The Establishment Of Patient Specific Induced Pluripotent Stem Cell Models And Animal Models For The Pathogenetic Study Of Paroxysmal Kinesigenic Dyskinesia

Paroxysmal kinesigenic dyskinesia(PKD)is a common type of paroxysmal movement disorder that caused by PRRT2(proline-rich transmembrane protein 2)gene mutations.The disease is characterized by recurrent,brief attacks of dyskinesia triggered by sudden voluntary movement,and the attacks usually occur during adolescence with short symptom durations.Given that PKD is a complex disorder with high genetic heterogeneity,the molecular mechanism underlying the pathogenesis of PKD still remains a mystery and the clinical treatment of the PKD disease needs further investigation.In this study,we report the successful generation of four PKD-induced pluripotent stem cell(iPSC)lines from two different PKD patients with Q163 X and G192 W PRRT2 mutations,respectively,through a somatic cell reprogramming process.To illustrate the features of neural conversion from PKD-iPSCs with mutant PRRT2,different neural induction approaches were applied to PKD-iPSCs.As a result,a step-wise neural induction study uncovered dysregulated alterations in cell morphology and severe defects in neural conversion from PKD-iPSCs.High throughput RNA-seq combined with bioinformatics analysis revealed aberrant transcriptional networks in neurogenesis of PKD-iPSCs compared with normal controls.Instead of the neural fate commitment,PKD-iPSCs showed a differentiation tendency to mesodermal development during the step-wise neural induction process.Simultaneously,we detected seven co-expressed gene modules that highly correlated with different stages during the step-wise neural induction of PKD-iPSC and further confirmed the aberrant gene regulatory networks in a step-wise neurogenesis of PKD-iPSCs by functional analysis of the co-expressed genes and the construction of hub gene networks.Additionally,we identified IRX3 and HAS2 as two key factors closely associated with the alterations found in the step-wise neural induction of PKD-iPSC.The results provide us with important clues for elucidating the molecular mechanisms underlying the pathogenesis of PKD.Furthermore,we established four types of conditional PRRT2 knockout mice,including global Prrt2 heterozygous-null mice,global Prrt2 homozygous-null mice,brain-specific Prrt2 heterozygous-knockout mice and brain-specific Prrt2 homozygous-knockout mice,basing on the techniques of homologous recombination,embryonic stem cell(ESC)blastocyst injection and embryo transfer.The behavioral tests of these conditional Prrt2 knockout mice displayed significantly aberrant behavior and movement disorders.Moreover,the content of some free amino acid neurotransmitters in the serum of these conditional knockout mice was strikingly different from the normal levels based on the detection results of high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method.Thus,we demonstrate that the loss of fuction of PRRT2 could disturb the fuctions of the nervous system,animal behavior and motor function.Taken together,our studies reveal the PKD related phenotypes and its pathogenetic mechanisms through establishment of PKD-specific iPSC models and Prrt2 related animal models.We uncover notable defects in neurogenesis of PKD-iPSC with mutant PRRT2 in a step-wise neural induction process.These results could help us with better understanding of the pathologic features of PKD and open new windows for the clinical treatment of the PKD disease.