The Role And Underlying Mechanism Of GABA Interneurons In The Cognitive Impairment Of Trim32 KO Mice

Autism spectrum disorders(ASDs)are a group of conditions characterized by deficits in social interaction,limited interests and activities,repetitive behaviors.It is generally believe that many individuals with ASD share the same cognitive problems.ASDs are highly heritable.More and more ASD genetic risk loci have been identified.Recently,rare CNVs affecting Trim32 at the 9q33.1 locus have been confirmed implicated in human ASD.Trim32 protein belongs to the tripartite motif(TRIM)family,act as the E3 ubiquitin ligase,has linked to multiple biological processes such as cell proliferation,neuronal differentiation,neurodevelopment,tumor suppression,apoptosis.Various mutations in the Trim32 gene caused several different genetic diseases,with different phenotypes that affect Bardet-Biedl syndrome,LGMD2 H,STM,epidermal carcinomas,psoriasis,Alzheimer and depression disorders.In animal and human being,the hippocampus is responsible for learning and memory.However,its anatomical basis and biological mechanisms remain unclear.Some phenotypes of ASD are supposed to be result from an imbalance of synaptic transmission between inhibitory and excitatory.GABAergic interneurons are important to regulate the synaptic activity by directly inhibit in the excitatory neurons in the hippocampus.Changes of the GABA level in presynaptic or the GABA receptors in postsynaptic result in a disturbance in excitatory/inhibitory circuit,which can cause epilepsy.However,whether Trim32 affects hippocampal GABAergic interneuron,synaptic acticity and the hippocampus-dependent learning and memory is poorly understood.Here,we investigate the role and underlying mechanism of GABA interneurons in the cognitive impairment using Trim32 KO mice.Part 1 Expression of Trim32 in the hippocampusWe analyzed the expression of Trim32 by immunofluorescence staining at P7 and 3 month,developmental expression of Trim32 was detected by western blot during development at ages of P0,P7,P15,P30 and P60,representing newborn,neonatal,adolescent,and adult stages,respectively.Results1.Trim32 belongs to the TRIM family and expressed in the neuron,GABAergic interneurons(but not in astrocyte)of neonatal and adult mouse brain hippocampus CA1,CA3 and DG region.2.Trim32 expressed at all time point from P0 to 2 month during the postnatal development,a minimum at P0 and a slight increase at P7,followed by an stable expression during adolescence in hippocampus.3.Trim32 also expressed in the hippocampal neural progenitor cells.Part 2 The role of Trim32 in mouse hippocampal neurogenesisWe performed Brd U labeling to assess the progenitor proliferation and neurogenesis in the hippocampus of Trim32 knockout(KO)and Trim32 wildtype(WT)littermates to verify whether Trim32 is involved in neurogenesis and related cognitive function in the hippocampus.Mice were administered with Brd U by intraperitoneal injection for 7 days to test the proliferation in the subgranular layer,and after 28 days without BrdU to test the neurogenesis in the granular layer.We also evaluated inmature and mature neurons and the programmed neuronal death by quantification of doublecortin-positive(DCX+)and NeuN+ and caspase-3+ cells respectively by immunohistochemistry in dentate gyrus(DG).Furthermore,using two hippocampus-dependent learning and memory tasks Morris water maze and new object recognition,we test Trim32 WT and Trim32 KO mice for associative,nonspatial memory as cognitive behavior.Results1.Trim32 KO mice showed an increased neural precursor proliferation and decreased neurogenesis in DG region of hippocampus,while the migration is not affected.2.Trim32 KO mice exhibited reduced apoptosis in DG region of hippocampus.3.Trim32 KO mice showed impairment in learning and memory tasks Morris water maze and new object recognition task.Part 3 Changes of GABA interneurons in hippocampus of Trim32 KO miceFind out how deficiency of Trim32 impact GABAergic interneurons is an crucial step for understand how Trim32 loss of function may lead to cognitive impairment.To study Trim32 function in GABA interneurons,We used immunocytochemical approaches to quantified subpopulations of GABAergic interneurons expressing the parvalbumin+,neuropeptide Y+,calretinin+,somatostatin(SST)+,GABA+ and one of the isoform for the GABA synthesizing enzyme GAD67 + within the DG and the CA1 and CA3 subfields of the hippocampus.Then we analyzed synaptic transmission in the CA3 region of hippocampus in adulthood.To investigate whether lack of Trim32 affected synaptic development,we quantitated the density of puncta of the presynaptic terminal marker GAD67 and presynaptic GABAergic inhibitory synaptic marker VIAAT in the hippocampal subfields CA3 of adult Trim32 WT and KO mice.To distinguish whether these reduction is caused by a decreased inhibitory synapse density or fewer number of VIAAT within existing synapses,we using electron micrography to counted perisomatic inhibitory synapses number of Trim32 KO and control mice.The concentration of neurotransmitter GABA was measured in hippocampus tissue by using HPLC method.To verify whether reduced GABA interneurons would lead to functional changes,we compared the changes of GABAA receptors mediated miniature and evoked whole-cell inhibitory postsynaptic currents(IPSCs)in hippocampus slices of 7w old Trim32 WT and KO mice.Record the pyramidal neurons in the CA1 region of hippocampus by the whole cell patchclamp technique.The decrease in GABAergic interneurons number and the reduced synaptic inhibition in hippocampus may result in neural hyperexcitability and develop a spontaneous epileptic phenotype.So we examined Trim32 KO and Trim32 WT mice for electrical activity by the video Electroencephalogram(EEG)monitoring.Astrocytes are closely related to the electrical activity of seizure.We use immunofluorescence staining to observe the expression of GFAP in hippocampal dentate gyrus.Results1.We observed significant reduction in expression of the diverse set of GABAergic interneuron molecular markers at late postnatal stages(P21)and adult(4 month)Trim32KO mice.2.At the light micrography level,both GAD67 and VIAAT-immunoreactive puncta is decreased in in CA3 region of hippocampus of Trim32 KO mice.3.Coincide with the result from light micrography,the number of symmetric synapses in Trim32 KO mice was reduced.4.Compare to WT mice,Trim32 KO mice containing lower GABA concentration in hippocampus.5.Trim32 deficiency decreases the frequency of spontaneous inhibitory postsynaptic currents(s IPSCs),amplitude of sIPSCs was not affect.No significant change in Miniature inhibitory postsynaptic currents(mIPSCs)frequency and amplitude were observed in Trim32 KO mice.While,evoked GABA mediated inhibitory postsynaptic currents(e IPSC)test showed an increased amplitude and 50 ms PPR is increased either.Electrophysiologic studies suggest the reduction of GABA transmitter released was caused by the deficit in presynaptic action potential firing,and the Ca2+-dependent probability of spike-dependent transmitter release is decreased.These changes would account for a reduction of GABA-mediated inhibitory synapse transmission in Trim32 KO mice.6.Trim32 KO mice exhibit enhanced neural hyperexcitability in EEG power.Loss of Trim32 results in more numbers of epileptic spikes detected in the hippocampus compared to control mice.7.Trim32 KO mice showed increased expression of astrocytes in DG subfield of hippocampus.Part 4 The role of clonazepam in the treatment of neural hyperexcitability and cognitive impairment in Trim32 KO mice.Cognitive impairment in Trim32 KO mice maybe result from decreased inhibitory neurotransmission in GABAergic interneurons.To further confirme these assumption,we administered a benzodiazepine antiepileptics drug clonazepam by intraperitoneal injection to adult Trim32 KO mice,to reverse reduced GABAergic tone.To evaluate the changes in electrical activity of the mice treated with clonazepam,we tested them by video Electroencephalogram(EEG)monitoring.To evaluate behavioral flexibility and persistence,we carried out the reversal water maze task.ResultThe neural hyperexcitability were rescued by pharmacologic inhibition of the clonazepam while the behavior dysfunction can not be rescued.ConclusionTaken together,Our results provide convincing evidence that1.TRIM family member Trim32 expressed in the neuron,GABAergic interneurons,and the neural progenitors(but not in astrocyte)of hippocampus during the postnatal development stablely.2.Trim32 KO mice showed increased neural precursor proliferation,decreased neurogenesis and reduced apoptosis in DG region of hippocampus,while the migration was not affected.Trim32 KO mice showed hippocampus-dependent cognitive impairment.3.Trim32 KO mice showed reduced of GABAergic interneuron number,reduced symmetric synapses,lower GABA concentration.Electrophysiologic studies suggest the Ca2+-dependent probability of spike-dependent transmitter release is decreased which is caused by the deficit in presynaptic action potential firing.These changes would account for a reduction of GABA mediated inhibitory synapse transmission,then Trim32 KO mice exhibit enhanced neural hyperexcitability.In addition,expression of astrocytes was increased.4.Treatment with antiepileptics drug clonazepam can rescu the neural hyperexcitability,but cannot rescue the cognitive impairment.We hypothesized the cognitive impairment in Trim32 KO mice are caused by reduced GABAergic interneurons and the disturbance in excitatory/inhibitory circuit.These data suggest a functional role for Trim32 in brain development and may help to better understand the comorbidity of Trim32 deficiency associated diseases such as ASD,or ADHA.