Study On The Molecular Mechanism Of XPR1 Causing Primary Familial Cerebral Calcification And Construction Of Striatum Slc20a2 Knockout Mouse Model

Background and ObjectivePrimary familial brain calcification?PFBC?is a group of neuropsychiatric disorders characterized by bilateral cerebral calcification with diverse neurologic or psychiatric symptoms.Most PFBC is an autosomal dominant inheritance.Five causative genes have been identified including SLC20A2,PDGFRB,PDGFB,ISG15 and,most recently,XPR1.The pathogenesis of PFBC may be related to the dysfunction of phosphorus transport,abnormal function of blood brain barrier and the excessive amplification of IFN-?/?immune signal in brain.However,XPR1 as the latest PFBC causing gene,the precise pathogenic mechanism has not been clear.Here,the entire coding region of the XPR1 gene was sequenced in PFBC patients and functional experiments were studied on the XPR1mutations to explore the pathogenic mechanism.Additionally,due to the complexity of pathogenesis and the lack of suitable animal model,there have been no effective treatments for PFBC.SLC20A2 is the main pathogenic gene of PFBC,but mice with Slc20a2 gene knockout could suffer from severe complications,which limits the study on the phenotype and pathogenesis of PFBC.Our studies aimed to construct striatum SLC20A2 knockout mice,in order to provide an ideal animal model for studying the pathogenesis and the potential therapy of PFBC.Methods1.We screened the mutations of XPR1 gene in the PFBC patients by PCR and sanger sequence.The identified variants were confirmed by cosegregation with the disease phenotype,compared with unrelated controls,predicted by computational programs and analyzed by homologous sequence alignment.Meanwhile,we summarized the clinical features of PFBC patients with XPR1 gene mutations according to the literature.2.Real-Time PCR,Western Blotting and immunofluorescence techniques were used to analyze the difference of RNA level,protein expression and localization between mutant and wild type of proteins.Meanwhile,the phosphate export in cells was analyzed by ELISA,and[Ca²⁺]_i concentration in cells was detected by Fluo-3AM.3.We used Real-Time PCR and Western Blotting to analyze the expression pattern of XPR1 gene,especially in different parts of brain.Immunoprecipitation was carried out to study the interaction between XPR1 protein and known pathogenic proteins?SLC20A2,PDGFRB,PDGFB?.4.Through CRISPR-Cas9 technology,we screened sgRNA targeting SLC20A2 gene,recombined sgRNA to AAV-CRE virus,transfered the virus to the Cas9-EGFP^fl/fll/fl mouse brain to construct striatum tissue-specific model by stereotactic methods.The knockout effects of Slc20a2 gene were identified on DNA,protein level,brain slices and imaging.Results1.We detected two novel variants of XPR1 gene,c.490G>T?p.D164Y?and c.1708C>T?p.R570C?,in two families respectively by screening mutations among 13 familial PFBC pedigrees and 122 sporadic patients.The frequency of XPR1 mutation in patients with PFBC was 1.5%?2/135?.Through analyzing the relationships between genotypes and phenotypes,we found that the calcified areas of PFBC patients with XPR1 gene mutations were large,especially in the cerebellum.And the most common clinical symptom was movement disorders,including dysarthria and facial dystonia.2.There was no difference at the transcriptional level between mutant and wild type of XPR1.At the protein level,c.490G>T?p.D164Y?mutant lead to the decrease of XPR1protein,and the inhibition of proteasome pathway could increase the expression of the mutant protein,the half-life was significantly shortened as well;The decrease of protein in c.1708C>T?p.R570C?was not obvious,but the stability of the protein was impaired.Mutants in XPR1 did not affect the cellular distribution of the protein,but reduced the function of phosphate export and increased the intracellular calcium concentration by upregulating store-operated calcium entry.3.XPR1 mRNA was highly expressed in the brain,lung and kidney of mouse,less in the heart and liver,and the least in the muscle.XPR1 protein was highly expressed in the cerebellum and striatum.XPR1 protein interacted with PDGFRB,and they both colocalized on the cell membrane.4.We confirmed that SLC20A2-targeted sgRNA showed vital cutting activity in vitro.AAV-SLC20A2-sgRNA-CRE had CRE recombinase activity after packaging.We also found that AAV virus expressing CRE recombinase in the striatum by stereotactic methods could stimulate the expression of GFP.Furthermore,the SLC20A2 gene was successfully edited according to the sequencing results.It revealed that the Pit2 protein was reduced by immunoblotting,which needs further detection of the intracranial calcification formation using imageology.Conclusions1.XPR1 gene is a rare pathogenic gene of PFBC in China.PFBC patients with XPR1gene mutations showed large calcification area accompanied with movement disorder.2.The variants,c.490G>T?p.D164Y?and c.1708C>T?p.R570C?,were identified as pathogenic mutations.XPR1 mutations might cause PFBC by reducing phosphate export and enhancing the intracellular calcium concentration through increasing store-operated calcium entry.3.The expression of XPR1 gene might explain the mechanism of the predilection site of calcification.PDGFRB might locate in the upstream of XPR1 gene.Mutations of PDGFRB might lead to abnormal signal pathway,leading to dysfunction of phosphate export of XPR1.4.Based on the CRISPR-Cas9 technology,it would be possible to construct striatum Slc20a2 knockout mice.