Pathogenesis And Mechanism Of Phospholipase D3 P.P22S In Spinocerebellar Ataxia

Objective:Spinocerebellar ataxia(SCA)is a type of hereditary ataxia that mainly affects the cerebellum,brainstem,and spinal cord.It usually occurs in young or middle-aged people,and the main clinical manifestations are cerebellar ataxia.In different subtypes,it can also be accompanied by ocular movement disorders,slow eye movements,optic nerve atrophy,retinal pigmentary degeneration,pyramidal tract signs,extrapyramidal symptoms,muscle atrophy,peripheral neuropathy,and dementia.SCA includes many subtypes,and SCA subtypes are mainly divided into polyglutamine repeat expansion SCA subtypes,non-coding region repeat expansion SCA subtypes,and pathogenic gene coding region non-ribonucleic acid repeat expansion mutations(point mutations,insertion/deletion mutations,etc.).The disease presents a high degree of clinical and genetic heterogeneity,accounting for 10%-15%of neurogenetic diseases.The pathogenesis of SCA is still unclear.Before diagnosing SCA in a patient with progressive ataxia,detailed medical history,physical examination,and laboratory tests should be performed to exclude acquired causes,and then molecular genetic testing should be initiated.There are currently more than40 SCA subtypes,and many genetic mutation sites have not been effectively explored.In 2017,the Nibbeling team discovered phospholipase D3(PLD3)p.L308 P as a pathogenic gene locus for SCA in a family through exon sequencing.Apart from this case,there are still few reports on the correlation between PLD3 gene and SCA.We found a family with ataxia in our clinical cases.After exon sequencing of all family members,we found that the three family members with obvious ataxia symptoms all carried the PLD3 p.P22 S variation site.There were no previous reports on the correlation between PLD3 gene and ataxia in China,so the purpose of this study was to preliminarily explore whether the PLD3 p.P22 S gene locus variation would affect cerebellar function,and then infer whether this locus might be a specific pathogenic gene locus for ataxia.Part1.Effects of Pld3 p.P22 S variation on memory motor ability and nervous systemObjective: Investigate whether the Pld3 p.P22 S variation may cause behavioral changes and pathological changes in the nervous system.Methods: The Pld3 p.P22 S gene-edited mice and wild-type(WT)C57BL/6 mice were subjected to behavioral tests such as water maze,rotating rod,and balance beam at 6/10/12 months of age to verify whether the gene-edited mice exhibit ataxia symptoms and memory impairment.The cerebellum and spinal cord tissue sections of the two groups of mice were stained at 6/10/12 months of age to clarify whether the gene-edited mice had neuronal and glial cell damage.Hematoxylin-eosin(HE)staining of the peripheral sciatic nerve and gastrocnemius muscle was performed to determine whether there were changes in the peripheral nerve and muscle tissue of the gene-edited mice compared to the WT of the same age.Results: 1.Our results showed that Pld3 p.P22 S gene-edited mice had a significantly reduced target approach rate in the water maze test compared to WT mice starting from 10 months of age(p<0.05),indicating memory impairment.2.Starting from 10 months of age,Pld3 p.P22 S gene-edited mice had a significantly longer time to pass the balance beam test compared to WT mice(p<0.05),and a significantly shorter distance to fall in the rotor rod test(p<0.05),suggesting impaired motor coordination and reduced tolerance to fatigue in Pld3 p.P22 S gene-edited mice.3.Pld3 p.P22 S gene-edited mice showed a significant reduction in the number of cerebellar and spinal neurons,disordered cell arrangement,increased extracellular spaces,and most neuronal nuclei were shrunken,deep-stained cytoplasm,and unclear nuclear membranes starting from 10 months of age,indicating necrotic neurons.4.Starting from 10 months of age,Pld3 p.P22 S gene-edited mice had a significantly reduced number of purkinje cells in the brain and spinal cord compared to WT mice(p<0.05).5.Starting from 10 months of age,Pld3 p.P22 S gene-edited mice had an increased number of microglia,astrocytes,and bergmann glial cells in the cerebellum and spinal cord compared to WT mice(p<0.05).6.Pld3 p.P22 S gene-edited mice did not show significant changes in peripheral muscle tissue morphology and muscle cell number compared to WT mice,and there was no significant difference in the arrangement of nerve fibers in the sciatic nerve between the two groups,which was neat and clear.Conclusion:1.Compared with WT mice,Pld3 p.P22 S gene-edited mice exhibited memory impairment,impaired motor coordination,and reduced fatigue tolerance.2.Pld3 p.P22 S gene-edited mice exhibited a typical neurodegenerative pathological change characterized by reduced neurons and increased glial cells.3.In comparison with WT,Pld3 p.P22 S gene-edited mice showed no significant changes in peripheral nerves and muscle tissues.Part 2 Specific effects of Pld3 p.P22 S variation on central neurodegenerationObjective: Explore whether the Pld3 p.P22 S variation may lead to typical neurodegenerative pathological changes such as cell apoptosis,inflammatory response,oxidative stress,and mitochondrial regulation disorder in the body.Methods: 1.The Pld3 p.P22 S gene-edited mice and WT C57BL/6 mice were subjected to Nissl staining and tunel staining at 6/10/12 months of age to verify whether the gene-edited mice have cerebellum and spinal cord neuronal cell apoptosis.2.The ribonucleic acid(RNA)of the cerebellum of Pld3 p.P22 S gene-edited mice and WT C57BL/6 mice at 12 months of age was extracted for real-time fluorescence quantitative polymerase chain reaction to verify whether the Pld3 p.P22 S gene-edited mice have changes of TNF-a,IL-6,IL-1β,TGF-β,IL-10,VEGF,SOD,HO-1,NQO-1BDNF,PSD95,SNAP25,NFL,ZO-1,Occludin,Claudin-1 and PGC-1a.3.The protein of the cerebellum of Pld3 p.P22 S gene-edited mice and WT C57BL/6 mice at12 months was extracted for immunoblotting to detect whether the Pld3 p.P22 S gene-edited mice have changes in bax/bcl proteins.Results: 1.Starting from the age of 10 months,the Nissl bodies in the cerebellum and spinal cord of Pld3 p.P22 S gene-edited mice were unclearly demarcated,with collapsed or wrinkled cell bodies and elliptical or triangular nuclei stained in light to dark blue,and most neurons showing edema.In contrast,WT mice of the same age still showed a large number of Nissl bodies,and the phenomenon of collapsed or wrinkled cell bodies was rare,with a statistically significant difference(p< 0.05).2.Compared with WT mice,the number of apoptotic cells in the cerebellum and spinal cord of Pld3 p.P22 S gene-edited mice increased significantly,with a statistically significant difference(p < 0.01).3.Compared with WT mice,TNF-a,IL-6,IL-1β were elevated in Pld3 p.P22 S gene-edited mice(p < 0.05);TGF-β,IL-10,and VEGF were reduced(p<0.05).4.Compared with WT mice,the expressions of oxidative stress markers such as SOD,HO-1,and NQO-1 in Pld3 p.P22 S gene-edited mice were elevated(p < 0.05).5.Compared with WT mice,the expression of BDNF in Pld3 p.P22 S gene-edited mice was reduced(p< 0.05).6.Compared with WT mice,the expressions of neural synaptic markers such as PSD95,SNAP25 were decreased,and NFL was elevated in Pld3 p.P22 S gene-edited mice(p< 0.05).7.Compared with WT mice,there were no significantly statistical differences in the expression of blood-brain barrier markers such as ZO-1,Occludin.8.Compared with WT mice,the expression of mitochondrial protein PGC-1a in the cerebellum of Pld3 p.P22 S gene-edited mice was reduced(p< 0.05).9.Compared with WT mice,Pld3 p.P22 S gene-edited mice showed an increase in bax/bcl protein expression(p < 0.05).Conclusion:1.The Pld3 p.P22 S gene-edited mice showed an increase in neuronal cell apoptosis compared to WT mice.2.In Pld3 p.P22 S gene-edited mice compared to WT mice,there were imbalances in inflammatory responses,oxidative stress damage,and mitochondrial regulation disorders in the cerebellum.3.In Pld3 p.P22 S gene-edited mice compared to WT mice,there was no change in the permeability of the blood-brain barrier.Part 3 Effects of Pld3 p.P22 S variation on cerebellomic component genes and metabolitesObjective: Transcriptomic sequencing and metabolomic sequencing were used to clarify the changes in the composition of genomic components and metabolites in Pld3 p.P22 S gene-edited mice compared to WT mice.Methods: 1.Select 3 12-month-old Pld3 p.P22 S gene-edited mice and WT mice of the same age made by Southern Pattern Company for transcriptome sequencing;2.Select 6 12-month-old Pld3 p.P22 S gene-edited mice and WT mice of the same age made by Southern Pattern Company for metabolomics sequencing.Results: 1.Pld3 p.P22 S transgenic mice and WT mice showed 430 upregulated genes and 114 downregulated genes,totaling 544 differentially expressed genes(p<0.05).2.At the cellular component(CC)level,differentially expressed genes were mainly enriched in cell protrusions,membrane-bound cell protrusions,extracellular regions,membrane regions,and intrinsic membrane components.At the molecular function(MF)level,differentially expressed genes were mainly enriched in transporter activity,transmembrane transporter activity,hormone activity,and ion transmembrane transporter activity.At the biological process(BP)level,differentially expressed genes were mainly enriched in multicellular organism development,pattern specification,process,system development,anatomical structure development,and embryonic skeletal system development.3.Our results showed that the most significant gene ontology(GO)Terms were anterior/posterior pattern specification,nervous system development,cell projection,neurogenesis,embryonic organ morphogenesis,and cell development process.4.The most significant pathways were environmental information processing,human diseases,metabolism,and organic systems,which were divided into four categories.Environmental information processing pathways include neuroactive ligand-receptor interactions,calcium signaling,and phosphatidylinositol signaling system.Human diseases include African trypanosomiasis,malaria,maturity-onset diabetes of the young,and choline metabolism in cancer.5.The most significant Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways were neuroactive ligand-receptor interactions,calcium signaling,thyroid hormone synthesis,nitrogen metabolism,and protein digestion and absorption.6.Protein-protein interaction(PPI)network analysis showed that compared to WT mice of the same age,the expression of Serpina3 n,Lhx4,Krt18,Cd55,Is11,Gog,etc.,in PLD3 p.P22 S gene-edited mice was increased;Shank2/3,SLc27a5,Ccn12,etc.,were reduced.7.Metabolomic results showed that compared to the WT group,2,4-dimethyl-1H-indole,cytidine,chenodeoxycholic acid disulfide,4-phospho-L-aspartate,dibutyl sulfide,trisilane heptadecanoic acid,malamide,phosphocreatine,arabinose,and stigmasterol B were relatively enriched in the Pld3 p.P22S gene-edited mouse group.Glycerol esters,melacon,3,5-tetradecadiene-2-amine,isopropylamine,pneumocystis,tenebrio toxin,shinan alkaloid,fenpropathrin,N-desmethylamoroloid,and phenazine grass ketone were relatively reduced.8.The top 10 KEGG metabolic pathways enriched in the two groups were sphingolipid signaling pathway,sphingolipid metabolism,retrograde endocannabinoid signaling pathway,phospholipase D signaling pathway,neuroactive ligand-receptor interactions,necrosis,linoleic acid metabolism,choline metabolism in cancer,unsaturated fatty acid biosynthesis,and alcohol intoxication.Conclusion: The Pld3 p.P22 S gene-edited mice and WT mice showed a variety of differences in genes and metabolites in their cerebellum,and these differential genes and metabolites played an important role in neural regulation,further verifying that this site variation will have a negative impact on brain function.