The Inhibition Of CREB Signaling Pathway In OPs Caused Cognitive Impairments

Organophosphorus insecticides (OPs) are the most widely used group of pesticides in the world. Because of their relatively low cost, OPs have been used in agriculture, homes, gardens, and in veterinary practice. The use of OPs around the world continues to produce detectable levels of OPs in the environment, thereby posing an ongoing risk for low-level exposure. Chronic exposures to OPs, especially the low-level envireonmental exposures, require greater attention.The epidemicology studies had reported memory impairments and behavioral alterations after long-term exposures to OPs, and the decrease cognitive function appeared not immediately after the OPs exposure but monthes later. However, there are a few animals studies available that focused on the mechanisam of cognitive impairments on chronic low-level OPs exposure. Some studies in which animals were used as experimental subjects had contradictory results on the cognitive and behavioral effects of OPs exposure. Therefor, the results and mechanisam of chronic low-level OPs exposure need further study.Chlorpyrifos (O,O'-dithyl-O-3,5,6-trichloro-2-pyrydyl phosphorothionate, CPF) is one of the most widely used OPs throughout the world. Despite the EPA (U.S. Environmental Protection Agency.2002) has entered into an agreement with the registrant to begin terminating residential CPF indoor uses since 2000, it is still one of the most widely used insecticides. With the elimination of acephatemet, methyl parathion, parathion, monocrotophos and phosphamidon in agriculture since 2007, CPF had been recommend as the low-toxic OPs replacement in agriculture in China.These days, CPF has become one of the major pesticides detected in farm products in China. In the present study, male rats were administrated CPF through intragastric for 12 weeks to evaluate the neurobehavioral impacts of CPF at the subthreshold doses(doses that do not produce overt signs of cholinergic toxicity, e.g. excessive salivation, urination, defecation, muscle fasiculations and so on). The neurobehavioral changes (especially the spatial memory) were studied and the and mechanisam of chronic low-level OPs exposure was explored.There were no effects on weight gain and food consumption detected during the 12 weeks exposures to CPF, and the daily dosages of 1.0, 5.0 and 10.0 mg/kg CPF for 12 weeks did not elicit any other signs of OPs toxicity, such as excessive salivation, urination, defecation, muscle fasiculations etc. However, the animals exhibited obvious neurobehavioral changes (especially the long-term spatial memory) after 12 weeks repeated exposures to CPF. The increased latency in the Morris water maze during the treated animals indicated that the chronic low-level CPF exposures can impair the long-term spatial memory. As there was no difference between the CPF treated groups and the vehicle treated group on the results from grip strength effects of CPF, and there were no effects on short-term spatial memory or learning. What's more, only the highest dosage (10mg/kg) had significant inhibition effects on AChE activity, the exhibition of impaired long-term spatial memory may exist non- cholinergic reasons.The hippocampus has become the most important method to study the higher cognitive function, since its special function in the spatial memory. In the present study, we found significant decrease of cell numbers in the regions of hippocampus, although the hippocampal regions from the CPF treated groups did not exhibit any obvious architectural difference from the vehicle control group on gross observation. However, the immunohistochemisty study exhibited that NeuN (mature neuron marker) positive cells in the dentate gyrus(DG)increased, and the western blot of the NeuN protein from the whole hippocampi also increased significantly. The results from immunohistochemisty and western blot study showed thatβ-3 tubulin(TUJ1)and NF68 (adult new born neuron marker) decreased significantly and the marker of neuron precursor cells, BrdU, also exhibited significant decrease in a dose-dependent manner. The phenomenon discussed above illustrated that the repeated exposures of CPF can affect the constitute of cells in hipocampus, in another words, repeated exposures of CPF lead to the decrease of the adult new born neurons, but have no effect on the mature neurons. The hurt to brain(hypoxia, infection,poisoning,et al.) often exhibited glial cell hyperplasia under normal circumstances, while the GFAP protein expression protein from the whole hippocampi and the GFAP positive cells in the dentate gyrus(DG)decreased simultaneously. The changes that discussed above indicated the decrease of the cell in DG may not the direct toxicity effect from CPF, but the regulation result of some signal pathway. What's more, the TUNEL positive cells did not show any increase in the treated animals, and apoptosis related regulator , Bax/Bcl-2,p53 and caspase-3 did not show any change after the repeated exposures to CPF. It means the apoptosis could not be the main reason of the cells decrease in the DG. The changes of the cell constitute in the DG indicate the repeated CPF exposures can affect the neurogensis of the hippocampus.More and more studies had proved that the DG region of the adult hippocampus can have neurogensis all the life, and the adult new born neurons have important function in the hippocampus related spatial memory, especially the long-term memory. The decrease of the new born neurons can result in the impairment of long-term memory. Then the significant decrease of the adult new born neurons in the DG region may be the main reason of the impaired long-term spatial memory in the CPF treated animals.The regulation of the adult neurogensis is the research focus of neurobiology studies, and the latest results indicated that the cyclic AMP responsive element binding protein (CREB) have an important function during the Memory Allocation. The phosphorylated CREB can activate the dowmstream gene transcription, which can induce the differentiation of the hippocampal precursor cells and promote the survival and proliferation of the adult new born neurons. The decrease of the phosphorylated CREB can lead to deficiency of the adult new born neurons. As CREB is one of the most prominent molecules involved in learning and memory processes. Evidence suggests that CREB may play a crucial role in hippocampal plasticity and consolidation of long-term memory, since CREB activates the transcription of genes required for long-term synaptic changes involved in memory formation. Animals with CREB protein mutations exhibit learning and memory deficits. While, the activation of CREB pathway can be the regulating results of Ca2+/calmodulin-dependent kinaseⅡ(CaMKⅡ),protein phosphakinase A (PKA),extracellular regulated protein kinases (ERK). CaMKⅡhas been regarded as the molecular switch during the leaning and memory function. The cAMP related protein kinases pathway was the first memory related signal pathway studied, and the PKA can active CREB directly. ERK, as the upstream kinases of CREB pathway have important function during the neuron differentiation and long-term potentiation(LTP).In the present study, the expression protein level of phosphorylated CREB and ERK1/2 in hippocampus from the CPF treated rats decreased significantly, but the CPF had no effects on the expression of PKA and phosphorylated CaMKⅡ. The dose-dependent decrease indicated that the inhibition of the ERK-CREB Signaling may be one of the important regulating pathway during the CPF caused spatial long-term memory impairments.In summary, the present study identified that repeated exposure to subthreshold doses of CPF can lead to the inhibition of ERK-CREB signaling, which regulated the neruogenesis of the hippocampal DG and induced the deficiency of adult new born neurons. The decreased young neurons lead to the impaired long-term spatial memory.