Mechanisms Of Cholinergic Gab1 Deficit In Mediating Cognitive Dysfunction

Background:The central cholinergic nervous system is widely distributed in the brain and participates in the regulation of multiple physiological processes including learning and memory,conscious sleep,attention and stress response.Dysfunctions of the cholinergic nervous system,including loss of cholinergic neurons and decreased synaptic function,have been suggested to play a critical role in the pathological process of cognitive dysfunction-related diseases.For example,the cholinergic hypothesis is one of the important hypotheses of the etiology of Alzheimer's disease(AD).However,the process that cholinergic system dysfunction results in cognitive impairment is very complex and the underlying mechanism has not been fully elucidated yet.Grb2-associated binding protein 1(Gab 1),is a docking/scaffolding molecule from Gab family.Upon activation of the upstream receptor tyrosine kinase,Gabl is recruited to the cell membrane,phosphorylated and functioned as a docking molecule to amplify downstream signals through a cascade reaction.Therefore,Gabl plays a critical role in physiological functions including cell growth,cell polarization,organism development,and immune regulation.However,the function of Gabl in the central nervous system has not been reported yet.Our preliminary data suggested that Gabl is specifically expressed in cholinergic neurons in the brain.Therefore,exploring the function of the Gabl in cholinergic neurons will further our understandings of how the cholinergic nervous system regulates cognitive function and provide a potential candidate target for cognitive dysfunction diseases.The research of current study mainly contains the following three parts:1.Exploring the expression distribution of Gabl in the brain and its association with Alzheimer's disease.Objective:To explore the distribution pattern of Gabl in the brain and the change of Gab1 in Alzheimer's disease.Methods and results:Immunofluorescence staining combined with GFP labeled transgenic mice were used to study the expression and distribution of Gabl protein in the central nervous system.Brain samples from AD patients and AD mouse models,were used to explore the expression changes of Gab1 protein under pathological conditions.The results suggested that Gab1 protein was expressed in the brain cholinergic neurons which are choline acetyltransferase(ChAT)positive or vesicular acetylcholine transporter(vAchT)positive.Majority of Gab1-positive cells expressed GFP in ChAT-eGFP mice.In the brain,more than 90%of ChAT-positive cholinergic neurons in the medial septum,striatum,and cortex also expressed Gab1,and the expression density of Gab1 was highest in the medial septum.Gab1 was not found in interneurons or excitatory neurons.In addition,in human brain samples,Gab1 was also expressed in ChAT-positive cholinergic neurons,but decreased in the brain cortex of an AD patient.Similarly,Gabl also decreased significantly in the medial septum of a mouse model of AD(APP see/PS1 mice).Conclusion:Gabl is specifically expressed in cholinergic neurons in the central nervous system.Gabl-positive cells have the highest density in the medial septum of the basal forebrain.Gabl decreases in cholinergic neurons of AD patient and AD mouse mode1.2.Exploring the role of cholinergic neuron expressed Gabl in regulating cognitive function.Objective:To explore the function of cholinergic neuron expressed Gabl.To be more specifically,to study whether loss of Gabl in cholinergic neurons affects mice behavioral performance,neuronal activity and other functions.Methods and results:Firstly Cre recombinase was expressed in the medial septum of adult Gabl floxed mice(Gab1 f/f)using adeno-associated virus(AAV),and behavioral tests were conducted to study the effects of Gab1 deficit on neurological function in mice.The results revealed that the loss of Gabl in the medial septum in adulthood significantly impaired the performance of mice in novel object recognition test(NORT)and Y-maze test,suggesting that the loss of Gab1 in the medial septum led to cognitive dysfunction.Then,by using the Cre-loxp system,cholinergic neuron Gab1 conditional knockout mice(ChAT-Cre;Gab1f/f)were generated.Behavior tests suggested that ChAT-Cre;Gab1f/f mice showed a decrease in the discrimination index in NORT test,a decrease of alteration rate in Y-maze test,an increase in escape latency in Morris water maze(MWM)test when compared with control mice.Consistent with behavioral performance,hippocampal synaptic plasticity was significantly impaired in ChAT-Cre;Gab1f/f mice,suggesting that Gab1 knockout in cholinergic neurons led to severe cognitive and memory impairment in mice.Further,the excitability of cholinergic neurons in the medial septum were recorded by whole-cell patch clamp.We found that Gab1 deletion increased the frequency of action potentials,decreased the amplitude of after-hyperpolarization(AHP),but didn't affect the passive membrane properties in cholinergic neurons.Using pharmacological intervention,we found that the increase in neuronal excitability in Gab1 knockout neurons was associated with dysfunction of SK channels.Conclusion:The proper level of Gab1 protein in the medial septum is necessary for the normal cognitive function.Deletion of Gab1 in cholinergic neurons leads to severe cognitive dysfunction and decreased hippocampal synaptic plasticity.Deletion of Gab1 in cholinergic neurons also increases excitability of cholinergic neurons in the medial septum,which is associated with dysfunction of SK channels.3.Improvement of the cognitive function by manipulating the level of cholinergic Gabl in medial septum.Objective:To explore whether overexpressing Gabl in medium septum by virus could improve the cognition in whole brain cholinergic neurons Gab1 deletion mice and Alzheimer's disease mice.Methods and results:Cre-dependent Gabl overexpression AAV was injected into medium septum of ChAT-Cre;Gab1 f/f mice,and behavioral tests were examined 3 weeks later We found that specifically re-expression of Gab1 in cholinergic neurons in medium septum could significantly improve the mice performance in MWM,NORT and Y-maze,and significantly reduced excitability of cholinergic neurons in medium septum.The results suggested that Gab1 protein in the medium septum played an important role in regulating the excitability of cholinergic neurons and maintaining the hippocampal-dependent cognitive function.In addition,since we found that Gab1 decreases in AD mouse model,to explore whether manipulation Gabl's level could improve the cognitive impairment of AD mice,we injected lentivirals into medium septum of APPswe/PS1 mice and performed behavior 3 weeks later.Our data suggested that overexpression of Gabl in medium septum of APPswe/PS1 mice effectively improved the spatial memory and restored hippocampal CaMKII autophosphorylation in AD model mice,but had no effect on brain glucose uptake.Conclusion:Overexpression of Gabl in the medial septum improves spatial memory and cognitive function in ChAT-Cre;Gabl f/f mice and improves cognitive dysfunction in AD model mice.