Verification Of The Causative Gene In PADMAL Variant And Pathological Mechanisms Of SMEK1 In Neuropathy

Part Ⅰ Cervical spinal involvement in a Chinese pedigree withPADMAL caused by a 3’UTR mutation of COL4A1 geneBackgroundPontine autosomal dominant microangiopathy and leukoencephalopathy(PADMAL)is a subtype of CSVD caused by mutations in COL4A1 3’ untranslated region(UTR).Several mutations of COL4A1 in translated regions have been reported responsible for the onset of small vessel disease in infants as well as adulthoods.However,PADMAL caused by mutations locating at 3’UTR is phenotypically distinguished from otherCOL4A1 mutation associated disorders.The genetic traits of PADMAL exhibit nolinks with cerebral autosomal dominant arteriopathy with subcortical infarcts andleukoencephalopathy(CADASIL)and retinal vasculopathy with cerebralleukodystrophy(RVCL),but shared many characteristics with Swedish hereditarymulti-infarct dementia(hMID)whether in clinical manifestations or causative geneThe clinical diagnosis of PADMAL largely relies on magnetic-resonance imaging(MRI)features;predominant lesions in pons but rare in the temporal lobe.Microbleeds were also uncommonly observed in PADMAL patients in contrast to CADASIL patients.Objectives1.To identify the genetic cause of a Chinese PADMAL pedigree2.To compare the genetic and clinical similarities and differences between PADMAL and Swedish hMID.Methodsbrain and spinal magnetic-resonance imaging(MRI)scanning,y,and whole-exome sequencing were performed on the patients in the pedigree.Suspected pathogenic variants were further confirmed by co-segregation analysis using Sanger sequencing in the family members.1.History collections and detailed physical examinations of patients in the pedigree2.DNA were extracted from family members.Whole exome sequencing was performed on patients.Sanger sequencing was carried out to confirm co-segregation3.Brain and spinal magnetic-resonance imaging(MRI)scanning were performed.4.skin biopsy was carried out in the proband to reveal angiopathy.Results1.Patients in the pedigree develop symptoms about 40 years old.Clinical manifestations are characterized by limb weakness and progressive dementia.2.Whole exome sequencing revealed a single nucleotide variant located at 3’ UTR of COL4A1(c.*32G>A).Sanger sequencing proved mutation co-segregation in the pedigree3.Brain and spinal cord MRI revealed lacuna lesions in white matter of brain and pons4.Skin biopsy showed fibril deposition between endothelial cells and vascular smooth muscle cells as well as narrowed lumen.Conclusions1.Identified the first Chinese PADMAL family carrying a mutation in 3’UTR of COL4A1(c.*32G>A).2.Discovered spinal cord involvement in PADMAL patients.Classified Swedish hMID and PADMAL into the same disease entity by comparing clinical features,radiography,pathological changes and causative genePart Ⅱ SMEK1 induced neurodegeneration via mediating microtubule stabilityBackgroundProtein phosphatase 4(PP4),a serine/threonine phosphatase,is a widely protein expressed that participates in various biological processes and signal pathways.including NF kappaB(Nuclear Factor kappaB),JNK(c-Jun N-terminal kinase),apoptosis,IRS4(Insulin Receptor Substrate 4)and mTOR(mechanistic Target Of Rapamycin Kinase)pathways.As a catalyzing enzyme complex,PP4 contains a catalyzing subunit,PP4C,and two regulatory subunits,PPP4R3A(Protein phosphatase 4 regulatory subunit 3A)and PPP4R3B(Protein phosphatase 4 regulatory subunit 3B).PPP4R3A,namely SMEK1(Suppressor of Mek1 null),encodes a conserved protein highly expressed in nervous system.Smekl is located at nucleus as well as perinuclear space and cytoskeleton.Studies have shown subcellular distribution of Smekl undergoes dynamic changes in mitotic cells depending on cycling stages,that is,Smekl locates at cytoplasm in prometaphase and anaphase while it enters nucleus during interphase and prophase.This characteristic depends on the nuclear localization domain.Previous studies have shown a pivotal role of Smek1 in neurogenesis.In neural stem cells and neural progenitor cells,Smek1 regulates differentiation and proliferation through Mbd3,Par3 and Ryk.To reveal the function of Smekl in adult nervous system,we established Smeklflox/flox C57BL/6 mice and knocked out Smekl constitutively via Cre-LoxP system.In the present study,we discussed Smekl’s function in neurodegenerative changes and its underlying mechanisms.Methods1.Investigation of SMEK1 expression in nervous system(1)Analyzing SMEK1’s spatial and temporal expression pattern in nervous system.(2)Confirmation of Smekl distribution and subcellular localization in particular region.2.Generation of Smekl knockout mice(1)Construction of targeting vector:Exon 2 in SMEK1 linearized vector was flanked by two loxP sites through recombination.(2)Recombinated vector was subsequently delivered to ES cells(C57BL/6)via electroporation,followed by drug selection,PCR screening,and Southern Blot confirmation.(3)Production of chimera mice:Correct targeted ES clones were delivered into blastocyst through microinjection,blastocyst was planted into utero of pseudopregnant mice to produce chimera.(4)Generation of heterozygous mice:Founders were confirmed as germline-transmitted via crossbreeding with wild type(5)Generation of homozygous mice:F1 mice were intercrossed to generate F2 Smeklflox/flox mice.(6)Generation of constitutive knockout mice:Smekl flox/flox were mated with Sox2-Cre C57BL/6.F1 mice were intercrossed to generate F2 mice.(7)Generation of C57BL/6-ICR mice:Smek1-/+ mice were mated with wildtype ICR,offspring were then intercrossed.3.Phenotypes and behavior examinations of Smekl knockout mice(1)Observation of birth rate and overall survival of knockout mice.Body weight was recorded each week in 4-12 weeks old mice.(2)Record of locomotion and cognition behavior in knockout mice:hindlimb clasp,rotarod test,treadmill examination,open field test,T-maze and nesting behavior.4.Pathological changes in Smekl knockout mice(1)NeuN,GFAP and MBP staining of brain and spinal sections in 12-month old mice(2)H&E staining of gastrocnemius from 12-month old mice.5.Smekl-deficiency effects on microtubule stability(1)Cell line construction of SMEK1 knockdown and overexpression in SH-SY5Y.(2)Co-staining of microtubule marker and Smekl in sciatic nerve to reveal subcellular localization.(3)Electron microscope analysis of sciatic nerve and optic nerve in aged mcie.(4)Confirmation of microtubule defects in SH-SY5Y,NHA and HM06 cell lines.6.The pathogenesis of neurodegeneration mediated by Smekl via regulating Kif2a and Tau(1)Co-IP and Duolink assay demonstrates interaction between Smekl and Kif2a.(2)Cell fractionation experiments identified characteristics of Kif2a subcellular localization.(3)Immunofluorescent staining revealed Kif2a expression in knockout mice.(4)Western blot examined PI3K-AKT alterations in transfected cell lines.7.Abnormal mitochondria transportation and apoptosis with Smekl deficiency(1)Locate mitochondria in SH-SY5Y transfected cell lines via mitoTracker.(2)Reveal the relationship between mitochondria and microtubule through electron microscope.(3)NADH staining of gastrocnemius from 12-month old mice.(4)Detection of cell proliferation and mitochondrial activity through CCK8.(5)TUNEL staining in aged mice brain.(6)PI staining to detect apoptosis in transfected cell lines.Results1.SMEKl is widely expressed in central nervous systemSMEK1is highly expressed in multiple regions including hippocampus,cortex,substantia nigra and striatum.Compared to young frontal cortex,SMEK1 is low expressed in aged frontal cortex2.SMEK1 deficiency affects mice development,growth and aging.After cross mating C57BL/6 SMEK1-/-mice,353 offspring with only 15 homozygous knockout mice were obtained.After cross mating SMEKr-/+ with C57BL/6 and ICR mixed background,90 offspring with 9 homozygous knockout mice were generated.Genotype ratios of the above two mating strategies deviated from Mendel’s law.This suggested that global SMEK1 deficiency might cause substantial prenatal death.Declined body weight and shortened life expectancy in SMEK1-/-mice were observed.Locomotion activity of heterozygous and homozygous knockout mice were impaired at 6-month old,while homozygous exhibited more severe.Locomotion in knockout mice declined apparently along with aging.Meanwhile,mice showed slight impairment in cognition after 6-month old.3.Brain staining revealed neurodegeneration in SMEK1 deficiency mice Loss of neurons in hippocampus,motor cortex and anterior horn of spinal cord,as well as activation of astrocytes were presented in 12-month old SMEK1 knockout mice.Senile plaques were also observed in aged knockout mice.SMEK1 deficiency also caused decreased neurotrophic factor expression.Gastrocnemius staining showed neurogenic changes.No obvious signs of demyelination in brain and spine.4.Microtubules are destabilized in SMEK1 deficiency axons Smekl colocalized with microtubule in sciatic nerve and SH-SY5Y.Neurofilaments were reduced in knockout mice brain,and microtubule in sciatic nerve and optic nerve of knockout mice were sparse.Meanwhile,knock down of Smekl in SH-SY5Y and NHA cell lines also exhibited reduced cytoskeleton and microtubule,which was rescued by overexpression of Smekl.5.Smekl mediates neurodegeneration via regulating Kif2a and Tau Kif2a mediate microtubule depolymerization in axons.Smekl interact with Kif2a in both nucleus and cytoplasm.Knockdown of Smekl impaired Kif2a nuclear localization.Smekl deficiency also triggered hyperphosphorylation in Tau and altered PI3K-AKT pathway.6.SMEK1 maintain the stability of microtubule,transportation and function of mitochondria,neuronal survival Electron microscope revealed abnormal mitochondria transportation in sciatic nerves in Smekl knockout mice.In SH-SY5Y,knock down of Smekl caused perinuclear aggregation of mitochondria while overexpressing Smekl could mediate mitochondria transportation to the distal end of cell process.Meanwhile,proliferation rate and mitochondria function were down-regulated in shSMEK1 cells.TUNEL staining revealed apoptosis signals in aged Smek1 knockout brain.PI staining in transfected cell lines showed more apoptosis cells when loss of Smekl.Conclusions1.SMEK1 deficiency in mice accelerated central nervous system aging,which were characterized as impaired locomotor ability and behavioral integrity.2.SMEK1 destabilizes microtubules and further impairs microtubule transportation and function.3.SMEK1 deficient displayed neuronal loss,axon reduction and gliosis in central nervous system and interrupted microtubules in peripheral nerves.Part Ⅲ SMEK1 deficiency exacerbated experimental autoimmune encephalomyelitisBackgroundExperimental autoimmune encephalomyelitis(EAE),mediated by CD4+T helper cells(Th),is a well-established animal model of multiple sclerosis(MS),an inflammatory demyelination disease in central nervous system(CNS).Interferon-y(IFN-y)secreted by Thl cells plays multiple roles during the pathogenesis of EAE/MS.Microglia plays a pivotal role in mediating inflammation during EAE and MS.IL-1β secretion intrigued by activation of microglia could cause pathological changes in central nervous system.Meanwhile,astrogliosis mediated by astrocytes also contributes to inflammatory responses.On the other hand,activation of astrocytes might disrupt the intactness of blood brain barrier(BBB)which could subsequently aggravate neuroinflammation.Studies revealed that both administration of IFN-γ and anti-IFN-y antibody could exacerbate clinical signs in EAE model and MS patients.Furthermore,injection of IFN-y modified dendritic cells(DC)into EAE animal models alleviated clinical scores via modulating indoleamine-2,3-dioxygenase-1(IDO1)expression.Indeed,DC participates in the pathogenesis of EAE and regulate immune responses through its antigen presenting role.Activation of aryl hydrocarbon receptor(AhR),in DC could induce immune tolerance and therefore suppresses EAE onset.Previous study has revealed the regulatory role of PP4 in rectal prolapse and colitis.While Smekl functions as the regulatory subunit of PP4,the effects of Smekl on the immune system and autoimmune diseases remain unclear.It is known that SMEK1 is constitutively expressed in immune cells and participates in neurogenesis.In this study,by establishing an CNS autoimmune disease model,we explored the role of SMEK1 in CNS immune response and inflammation.Objectives1.Demonstrate the expression pattern of SMEK1 in MS and explore the effects of Smekl deficiency in EAE clinical manifestations and pathological changes2.Investigate the effects of SMEK1 on immune system and inflammation in central nervous system.Unveil the EAE pathogenesis in Smekl deficiency mice.Methods1.Analyze SMEK1 expression level in peripheral blood mononuclear cells(PBMC)and brain tissues from Gene Expression Omnibus(GEO).Establish EAE model by MOG35-55 subcutaneous injection and PTX intraperitoneal administration in 6-8 weeks old female C57BL/6.The day of immunization was marked as day 0.Body weight and clinical score were recorded every day after immunization.2.EAE mice were sacrificed at peak stage of disease and spinal cervical enlargements were dissected,fixed,paraffin-embedded and sliced.The serial slices were stained with H&E staining,immunohistochemical staining and immunofluorescent staining.3.Spleens and inguinal,axillary lymph nodes from EAE mice at peak stage of disease were isolated to prepare single cell suspension.According to specific purposes,anti-CD11c,CD80,CD86,MHC-Ⅱ antibodies were added to detect DC;Anti-CD4,IFN-γantibodies were added to detect Thl cells;Anti-CD4,IL-4,IL-10 antibodies were added to detect Th2 cells;Anti-F4/80,IL-1β antibodies were added to detect macrophages.4.Single cell suspension were prepared by using spleens from non-immunized C57BL/6 mice,and treated with 1640 containing anti-CD3 and anti-CD28,anti-IFN-γ,LPS respectively.Real-time PCR(qPCR)and Western blot were used to detect cytokine transcriptional alterations and pathway changes.5.Spinal cord slices were used for MMP9,GFAP and IBA1 staining to reveal MMP9 deposit and glial activation.Sodium fluorescent were administered to non-immunized and MOG35-55-immunized C57BL/6 mice to reveal BBB permeability.HM06 cell line and spinal cord slices were used for AhR staining.6.mRNA and protein were extracted from brain tissues from EAE mice at peak stage of disease.mRNA was used for reverse transcription and qPCR.Protein were quantified and underwent Western blotting.Blood serum and brain tissue homogenate were used to detect IL-1β level through ELISA kit.7、Single-cell sequencing of brain tissue from Smekl knockout mice was used for analyzing microglia differentiation.Results1.Smekl deficiency caused worse symptoms in MS patients and EAE modelSMEK1 expression level was decreased in PBMC and brain tissues from MS patients.Smekl heterozygous knockout mice exhibited more severe symptoms in EAE model.2.Smekl deficiency inhibited IFN-y production and induced macrophage activation in EAEIn EAE,spinal cords of Smekl-/+mice had more infiltrated cells but small proportions of CD4-possitive cells.IBA1-possitive cells were significantly increased in Smekl-/+mice.Flow cytometry revealed downregulated expression of IFN-y but increased level of IL-1β.3.Smek1-/+ mice showed more activated microglia and astrocytes in EAEIn EAE SMEK1-/+ mice,astrocytes and microglia were more activated.IL-1βsecretion was elevated and NF kappaB pathway was activated in CNS.BBB permeability was increased in Smek1-/+ mice.4.Single-cell sequencing discovered a novel microglia cluster in Smekl-/+ brainWe discovered two microglia subcluster with different expression pattern,among which one cluster exhibited high level of inflammatory cytokines.Further analysis showed the formation of this pro-inflammation microglia was due to abnormal differentiation5.Low IFN-y mediated the dysfunction of the kynurenine-AhR pathway in microglia and lymphocytesAhR-IDO1 pathway was suppressed in splenic lymphocytes and CNS microglia.IL-10 transcriptional level,mediated by AhR,was down-regulated in accordance with severe clinical scores.In vitro culturing splenic cells from Smek1-/+ mice with anti-CD3 and anti-CD28 downregulated IFN-y expression as well as IL-10 mRNA level.Conclusions1.IL-1β is upregulated in SMEK1 knockout macrophages.macrophages were hyperactivated after immunization in experimental autoimmne encephelomyelites model.2.A novel differentiated SLC2A5-CSF1+ microglia cluster was identified in SMEK1 knockout brain.3.Immunosuppression pathway IDO1-AhR is impaired in immunized SMEK1 knockout mice.