Mechanism Research Of Abnormal Dopaminergic Neuron-like Cell Caused By COG5 Deficiency

Objective:COG5 is one of the constituent subunits of heterooctameric conserved oligomeric Golgi（COG）complex,which is responsible for tethering vesicles and coordinating various transport molecules during the retrograde transport of Golgi apparatus.Deficient in COG5 leads to the abnormal function of COG complex,and results in insufficient recovery of glycosyltransferase and glycosidase in Golgi,which leads to the abnormal glycosylation of secretory proteins,and further affects its structure and function.Patients with COG5 deficiency showed multiple organ system involvement,among which nervous system symptoms were the most intractable,including growth retardation,dystonia,mental retardation,cerebrum/cerebellar atrophy,microcephaly,ataxia and so on.At present,there is no corresponding treatment and no clear pathogenesis has been reported.Taking advantage of the multidirectional differentiation potential of human embryonic stem cell（h ESC）,the constructed COG5-deficient h ESC was induced to differentiate into neural cell and explore the mechanism of COG5 in it,in order to provide a theoretical basis for analyzing the pathogenic mechanism of patients with COG5 deficiency and screening key therapeutic targets.Methods:The COG5 gene of h ESC was targeted knockout by CRISPR/Cas9 gene editing system,and the deletion of COG5 was verified by Sanger sequencing and western blotting at gene and protein level.Furthermore,the pluripotency and multi-differentiation potential of COG5-deficient h ESC were identified by the expression level of pluripotent markers and the formation of three germ layers in spontaneous differentiation.Subsequently,the structural and functional phenotypes of Golgi apparatus in COG5-deficient h ESC were identified by transmission electron microscope observation and Golgi migration test.Finally,COG5-deficient h ESC and wild-type h ESC were induced to differentiate into dopaminergic neuron-like cell（DNl C）,and the protein glycosylation level of DNl C from the two sources was analyzed by N-glycoproteomic,to find out the key differential glycoproteins that may lead to nervous system abnormalities.Results:After Sanger sequencing and western blotting verification,COG5-deficient h ESC was successfully constructed(h ESC-COG5^-/-).h ESC-COG5^-/-not only maintained its pluripotency and multidirectional differentiation ability,but also showed abnormal structure and migration ability of Golgi apparatus.The positive signal of dopaminergic neuron marker tyrosine hydroxylase（TH）could be detected in the early stage of DNl C derived from h ESC-COG5^-/-,but the signal was lost in continuous culture.The results of N-glycoproteomic analysis showed that the glycosylation level of proteins involved in receptor activity,channel activity and synaptic organization was significantly down-regulated in DNl C-COG5^-/-,including multiple subunits of glutamate receptors NMDAR and AMPAR,which play a key role in the plasticity of the central nervous system.Conclusion:In Parkinson’s disease,TH reduction is thought to be secondary to dopaminergic neuron degeneration and death.Therefore,it can be speculated that the deficiency of COG5 did not affect the differentiation of h ESC into DNl C,but may cause the failure and death of the generated DNl C.This may be due to insufficient glycosylation of glutamate receptors NMDAR and AMPAR,which blocked the neurotransmitter-receptor interaction.This may be one of the important mechanisms of COG5 deficiency leading to neurological symptoms.