Correlation Between The Neuroprotective Effect Of EGb761 Against Glutamate-induced Excitotoxicity And The Snk-SPAR Pathway

Background and objective:The glutamate-induced excitotoxicity has been implicated in the etiology of many CNS diseases such as stroke, epilepsy, Alzheimer's disease and amyotropic lateral sclerosis. N-methy-D-aspartate (NMDA) subtype of ionotropic glutamate receptors is proved to play a critical role in excitotoxicity. Accordingly, a significant effort had been made to develop therapeutic strategies against excitotoxicity, but successful therapy in humans was limited by many factors. The study on strategy against excitotoxicity needs to be found new target. In this circumstances, a new target is the effect of Snk (serum-inducible kinase)–SPAR (spine-associated Rap GTPase activating protein) pathway on dendritic spine regulation. There are about 90% excitatory glutamatergic postsynaptic parts in the brain are located on dendritic spines. Spines are small protrusions on dendritic shafts that reconstitute the primary loci of excitatory synaptic transmission in the mammalian CNS, and synaptic transmission are thought to represent the physiological basis of learning and memory. SPAR located in postsynaptic density of dendritic spine, formed complex with NMDAR and the scaffold protein PSD-95, is an important candidate for mediating activity-dependent remodeling of synapses, and is critically involved in spine maturity, especially in the mature spineformation and the maintenance of spine maturity. Snk, one of the polo-like kinases, induced by synaptic activity and was targeted to dendritic spines, eliminates SPAR protein, and causes loss of mature dendritic spines and synapses. Therefore, the Snk-SPAR pathway probably participates in excitotoxicity in CNS through regulation the stability of dendritic spine, but now, we know little about it.Ginkgo biloba extract contains multiple compounds such as flavonoids and terpenoids, and it has many neuroprotective effects such as removing free radicle, protecting neurons against apoptosis induced by ischemia injury, inhibiting the accumulation ofβ-amyloid, and influencing the transport of neurotransmitters. Ginkgo biloba extract has been therapeutically used for several decades to increase peripheral and cerebral blood flow as well as for the treatment of cognitive speed, dementia, and aging damages, but the mechanism still remains elusive.The objective of this study is to observe the relationship between excitotoxicity induced by glutamate in cultured rat hippocampal neurons and Snk-SPAR pathway, and to explore the effect of EGb761 on Snk-SPAR pathway and morphology of dendritic spine in excitotoxic condition, furthermore discuss the molecular mechanism of neuroprotective effect of EGb761, and hope to find a new strategy for neuroprotection and neurogenerative regulation.Methods:The primary hippocampal cultures from neonatal rat were treated with 100μM glutamate and 10μM glycine at 18 day in vitro (DIV). Trypan blue staining, fluorescent TUNEL and LDH assay were used to study the glutamate-induced injury in neurons. In the glutamate-induced excitotoxicity model, RT-PCR was used to detect the changes in Snk/SPAR mRNA levels. Western blot was used to detect the changes in Snk/SPAR protein levels. After the hippocampal neurons were pretreated with EGb761 and MK-801, Trypan blue staining, fluorescent TUNEL and LDH assay were used to study the neuroprotective effect of EGb761 and MK-801 on neurons injured by glutamate, and RT-PCR, western blot, fluorescent in situ hybridization, and double immunofluorescent staining were used to observe the effect of EGb761 and MK-801 on glutamate-induced expression anddistribution changes of Snk and SPAR in cultured hippocampal neurons. The green fluorescence protein contained vector was transfected into hippocampal neurons to observe the protective effect of EGb761 and MK-801 on dendritic spine injury induced by glutamate exposure.Results:1. Changes in Snk/SPAR mRNA and protein levels in glutamate-induced excitotoxicity in cultured hippocampal neurons(1)The primary hippocampal neurons at 18 DIV were treated with 100μM glutamate and 10μM glycine for 10min, 18h later, cell viability, cell apoptosis, and LDH efflux were detected. Mortality rate was about 35%, apoptosis rate was about 37.2%, and LDH efflux in glutamate group was 3.1-fold compared with control group.(2)In control group, the basal level of Snk mRNA was low, but 1h after glutamate exposure, Snk mRNA had begun to elevate. This elevation was continued and reached the highest peak at 6h, at this time point, Snk mRNA was increased about 4.2-fold compared with the basal level, and mRNA level returned to baseline by 12h. In control neurons, the expression level of SPAR mRNA was much higher than Snk, but after glutamate insult, the expression tendency of SPAR mRNA was contrary to Snk. In 6h after glutamate stimulation, SPAR mRNA was about 34% compared with basal level.(3)Identical with the mRNA level, in control hippocampal neurons, Snk was expressed at low basal level but the expression level of SPAR was high. 3h after glutamate stimulation, level of Snk began to increase, and reached the highest peak by 36h, at this time point, Snk was increased about 3.3-fold compared with the basal level. In the period of 6h after glutamate stimulation, level of SPAR protein decreased slowly, but after the time point of 6h, SPAR decreased quickly, and became barely detectable by 36h.2. The Snk-SPAR pathway in neuroprotective effect of EGb761(1)Both EGb761 and MK-801 pretreatment can protect neurons against glutamate-induced excitotoxicity. Compared with glutamate group, the neuronal mortality rates in EGb761 and MK-801 pretreatment group decreased 31.4% and 67.8% respectively; the apoptosis rates decreased37.8% and 68.2% respectively; LDH effluxes decreased 32.6% and 60.6% respectively.(2)6h after glutamate exposure, compared with glutamate group, Snk mRNA levels in EGb761 and MK-801 pretreatment group decreased 39% and 46% respectively, and SPAR mRNA levels in EGb761 and MK-801 pretreatment group increased 47% and 65% respectively.(3)36h after glutamate exposure, Snk protein levels in EGb761 and MK-801 pretreatment group decreased 48.7% and 52.5% respectively compared with glutamate group, and SPAR protein levels in EGb761 and MK-801 group increased into 47% and 65% of basal level respectively.(4)EGb761 and MK-801 pretreatment could inhibit the increase of Snk and decrease of SPAR distributed in neuronal dendrites induced by glutamate stimulation.(5)There are visible destroy effect with GFP-transfected neurons treated with glutamate such as the breakage of dendrities, the degeneration and loss of dendritic spines. Compared with control group, the number of dendritic spines in glutamate group was decreased about 70.5%. EGb761 and MK-801 pretreatment could protect dendritic spines against glutamate insult, and the number of dendritic spines increased 56.7% and 60.6% compared with glutamate group respectively.Conclusions:(1)In mRNA and protein levels, the expression of Snk/SPAR was changed in glutamate-induced exicitotoxicty. It proves that the excitotoxicity induced by glutamate is related to Snk-SPAR pathway closely.(2)EGb761 pretreatment can protect neurons against glutamate-induced excitotoxicity, but this effect is weak compared with MK-801.(3)EGb761 could block the activity of Snk-SPAR pathway after neuronal activity, and could also inhibit the increase of Snk and decrease of SPAR distributed in neuronal dendrites induced by glutamate stimulation. These suggest that the neuroprotective effect of EGb761 is relevant to Snk-SPAR pathway.(4)The mechanism of dendritic spine injury induced by glutamate is possibly concerned with Snk-SPAR pathway that regulates the morphology and structure of dendritic spines.(5)EGb761 pretreatment can protect dendritic spine against glutamate-induced injury, and the mechanism of it possibly correlate with Snk-SPAR pathway.