Studies On The Disease Mechanism Of Retinitis Pigmentosa-related Genes

Retinitis pigmentosa(RP)is a group of complex retinal diseases with a high degree of genetic heterogeneity,characterized by degeneration of the retinal pigment epithelium and progressive degeneration of photoreceptors.The disease has an insidious onset at the early stage and presents as night blindness with impaired peripheral vision,which then progressively worsens until blindness.Most of RP cases are hereditary diseases.To date,over 90 genes have been identified to be associated with RP.These genes encode proteins involved in a variety of functional pathways in the retina,including phototransduction,photoreceptor development,and intracellular protein transport.The clinical manifestations and severity of RP are highly heterogenous,depending on pathogenic genes.Therefore,the pathogenic mechanisms of RP are highly complicated.Currently,there is no effective treatment for retinitis pigmentosa except that gene therapy shows some effect on certain types of RP at specific stages.So,it is of great clinical significance to explore the pathogenic mechanisms and develop effective treatment methods for RP.In this dissertation,the function of CCP5 and REEP6 in photoreceptors and determined the detrimental role of c GMP in the pathogenesis of rd1 mice with a spontaneous mutation in the Pde6 b gene were investigated.In vivo and in vitro experiments were employed to elucidate the mechanism of these genes in retinal degeneration,providing insight into the pathogenesis of RP and the basis for the potential treatment of RP.The main contents of the thesis are as follows:1.Explore the mechanism of the CCP5 gene in RP.Retinal photoreceptors are specialized ciliated cells.The main component of the axoneme in the photoreceptor connecting cilium is the microtubule.Microtubules in connecting cilia harbor a variety of post-translational modifications,including polyglutamylation.CCP5 is an enzyme gene that regulates microtubule deglutamylation.Mutations in CCP5 cause RP,but the role of the CCP5 gene in the retina and the pathogenic mechanism of its mutations leading to RP remain unclear.To study the function of the CCP5 gene in photoreceptors,the Ccp5 knockout mouse model was constructed by using the CRISPR/Cas9 technology.The Ccp5 knockout mice showed impaired retinal function with a significant reduction in responses to light stimuli under dark-adapted and light-adapted conditions,and exhibited progressive photoreceptor degeneration.Compared with wild-type mice,the level of polyglutamylation was significantly enhanced in Ccp5 knockout mice.Transmission electron microscopy showed that the arrangement of axonemal microtubules in knockout mice was disturbed and the boundaries were blurred,indicating that the structure of axoneme microtubules was affected.Immunoblotting and immunohistochemistry showed that the expression of key phototransduction proteins,including RHO,GC1,GC2,and GRK1,was down-regulated.Cone opsins were mislocalized in inner segments and outer nuclear layers.The structure of the membrane disk in photoreceptors was disorganized.The above results revealed that the deletion of Ccp5 led to retinal degeneration by causing excessive glutamylation of microtubules and affecting the structure of axoneme microtubules,which in turn interfered with the transport of phototransduction proteins to outer segments2.Explore the mechanism of REEP6-associated RP.The REEP6 protein(encoded by REEP6),a member of the receptor expression enhancing proteins family,has been previously implicated to be critical for the function of photoreceptors.Genetic studies showed that its mutations caused RP and deletion of Reep6 caused retinal degeneration,but its pathogenic mechanism is not fully understood.To further investigate the pathogenic mechanism of RP associated with REEP6 mutations,it was found that overexpression of REEP6 in cultured cells could significantly increase the expression of rhodopsin in vitro.It was also found that over-expression of REEP6 changed the morphology and distribution of mitochondria,and also affected the morphology of the endoplasmic reticulum and Golgi apparatus,indicating that REEP6 has a regulatory effect on the morphology and function of the mitochondria,endoplasmic reticulum,and Golgi apparatus.Then,the Reep6 knockout mouse model was generated.The study revealed a significant decrease in the scotopic response to flash light in 6-month-old knockout mice.With the increase of mouse age,both the outer and inner segments showed progressive degeneration,indicating that Reep6 gene plays an important role in the development and function maintenance of the morphological integrity of photoreceptors.Furthermore,western blot and immunohistochemistry revealed that the expression of the membrane proteins in phototransduction,including RHO,GC1,GC2,and GRK1 were significantly down-regulated,and the morphology of the Golgi apparatus was abnormal,indicating that REEP6 regulates the expression of phototransduction proteins,possibly by modulating the function of the Golgi apparatus.In addition,the ATP level in the retina of knockout mice was increased,which confirmed the regulatory role of REEP6 on mitochondrial function.RNA-seq data showed that the deletion of Reep6 induced complement factor-mediated inflammatory responses in the retina.Taken together,the above experimental results showed that the mechanism of REEP6-associated retinal degeneration might be due to multiple effects,including aberrant mitochondrial function,ER and Golgi apparatus dysfunction,down-regulation of phototransduction proteins,and retinal inflammation,which eventually lead to the death of photoreceptors.3.To determine the role of c GMP in retinal degeneration by expression of PDE5 in photoreceptors of the rd1 mice.PDE6 is a key enzyme in the phototransduction signal pathway in photoreceptors,which catalyzes the hydrolysis of c GMP after activation.The rd1 mouse carries a naturally occurring nonsense mutation in the Pde6 b gene.The rd1 mouse retina rapidly degenerates and fails to form rod photoreceptor outer segments due to the elevated c GMP level.To determine the role of c GMP in photoreceptor degeneration,we tested whether expression of non-photoreceptor specific PDE5 by electroporation in neonatal rd1 mice can rescue photoreceptors.PDE5 is closest to PDE6 in terms of structure and function.The PDE5 expression plasmid was used to transfect neonatal C57 and rd1 mice by subretinal injection followed by electroporation and found that PDE5 was distributed in inner segments and synaptic terminals.The analysis of the retinal sections at P16 showed that the outer nuclear layer of the retina had six to eight rows of nuclei in the transfected area,and formed the outer segments in rd1 mice.In vitro,coexpression of PDE5 and rhodopsin in cultured cells showed enhanced rhodopsin expression.These results indicated that reducing the level of c GMP in photoreceptors protected photoreceptors and promoted rhodopsin expression in rd1 mice,which contributed to the formation of photoreceptor outer segment.These results reveal the critical role of c GMP in retinal degeneration in rd1 mice as well as the additional role of c GMP in regulating protein expression in photoreceptors.In conclusion,this thesis explored the important role of CCP5 and REEP6 in the retina.It revealed the molecular mechanism of retinal degeneration caused by functional impairment of CCP5 or REEP6,and demonstrated that the hydrolysis of c GMP by PDE5 was sufficient to rescue photoreceptors in rd1 mice.These studies provided a preliminary basis for the further study and intervention of RP.