Exploring the modulation of synaptic/extrasynaptic NMDAR balance as a novel therapeutic strategy in Alzheimer's disease and other neurodegenerations

兴奋性毒性被定义为谷氨酸能过量引起的神经元功能/结构的恶化 刺激。它是许多神经退行性疾病(ND)的共同主要病理特征，包括 阿尔茨海默病(AD)、亨廷顿病(HD)和肌萎缩侧索硬化症(ALS)。兴奋性毒性是 主要由NMDA型谷氨酸受体(NMDARs)的激活所介导。然而，NMDAR 正常的神经功能离不开功能。这一难题的解释是，NMDAR 分离于两个种群：突触(SNMDAR)和突触外(ExNMDAR)。而sNMDAR是 ExNMDARs的过度激活与促生存信号有关，可触发兴奋性毒性。因此，exNMDAR是 在ND的大范围内有明显的药理靶点，事实上，强烈地阻断了NMDAR的活性 改善AD和HD小鼠模型的认知缺陷。然而，选择性抑制exNMDARs是 具有挑战性，绝大多数NMDAR拮抗剂因其介导的副作用而在临床上失败 SNMDAR封锁。我们建议测试一种新的治疗策略，基于以下事实：S和exNMDAR 并不是独立的种群。相反，S和ExNMDAR池通过以下方式进行生理连接 横向扩散。我们假设，将S/exNMDAR平衡转向突触表达 有益的两个方面：(I)促进存活级联反应(sNMDAR介导)和(Ii)减少促死亡信号 (exNMDAR调解)。我们非常适合测试这一策略，因为我们之前已经确定了几个 控制S/exNMDAR平衡的机制。这些包括不同的蛋白质与 GluN2B-NMDAR的亚基和GluN2B上的一个特殊的磷酸化(位于S1480)，它促进sNMDAR 突触外部位的清除和受体稳定。这项提案的目标是验证通过防止sNMDAR清除和/或促进exNMDAR重新插入来减少兴奋性毒性的原则证明 进入突触部位是ND的有效治疗策略。在目标1中，我们将评估新的分子工具以 在培养和体内调节S/外源性NMDAR平衡，包括(I)小干扰肽(Sip)和(Ii) 调节GluN2B磷酸化的药理作用。我们的研究包括改变一种抗肿瘤药物的用途 目前处于临床试验的1/2阶段。此外，我们将使用蛋白质组学来比较翻译后修饰 S与exNMDARs的对比分析，旨在找出调节这种平衡的新机制。在目标2中，我们将 评估该策略作为ND常见治疗策略的适宜性。首先，我们要测试一下它的疗效 我们在几种AD模型中改善兴奋性毒性介导的病理结果的工具，这两种模型都在培养中 在活体内。最后，我们将在HD模型的原代培养中使用我们的策略(与兴奋性毒性相关)。 和帕金森氏症(兴奋性毒性不是主要的致病机制)。如果成功，这项提案将基于 通过调节NMDAR转运而不是通过抑制NMDAR功能来减少兴奋性毒性，将具有 突破性的翻译对识别各种ND的创新疗法的影响。

Excitotoxicity is defined as the deterioration of neuronal function/structure caused by excessive glutamatergic stimulation. It is a shared major pathological hallmark in many neurodegenerative diseases (ND), including Alzheimer’s disease (AD), Huntington’s disease (HD), and Amyotrophic Lateral Sclerosis (ALS). Excitotoxicity is mostly mediated by the activation of the NMDA-type of glutamate receptors (NMDARs). However, the NMDAR function is indispensable for normal neuronal function. This conundrum is explained by the fact that NMDARs are segregated in two populations: synaptic (sNMDARs) and extrasynaptic (exNMDARs). While sNMDARs are linked to pro-survival signaling, over-activation of exNMDARs triggers excitotoxicity. Therefore, exNMDAR are obvious pharmacological targets in a broad range of ND and, in fact, blocking NMDAR activity strongly ameliorates cognitive defects in AD and HD mouse models. However, selective inhibition of exNMDARs is challenging, and the vast majority of NMDAR antagonists have failed in clinic due to side effects mediated by sNMDAR blockade. We propose to test a novel therapeutic strategy based on the fact that s- and exNMDARs are not independent populations. On the contrary, s- and exNMDARs pools are physiologically connected via lateral diffusion. We hypothesize that shifting the s/exNMDAR balance towards synaptic expression would be beneficial two-folds (i) promoting survival cascades (sNMDAR-mediated) and (ii) decreasing pro-death signaling (exNMDAR-mediated). We are ideally suited to test this strategy because we have previously identified several of the mechanisms controlling s/exNMDAR balance. Those include different protein interactions with the GluN2B-subunit of NMDARs and a particular phosphorylation on GluN2B (at S1480) that promotes sNMDAR clearance and receptor stabilization at extrasynaptic sites. The goal of this proposal is to validate the proof-of-principle that reducing excitotoxicity by preventing sNMDAR clearance and/or promoting exNMDAR reinsertion into synaptic sites is an effective therapeutic strategy in ND. In Aim 1, we will evaluate novel molecular tools to modulate s/exNMDAR balance in culture and in vivo, including (i) small interfering peptides (sIPs) and (ii) pharmacology to modulate GluN2B phosphorylation. Our study includes the repurposing of an anti-tumoral drug currently in phase 1/2 of clinical trial. Also, we will use proteomics to compare the posttranslational modification profile of s- vs. exNMDARs, aiming to identify novel mechanisms regulating the balance. In Aim 2, we will evaluate the suitability of this strategy as a common therapeutic strategy in ND. First, we will test the efficacy of our tools in ameliorating excitotoxicity-mediated pathological outcomes in several models of AD, both in culture and in vivo. Finally, we will use our strategy in primary cultures from models of HD (associated by excitotoxicity) and Parkinson’s disease (excitotoxicity is not a primary pathomechanism). If successful, this proposal, based on reducing excitotoxicity by regulating NMDAR trafficking but not by inhibiting NMDAR function, will have a groundbreaking translational impact on the identification of innovative therapeutics for a wide range of ND.