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

硫化氢（H2S）由哺乳动物细胞（包括海马中的神经元和星形胶质细胞）中的胱硫醚β-合酶（CBS）、胱硫醚γ-裂解酶（CSE）和3-巯基丙酮酸硫转移酶（MST）合成。它通过激活N-甲基-D-天冬氨酸（NMDA）受体促进突触可塑性（长时程增强，LTP）。迄今为止，含硫氨基酸限制（SAAR），即减少饮食中蛋氨酸和半胱氨酸的含量，对认知功能的影响以及H2S在其中的作用尚不清楚。 目的：探讨SAAR对认知功能的影响及H2S介导的海马可塑性的机制。我们推测，SAAR触发整合的应激反应，以增加内源性H2S的产生，这有利于海马可塑性和记忆通过激活NMDA受体。 科学方法：雄性C57 BL/6小鼠将喂食正常小鼠饮食作为对照。采用不同的喂养方案，将监测体重、%脂肪量、血清甘油三酯、血脂谱和血糖、摄食量、血浆和海马蛋氨酸、半胱氨酸和同型半胱氨酸水平。目标1。为了确定SAAR饮食对记忆的影响，我们将用SAAR饮食（蛋氨酸和半胱氨酸含量减少20%、35%和50%）或增强的SAA饮食（正常饮食的SAA水平的125-200%）短期（1-2周）和长期（1-4个月）喂养实验小鼠。此后，我们将测试记忆相关的行为和海马突触电流和可塑性。 目标2.为了探讨SAAR增加内源性H2S产生的机制以及H2S在SAAR增强记忆中的作用，在SAAR饮食处理后，将在小鼠海马组织中使用Western印迹或qRT-PCR进行以下测量：a）整合应激反应的两个调节因子，一般对照非抑郁因子2和转录激活因子4的表达和活性;和B）产生H 2 S的酶（CSE、CBS、MST）的表达。H2S是否介导SAAR的认知效应将通过直接施用H2S盐或通过选择性抑制内源性H2S产生来测试。 目标3。为了研究H2S对小鼠海马可塑性影响的机制，在SAAR或外源性H2S应用后，我们将检查i）NMDA受体的S-巯基化; ii）PKA和cAMP的磷酸化，这是晚期LTP所必需的;以及iii）TRPA 1通道介导的Ca 2+内流。新奇和预期的意义：这个程序将有助于理解SAAR对记忆的影响。通过选择性地关注SAAR，不仅可以识别负责完全饮食限制的神经元效应的关键分子，而且可以避免完全饮食限制的营养不良的不良影响。可以设计增加脑中内源性H2S水平的新营养方法，以增强记忆或防止不同生物和生理过程中的记忆丧失。

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.