The Role Of GLUT4 In Lead-induced Learning And Memory Impairment And The Underlying Mechanism

Background: Lead is a widespread environmental heavy metal poison with significant neurotoxicity,especially irreversible brain damage caused by lead exposure during development is a refractory,global environmental health issue.Mechanism research has always been a hot and difficult issue in the field of environmental neurotoxicology.As the integration center of brain learning and memory,hippoca mpus is the main target of lead neurotoxicity.Synaptic plasticity in the hippocampus is the neurobiological basis for the formation of learning and memory,including transfer efficiency associated functional plasticity and information storage related stru ctural plasticity.Previous studies have suggested that the effect of lead exposure on hippocampal synaptic plasticity is the core link of its neurotoxicity,and its underlying mechanism is not well understood.Therefore,an in-depth study of the mechanism of the effect of lead on hippocampal synaptic plasticity may be of great significance for elucidating the mechanism of lead-induced learning and memory impairment.Glucose,the most important energy substance in the brain,plays a crucial role in maintaining the integrity of the structure and function of the brain’s nerve cells.Studies have shown that abnormal brain glucose metabolism can lead to changes in synaptic structure and functional plasticity of neurons,and then affect learning and memory function.Exposure to lead can cause decreased levels of glucose metabolism in the brain and result in brain energy metabolism disorders.Some lead poisoning based animal experiments have confirmed that brain dysfunction is related to the disorder of energy metabolism,and it is proposed that the abnormal glucose metabolism caused by lead exposure is the root cause.Therefore,studying the change of glucose metabolism in hippocampus after lead exposure and its effects on synaptic plasticity and related mechanisms may be a new breakthrough for revealing the mechanism of lead injury learning and memory function.Glucose can’t be synthesized or stored in brain nerve cells and needs to be transported into neurons through glucose transporters（GLUTs）.Studies have found that GLUT4 mediated glucose supplement plays a key role in hippocampal-dependent learning and memory.Insulin in the brain induces translocation of GLUT4 to the neuronal membrane by activating the PI3K-Akt signaling pathway,providing more glucose supply to neurons during learning and learning to maintain neuronal structure and functional integrity,whereas the effect of lead exposure on GLUT4 is not yet clear.Therefore,the interference of lead with GLUT4 may cause insufficient supply of glucose during learning and memory in the hippocampus,thereby leading to neuronal synaptic plasticity impairment and eventually inducing the impairment of learning and memory function.This will provide a new theoretical basis for further elucidating the mechanism of lead-induced learning and memory impairment.Aims: In this study,a lead-exposed rat model and a primary cultured hippocampal neuron model were established to observe the effect of lead exposure on synaptic plasticity and glucose uptake in the brain,and to investigate the effect of glucose uptake on synaptic plasticity of neurons,and to further explore the role of GLUT4 in lead-induced learning and memory impairment.The research results will provide a new theoretical basis for further elucidating the mechanism of lead-induced learning and memory impairment.Methods: 1.Establishment of lead exposure model.1).Female SD rats were exposed to water containing 0 or 0.02% of lead acetate（PbAC₂）beginning 2 weeks before mating,throughout gestation,and until PND10.2).Primary cultured rat hippocampal neurons were treated with Neurobasal mediem containing a dose of 0 or 1μM PbAC₂ on day in vitro（DIV）7,and tested on DIV 12.2.Atomic absorption spectrophotometry was employed to detect the blood lead levels,and the Morris water maze was performed to detect the effect of lead exposure on spatial learning and memory function in rats.3.Patch-clamp recordings were performed to observe the effects of lead exposure on LTP induction in rat hippocampus,and Golgi staining was employed to detect the effect of lead exposure on dendritic spines of hippocampal pyramidal neurons 4.PET-CT scanning and PMOD software were employed to analyze the effect of lead exposure on glucose metabolism in rat brain.5.Western blotting was performed to observe GLUT4,Akt and p-Akt protein expression in rat hippocampal and primary cultured rat hippocampal neurons.6.Immunofluorescence staining was performed to detect the protein expression of GLUT4 in rat hippocampal neurons and astrocytes.7.2-NBDG combined with flow cytometry was employed to analyze the effect of lead exposure on glucose uptake in primary cultured rat hippocampal neurons.8.Lentivirus infection combined with laser confocal microscopy live cell workstation was performed to observe the effects of lead exposure on dendritic spines in primary cultured rat hippocampal neurons.9.Plasmid transfection was performed to overexpress GLUT4 in primary cultured rat hippocampal neurons,and the effect of changes in glucose metabolism on synaptic plasticity in neurons was observed.10.Stereotactic injection of adenovirus was employed to overexpress GL UT4 in rat hippocampus,and the effects of changes in glucose metabolism on hippocampal neuronal learning and memory function were observed.Results: 1.Effects of low-level gestational lead exposure on learning and memory,and hippocampal synaptic plasticity in rats 1)After lead exposure,compared with the control group,lead exposure group PND0,10 and 21 rat blood lead level significantly increased with a dose-response relationship,whereas in the PND30 lead-exposed rats the blood lead levels had no significant difference.2)The results of Morris water maze suggested that compared with the control group,the arrival latency of rats in the PND30 lead exposure group significantly prolonged and the target quadrant retention time significantly decreased.3)The results of patch-clamp recordings showned that compared with the control group,the f EPSP slope of LTP in the hippocampus of lead-exposed rats was significantly reduced.4)The results of Golgi staining suggested that compared with the control group,the spine densities of basal and apical dendritic in pyramidal neuron of lead-exposed rats hippocampus were significantly reduced,and the numbers of dendritic spines of the Thin and Mushroom-shaped were significantly reduced.2.Effect of low-level gestational lead exposure on glucose uptake in rat brain 1)PET-CT results suggested that compared with the control group,the lead exposure group rats significantly reduced the level of glucose metabolism in the hippocampus,amygdala,and olfactory.2)Western blot results suggested that compared with the control group,the protein expression of GLUT3 and GLUT1 in the hippocampus of lead-exposed rats showed no significant changes,while the protein expression of GLUT4 was significantly reduced.3)Immunofluorescence staining showed that GLUT4 and Neu N were significantly double-labeled in the CA1 and DG regions of hippocampus in rats,and double-marking in the lead-exposed group was significantly reduced compared to the control group;whereas GLUT4 and GFAP were no significant double-marking in hippocampal CA1 and DG regions of rats.4)Western blot results showed that compared with the control group,the protein expression of p-Akt and PM-GLUT4 in the hippocampus of the lead-exposed rats were significantly decreased.5)Western blot results suggest that insulin stimulation significantly increased the expression of p-Akt and PM-GLUT4 protein in rat hippocampus,while in the lead-exposed group,insulin stimulation did not reverse the decrease of p-Akt and PM-GLUT4 protein expression.3.Effect of abnormal glucose metabolism on synaptic plasticity in rat hippocampus after lead exposure 1)Flow cytometry results showed that compared with the control group,glucose uptake was significantly reduced in primary cultured hippocampal neurons after treatment with 1 μM lead acetate.2)Western blot results showed that the expression of GLUT4,PM-GLUT4,and p-Akt protein in primary cultured hippocampal neurons was significantly reduced in the lead exposure group compared with the control group.3)Western blot results showed that the level of p-Akt and the expression of PM-GLUT4 protein were significantly increased after insulin stimulation,while in the lead-exposed group,insulin stimulation did not reverse the decrease of p-Akt and PM-GLUT4 protein expression.4)After over-expression of GLUT4,PM-GLUT4 protein expression and glucose uptake in the primary hippocampal neurons in the lead-exposed group turned back;at the same time,both m EPSC amplitude and dendritic spine densities were reversed.4.The role of GLUT4 in lead-induced learning and memory impairment 1)Western blot results showed that after overexpression of GLUT4 in the rat hippocampus,the expression of PM-GLUT4 protein in the hippocampus of rats exposed to lead was reversed.2)PET-CT results showed that after overexpression of GLUT4 in the rat hippocampus,the level of glucose metabolism in the hippocampus of rats exposed to lead was reversed.3)The results of Golgi staining showed that after overexpression of GLUT4 in the rat hippocampus,the spine densities of basal and apical dendritic in pyramidal neuron of lead-exposed rats hippocampus turned back,and the numbers of dendritic spines of the Thin and Mushroom-shaped were reversed.4)Patch clamp results showed that after overexpression of GLUT4 in the rat hippocampus,the slope of f EPSP in the hippocampus of rats exposed to lead was reversed.5)The results of Morris water maze suggested that after overexpression of GLUT4 in the rat hippocampus,the arrival latency of rats in lead exposure group turned back and the target quadrant retention time was reversed.Conclusion: 1.Lead exposure causes hippocampal synaptic functional plasticity and structural plasticity injury,which may be one of the mechanisms of lead-induced learning and memory impairment.2.Lead interferes with glucose uptake in rat hippocampal neurons by interfering with GLUT4 protein expression,and inhibiting PI3K-Akt signaling pathway and the translocation of GLUT4.3.GLUT4 plays an important role in synaptic plasticity in rat hippocampus.4.Inhibition of GLUT4 protein expression is one of the mechanisms of lead-induced learning and memory impairment.In summary,the current study confirms that lead interferes with GLUT4 molecules,thereby affecting glucose uptake during learning and memory in hippocampal neurons of rats,which in turn leads to decreased synaptic plasticity in neurons and ultimately leads to learning and memory impairment in rats.