The Abnormal Expressions Of Gene Related To The Functions Of Lysosomal, Mitochondrial, Synapse In The Intractable Epilepsy

Background: Not only an alteration in neuronal excitability is a primary prerequisite for epileptogenesis, but the synchronous activation of large population of hyperexcitable neurons is indispensable to it. A sezure is a transient disturbance of cerebral function caused by an abnormal neuronal discharge.Abnormal expression of gene underly the abnormal neuronal discharge. It may result in disturbance of mitochondrial, lysosomal, synapse function, necrotic and/or apoptotic neuron, mossy fibre sprouting and neuronal circuitry rearrangements, pathological neuron connections, location of drug substrate disappear. So in about one-third of patients, epilepsy remains uncontrolled, despite a variety of AEDs being prescribed. It is important to investigate the expressions of genes to definite the epileptogenesis.Objective: To investigate the expression of mitochondrial malate dehydrogenase(mMDH), NADH dehydrogenase ubiquinone FC2(NDUFC2) in relation to mitochondrial function, postsynaptic density(PSD)-93, N-methyl-D-aspartate receptors subunit 2B(NR2B) related synapse function and lysosomal proteinase cathepsin D(Cath D) in the brain tissue from the patients with intractable epilepsy and non-intractable epilepsy. So as to explore the possible roles of them in the pathogenesis of epilepsy.Methods: By random sampling from brain bank, brain tissue samples of 48 intractable epilepsies(IE), 8 non-intractable epilepsies (NIE) and 8 control cases were paired up to three groups. Then scanning the cDNA microarrays. After achieved the positive results, subsequently, partial abnormal expression gene ( PSD-93,NR2B,Cath D)was evaluated by reverse transcription polymerase chain reaction(RT-PCR) (GAPDH gene, internal control), mMDH was analysed by fluorescent quantitative reverse transcription polymerase chain reaction(FQ-PCR,18s gene, internal control) ,Expression ratio (target gene/GAPDH or target gene/18s) was used to evaluate each gene relative expression level. After increasing the number of control samples, immunohistochemistry and immunofluorescence were used to definite the expression of Cath D.Results: The cDNA microarray analysis showed that the expressions of mMDH , NDUFC2 and Cath D were significantly lower in IE than control, the Cy5/Cy3* value was 0.349,0.373,0.433,respectively. up-regulated expression of PSD-93 was found both in IE and in NIE, the Cy5/Cy3*value was 2.124,5.550,respectively. The results of FQ-PCR and RT-PCR were consistent with those of the cDNA microarrays(p<0.05). The relative expression ratio of PSD-93 in patients with non-epileptogenic control, NIE and IE was 0.159, 0.368 and 0.341, respectively. Correspondingly, that of NR2B was 0.198, 0.738,0.903, respectively. The expressions of PSD-93 and NR2B in the NIE and IE were significantly higher than those of control, respectively(P<0.05). However, there was no significantly difference the expression of PSD-93 between NIE and IE(P>0.05), neither do that of NR2B (P>0.05).The expression of Cath D mRNA in the experimental group was significantly different from those of the control group(P<0.05),the median of relative gray value of RT-PCR was 0.3084, 0.8370, respectivly. In the hippocampal and temporal lobe neocortex tissue, there were the expression of Cath D, localed at the neuron body, but no expression at the glial cells. The result of immunohistochemistry showed the expression of Cath D in the temporal lobe and hippocampal of experimental group(0.0609±0.03550,0.0688±0.02500)were significantly lower than those in control group(0.1542±0.02234 , 0.1667±0.02082 , P<0.005). The results of immunofluorescence were well concordant with immunohistochemistry data. There was no significant difference between the temporal lobe and hippocampal in each group(P>0.05).Conclusions:While screening the gene changes of IE and NIE, cDNA microarray is characterized by high speed, large capacity and high agility. Because cDNA microarray maybe results in pseudo-positive and pseudo-negative findings, it is indispensable to use other method to verify its result.Impaired energy metabolism and changing membrane ion channels are associated with epileptogenesis. They may cause neuron necrosis and/or apoptotic, neuronal circuitry rearrangements, pathological neuron connections and location of drug substrate lost. These may result in IE.The downregulated expression of mMDH,NDUFC2 may involved in impaired energy metabolism, which caused a variety of neurotransmission injured and activated a serial of response, even brought neuron dead.The upregulated expression of PSD-93,NR2B may accelerate the release of excitatory toxic neurotransmission and the synchronous activation of large population of hyperexcitable neurons. Decreased expression of Cath D suggested that Cath D may play special roles in the pathogenesis of intractable epilepsy.These abnormal expression genes may underly further exploring pathogenesis of IE and NIE and exploiture new drug.