The Cognitive Function In Temporal Lobe Epilepsy Rats And The Expression Of Synaptic Plasticity-Related Proteins

BackgroundEpilepsy is one of the most common chronic neurological disorders affecting people of all ages, and it is prone to cause cognitive and neurobehavior impairment. At present, there are about 50 million of epilepsy patients worldwide. They suffer a lot from this disease and become big burdens to family and society. Temporal lobe epilepsy (TLE), the most common type of epilepsy in human, is characterized by spontaneous recurrent seizure (SRS), and is also be associated with memory impairment and behavioural problems, including depression, anxiety and psychoses. The clinical symptom of TLE is associated with aesthema, emotion, mind, and locomotion. SRS may be caused by lesion in hippocampus and other limbic system, and the hippocampal formation participates in the form of learning, memory, emotion, behavior and other cognitive function. Therefore, it is important to study the mechanism of cognitive impairment caused by SRS. The cognitive function belongs to higher nervous activity in human and animal, including every aspects of mind and intellect activity, which are aesthema, perception, learning and memory. It is very complicated to form learning and memory. Recent studies indicate that hippocampal formation, synaptic plasticity, inhibited amino acids receptor, calcium ion, nitrogen monoxidum (NO) all participate in the formation of learning and memory. A few of synaptic plasticity-related proteins are known as principal regulators in synaptic plasticity. Until now, only few studies report the mechanism underlying synaptic plasticity-related proteins in seizure activity. But the mechanism underlying synaptic plasticity-related proteins in behavioral deficits induced by SRS is still unclear. Moreover, there are still limited strategies to protect cognitive impairment induce by SRS.Some studies confirm that the hippocampus is important encephalic region, which plays a central role in learning and memory. Long-term potentiation (LTP) in the hippocampus is regarded as an ideal model to study synaptic plasticity, also as "a synaptic model of memory" or "neuronal mechanism of memory". The NMDA receptor, one of excitatory amino acids receptors—glumatic acid receptor, is known as a principal regulator in synaptic plasticity and LTP in hippocampus and cortex. The NR2B subunit, which is expressed at its highest levels in the olfactory tubercle, hippocampus, olfactory bulb, and cerebral cortex, has been implicated as a principal player in learning, memory, synaptic plasticity, neural development, ischemic brain injure, neurodegeneration, epilepsy and tumor. PSD-95 (postsynaptic density protein 95), i.e. SAP90 (synapse associated protein 90), is a scaffold protein and one of guanylate kinase superfamily exclusively localized to glutamatergic synapses and is characterized by three PDZ (Dlg, ZO-1/Dlg-homologous region) domains, a SH3 (SRC Homology 3) domain and a GK (Guanylate kinase-like) domain. The PDZ domains of PSD-95 specifically bind to the C terminus of the NR2B subunit. It has been shown that the complex of NR2B and PSD-95 plays an important role in several neurodegenerative diseases, such as ischemic stroke, aged-learning deficits, depression, Alzhemer's disease (AD), epilepsy, Huntington disease and schizophrenia. Synaptophysin (Syp) is a 38-kDa calcium-binding glycoprotein found in the membranes of neurotransmitter-containing presynaptic vesicles. Syp may regulate release of endogenous glumatic acid to influence synaptic plasticity through phosphorylation after protein tyrosine kinase (PTK) activation. As the loss of synaptic marker, it is better parameter that correlates with memory dysfunction in AD and age-related memory. One study also indicates that induction of LTP and synthesis of Syp may be link-coupled. For example, LTP and Syp in old age animal is synchronously decreased. Together with other proteins including the synaptic vesicle protein VAMP (vesicle associated membrane protein, also called synaptobrevin) or the plasma membrane protein syntaxin (also HPC-1), SNAP-25 (synaptosomal-associated protein of 25 kDa) forms the so-called SNARE (soluble Nethylmaleimide-sensitive factor attachment receptor) protein complex. SNAP-25 is an integral component of the synaptic vesicle-docking/fusion core complex and plays an essential role in exocytosis/release of neurotransmitter. It plays a critical role in axon growth, dendric formation, neurotransmitter release and expression of hippocampal LTP. Recent study suggests SNAP-25 is highly expressed by neurons in hippocampus and play a major role in learning and memory. Synaptotagmin (Syt) is a protein located principally in secretory vesicle membrane of neuron and endocrine cell. There are 16 subtypes identified in mammal, and Syt 1 is the most abundant in hippocampus. As a calmodulin, Syt 1 binds to cellular membrane by calcium-dependent way and correlates intimately with release of neurotransmitter. Syt 1 inserts synaptic vesicle membrane by membrane spaning domain at N terminus. Syt 1 possesses two C2 domains, C2A and C2B, both being capable of calcium-dependent phospholipid binding in in vitro reactions. According to the available data, Syt 1 is a promoter of the synaptic vesicle fusion, mainly functioning as a fast calcium sensor for synchronous release of neurotransmitter via facilitating exocytosis and endocytosis. Now, some studies demonstrate that Syt 1 may act as a calcium sensor to mediate stimulus-coupled fast chemical synaptic transmission, and the domain carrying out the function is C2B. In addition, Syt 1 may bind to SNARE protein receptor. Syt 1 is important to elucidate the pathogenesy and therapy of refractory nerver diseases. Recent studies suggest that Syt 1 correlates with AD and aged-memory deficits.To our knowledge, the role of NR2B, PSD-95, Syp, SNP-25, Syt 1 has not been studied in TLE animal model with behavior deficits. Thus, in this study we try to investigate whether SRS might decrease expression of NR2B, PSD-95, Syp, SNP-25, Syt 1 and consequently cause behavior deficits in rats with SRS induced by kainic acid (KA). Therefore, part of the molecular mechanism of cognitive impairment might be revealed.PartⅠThe cognitive impairment in temporal lobe epilepsy rats and the abnormal expression of synaptophysin, SNAP-25,synaptotagmin 1 in the hippocampus.ObjectiveTo evaluate cognitive impairment in TLE rats induced by KA, investigate the expression of Syp, SNAP-25, Syt 1 in the hippocampus of rats with behavior deficits, and explore the partly possible molecular mechanism of cognitive impairment caused by TLE.Method1. Seizures were induced by KA through intraperitoneal injection. Rats were randomly divided into control group, KA group. Next, according to whether to develop SRS, KA group rats were divided into KA+SRS group and KA-SRS group.2. To evaluate cognitive function in every experiment groups by behavior tests.3. To evaluate pathology features of hippocampal neurons of KA+SRS and KA-SRS groups at 6w. Haematoxylin & Eosin staining and Nissl staining by Toluidine Blue were performed.4. Immunohistochemistry staining was used to detect expression of Syp, SNAP-25, Syt 1 proteins at different experiment groups by electron microscope.5. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was used to detect expression of Syp, SNAP-25, Syt 1 mRNA at different experiment groups.6. Western blot analysis was used to detect expression alterations of Syp, SNAP-25, Syt 1 proteins at different experiment groups.Result1. Morris water maze, elevated-plus maze and open field analyses showed the cognitive function of KA+SRS group became worse (P<0.05), when compared with control or KA-SRS group. 2. No extensive destruction was noted in the dentate gyrus, CA1 and CA3 subfields of hippocampus of rats post KA, areas of neuronal cell loss and gliosis were seen in hippocampus of KA+SRS group rats.3. Immunohistochemistry staining of Syp, SNAP-25, Syt 1 showed there were lots of trachychromatic brown Syp, SNAP-25, Syt 1 positive neurons in control group rats. Compared with Syp, SNAP-25, Syt 1 positive neurons in control group, those of KA+SRS group were fewer and shallower. But there was no significant difference between control group and KA-SRS group.4. RT-PCR analysis showed that expression of Syp, SNAP-25 mRNA in KA+SRS group were decreased (P<0.05), and expression of Syt 1 mRNA in KA+SRS group was not significantly changed, when compared with those in control or KA-SRS group.5. Western blot analysis showed that expression of Syp, SNAP-25, Syt 1 protein in KA+SRS group were decreased (P<0.05), when compared with those in control or KA-SRS group.Conclusion1. SRS induced by KA may lead to cognitive impairment.2. At the present experiment, no extensive destruction was noted in hippocampus of rats post KA, areas of neuronal cell loss and gliosis were seen in hippocampus of rats with SRS.3. SRS induced by KA may decrease Syp and SNAP-25 expression, and increase Syt 1 expression, consequently cause cognitive impairment.PartⅡThe evaluation of cognitive function in temporal lobe epilepsy rats and the expression of NR2B/PSD-95 with time in the hippocampus ObjectiveTo evaluate cognitive function in TLE rats induced by KA, investigate the alterations of NR2B/PSD-95 in the hippocampus of rats with behavior deficits with time, investigate the role of antiepileptic VPA for cognitive function and expression of NR2B/PSD-95 in the hippocampus, and explore the possible molecular mechanism of cognitive impairment caused by SRS.Method1. SRS were induced by KA through intraperitoneal injection. Rats were randomly divided into control group, KA group and KA+VPA group. Next, according to the time point of behavioral tests, the above three groups were randomly divided into 2 w, 4w and 6w subgroups, respectively.2. To evaluate cognitive function in every experiment subgroups by behavioral tests.3. To evaluate pathology features of hippocampal neurons of KA and KA+VPA subgroups at 6w. Nissl staining by Toluidine Blue were performed.4. Immunohistochemistry staining was used to detect expression of NR2B and PSD-95 proteins with time at different experiment subgroups by electron microscope.5. RT-PCR analysis was used to detect alterations of NR2B and PSD-95 mRNA with time at different experiment subgroups.6. Western blot analysis was used to detect alterations of NR2B and PSD-95 proteins with time at different experiment subgroups.Result1. Morris water maze, elevated-plus maze and open field analyses showed the cognitive impairment in three KA subgroups gradually became more serious with time (P<0.05), while there was no difference among KA-2w, control and KA+VPA subgroups at any time.2. No extensive destruction was noted in the dentate gyrus, CA1 and CA3 subfields of hippocampus of rats post KA, areas of neuronal cell loss and gliosis were seen in hippocampus of KA-6w subgroup rats.3. Immunohistochemistry staining of NR2B and PSD-95 showed there were lots of trachychromatic brown NR2B and PSD-95 positive neurons in control group rats. Compared with NR2B and PSD-95 positive neurons in control group, those in KA-6w subgroup were fewer and shallower. But there was no significant difference between control subgroup and KA+VPA subgroup at any time.4. RT-PCR analysis showed that expression of NR2B and PSD-95 mRNA in three KA subgroups were gradually down-regulated (P<0.05), while there was no difference among KA-2w, control and KA+VPA subgroups at any time.5. Western blot analysis showed that expression of NR2B and PSD-95 protein in three KA subgroups were gradually down-regulated (P<0.05), while there was no difference among KA-2w, control and KA+VPA subgroups at any time.Conclusion1. SRS induced by KA may lead to cognitive impairment, which gradually become more serious with time.2. At the present experiment, no extensive destruction was noted in hippocampus of rats post KA, areas of neuronal cell loss and gliosis were seen in hippocampus of KA-6w subgroup.3. SRS induced by KA may decrease NR2B and PSD-95 expression, and consequently cause cognitive impairment.4. VPA might improve cognitive impairment by maintain NR2B and PSD-95 expression.SignificanceUsing a rodent TLE model induced by KA and evaluating behavioral deficits caused by SRS, we investigated and confirmed that synaptic plasticity-related proteins, such as NR2B, PSD-95, synaptophysin, SNAP-25, synaptotagmin 1 played an important role in cognitive impairment caused by SRS. Furthermore, we showed that VPA might improve cognitive impairment by maintain NR2B and PSD-95 expression. Studies on synaptic plasticity-related proteins will undoubtedly help us to know more about the possible mechamism of behavioral deficits caused by SRS. Thus, this study may provide new insights into therapeutic target and develop new neuroprotective drug improving cognitive impairment.