A Role Of SrGAP3 In NMDA-induced Excitotoxic Cortical Neuronal Damage

The concept of excitotoxicity was first proposed by Dr Olney in 1969 to state the death of neurons induced by over-stimulation of glutamate receptors. Nearly half of a century passed, a lot of studies proved that excito toxic ity contributes to neurological deficits and neurodegeneration, such as stroke, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, epilepsy. Although the molecular mechanisms involved in excitotoxicity are still not so clear at the present, there is a wealth of evidences suggesting that over-stimulation of glutamate receptors produces multiple adverse effects including endoplasmic reticulum stress, impairment of intracellular calcium homeostasis, mitochondrial dysfunction, lysosome instability, free radicals formation, NO oxidative damage, transcription factors activated, pro-death genes expressed, protein kinases activation and proteases releasing, that leading to the autophagy, apoptosis, necrosis of neuron in the end. So,various pharmacological attempts have been made to stop those molecular pathways and attenuate neuronal damage, including glutamate receptor antagonists such as MK-801(Dizolcipine), Memantine, NBQX; Glutamate release blockers BW619C89, Riluzole; Free radical scavengers and antioxidants Alpha-phenyl-N-tert-butyl-nitrone (PBN), Tirilazad; Nitric oxide synthase inhibitors 7-nitroindazole. Many studies have confirmed the effect of these drugs on cell death pathways in various animal models, but in the end, most of the clinical development was abandoned for safety concerns or due to a lack of clinical efficacy, except Memantine and Riluzole. Memantine is currently indicated for severe Alzhermer's disease and Riluzole for ALS, but no clinical trials for stroke or traumatic brain injury are underway currently. So far, unfortunately, we fail to provide effective neuroprotection in the clinical setting.Rho GTPase is one subfamily member of small G protein superfamily, including main members:Racl, Cdc42, RhoA. They exist in all kinds of eukaryote and regulate a wide range of cellular processes involved in transformation, including adhesion, polarity, migration, proliferation and cell survival. Like other members of the Ras superfamily, Rho GTPases cycle between an active (GTP-bound) conformation and an inactive (GDP-bound) conformation. Cycle activation is catalysed by guanine nucleotide exchange factors (Rho GEFs) that promote exchange of GDP with GTP, while inactivation is induced by GTPase-activating proteins (Rho GAPs) that stimulate intrinsic GTPase activity. Guanine nucleotide exchange inhibitors (GDIs) extract the inactive GTPase from membranes. It has been suggested that Rho GTPase involve in neuronal survival. The overexpression of dominant-negative forms of cdc42 and Rac is reported to protect neurons against the withdrawal of trophic support, indicating that Rac/cdc42-associated proteins might contribute to neuronal death resulting from such withdrawal. RhoA mediates calcium-dependent activation of p38? and following neuron death. These studies illustrate the role of Rho GTPase in neuron survival in whole cell level, but its activation in subcellular remains to be investigated.srGAPs, the Slit-Robo GTPase activating protein, is a subfamily of Rho GAP that acts downstream of the Robo receptor. Its main members include srGAP1, srGAP2, srGAP3. srGAPs play a vital role in mammalian neurodeve lop mental processes, dynamically regulate cytoskeletal reorganization including neurite outgrowth, dendritic spine and axonal pathway formation, neuronal migration. Tissue culture studies indicate that the SH3 domin of srGAP1 interact with CC3 domian of Robo1, transduces the Slit repulsion signal affecting neuronal migration through downregulation of Cdc42 and activation of RhoA, while srGAP2 is involved in axon regeneration and neuronal migration through its Rho GAP domin. srGAP3 consists by three domains, F-BAR, GAP, SH3. In NIH3T3 cells, srGAP3 localizes to the plasma membrane through its F-BAR domain and to focal adhesions through its SH3 domain. Its expression downregulates Racl activity through its GAP domain, and thereby impairs actin and microtubule dynamics, and the formation of protrusions and focal adhesions. srGAP3 also named MEGAP (Mental disorder-associated GAP protein), because its haploinsufficienc in nervous system 3p syndrome patient. In addition, srGAP3 binds to the Arp2/3 activating protein Wave-1 and the Wave-1/srGAP3 complex facilitates the initiation of spine development by an inverse F-BAR domain, and its loss impairs long-term memory. srGAP3 expresses in many brain region and highly expresses in cortical neurons. In recent years, many other functions of srGAP3 have been reported, it may play a tumor suppressor role in mammary epithelial cells; nuclear-localized srGAP3 interacts with SWI/SNF chromatin remodeler Brgl implicating a novel role in regulation of gene expression; Depletion of srGAP3 may induces apoptosis in neural stem cells/neural progenitor cells. The role of srGAP3 in neuron survival is still unclear.Our interesting problems include:whether expression of srGAP3 is changed in excitotoxicity? Whether the neuronal death will be reversed after its expression disturbed? Whether its downstream is Rho GTPase in excitotoxicity?To solve these problems above, first, western blot analyses were performed to examine the expression of srGAP3 protein from whole-cell extracts at 0,1,3,6h after NMDA treatment. We found that srGAP3 protein levels began to decrease at 1h and continued to decrease in a time-dependent manner.Next, we used a lentivirus-based shRNA delivery system to infect cultured cortical neurons with short interfering RNAs targeting srGAP3. Knockdown of srGAP3 did not induce neuronal death in the absence of NMDA treatment, but significantly reduced NMDA-induced neuronal death.It has been shown that srGAP3 protein may localize both in the cytoplasm and in the nucleus of cortical neurons. Therefore, we further examined the expression changes of srGAP3 protein overtime at subcellular levels. Cytoplasmic and nuclear pools of srGAP3 protein were examined by Western blot analyses separately. After NMDA treatment, srGAP3 protein levels in the cytoplasm began to decrease at lh and further decreased at 3h and 6h; however, srGAP3 protein levels in the nucleus transiently increased at Oh and 1h and recovered to the normal level thereafter. These results suggest that srGAP3 might play different roles in the cytoplasm and in the nucleus.And then, the activation level of Racl, Cdc42, RhoA in the cytoplasm and in the nucleus were checked respectively. At 1h after NMDA treatment, a significant increase of cytoplasmic RhoA activity and a robust decrease of nuclear RhoA activity were observed. Meanwhile, the activities of Racl and Cdc42 remained unchanged. After knockdown of srGAP3, the activation of RhoA was reversed in the nucleus but no impact in the cytoplasm. These results indicate that the neuroprotection by srGAP3 knockdown is mediated by inhibiting the nuclear RhoA inactivation, but not by regulating the cytoplasmic RhoA activity.Next, we want to figure out that what is the downstream molecule of srGAP3 in cytoplasm.Akt has been well recognized as a pro-survival signaling protein in neurons subjected to excitotoxic stimuli. Recent findings suggest that Akt prohibits neuronal death by both impinging on the cytoplasmic cell death machinery and by regulating nuclear proteins We therefore detected the activation of Akt in the cytoplasm and in the nucleus by Western blot. At 1h after NMDA treatment, phosphorylation of Akt in the cytoplasm significantly decreased, and knockdown of srGAP3 further decreased the activation of Akt. This suggests that the presence of srGAP3 in the cytoplasm plays a pro-survival role through the Akt pathway. In the nucleus, the phosphorylation of Akt was not significantly altered, supporting the compartmental role of srGAP3 and Akt.We next determined whether knockdown of srGAP3 protects against NMDA-induced excitotoxic injury in vivo. An AAV vector srGAP3 shRNA was used to specifically downregulate the srGAP3 expression in cortical neuron of mice. The animals were killed 24 h after NMDA injection, and cortex injury was assessed by cresyl violet staining. srGAP3 knockdown significantly reduced the lesion volume induced by NMDA, suggesting that srGAP3 may represent a newly identified target for neuroprotection.Our studies first prove that srGAP3 involves in neuronal excitotoxicity. shRNA mediated depletion of srGAP3 plays a neuronal protective role in vitro and in vivo. srGAP3 plays a different role through different molecule mechanisms in the cytoplasm and in the nucleus. In the cytoplasm, the presence of srGAP3 plays a pro-survival role through the Akt pathway. In the nucleus, the neuroprotection by srGAP3 knockdown is mediated by inhibiting RhoA inactivation. We not only find a novel molecular pathway, but also a potential approach for therapeutic strategy in excitotoxicity.