Toxic Effects Of TMT On Primarily Cultured Hippocampal Neurons And Protection By BDNF Treatment

BackgroundTMT is an important occupation poison, and is a risk factor of environmental pollution.Acute TMT exposure could cause multi system including nerve system damage. The maindamage caused by TMT in the central nervous systemincludes apoptosis of nerve cells,neuronal inflammation and oxidative damage of the nervous tissue. TMT can selectivelyinduce the limbic system, especially caused the hippocampal neuronal death effect. BDNFplays a main role in regulating hippocampal synaptic plasticity in the central nervoussystem. Rat brain exposed to TMT showed reduced BDNF expression and the neurons overexpression of BDNFcould antagonist the neurotoxicity of TMT. But the mechanism ofTMT damage to the brain neurons especially the damage to neuronal dendritic spines isunclear, and the mechanisms of BDNF antagonist TMT damage effect remains to be furtherstudied.Therefore, this paper chooses BDNF and TMT as the research content, trying tounderstand TMT damage to the neural synaptic plasticity and the molecular mechanism ofBDNF protective effect, in order to provide clues for the treatment and prevention of TMToccupation exposure on synaptic plasticity damage.Synaptic plasticity refers to the functional synaptic efficiency enhanced or reduced, atthe same time transmission of neural signal intensity changes accompanied by structuralchanges in neuronal synapse. Moreover, brain derived neurotrophic factor involved in thedevelopment and plasticity of dendritic spines of regulating activity dependent. Recentstudies show that BDNF can be used as the adult brain activity dependent regulation offactors involved in the changes of structure and function of regulating nerve. BDNFsecreted from the dendrites, immediately combined with neurotrophic factor receptor TrkB,and then promote the presynaptic neurotransmitter release and increase the level oftranscription and translation of the postsynaptic protein. Tau protein was mainly expressedin the neuronal dendritic shaft, independent or associated with other tubulin co regulation ofdendritic shaft microtubule function. In nerve cells, tau protein and cell membrane associated or interact with microtubules. Expression of Tau protein or structure may affectthe stability of microtubule protein function. Under physiological conditions, tau proteinphosphorylation changes may occur at different sites, thereby affecting its microtubulestabilizing function. Mature neurons can use dynamic microtubules maintain cell system, toadapt to the changes of neural network place.MethodsPart I: To observe the neurotoxity of TMTPrimary cultures of embryonic day18hippocampal neurons were used as model. Afterthe treatment with TMT, the protective effect of BDNF was observed by CCK-8assay;neuronal dendritic spine morphology were observed after treatment with TMT and TMTplus BDNF.We would like to address the characteristic of TMT toxicity on dendritic spine.Part II: To observe the effect of BDNF in the regulating synaptic plasticityPrimary cultured embryonic day18(E18) hippocampal neurons of embryonic day18was treated by BDNF, the effect of the hippocampal synaptic growth and dendritic spineswas observed; GFP, RFP plasmids were transfected into primary hippocampal neurons tolabel dendritic spines; colocalization of tau protein and the microtubules was examined byimmunocyochemistry; synaptic complex was measured after treatment with BDNF; tauprotein expression and phosphorylation was detected by Westernblot. These experimentsare set to investigate the role of tau protein in neuronal morphology changes in synapticplasticity of hippocampal neurons.Part III: To observe the effect of BDNF on Tau phosphorylation and celldistributionRetinoic acid (RA) differentiated human neuroblastoma SH-SY5Y and primary cultureof embryonic day18hippocampal neurons were used as a model. After the treatment withBDNF, the subcellular distribution of tau protein was observed by immonocytochemistry;SH-SY5Y cell outgrowth was measured by Leica software; expression of tau protein wasdetected by Westernblot. These experiments were set to address the possible relationshipbetween the cellular distribution of tau protein and BDNF treatment during cell outgrowth.Results：Part I: TMT could induce abnormal morphology of dendritic spines 14DIV Primary cultured hippocampal neurons were treated by TMT for24h; cellviability was decreased significantly by CCK-8assay; while the application of BDNFtogether with TMT, cell viability does not increase significantly. Primary culturedhippocampal neurons were transfectd of GFP plasmid at7DIV and then were cultured to20days. Then it was treated by TMT and/or BDNF for24h. Confocal microscopy showedthat TMT treatment could cause the occurrence of ball-like morphological abnormalities ofdendritic spine. The application of BDNF could reduce the morphological abnormalities ofdendritic spine to some extent. BDNF and LiCl co-stimulation can also mimic this dendriticspine abnormality, which share great similarity of that treated by TMT. Westerbnlotshowed that BDNF treatment could both phosphorylated Akt and ERK in primary culturedhippocampal neurons (Figure2). Lithium alone does not seem to affect the phosphorylationstatus of both Akt and ERK. When adding the lithium together with BDNF, thephosphorylation of ERK induced by BDNF was not affected. But lithium inhibited thephoisphorylation of Akt induced by BDNF. TMT induced abnormal enlargement ofdendritic spine as well as that induced by BDNF and LiCl cotreatment might result from theimbalance of the cross-talk between ERK and Akt.Part II: BDNF regulating morphological synaptic plasticity through tau protein14day in vitro (DIV) hippocampal neurons were treated by BDNF for24h. Tauprotein expression was significantly increased and phosphorylation at Ser262wasdecreased after BDNF stimulation. Immunocytochemistry showed that colocalization of tauprotein and tubulin was enhanced by BDNF treatment. The increased tau protein expressionwas also consistent with the increase of dendritic spine density after BDNF treatment.Application of shRNA methods in cultured neurons at7days to down-regulate tau proteinexpression level, and then we observe at21day that hippocampal dendritic spine densitywas decreased significantly. And in hippocampal neurons whose tau protein was thedown-regulated by shRNA plasmid,24h BDNF stimulation could not increase the densityof dendritic spines of neurons.Part III: BDNF regulation of tau protein phosphorylation and subcellulardistributionBy immunocytochemical method, we found that in undifferentiated SH-SY5Y cellslacking neurites, tau protein could form a sphere. In contrast, in SH-SY5Y cells differentiated by retinoic acid for5days, tau protein was distributed in the neuronalprocesses and the cell body. Through the Western blot detection method, we found thatretinoic acid also increased total tau protein levels and decreased tau protein levels ofSer262phosphorylation. The up-regulated expression of tau protein and tau proteinphosphorylation level of ser262reduction was associated with the neuronal processes (R=0.94and R=-0.98). When the primary cultured E18hippocampal neurons were treated withthe microtubule depolymerizing agent nocodazole, neuronal loss occurred and new tauprotein was transferred to the cell body. This process can be reversed the application ofBDNF to some extent.Conclusion：Based on the above results, we can draw the following conclusions that: TMTtreatment could cause the occurrence of ball-like morphological abnormalities of dendriticspine, which might be an important character of TMT neurotoxicity. The application ofBDNF showed potential protection against this damage. TMT induced abnormalenlargement of dendritic spine as well as that induced by BDNF and LiCl cotreatmentmight result from the imbalance of the cross-talk between ERK and Akt. BDNF canregulate the expression of tau protein in primary hippocampal neurons, and tau protein maybe involved in the signal pathway of BDNF regulating the synaptic plasticity, and theregulation effect may be achieved through regulation of tau protein expression andmodulation of tau protein microtubule stabilizing ability; tau protein Ser262sitephosphorylation, total tau protein expression and subcellular distribution of tau proteinchanges were closely related to the neuronal outgrowth; Our experiments provide newevidence for the neurotoxicity of TMT to hippocampal neurons, and also provide a newclue in revealing the role of tau in BDNF signaling pathway in regulating neuronaloutgrowth and dendritic spine morphology.