Relationship Between Chronic Lead Exposure And Hippocampine CaMKⅡ, PCREB, NOS, NO, IEG, Learning And Memory In Mice

ObjectiveThe damage of low level lead exposure to the nervous system of children in the environment, particularly to the impairment of learning and memory and mentality functions in children,is an important public health concern. Because of incompletely developed blood brain barrier, children who are exposure to lead as low as 100 μg/L (blood lead level) show significant deficits of learning and memory and significant behavioral abnormal. And cause apparently irreversible damage to the nervous system. It is a matter of great import to prevent and treat lead poisoning, which related to three hundred millions children body and mind health in China and our offspring's wealthy and strong. So it is urgent to probe into lead nerve poisoning.The effect of lead on learning and memory function is not clear, but it is sure that lead can impact learning and memory function by means of the nervous system multi - levels damage. CaMK â…¡ is the most abundant protein kinase in hippocampus and postsynaptic density (PSD). Both behavioral training and the induction of long term potentiation(LTP) can trigger αCaMK â…¡ autophosphorylation at Thr286, which suggest that this mechanism is critical for synaptic plasticity and behavioral training. Calcium influx through NMDA receptors also activate CaMK II and Thr286 autophosphorylation. This autophosphorylation allows the kinase to be active even in the absence of calcium. In addition, Thr286 phosphorylation promotes association with the postsynaptic density by binding to the NMDA receptor. So a transient increase in calcium can dramatically change the activity and the localization of this kinase. And the characteristics of CaMKâ…¡ are molecule basis of learning and memory. There are common molecule mechanisms on encoding of long - term memories from invertebrate to mammalia. CREB is the key mediator in long - term memory. CaMK â…¡ is one of protein kinases for CREB phosphorylation. CaMK â…¡, CREB and lead poisoning impair learning and memory, but the effects of lead on them and their relations have not been defined. The expression of CaMK â…¡ mRNA, CaMK â…¡ and CREB proteins in hippocampus of chronic lead - poisoned mice were investigated by means of RT - PCR( Reverse Transcription - Polymerase Chain Reaction) and western blotting in the study. We combine behavior experiment and cell signal molecules mechanism research, and to set up chronic lead exposure model in the study. The ability of spatial learning and obtaining or maintaining ability of passive - a-voidance reaction are measured by means of water maze paradigm and jumping stand test.As cell signal molecule of the nervous system, Nitric Oxide can impact learning and memory function in hippocampus. Only NO synthase (NOS) can catalyse endogenesis NO, and determine its biology effect. NOS is one of target molecules in lead poisoning. The abnormal changes of NOS and NO might impact synaptic form ; c - fos and c - jun normal expression in pallium and hippocampus at the same time. New protein and transcription of c - fos and c - jun are required for learning and memory, particularly for long - term memory. LTP inducing stimulation protocols related to the expression of c - fos and c - jun. The effects of chronic lead exposure on NOS and NO are observed. And c - fos, c - jun mRNA are observed by means of RT - PCR in order to explore the effect of lead on them.The paper investigate the biochemistry mechanism of the effects of lead exposure on learning and memory from the view of cell signal transduction. Chronic lead exposure result in CaMK â…¡ mRNA decrease and CaMK â…¡ activity ab-nomal; disturb NOS activity and NO content; lead to pCREB decrease; and further to impact c - fos , c - jun mRNA expression and learning and memory function. New point of view is advanced for the mechanism of lead poison.Materials and MethodsThe protocols for chronic exposure to lead were as follow: Kunming mice were purchased from animal department of china medical university and were individually housed in plastic cages and one male mouse was mated with two female mice of same strain. Mice were randomly and equally divided into four groups. On postnatal day 1, the dam began to be exposed to lead at 2. 4,4. 8,9. 6mmol. L^-1in their drinking water. Since weaning at postnatal 21d, the pups were directly provided lead in drinking water. Food and water were free provided to all animals. At postnatal 7d, 14d, 21d, 28d, 42d,56d individually the hippocampus were got from mice in different groups and stored at fluid nitrogen and then stored at -70℃ refrigerator. Since postnatal 42d the mice began to train in a spatial learning task using a water maze paradigm and in a passive escape reaction task using jumping paradigm. The expression of CaMK â…¡ , pCREB were determined by western blots and the expression of CaMK â…¡ , c - fos and c - jun mRNA were determined by RT - PCR. The activities of NOS and the content of NO were determined by their reagent.Results1. Chronic Lead exposure impairs spatial and passive - avoidance learning and memory in miceIn the Morris water maze task, results indicated that mice having drink lead water displayed significant impairment in their performance, and this extent of impairment showed in lead concentration dependent manner. In the jumping stand test, results suggested that lead exposure significantly reduced ability of acquiring and maintaining passive - avoidance reaction and that was correlated with concentration of drinking lead.2. Effect of chronic lead exposure on the mRNA level of CaMK â…¡ Expression of CaMK â…¡ gene in hippocampus of mice chronic exposured tolead were determined by using RT - PCR. The results indicated that decreasedexpression of CaMK â…¡ gene was found in hippocampus after Pb²⁺ treatment in dose - dependent manner. Compared with control group, there were statistical change in the expression of CaMK â…¡ gene in the hippocampus of Pb²⁺ treated groups.3. Effect of chronic lead exposure on the protein level of CaMK â…¡The protein level of CaMK â…¡ were determined by using western blot analysis in the hippocampus of mice aged postnatal 14, 28 ,42,56days after in vivo exposure to Pb²⁺ acetate. Compared with control group, CaMK â…¡ protein expression of chronic lead exposure mice in the hippocampus was downtrend , which exhibited in a dose - dependent and developmental expressed differences manner. The low concentration lead exposure could make the protein expression at 14 days higher than that of control (p <0. 01) , but at follow days lower than that of control. As to mid - and high concentration, the protein expression was always lower than that of control. The results showed lead could disturb the normal change of CaMK II protein expression.4. Effect of chronic lead exposure on the protein level of pCREBThe protein level of pCREB were determined by using western blot analysis in the hippocampus of mice aged postnatal 14, 28 ,42,56days after in vivo exposure to Pb²⁺ acetate. Compared with control group, pCREB protein expression of chronic lead exposure mice in the hippocampus was downtrend , which exhibited in a dose - dependent and developmental expressed differences manner. The low concentration lead exposure could make the protein expression at 14 days higher than that of control ( no statistical change ) , but at follow days lower than that of control. As to mid - and high concentration, the activities always lower than that of control. The results showed lead could disturb the normal change of CaMK II protein expression.5. Effect of chronic lead exposure on NOS activity and NO contentThe lead exposure could make the NOS activity at 7d higher than that of control ( p <0.01 ) , but NO content lower than that of control. The NOS activity of control was uptrend and that of lead exposure mice was downtrend. Along with lead exposure prolong and lead level arise, NOS activity decreased invariably and lower than that of of control. Then No also decreased along with them.6. Effect of chronic lead exposure on the mRNA level of c - fos and c - jun Expression of c - fos gene in hippocampus of chronic lead exposure mice was determined by using RT - PCR. The results indicated that decreased expression of c - fos and c - jun gene was found in hippocampus after Pb²⁺ treatment in dose - dependent manner. Compared with control group, there were statistical change in the expression of c - fos and c - jun gene in the hippocampus of mid - and high Pb²⁺ treated groups .DiscussionCaMK II was able to be activated by Ca²⁺ influx, so it enhanced the ability of ion transmition of AMPA(α - amino - 3 - hydroxyl -5 - methyl -4 - isox-azole — propionate ) receptor. CaMK â…¡ could make cytoskeletal proteins phosphorylation in presynaptic and postsynaptic . CaMK â…¡ could keep Synapsin â…  and MAP2 activity and regulate nuclear protein and signaling proteins. SynGAP was a Ras - GTPase activating protein, which was highly enriched in excitatory synapses , and the GTPase activating ability of SynGAP was inhibited by phosphorylation by CaMK â…¡. Since the Ras protein was active only when bound to GTP, the RasGAP proteins, by stimulation of Ras - GTPase activity; in fact function as "switch off" of the Ras signaling pathway. Therefore, it was quite conceivable that inactivation of SynGAP by CaMK â…¡ , which activated by Ca²⁺ influx, lead to release of a brake on "switch off" of the Ras signaling pathway. CREB phosphorylation, by the Ras signaling pathway and the PKA signaling pathwayetc, regulated c -fos, c - jun activation. And they all related to LTP happen, synaptic plasticity and memory form.The results showed that the effects of developmental lead exposure on CaMK â…¡ protein expression vary with different lead concentration. Lower lead concentration could increase CaMK â…¡ protein expression at 14 days of ages and keep this level till; but middle and high lever lead exposure could always keep this kinase in lower level. The present study showed that the downtrend of CaMK II gene and protein expression was found in hippocampus of mice exposed to chronic lead acetate, and the changes of CaMK â…¡ and pCREB were similar inhippocampus of mice exposed to chronic lead acetate. And decreased expression of c - fos and c - jun gene was found in hippocampus after Pb²⁺ treatment in dose - dependent manner.Some studies suggested that the inhibition of learning and memory because of lead poisoning related to the changes of NOS. lead could inhibit NOS activity. NOS determined the biology effect in great degree. NO was a matter of great import in the formation and maintain of LTP and the adjustment of neurotransmit-ter release. LTP of synaptic transmission is the basis for memory formation. After one or several pairings of stimuli, neurotransmitter glutamines are released from dendrite spine in presynaptic terminal. The postsynaptic membrane of typical spine contains glutamine receptors concentrate at the site of contact with the presynaptic terminal. N - methyl - D - aspartate (NMDA) receptors are ligand -gated ion channels. However, opening of their larger channel does not occur when glutamate binds to it, unless the membrane is strongly depolarized to relieve blockade of the channel by extra cellular magnesium. When two conditions of glutamine binding and strong depolarization are met, the NMDA receptor channel opens and allows the flow of sodium and calcium ions into the cell. The resulting influx of calcium ions is a powerful trigger that initiates a series of biochemical changes and adjusts synaptic plasticity.NOS was able to be activated by Ca²⁺ influx and the NO level of nerve cell increased. No activated sGC and ADPRT. Activated ADPRT acted on other enzymes and further to make ion channels including non N - methyl - D - aspartate receptors activity change or increased the sensitivity of Ca²⁺. And resulted in neurotransmitter release increase and LTP formation. NO combined the Fe²⁺ in the activity center of GC and resulted in the increase of GC activity and cGMP synthesization. As a new messenger molecule, cGMP leaded to protein phospho-rylation and exerted manifold biology effects. It could induce c -fos, c - jun expression and their protein, FOS and JUN, entered nucleus. They combined to AP - 1 of DNA and started related gene expression. So a series of physiology functions were brought into play.Our study suggested that the lead exposure could make the NOS activity at 7 days higher than that of control (p <0.01) , but NO content lower than that ofcontrol. Along with lead exposure prolong and lead level arise, NOS activity decreased invariably and lower than that of of control. Then No also decreased a-long with them. The results suggested that the effects of lead on NOS activity and NO content were sticking points of the inhibition of learning and memory because of lead poisoning.And decreased expression of c - fos and c - jun gene was found in hippocampus after Pb²⁺ treatment in dose - dependent manner. We thought that chronic lead exposure result in CaMK â…¡ mRNA decrease and CaMK â…¡ activity ab-nomal; disturb NOS activity and lead to NO content decrease; lead to pCREB decrease; and further to impact c - fos , c - jun mRNA expression and learning and memory function.The latent period of escape in the first and the second stage of jumping stand test were prolonged, whereas the latent period of stay in the same stage were shortened significantly respectively in lead acetate treatment group compared with the control group. The results suggested that chronic lead acetate expose reduced ability of passive - avoidance learning in mice including acquiring and maintaining ability of passive - avoidance reaction. In the place navigation version of the Morris water maze task, mice having lead acetate water displayed significant impairment in spatial learning and memory and lower ability of spatially locate and move. So the disorder of cell signal transduction route was one of the impairment reasons of learning and memoryConclusion1. Chronic Lead exposure impairs the ability of spatial learning and memory and passive - avoidance reaction learning.2. Chronic lead exposure can disturb the normal activity changes of CaMK E and pCREB. Chronic lead exposure can remarkable decrease CaMK II mRNA level. And the impairment of learning and memory relate to the changes.3. The impairment of learning and memory relate to the abnormal changes of NOS activity and NO content induced by chronic lead exposure4. The impairment of learning and memory relate to the decrease of c - fos...