Nuclear Modifier Gene PRICKLE3 Manifests The Tissue-specific Defects Of Retinal Ganglion Cells Bearing LHON-associated MtDNA Mutation

Leber’s hereditary optic neuropathy(LHON)is one of the most common maternal inheritances of eye diseases.This disorder is characterized by bilateral,painless,subacute,central visual loss in young adults.The degeneration of retinal ganglion cells(RGCs)is the primary defect in the retina.Mutations in mitochondrial DNA(mt DNA)have been linked to LHON with varying degrees of contribution to pathogenesis.Of these,m.3460G>A(MT-ND1),m.11778G>A(MT-ND4)and m.14484T>C(MT-ND6)mutations affecting essential subunits of NADH ubiquinone oxidoreductase(complex I)were responsible for the majority of LHON cases worldwide.The primary defects in these LHON-linked mt DNA mutations appeared to be a failure in the activity of complex I,thereby causing respiratory deficiency,diminishing ATP synthesis,and an increased generation of reactive oxygen species(ROS).However,only relatively mild mitochondrial dysfunctions,especially the reduced activity of complex I,were observed in mutant cells carrying the mt DNA mutations.Furthermore,there were marked variations in the penetrance and gender bias in the LHON pedigrees carrying primary mt DNA mutations,reflecting the complex etiology of this disease.X-linked modifier genes were proposed to modulate the phenotypic expression of LHON-associated mt DNA mutations.However,the pathophysiology of these LHON-linked mt DNA mutations,particularly for tissue-specific defects in RGC,has not been well understood.The previous studies in Dr.Guan’s lab identified the X-linked LHON modifier(c.157C>T,p.Arg53Trp)in the PRICKLE3 encoding a mitochondrial protein linked to the biogenesis of ATPase in three Chinese families bearing LHON-linked mt DNA mutations.They showed that the c.157C>T mutation acted in synergy with m.11778G>A mutation and deteriorated mitochondrial dysfunctions necessary for the expression of LHON.Prickle3 knock-out mice recapitulated LHON phenotypes with retina deficiencies including degeneration of retinal ganglion cells and abnormal vasculature.However,the lack of an LHON-associated ND4 mutation animal model makes us difficult to elucidate the pathogenic mechanism underlying the interaction between the ND4 and Prickle3 mutations that manifests the optic neuropathy in vivo.The technology to generate induced pluripotent stem cells(i PSC)from somatic cells provided the solution to this problem.Somatic cells could be reprogramed to be i PSC after transferred to defined factors,and i PSC could differentiate into nearly all cell types ex vivo.To further elucidate the tissue-specific effects of LHON,we generated i PSC from fibroblasts derived from the members of one Han Chinese family carrying only m.11778G>A,or PRICKLE3 c.157C>T mutation,and both m.11778G>A with heterozygous or hemizygous PRICKLE3 c.157C>T mutations and control subject lacking these mutations.These i PSCs were stepwise differentiated to neural progenitor cells and subsequently RGC-like cells.The m.11778G>A mutation affected the differentiation of i PSC into RGC-like cells but not neural progenitor cells,while there was no effect of PRICKLE3 c.157C>T mutation on the differentiation of i PSC into RGC-like cells.Strikingly,these i PSCs bearing both m.11778G>A and PRICKLE3 c.157C>T mutations exhibited severe defects in the differentiation of i PSC into neural progenitor cells and RGC-like cells.RGCs-like cells harboring only m.11778G>A mutation revealed the loss of tiny neurites,and those carrying the PRICKLE3 c.157C>T mutation were comparable with those in controls.Notably,RGC-like cells bearing both m.11778G>A and PRICKLE3 c.157C>T mutations exhibited abnormal morphologies including defects in the lengths and sizes of neurites.The PRICKLE3 c.157C>T mutation exacerbated the electrophysiological abnormalities caused by m.11778G>A mutation,including the decrease of sodium current,the increase of resting potential,and evoked APs number.Furthermore,the PRICKLE3 c.157C>T mutation enhanced RGC-likes cells apoptosis associated with m.11778G>A mutation.Moreover,both PRICKLE3 c.157C>T and m.11778G>A mutations led to more severe mitochondrial dysfunctions in RGC-likes cells than in these parental fibroblasts,i PSCs and neural progenitor cells.In summary,our findings demonstrated the tissue-specific effects of m.11778G>A mutation and PRICKLE3 c.157C>T mutation on RGC-liked cells.Our findings provide new insights into the pathogenic mechanism of LHON and open a new revenue for the intervention and treatment of this disorder.