Molecular Mechanism Of NADH In Regulating Synaptic Plasticity Gene Expression

Synaptic plasticity is an adaptive change in synaptic strength or efficacy between neurons that undergoes various environmental changes.It is one of the most critical characteristics of human and animal brains.Not only does it play an important role in the development and formation of neural circuits,it is also closely related to learning and memory and the occurrence and development of various neurogenic and psychiatric diseases.Synaptic plasticity is the result of multiple signaling molecules,receptor activation,and gene expression regulation.It is generally believed that the activation of N-methyl-D-aspartate receptors(NMDA receptors)in the postsynaptic membrane of nerons plays an important role in the formation of synaptic plasticity.NMDA receptor activation triggers calcium influx and further activates a variety of protein kinases,such as extracellular regulated protein kinases(Erkl/2),calcium/calmodulin-dependent protein kinase 2(CaMKII)and protein kinase A(PKA),and then promote the expression of genes related to synaptic plasticity.Synaptic plasticity changes are often thought to be related to the expression of a group of immediate-early response genes(IEGs).IEGs are often expressed first after cells are stimulated,including C-Fos,activity regulated cytoskeleton associated protein(Arc)and early growth response protein 1(Egr1).C-Fos is a kind of nuclear protein transcription factor,which not only regulates the growth and differentiation of cells,but also often acts as a marker molecule for neuron activation.The expression of C-Fos is also related to the extension of dendrites.Arc is also an early response factor,which is quickly produced after neurons are stimulated and accumulates at the synaptic active site that activates dendrites of neurons,and participates in regulating early synaptic plasticity.Egrl is also an important transcription factor that regulates synaptic plasticity.Recent studies have found that the induction of long-term potentiation in the hippocampal dentate gyrus requires rapid transcription of Egrl in neurons.Of course,some late-response genes such as brain-derived growth factor and cyclophosphadenosine-responsive element binding protein are also involved in the regulation of synaptic plasticity.Although these genes have many other functions,they are considered to be the key genes regulating synaptic plasticity.These genes often participate in neural activities such as learning and memory by regulating synaptic plasticity processes,and are associated with the occurrence of various neurogenic diseases.Nicotinamide adenine dinucleotide(NADH)is a reduced molecule produced in the process of glycolysis,tricarboxylic acid cycle and lactate to pyruvate,not only involved in electron transfer in the respiratory chain,It can also promote the synthesis and metabolism of nutrients in the body by activating the multi-enzyme system in the body.Therefore,NADH is widely involved in the regulation of cellular redox levels and energy metabolism.When more NADH is produced,the ratio of NADH/NAD+in the cell increases,and the neuron is in a reduced state.At this time,the NR1 subunit on the NMDA receptor that is sensitive to redox may be activated,and the activity of the NMDA receptor is enhanced to exert physiological functions.In addition to the above functions,NADH can also be used as a signaling molecule to regulate various biological processes such as calcium homeostasis and gene expression.Recent studies have suggested that NADH may be involved as a signaling molecule in the regulation of synaptic plasticity and related IEGs gene expression.First,regulation of NADH/NAD+biosynthesis can regulate changes in synaptic plasticity.For example,nicotinamide phosphoribosyl transferase(Nampt)is a necessary substance in its synthesis,and the long-term depression and learning and memory of Nampt knockout mice are obviously impaired.Second,regulating the decomposition of NADH/NAD+also regulates synaptic plasticity.For example,a NAD+-dependent deacetylase(SIRT1)can directly promote the changes of synaptic plasticity in neurons.Recent studies have also found that lactate regulates synaptic plasticity mainly through NADH produced by lactate metabolism,rather than pyruvate,to carry out signal transmission of synaptic plasticity and IEGs expression.These studies suggest that NADH may be used as a signaling molecule to regulate synaptic plasticity changes,so the possible molecular mechanism needs to be further elucidatedIn view of the above research background,we assume that NADH molecules may be used as signaling molecules to regulate neuronal synaptic plasticity changes.NADH can activate NMDA receptors,promoting Ca2+signaling molecules and related molecular pathways,which in turn promotes the expression of IEGs,causing synaptic plasticity changes.In order to verify our assumptions,the following experiments were carried out:(1)Added different concentrations of NADH(1,2,4,8,16mM)to culutured cortical neurons.1 hour later,the expression of IEGs(C-Fos,Arc,Egrl)was detected by RT-PCR for choosing the appropriate concentration of NADH.Then at different times of NADH(20,40,60,120,240 min),the changes of IEGs mRNA and protein levels were detected.(2)The noncompetitive antagonist NMDA receptor MK-801 specifically blocked the NMDA receptor,and the mRNA and protein expression of IEGs after NADH was detected by RT-PCR and western blot experiments,respectively.Sholl analysis was employed to detect neuronal morphological changes.(3)Further analysised of Ca2+and downstream molecular Erk1/2 protein changes.After incubating neurons with Fluo-4AM dye,the real-time course of intracellular calcium changes under different treatment conditions was recorded under a laser confocal microscope.Then the expression of Erk1/2 protein downstream of Ca2+signal was detected After U0126(10μM)specifically blocked Erk1/2 molecule,the expression change of IEGs was further analyzed.(4)Moreover,NADH(8mM)was first injected into the frontal cortex of mouse through a brain stereotaxic instrument.After 1 hour,brain tissue was taken to detect the expression of IEGs by RT-PCR.Then,the calcium indicator GCaMP6 adeno-associated virus was injected into the frontal cortex.After 2w,8mM NADH was injected at the same location.1h later,brain slices were taken to observe changes in Ca2+signal under a fluorescence microscope.An experiment was also designed to inject the C-Fos promoter-containing viral vector GV581 into the frontal cortex,and then analyzed the effect of NADH on neuron activation.The experimental results show that:(1)NADH could promote the expression of IEGs of cortical neurons,including C-Fos,Arc and Egr1,and was in a concentration-and time-dependent manner.When the NADH concentration was 1,2,4,8 mM,the expression level of IEGs gradually increaseed with the increase of NADH concentration,the expression level of IEGs peaks at 8 mM.The expression level of IEGs also increased with the prolonged action time of NADH,reaching a peak at about 1h and returning to normal levels at 2h.(2)After MK-801 was given to specifically block the NMDA receptor,it almost completely inhibited the expression-promoting effect of NADH on IEGs,and the mRNA and protein expression of C-Fos,Arc,and Egr1 all returned to the level of the control group.Sholl analysis showed that when MK-801 and NADH co-treated cortical neurons,the numbers of primary neurites and dendritic branches of the neurons were significantly reduced compared with the NADH treatment group.3)Ca2+time course detection showed that after adding 8mM NADH,the Ca2+fluorescence signal in neurons increased sharply,and the neuron cell body showed bright green fluorescence.However,MK-801 was given 200s before NADH was added,and no significant change in fluorescence intensity was detected.In addition,when the extracellular fluid did’n contain calcium,the effect of NADH on increasing intracellular Ca2+concentration was also blocked.Further research inhibited Erk1/2,a related molecule downstream of NMDA receptors,and found no significant change in the expression of IEGs after NADH,indicating that Ca2+/Erk1/2 was the main molecular pathway through which NADH regulates synaptic plasticity.4)The in vivo experiment resulted show that the injection of 8mM NADH into the mouse frontal cortex can significantly increase the expression of IEGs,of which the expression of Egr1 increased most obviously,about 17.5 times of the control group;the expression of C-Fos and Arc was also increasing significantly,6.3 and 5.9 times of the control group,respectively.Intracranial injection of calcium indicator GCaMP6 showed that NADH significantly increased the calcium signal of cortical neurons.After the GV581 virus was injected into the mouse brain,it was also found that NADH significantly increased the expression of C-Fos and promoted the activation of neurons.In summary,the results of this experiment indicate that the NADH produced by cell metabolism can promote the expression of IEGs and regulate the synaptic platicity.The possible mechanism is achieved through the NMDAR/Ca2+/Erk1/2 molecular pathway.This study provides an experimental basis for understanding the regulatory role and mechanism of NADH in synaptic plasticity.