The Mechanisms Of Poly （ADP-ribose） Polymerase On Epileptic Hippocampal Neuron Death Of Rats Induced By Mg²⁺-free Treatment

Epilepsy is a common and diverse set of chronic clinic syndrome characterized by paroxysmal, transient, repetitive and inflexible neurological disorders caused by hyper-synchronous neuronal discharges in the brain. Epilepsy is one of the most common neurological diseases which affects about50million patients around the world and9million in China. And about25%of patients suffer from intractable epilepsy with poor effects of medication and surgery. The pathophysiologic mechanisms are complicated. It has been suggested that the behavior, electroencephalogram (EEG) and the hippocampal neuronal injury of seizures induced by lithium-pilocarpine and kainic acid in rats is similar to that in patients with temporal lobe epilepsy. So the two methods have been the most frequently used animal models to research human epilepsy. Sombati established the hippocampal neuronal culture (HNC) model of acquired epilepsy (AE) which displayed spontaneous recurrent epileptiform discharges (SREDs) induced by Mg2+-free treatment in1995. It is convenient to observe the electrophysiological changes and drug effect in this model. Moreover, the pharmacological interference of epileptogenic drugs can be properly excluded to reveal the underlying mechanisms of epilepsy.Poly (ADP-ribose) polymerase-1(PAR.P-1) is a kind of non-histone chromosomal proteins which exists in the nucleus of eucaryotes, which is proved to be involved in DNA repair, gene transcription. DNA duplication and cell death. In physiological conditions, PARP-1activated by DNA breaks cleaves nicotinamide adenine dinucleotide (NAD) into nicotinamide and ADP-ribose. The latter can be synthesized into long-branching poly(ADP-ribose)(PAR) polymers which covalently attach to acceptor proteins including histones, DNA repair enzymes, transcription factors and PARP-1itself, facilitating DNA repair and cell survival. However, PARP-1can be hyper-activated by excessive DNA damage, triggering apoptosis or necrotic cell death which depletes a large amount of NAD+and ATP.Cumulative evidence suggested that the inhibition of PARP-1has cytoprotective effects against neurotoxicity, central nervous system injury, ncurodcgcnerative disorders, inflammation and epilepsy. The mechanisms of neuronal damages mediated by hyper-activation of PARP-1are complicated, including classical caspase-dependent apoptosis pathway, caspase-independent cell death pathway, accumulation of inflammatory mediators, hyper-activation of Ca2+/Mg2+-dependent endonuclease and energy failure. It is noteworthy that PARP-1-dependent cell death is unique in terms of its biochemical and morphological features, which is called "parthanatos". The biochemical features of parthanatos are distinct from classically defined pathways of cell death and involve rapid PARP-1activation, early PAR accumulation, mitochondrial depolarization, early nuclear apoptosis-inducing factor (AIF) translocation, cellular NAD+and ATP consumption and late caspase activation. And silent mating type information regulation2homolog1(SIRT1) has recently confirmed to be related to cell survival conditions. Therefore, it will be prospective to invent new therapies of epilepsy by regulating the target proteins involved in the pathway.In this study, the I INC model of AE was established to mimic epileptic conditions caused by hippocampus damages. Based on this, we examined the response of related cell signaling pathway and critical proteins to reveal the underlying mechanisms of caspase-independent cell death mediated by PARP-1hyper-activation. PART I A study of morphological and electrophysiological characteristics of neurons in the HNC model of AE in ratsObjectiveTo establish the HNC model of AE induced by Mg2+-free treatment in rats, and investigate the morphological and electrophysiological characteristics of neurons in this model.MethodsHippocampal cells were separated from24h postnatal Sprague-Dawley rats, plated and maintained in proper conditions. The purity of neurons in primary cultures was detected by immunocytochemical technique using primary antibodies to NSE. The growth and morphological features were carefully observed through the process. Two weeks later, the neuron cultures were exposed to Mg2+-free solution for3h and then returned to normal maintenance medium. Whole-cell patch clamp (WCC) recordings were performed at different time points to investigate the electrophysiological characteristics of neurons and verify establishment of the HNC model of AE.Results1. Morphological characteristics of neuron culture:The isolated round hippocampal neurons adhered to the plate surface with growing neuritis after12-24h. On the7th day. the neurons became more stereoscopic with classic axons and dendrites. Synaptic connections were observed on the10th day, which became intensive network during the10～14th day. Part of the neurons were cracked on the25～28th day.2. Purity of neuron culture:The cytoplasm and neuritis of neurons were stained by NSE antibody on the14th day. The purity of neuron culture was92.4%. 3. Electrophysiological characteristics of neurons:WCC recordings of control neurons revealed "normal" baseline recordings, displaying spontaneously occurring action potentials, excitatory and inhibitory postsynaptic potentials. During the3h Mg+-free exposure, neuronal firing behavior changed to continuous tonic high-frequency burst disclarges with "wedge-shaped depolarization". And WCC recordings demonstrated recurrent seizure activity with paroxysmal depolarizing shifts (PDSs) and sustained spike discharges alter the restoration of the normal Mg" concentration. The SREDs could be detected at96h after Mg2+-free treatment.Conclusions1. The hippocampal neurons of rats in this model were suitable for patch clamp studies because of the smooth surfaces, stereoscopic shapes and high purity.2. The hippocampal neurons in this model could form intensive synaptic connections in vitro, which provided the base of studies of information transformation.3. The electrophysiological characteristics of this model suggested that the SREDs induced by Mg2+-free treatment were similar to electrical activities of epileptic seizure. This neuronal culture model of AE provided a useful tool to epilepsy study. PART Ⅱ The effects of PARP-1on AIF translocation and PI3K/Akt signal pathway in the HNC model of AE in ratsObjectiveTo investigate the neuronal survival conditions and the effects of PARP-1on regulating mitochondria-nucleus translocation of AIF and PI3K/Akt signal pathway in the HNC model of AE in rats.Methods1. The hippocampal neurons of rats were randomly divided into control, Mg-free, Mg-free+Z-VAD-FMK (Caspase inhibitor), Mg-free+DPQ (PARP-1inhibitor) groups. The conditions of neuronal death were detected by TUNEL and LDH detection at1and24h after Mg2+-free treatment.2. The hippocampal neurons were randomly divided into control, Mg-free, Mg-free+DMSO, Mg-free+DPQ, Mg-free+WTN (PI3K inhibitor) groups. Proteins were extracted from whole cells, nuclei or mitochondrial fractions, which were used to detect the expressions of PARP-1, AIF,(p-)Akt and (p-)Gsk3p by Western blot analysis.3. The distributions of AIF were detected by cell immunofluorescence and confocal laser scanning microscopy.Results1. The TUNEL-positive nuclei ratio was71.2%at24h after Mg2+-free treatment, which decreased to45.25%after pretreatment of Z-VAD-FMK and26.88%by the addition of DPQ.2. PAR expression was elevated intensively after Mg2+-free treatment with decreased expression of AIF in mitochondria and increased amount in nuclei (P<0.05). And DPQ inhibited the expression of PAR, made more amount of AIF in mitochondria and less in nuclei than Mg-free+DMSO group (P<0.05).3. Phosphorylation of Akt and Gsk3β temporally decreased at1h after Mg2+-free treatment, then returned to the control level at12h. The phosphorylation was increased by DPQ addition and decreased by WTN (P<0.05). WTN also made less amount of AIF in mitochondria and more in nuclei than Mg-free+DPQ group (P<0.05).ConclusionsPARP-1was hyper-activated by Mg2+-free treatment, which resulted in mitochondria-nucleus translocation of AIF and neuronal death. And the PARP-1inhibitor, DPQ, could block the AIF translocation and protect neurons from cell death by activating the PI3K/Akt pathway. PART Ⅲ The effects of PARP-1on cellular NAD+and SIRT1levels in the HNC model of AE in ratsObjectiveTo investigate the linkage among PARP-1-mediated neuronal death, cellular NAD+level, expression and deacetylase activity of SIRT1, and the effect of NAD+on mitochondria-nucleus translocation of AIF in the HNC model of AE in rats.MethodsThe hippocampal neurons were randomly divided into control, Mg-free. Mg-free+DPQ, Mg-free+NAD (5,10mM). Mg-free+resveratrol (activator of SIRT1) and Mg-free+sirtinol (inhibitor of SIRT1) groups. Cellular NAD+content. expression and deacetylase activity of SIRT1were detected at1h,3h,6h,12h and24h after Mg2+-free treatment. The effects of DPQ or NAD on cellular NAD+content. TUNEL positive ratio. PARP-1conditions. AIF translocation. expression and activity of SIRT1were studied at24h after Mg2+-free treatment.Results1. Cellular NAD’ content was decreased with time alter Mg2+-free treatment. Compared with Mg-free group, DPQ pretreatment increased cellular NAD content and attenuated TUNHL positive ratio significantly (p<0.05).2. The data of cell immunofluorescence and Western blot demonstrated that All’ expression was decreased in nuclei and increased in mitochondria significantly (p<0.05), compared with Mg-free group, at24h after Mg2+-free treatment.3. The mRNA levels of SIRT1were gradually elevated with time after Mg2+-free treatment (p<0.05), but protein levels remained stable. The mRNA levels were significantly decreased by DPQ or NAD addition without changes of protein levels, compared with Mg-free group (p<0.05).4. The deacetylase activity of SIRT1was elevated by more than3times by addition of DPQ and NAD, accompanied by lower TUNEL positive ratio, compared with Mg-free group. And Mg-free+sirtinol group was quite the opposite.5. The PAR protein level was markedly decreased in Mg-free+DPQ group (p<0.05), and had no distinct changes in Mg-free+NAD group, compared with Mg-free group. There were no significant changes of PARP-1protein levels in each group.ConclusionsMg2+-free treatment hyper-activated PARP-1, depleted cellular NAD+, attenuated deacetylase activity of SIRT1, induced AIF translocation and led to severe neuronal death. And NAD+repletion and SIRT1activation could both protect neurons from cell death mediated by PARP-1activation, providing another pathway to reveal the mechanisms of neuronal death in the HNC model of AE.