Abnormal Glutamate And GABA Release From Astrocytes Influences Neuronal Development In Fmr1 Knockout Mice

Background and objectiveFragile X syndrome(FXS)is a form of inherited mental retardation that results from the absence of FMRP,the product of the Fmr1 gene.Numerous studies have shown that FMRP expression in astrocytes is important in the development of FXS.Although astrocytes affect neuronal dendritic development in Fmr1 KO mice,the factors released by astrocytes are still unclear.Methods1.Primary cultured the cortex astrocytes of WT or Fmr1 KO mice.After further purification,we cultured WT cortical neurons in collected astrocyte-conditioned medium(ACM)from WT or Fmr1 KO mice.2.Immunofluorescence was performed to detect the dendritic growth of neurons cultured in WT or Fmr1 KO ACM.3.Glutamate and GABA levels were determined by using HPLC.4.Western blot was used to evaluate the influence of WT or Fmr1 KO ACM for the synapse related proteins expression of the neurons,the expression of enzymes related to synthesis of glutamate and GABA in WT or Fmr1 KO astrocytes.5.VGB was added in cultured astrocytes,and western blot was used to detect the level of GABA-T in astrocytes.The concentration of glutamate and GABA was measured by using HPLC.6.Locomotion and trace fear memory of Fmr1 KO mice were tested after oral administration of VGB.Results1.Immunofluorescence results showed that dendrite branching complexity of neurons cultured with Fmr1 KO ACM was decreased,compared with WT group.2.HPLC results showed that an increased release of glutamate and decreased GABA level from Fmr1 KO astrocytes.3.Exogenous glutamate was added into WT ACM and neuronal morphology was examined.It showed robust signs of dendritic damage including shorter dendrites and stunted arborization,which was similar to KO ACM cultured neurons.Meanwhile,synapse-associated proteins such as PSD95,MAP2 and GluR1 were decreased after added glutamate.4.We found increased glutaminase and GABA-T expression,decreased MAOB level in Fmr1 KO astrocytes.The elevated levels of glutamate contributed to oxidative stress in the cultured neurons.5.VGB,a GABA-T inhibitor,reversed the imbalance release of glutamate and GABA in Fmr1 KO astrocytes.6.Oral administration of VGB decreased the locomotion activity,improved learning and memory of Fmr1 KO mice.ConclusionThe present study provides a possible explanation for the role of glia in the abnormal neurodevelopment of FXS.The data demonstrated that the aberrant release and uptake of neurotransmitters by Fmr1 KO astrocytes,which was caused by increased glutaminase and GABA-T expression and decreased MAOB level,influenced neuronal dendritic development.A GABA-T inhibitor VGB reversed abnormal morphology of neuronal dendrites by inhibiting the high glutamate release in KO astrocytes.The oral administration of VGB rescued the abnormal behaviors of Fmr1 KO mice.Our results represent a new experimental approach for FXS treatment.Further research is required to clarify this issue.