functional analysis of the genetic modifier for the SCA3 familyTo validate the modification effect of the PPP4R3A variant on the phenotypes of SC A3 in vivo,we constructed the digenic MJD84.2/SCA3 mouse model carrying Ppp4r3aK585T/+variant.Through behavioral tests and pathological analysis,we demonstrated that the Ppp4r3aK585T/+variant leads to severer motor dysfunction,broader distribution of the pathological aggregates,and aggravated neurodegenerative changes in MJD84.2 mice.Molecular mechanisms revealed that the PPP4R3 A variant increased the neuronal pressure in eliminating misfolding proteins,leading to the upregulation of the components of protein quality control system in the SC A3 patient and Ppp4r3aK585T/+MJD84.2 mice.Then,apoptotic-and autophagy-associated factors were upregulated,leading to neuronal death.Therefore,we proposed the PPP4R3A variant as the genetic modifier for this SC A3 family.Significance(1)Introduced a rare SC A3 family with complex clinical and pathological phenotypes,further demonstrating the high heterogeneity of disease phenotypes in SCA3.(2)Identified that PPP4R3A(C.1754A>C)was the genetic modifier of the SCA3 family,and explained the variant with unknown clinical significance by biological functional analysis,which was invaluable for the realization of precision medicine for family patients.(3)Enriched the role of PPP4R3A in neurodegenerative diseases,suggesting that the broader function of PPP4R3A in the maintenance of brain funnction needed to be explore.