Analyzing The Effect Of Depletion Of Cul4b From Mouse Nervous System On Cognitive Ability From The Perspective Of Hippocampus Neuronal Excitability

Mental retardation(MR)is a common disease,characterized by intelligence quotient(IQ)lower than 70,limited adaptive behavior,and deficits in social skills.The incidence of MR is as high as 1-3%.Among genetic factors,mutations of genes on the X chromosome are closely related to mental retardation.This type of mental retardation is called X-linked mental retardation(XLMR),which accounts for about 10%of all mental retardation cases.We and a British research team simultaneously reported that mutations in CUL4B caused XLMR in 2007.Since then,CUL4B mutations have been confirmed in more patients in XLMR families.CUL4B gene has become the third most common XLMR-causing gene,after FMR1 and ATRX.CUL4B belongs to the Cullin family,the members of which are scaffolding proteins of Cullin-RING E3 ligase complexes(CRLs),the largest known class of ubiquitin ligases in eukaryotes.The CRL complex formed by CUL4B as a scaffolding protein is called Cullin 4B-RING E3 ubiquitin ligase complex(CRL4B),which catalyzes the polyubiquitination or monoubiquitination of substrate proteins and plays important roles in a variety of tissues and a variety of pathophysiological processesStudies have shown that CUL4B has important functions in the central nervous system.The Cul4b global knockout mice(Cul4bSox2-Cre mice)exhibited impaired spatial learning and memory,increased epileptic susceptibility,decreased parvalbumin-positive GABAergic interneurons in the hippocampus,and abnormal dendritic complexity and spine density in the hippocampal neurons.We previously found that the nervous system-specific Cul4b knockout mice(Cul4bNestin-Cre mice)exhibited spatial learning and memory deficits.The GFAP+cells were found to be more abundant in the brains of Cul4bNestin-Cre mice,and the increased generation of GFAP+cells was mediated by an upregulation of PTGDS.In addition,we previously found that neuron-specific Cul4b knockout mice(Cul4bNestin-Cre mice)exhibited spatial learning and memory impairment,while astrocyte-specific Cul4b knockout mice(Cul4bGFAP-Cre mice)did not show spatial learning and memory impairment,suggesting that the spatial learning and memory impairment of Cul4b knockout mice is caused by neuronal abnormalities.Further studies found that loss of Cul4b in primary neurons led to slower growth of neurites,shorter total dendrite length and reduced branches of dendrities.At present,we still know little about the pathogenic mechanism of XLMR caused by mutation of CUL4B gene.In order to further explore the function of CUL4B in the central nervous system and the pathogenic mechanism of CUL4B mutation in human mental retardation syndrome,we used a series of experimental methods,such as behavioral tests,histology and patch clamp,and achieved the following results(1)The results of Y-maze showed that Cul4bNestin-Cre mice exhibited working memory impairment.The results of three-chamber interactive experiment showed that Cul4bNestin-Cre mice displayed impaired social interaction.The results of open field test showed that Cul4bNestin-Cre mice displayed normal locomotor activity.Thus the results in Y-maze and three-chamber interactive experiments were not caused by the abnormal motor ability of Cul4bNestin-Cre mice.The results also showed that the anxiety levels were normal in Cul4bNestin-Cre mice(2)Nissl staining results showed that the numbers of neurons in the hippocampal CA1 and CA3 were significantly reduced in Cul4bNestin-Cre mice.The results of transmssion electron microscopy showed that the width of synaptic cleft of Cul4bNestin-Cre mice was increased(3)In order to explore the effect of CUL4B on the function of the mice central nervous system,we used patch clamp to detect the excitability of the hippocampal circuit,and found that the excitability of the hippocampal circuit of Cul4bNestin-Cre mice was increased.To further explore the reasons for the increased excitability of the hippocampal circuit of Cul4bNesNestin-Cre mice,we did the following investigation.1)As for the intrinsic excitability of neurons,we observed the increased number of action potentials evoked by depolarizing current injections of hippocampal CA1 neurons of Cul4bNestin-Cre mice with the elevated initial frequency and the signif:cantly increased input resistance,indicating that loss of Cul4b led to increased intrinsic excitability of neurons in mice hippocampal CA1.2)As for the excitatory synaptic transmission,we examined the expression of vesicular glutamate transporter 1(vGLUT1),an excitatory synaptic marker on pre-synaptic membrane,by immunofluorescence,and found that the expression of vGLUT1 in the hippocampal CA1 of Cul4bNestin-cre mice was significantly decreased.The expressions of excitatory receptors were also determined.It was found that GluAl,NMDAR1 and GRIN2A in hippocampal CA1 of Cul4bNestin-Cre mice were significantly increased,and the expression of GluA2 did not change significantly.Subsequently,we recorded the miniature excitatory postsynaptic currents(mEPSCs)of mice hippocampal CA1 neurons by patch clamp technology,and found that the mEPSCs frequency of Cul4bNestin-Cre mice hippocampal CA1 neurons did not change significantly,but the mEPSCs amplitude increased significantly,suggesting that the excitatory basal synaptic transmission of hippocampal CA1 neurons in Cul4bNestin-Cre mice was increased.3)As for the inhibitory synaptic transmission,we evaluated the expression of presynaptic membrane inhibitory marker vesicle y-aminobutyric acid transporter(vGAT)by immunofluorescence,and found that the expression of VGAT in the hippocampal CA1 of Cul4bNestin-Cre mice was significantly reduced.Subsequently,the mRNA expression of ionotropic and metabotropic inhibitory receptors(GABA receptors:GABAAα2,GABAAα3,GABAAα5,GABAAβ1,GABAAβ2,GABAAγ2,GABAB1 and GABAB2)were determined by RT-qPCR.mRNA expressions of GABA inhibitory receptors were normal in Cul4bNesNestin-Cre mice hippocampus.The miniature inhibitory postsynaptic current(mIPSCs)of mice hippocampal CA1 neurons was recorded by patch clamp technology.The mIPSCs frequency of Cul4bNestin-Cre mice mice hippocampal CA1 neurons was significantly decreased,but the mIPSCs amplitude did not change significantly,suggesting that the inhibitory basal synaptic transmission of hippocampal CA1 neurons in Cul4bNestin-Cre mice was decreased4)Based on the increased mEPSCs amplitude and decreased mIPSCs frequency in Cul4bNestin-Cre mice hippocampal CA1 neurons,we analyzed the ratio of mEPSCs/mIPSCs of mice hippocampal CA1 neurons,and found that the ratio of mEPSCs/mIPSCs amplitude was significantly increased in Cul4bNestin-Cre mice hippocampal CA1 neurons.At the same time,the ratio of Glu/GABA content in the brain tissue of Cul4bNestin-Cre mice was significantly increased through LC-MS experiments.The above results indicated that the hippocampal CA1 neurons of Cul4bNestin-Cre mice were excitatory/inhibitory(E/I)imbalancedIn summary,this study confirmed that loss of Cul4b leads to impaired working memory and social interaction in mice.We found that loss of Cul4b leads decresed number of neurons and increased width of synaptic cleft in hippocampus.We also found that loss of Cul4b leads to increased excitability of the hippocampal circuit.The hyperexcitability is due to the increased intrinsic excitability,increased excitatory basal synaptic transmission and decreased inhibitory basal synaptic transmission of hippocampal neurons.This study is of great value for analyzing the pathogenic mechanism of XLMR caused by CUL4B gene mutation,and has important theoretical and practical significance for understanding diseases related to nervous system development.