| Background: Alzheimer’s disease(AD)is the most common neurodegenerative disease and the leading cause of dementia.The pathogenesis of AD is complex,which is related to various factors such as genetics,environment,and lifestyles.Previous studies have shown that both epigenetic and epitranscriptomic modifications could contribute to learning and memory function and AD pathogenesis.Methyltransferaselike protein 7B(METTL7B)belongs to the methyltransferase-like(METTL)proteins family and could function as DNA or RNA methyltransferase and participate in epigenetic or epitranscriptomic modification.It has been reported that members of METTL proteins family,such as METTL3 and METTL14 were involved in learning and memory regulation and AD pathogenesis through epitranscriptomic modification.METTL7 B is mainly expressed in the hippocampus and entorhinal cortex of primates,and its protein expression level was reported to be elevated in brains of AD patients,suggesting that it may play an important role in learning and memory and AD pathogenesis.Objective: To explore the roles of METTL7 B protein in learning and memory function in both wildtype mice and AD mouse model as well as its regulatory mechanism.Methods:(1)Human METTL7 B was overexpressed in hippocampus of wildtype mice at around 2 months old by injecting adeno-associated virus(AAV).Learning and memory-related behavioral tests were performed using the METTL7 B overexpression(OE)mice one month and six months after AAV injection and 8-month-old nervous system-specific Mettl7 b conditional knockout(CKO)mice to explore whether METTL7 B could impact cognitive function in mice.Gene expression profiling was performed using the hippocampus of METTL7 B OE mice one month and six months after AAV injection and 3-month-old Mettl7 b CKO mice to determine the impact of METTL7 B on mouse hippocampal transcriptome.(2)The METTL7 B OE mouse hippocampus tissues or human neuronal progenitor cell(h NPC)were used for immunoprecipitation followed by mass spectrometry to determine the METTL7B-interacting proteins in mouse hippocampus and h NPC and to explore the potential mechanism of METTL7B-mediated regulation of learning and memory.Then the interaction of METTL7 B with proteins of interest were verified.(3)Human METTL7 B was overexpressed in hippocampus of 5x FAD mice at around 2 months old by injecting AAV,then learning and memory-related behaviors,pathology,and gene expression profiling were performed six months later to determine the effect of overexpressing METTL7 B on the behavioral,pathological,and transcriptomic changes in 5x FAD mice.In addition,gene expression profiling was performed in METTL7 B OE h NPC to investigate the transcriptomic changes from METTL7 B overexpression in neuronal cells.Results:(1)In Morris water maze test,METTL7 B OE mice one month and six months after AAV injection travelled longer time and distance before reaching the hidden platform in training session and spent less time in the platform quadrant in probe test than control mice(P <0.05),indicating impaired spatial learning of the above mice.In fear conditioning tests,METTL7 B OE mice six months after AAV injection exhibited a reduced percentage of immobility time during fear training and associative tests compared to control mice(P < 0.05),indicating impaired conditional associative memory.However,mice from other research groups did not show significant differences from control mice in the above tests.Gene expression profiling revealed 1566 differentially expressed genes(DEGs)in METTL7 B OE mice one month after AAV injection compared to controls(FDR < 0.05),of which 891 up-regulated genes,such as Igkv4-59 gene,were mainly clustered in immune-related pathways,and 675 down-regulated genes,such as Wnt9 a gene,were mainly clustered in nervous system function-related pathways.METTL7 B OE mice six months after AAV injection and 3-month-old Mettl7 b CKO mice had fewer DEGs(P < 0.05),and the dysregulated genes gathered in similar pathways with those of METTL7 B OE mice one month after AAV injection.(2)The 657 METTL7B-interacting proteins were identified in mouse hippocampus,enriched with the biological processes of protein transport,signal transduction,lipid metabolism,and ion transport,and pathways of signal transduction and neurodegenerative disorders such as AD,whilst 38METTL7B-interacting proteins in h NPC were mainly enriched with biological processes of cytoskeleton,protein metabolism,and immune response,and pathway of immune response and neurodegenerative disorders such as AD.Co-transfection and co-immunoprecipitation verified that METTL7 B interacted with FAF2 and HSPB1 but may not interacted with HSPA1 A.(3)There was no significant difference between METTL7 B OE 5x FAD mice and their controls in Morris water maze test and fear conditioning test.METTL7 B overexpression increased Aβplaques and GFAP-labeled astrocytes in 5x FAD mice.METTL7 B OE5x FAD mice had 46 DEGs compared with controls(FDR < 0.05),of which 40 down-regulated genes,such as Neurod6 gene,were mainly clustered in pathways related to nervous system function.In addition,METTL7 B OE h NPCs had 1127 DEGs(FDR < 0.05)compared to controls;770 up-regulated genes,such as HSPA6 gene,were mainly clustered in immune response,endoplasmic reticulum stress-related pathways,and 357 down-regulated genes,such as HIST1H1 B gene,were mainly clustered in cell cycle-related pathways.Conclusions:(1)Overexpression of METTL7 B impairs spatial learning and conditional associative memory in C57BL/6 mice possibly by regulating the expression of genes related to immune response and nervous system function.(2)METTL7B may regulate learning and memory and AD pathogenesis by interacting with the proteins involved in lipid and protein metabolism,cytoskeleton,signal transduction,and immune response.(3)Overexpression of METTL7 B did not affect impaired learning and memory-related behaviors but enhanced hippocampal Aβ deposition and astrogliosis possibly by dysregulating expression of genes related to brain development in 5x FAD mice. |
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