The role of hydrogen sulfide in cognitive benefits of sulfur amino acids restriction diet

硫化氢(H_2S)是哺乳动物细胞(包括海马神经元和星形胶质细胞)中由胱硫醚-β-合成酶(CBS)、胱硫醚-γ-裂解酶(CSE)和3-巯基丙酮酸硫转移酶(MST)合成的。它通过激活N-甲基-D-天冬氨酸(NMDA)受体促进突触可塑性(LTP)。到目前为止，硫磺氨基酸限制(SAAR)，即降低饮食中蛋氨酸和半胱氨酸的含量，对认知功能的影响以及H_2S在其中的作用尚不清楚。目的：阐明SAAR对认知的好处以及H_2S介导的海马可塑性的潜在机制。我们假设SAAR通过激活NMDA受体，触发整合的应激反应，增加内源性硫化氢的产生，从而促进海马可塑性和记忆。科学方法：雄性C57BL/6小鼠以正常小鼠饲料喂养作为对照。在不同的喂养方案下，将监测体重、脂肪百分比、血清甘油三酯、血脂和血糖、食物摄入量、血浆和海马体中蛋氨酸、半胱氨酸和同型半胱氨酸的水平。目的1.采用短期(1-2周)和长期(1-4个月)SAAR饲料(蛋氨酸和半胱氨酸含量分别降低20%、35%和50%)和强化SAA饲料(相当于正常饲料SAA水平的125-200%)喂养小鼠，观察SAAR饮食对小鼠记忆的影响。之后，我们将测试记忆相关行为以及海马区突触电流和可塑性。目的2.为了探讨SAAR增加内源性H_2S生成的机制以及H_2S在SAAR增强记忆中的作用，采用Western印迹或qRT-PCR方法对SAAR饮食治疗后的小鼠海马区组织进行了以下检测：a)两种整合应激反应调节因子--非抑制性2和激活转录因子4的表达和活性；b)H_2S产生酶(CSE、CBS、MST)的表达。通过直接使用硫化氢盐或选择性地抑制内源性硫化氢的产生，将测试硫化氢是否介导了SAAR的认知效应。目的3.为了探讨H_2S对小鼠海马可塑性的影响机制，在SAAR或外源性H_2S作用后，我们将检测：(1)N-甲基-D-天冬氨酸受体的S水合作用；(2)与晚期长时程增强有关的PKA和cAMP的磷酸化；(3)TRPA_1通道介导的钙内流。新颖性和预期意义：这个程序将有助于理解SAAR对记忆的影响。通过选择性地聚焦于SAAR，不仅可以确定与完全饮食限制的神经元效应有关的关键分子，而且还可以避免完全饮食限制对营养不良的不良影响。可以设计一种新的营养方法来增加大脑中内源性H_2S的水平，以增强记忆或防止在不同生物和生理过程中记忆的丧失。

Hydrogen sulfide (H2S) is synthesized by cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (MST) in mammalian cells, including neurons and astrocytes in the hippocampus. It facilitates synaptic plasticity (long-term potentiation, LTP) via activation of N-methyl-D-aspartate (NMDA) receptors. To date, the effect of sulfur amino acid restriction (SAAR), i.e. reducing the content of methionine and cysteine in diet, on cognitive functions and the role of H2S therein have been unclear.  Objective: To elucidate the cognitive benefits of SAAR and the underlying mechanism for H2S-mediated hippocampal plasticity. We hypothesize that SAAR triggers integrated stress response to increase endogenous H2S production, which facilitates hippocampal plasticity and memory by activating NMDA receptors. Scientific Approach: Male C57BL/6 mice will be fed with normal mouse diet as control. With different feeding schemes, body weight, % fat mass, serum triglycerides, lipid profile and blood glucose, food intake, plasma and hippocampal levels of methionine, cysteine and homocysteine will be monitored. Aim 1. To determine the effects of SAAR diet on memory, we will feed experimental mice for short-term (1-2 weeks) and long term (1-4 months) with SAAR diets (20%, 35%, and 50% reduction of methionine and cysteine content) or enhanced SAA-diet (125-200% of SAA level of the normal diet). Thereafter, we will test memory-related behaviors and hippocampal synaptic currents and plasticity. Aim 2. To explore the mechanism of SAAR-increased endogenous H2S production and the role of H2S in SAAR-enhanced memory, after SAAR diet treatment, the following measurements will be made in mouse hippocampal tissues using Western blot or qRT-PCR: a) the expression and activities of two regulators of integrated stress response, general control non-depressible 2 and activating transcription factor 4; and b) the expression of H2S-producing enzymes (CSE, CBS, MST). Whether H2S mediates the cognitive effects of SAAR will be tested by directly administering H2S salt or by selectively inhibiting endogenous H2S production. Aim 3. To investigate the mechanisms for the effects of H2S on mouse hippocampal plasticity, after SAAR or exogenous H2S application, we will examine i) the S-sulfhydration of NMDA receptors; ii) the phosphorylation of PKA and cAMP, essential for late LTP; and iii) the Ca2+ influx mediated by TRPA1 channels. Novelty and expected significance: This program will help understand the effect of SAAR on memory. By selectively focusing on SAAR, not only the key molecules responsible for the neuronal effect of total diet restriction can be identified, but also the unwanted effects of malnutrition of total diet restriction avoided. Novel nutritional approach to increase endogenous H2S levels in the brain may be devised to enhance memory or prevent the loss of memory in different biological and physiological processes.