Congenital disorders of glycosylation(CDG)are a cluster of monogenic disorders resulting from defects in glycosylation.FCSK encodes fucokinase,an enzyme that catalyzes the phosphorylation of L-fucose to generate fucos...Congenital disorders of glycosylation(CDG)are a cluster of monogenic disorders resulting from defects in glycosylation.FCSK encodes fucokinase,an enzyme that catalyzes the phosphorylation of L-fucose to generate fucose-1-phosphate,an important step in fucosylation.Mutations in FCSK lead to CDG with an autosomal recessive inheritance pattern,primarily manifesting as developmental delay,hypotonia,and brain abnormalities.However,no fcsk mutant animal models have yet been established.This study constructed the first fcsk knockout(fcsk^(-/-))zebrafish model using CRISPR/Cas9 technology.Notably,fcsk^(-/-)zebrafish exhibited impaired growth,characterized by delayed epiboly and DNA accumulation during early embryonic development,as well as brain atrophy in adulthood.Larval-stage fcsk^(-/-)zebrafish displayed locomotor deficits and increased susceptibility to pentylenetetrazole-induced seizures.In adulthood,fcsk^(-/-)zebrafish showed neurodevelopmental abnormalities,including increased anxiety,decreased aggression,reduced social preference,and impaired memory.Additionally,total protein fucosylation was markedly reduced in fcsk^(-/-)zebrafish,accompanied by decreased expression of pofut2,which encodes protein Ofucosyltransferase 2,an enzyme involved in the fucosylation salvage pathway.Apoptotic activity was elevated in the midbrain-hindbrain boundary(MHB)of fcsk^(-/-)zebrafish.Supplementation with GDP-L-fucose or the human FCSK gene restored developmental defects and total protein fucosylation in fcsk^(-/-)zebrafish.RNA sequencing revealed dysregulated gene expression associated with glycosylation,apoptosis,and neurodegenerative diseases.These findings suggest that fcsk^(-/-)zebrafish exhibit neurodevelopmental disorders,providing the first fcsk gene knockout animal model and offering a platform for investigating the molecular underpinnings of the disease and facilitating drug screening efforts.展开更多
基金supported by the National Key R&D Program of China(2023YFC2706302)National Natural Science Foundation of China(81000079,81170165 and 81870959 to X.Z.)+1 种基金Program of HUST Academic Frontier Youth Team(2016QYTD02)Fundamental Research Funds for the Central Universities(HUST:2019JYCXJJ035)。
文摘Congenital disorders of glycosylation(CDG)are a cluster of monogenic disorders resulting from defects in glycosylation.FCSK encodes fucokinase,an enzyme that catalyzes the phosphorylation of L-fucose to generate fucose-1-phosphate,an important step in fucosylation.Mutations in FCSK lead to CDG with an autosomal recessive inheritance pattern,primarily manifesting as developmental delay,hypotonia,and brain abnormalities.However,no fcsk mutant animal models have yet been established.This study constructed the first fcsk knockout(fcsk^(-/-))zebrafish model using CRISPR/Cas9 technology.Notably,fcsk^(-/-)zebrafish exhibited impaired growth,characterized by delayed epiboly and DNA accumulation during early embryonic development,as well as brain atrophy in adulthood.Larval-stage fcsk^(-/-)zebrafish displayed locomotor deficits and increased susceptibility to pentylenetetrazole-induced seizures.In adulthood,fcsk^(-/-)zebrafish showed neurodevelopmental abnormalities,including increased anxiety,decreased aggression,reduced social preference,and impaired memory.Additionally,total protein fucosylation was markedly reduced in fcsk^(-/-)zebrafish,accompanied by decreased expression of pofut2,which encodes protein Ofucosyltransferase 2,an enzyme involved in the fucosylation salvage pathway.Apoptotic activity was elevated in the midbrain-hindbrain boundary(MHB)of fcsk^(-/-)zebrafish.Supplementation with GDP-L-fucose or the human FCSK gene restored developmental defects and total protein fucosylation in fcsk^(-/-)zebrafish.RNA sequencing revealed dysregulated gene expression associated with glycosylation,apoptosis,and neurodegenerative diseases.These findings suggest that fcsk^(-/-)zebrafish exhibit neurodevelopmental disorders,providing the first fcsk gene knockout animal model and offering a platform for investigating the molecular underpinnings of the disease and facilitating drug screening efforts.
