Effect Of TRIM32 Gene Knockout On Synaptic Plasticity In The Hippocampus Of Mice And Its Mechanism

Introduction:Tripartite motif-containing protein 32(TRIM32)is a member of the tripartite motif protein family,a neural stem cell inhibitory protein as well as a transcription factor.Reports show that it has an E3 ubiquitin ligase activity.The presence of a RING domain gives most members of the TRIM family their E3 ligase activity and this makes them undergo ubiquitination of specific substrates,one of the means by which they exert their biological functions.Physiologically,E3 ubiquitin ligases control homeostasis,cell cycle,as well as several DNA repair pathways.The E3 ubiquitin ligases are engaged mainly in modifying their targets post-translationally through a myriad of ubiquitin-modifying reactions which eventually produces a conjugation of a ubiquitin moiety onto a target substrate.Nicklas et al.observed that TRIM32 initiates differentiation of neurons and selfrenewal is suppressed by ubiquitination of the c-Myc transcription factor and certain micro RNAs activation.TRIM32 protein was reported to be almost absent in NSCs both in the SVZ and in the DG.The NSCs that are found in the inner zone of the DG show higher nuclear expression of TRIM32 as soon as they become neuroblasts or immature neurons and come to the outer layer of the DG.This is an indication that TRIM32 is upregulated upon differentiation of the NSC.TRIM32 plays other important roles including neurogenesis,inhibition of nerve cell proliferation and restriction of apoptosis.Many abnormalities in some protein expressions in the brain have been linked to TRIM32 overexpression or downregulation.There has been a strong association between TRIM32 regulation and the development of certain neurological disorders such as depression,anxiety,Alzheimer’s Disease,Autism Spectrum Disorder(ASD),Attention deficit hyperactivity disorder(ADHD).Many of these neurological disorders have been suggested by numerous studies to have learning and memory impairments as one of their resultant outcomes.Though there has not been a direct study on the effect of TRIM32 deficiency on synaptic plasticity,it has been speculated that the absence of TRIM32 does impair synaptic plasticity because of its links to these neurological disorders.With the numerous research advances on the functions of TRIM32,it remains unclear the effect of TRIM32 on synaptic plasticity.This study,therefore,proposes that TRIM32 can influence synaptic plasticity and by extension,learning and memory.Methods:The study included both in-vivo and ex-vivo experiments.TRIM32 KO and their wildtype littermates were used for the experiments.Long-term potentiation(LTP)-excitatory postsynaptic potential(EPSP),input/output(I/O)curve and paired-pulse facility(PPF)were measured using electrophysiology to determine the changes in synaptic plasticity.Western Blotting was used to determine protein expression levels of Synaptic proteins(Synaptophysin,Synapsin I,GAP-43 and PSD95),AMPA receptors,NMDA receptors,GABA A receptors,Excitatory and inhibitory neurotransporters(EAAT2 and SLC32A1 respectively).Also,Notch and its related genes were done using Western Blotting.Immunostaining was used to confirm the protein expressions of some of the proteins did with western blotting.m RNA expression levels were determined using RTq PCR and real-time PCR.Nissl staining was used to observe the changes in the estimated number of neurons in brain slices of TRIM32 KO.Golgi staining was also done to estimate the number of spine density in TRIM32 KO.Neural signals were recorded in a wide frequency band(0.01-40,000 Hz)using Plexon equipment and were analyzed for Spike frequency,Local field potentials(LFPs)as well as various Oscillatory bandwidth(Delta,Theta,Alpha,Beta,Gamma).Meanwhile,in the ex-vivo experiments,Primary cultured neurons were used for all the experiments.DAPT,a Notch inhibitor,was used to inhibit notch at various concentrations and their effects determined using some of the above techniques.In the statistical analysis,Unpaired t-test was used to analyze differences between 2 groups,and one-way ANOVA was used to analyze more than 2 groups(Turkey’s posthoc analysis was used to compare differences).Results:LTP is an important form of synaptic plasticity which has an important role in learning and memory.Recording LTP after high-frequency stimulation showed a significant reduction in field excitatory postsynaptic potential(f EPSP)slope and amplitude in TRIM32 knockout mice compared with wild type littermates.The difference was significant(p<0.05).The input/output(I/O)curve mainly reflects the basic synaptic transmission at the Schaffer collateral-CA1 synapses.There was no significant difference in the normalized I/O%.This could be an indication that TRIM32 does not affect the basal synaptic response.This suggests that there was no change in the basic transfer efficiency of synapses of neurons in TRIM32 knockout mice.PPF(double-pulse facilitation)is a short-term plasticity model.In TRIM32 knockout mice and wild type mice,marginal reduction(no significant difference)was found in their PPF curve,suggesting that interference in the probability of basal synaptic vesicle release from the presynaptic terminals may occur in TRIM32 KO.Observation made showed that,even though the average number of dendritic branches were increased in TRIM32 KO mice,the dendritic spines in CA1 were significantly reduced compared to WT littermates.The dendritic spine morphology and density observed looked thinner in the spine stem as well as smaller head in TRIM32 KO compared to their WT littermate which had bulge-headed spines and thicker stems.The mechanisms occurring at both presynaptic and postsynaptic sites are established to be very important in LTP.There were reductions in the protein and m RNA expressions of Syp,Syn1,GAP-43,and PSD-95.Observation made in the western blot showed that the protein expression levels of AMPA receptors were significantly reduced in the TRIM32 KO mice compared to WT.This was corroborated by the immunostaining results.Investigation on the protein expression level of GABAA receptors was carried out and the results revealed that these receptors were significantly reduced in the TRIM32 KO mice.This study’s findings revealed that the protein expression levels of NR1,NR2 A,NR2B were increased in TRIM32 KO mice.Even though the increase in NR1 was marginal,those of NR2 A and NR2 B were significant.The results were further confirmed with the immunostaining of brain slides of TRIM32 KO and their WT littermates.It was observed that NR2 A and NR2 B staining were increased in the TRIM32 KO.m RNA expression results obtained confirmed the increase that was reported for the protein expression.Results from the western blot results showed that protein expression levels of EAAT2 increased significantly whereas SLC32A1 decreased significantly in the TRIM32 KO mice compared to their WT littermates.This was corroborated by double staining EAAT2(red)and SLC32A1(green)of some areas in the hippocampus and Cortex.Using EEG recording,an analysis was done on the brain spike activity in the TRIM32 KO mice.The results showed the number and frequency of spikes occurring during an estimated period of 4000 s were increased significantly in the TRIM32 KO mice compared to their WT littermates confirming the reality of overexcitation(imbalance)in neural activities of TRIM32 KO.With neurons being the structural and functional units of the nervous system,Nissl staining was done to observed the neuron integrity after detecting overexcitation and imbalance.The number of neurons significantly decreased in all these brain regions estimated from(in the cortex and the hippocampus).The overall LFP(0.1-300Hz),is a representative recording from synapses that are formed.Results from the filtering showed a significant reduction in LFPs in the TRIM32 KO mice.Neurons are believed to give off some transmembrane currents which are believed to be local field potentials.Hence,a reduction in the LFP confirms the decreased number of neurons observed earlier in TRIM32 KO mice.Further analyses showed that the power of Delta and Beta waves were significantly increased in TRIM32 KO mice whereas the power of Theta and Gamma waves was significantly reduced in TRIM32 KO mice.Western blots results showed that in TRIM32 KO,the expression of notch related genes(Notch1,Hes1,NGN3)was significantly higher as compared to their WT littermates.RT-q PCR was also done to confirm the m RNA expression levels of these genes.The NGF protein and m RNA expression levels in TRIM32 KO mice were looked at and the results showed significant downregulation in NGF compared to their WT littermates.DAPT(a gamma-secretase inhibitor),is well-known to inhibit notch.Treating primary cultured neurons with DAPT,revealed a concentration-dependent downregulation of notch1,hes1,ngn3 m RNA and protein expressions.Neurotrophic factor,NGF remained unchanged following treatment with DAPT(in all concentrations).Mash1 was also found to be upregulated in a concentration-dependent manner.Protein expressions of AMPA and NMDA(NR1,NR2 A,NR2B)receptors were significantly downregulated in DAPT treated neurons.GABAA receptors’ expression was also down-regulated but could not be compared to the decrease in the AMPA and NMDA receptors.The overall effect of the results above,on the excitatory(EAAT2)and inhibitory(SLC32A1)transporters,were also determined.EAAT2 and SLC32A1 were shown to be downregulated and upregulated respectively after treatment with DAPT.Conclusion:This study demonstrates that the absence of TRIM32 impairs synaptic plasticity.Here,LTP impairment,reduced dendritic spines,down-regulation of synaptic plasticity-related proteins,imbalance in excitation,and inhibition phenotypes leading to decreased neuronal numbers,all contribute to synaptic plasticity impairment.Changes in EEG waves have been explored to support the hypothesis that TRIM32 knockout impairs synaptic plasticity.Finally,this study provides evidence that the expression of notch and its related elements are altered in one way or another in TRIM32 knockout mice.The data from this study also support the hypothesis that Notch signaling is responsible for impaired synaptic plasticity via an excitatory-inhibitory imbalance in TRIM32 KO mice.