Organellar Na+/H+ Exchangers and Intracellular pH Regulation in Schizophrenia Brain

精神分裂症脑细胞器 Na /H 交换器和细胞内 pH 调节

• 批准号: 10448961
• 负责人: Brandon Scott Pruett
• 金额: $ 19.14万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10448961
• 关键词: AMPA Receptors; Acidity; Acute; Affect; Astrocytes; Autopsy; Biology; Brain; Calibration; Cell Culture Techniques; Cell Hypoxia; Cell membrane; Cells; Closure by clamp; Co-Immunoprecipitations; Confocal Microscopy; Development; Diagnosis; Disease; Disease model; Doctor of Philosophy; Endosomes; Energy Metabolism; Enrollment; Enzymes; Excision; Fluorescence; Funding; Future; Glycolysis; Goals; Health; Hypoxia; Individual; Knowledge; Label; Lead; Learning; Measures; Mediating; Mentorship; Metabolic; Methods; Molecular; Neurons; Neurotransmitter Receptor; Organelles; Patients; Peripheral; Pharmaceutical Preparations; Positioning Attribute; Post-Translational Protein Processing; Pre-Clinical Model; Prefrontal Cortex; Proteins; Psychiatry; Psychopathology; Regulation; Reporting; Research Personnel; Research Project Grants; Residencies; Resolution; Role; Scaffolding Protein; Schizophrenia; Sodium-Hydrogen Antiporter; Stress; Subcellular Fractions; Synapses; Techniques; Testing; Time; Tissues; Training; Transfection; Universities; Visualization; Work; base; brain tissue; cancer cell; career; cell type; cost; economic cost; experience; experimental study; first episode schizophrenia; high throughput screening; induced pluripotent stem cell; mortality; multimodality; neuroimaging; new therapeutic target; novel; novel therapeutics; professor; protein distribution; protein transport; ratiometric; receptor; response; severe mental illness; skills; stem cell differentiation; stressor; trafficking

Candidate: I am an Assistant Professor in UAB’s Department of Psychiatry with a background in molecular methods used in preclinical models and the role of intracellular pH dysregulation in neurodevelopmental illnesses. Additionally, I have expertise in diagnosis and treatment of psychopathology having received my MD- PhD from LSU Health Shreveport and completed a Psyhciatry Residency at Brown University. Career Goals and Development: I hope to gain expertise in assessing schizophrenia (SZ)-associated molecular disruptions in postmortem brain, in generating SZ patient-derived induced pluripotent stem cells (iPSCs) and differentiating them into disease relevant cell types, and in measuring cellular trafficking and luminal pH through the use of fluorescently-tagged protein constructs. By acquiring these skills and completing the studies laid out in this proposal, I will be well positioned and competitive for independent funding. Research Project: Deficits in protein post-translational modifications (PTM) and trafficking are reported in schizophrenia (SZ) brain, but the underlying cause is unknown. The function of organelles involved in PTM and trafficking is greatly impacted by pH disruptions, and Na+/H+ Exchangers (NHE) 6-9 are major regulators of organelle pH. In cancer cells, hypoxia causes altered energy metabolism and redistribution of NHE6 from endosomes to the plasma membrane. Similar metabolic alterations are reported in SZ brain suggesting that NHE6-9 intracellular distribution may also be affected, which could contribute to disrupted protein PTM and trafficking. So far, I have found that NHE7/8 expression is decreased in SZ dorsolateral prefrontal cortex (DLPFC) while NHE6/9 is unchanged. Still, NHE6/9 show increased expression in a tissue fraction enriched for synapses suggesting altered distribution of these proteins. Here, I propose to more extensively determine the expression and distribution of NHE6-9 first in SZ postmortem DLPFC and then in excitatory cortical neurons and astrocytes differentiated from patient-derived iPSCs. I will also determine how the introduction and removal of an acute stressor (hypoxia) affects the distribution of these proteins in these cells. Finally, I will transfect cells with fluorescently-tagged protein constructs to measure NHE6-9 and neurotransmitter receptor trafficking as well as organelle pH in live cells. These studies could help identify novel treatment targets for SZ and lead to high throughput assays to identify drugs that reverse SZ-associated molecular disruptions. Mentorship: The primary mentorship team for this proposal consists of Dr. James Meador-Woodruff, a world renowned expert in molecular disruptions in schizophrenia brain and analysis of postmortem brain tissue, Dr. Marek Napierala, an expert on molecular mechanisms of repeat expansion disorders and of modeling these illnesses using iPSCs differentiated into a variety of cell types including cortical neurons, and Dr. Vladimir Parpura, an expert in glial biology and visualization of vesicular trafficking in live cells through the use of fluorescently-tagged protein constructs.

候选人：我是阿拉巴马大学精神病学系的助理教授，具有分子生物学背景 临床前模型中使用的方法以及细胞内 pH 失调在神经发育中的作用 疾病。此外，我在获得医学博士学位后，拥有精神病理学诊断和治疗方面的专业知识- 路易斯安那州立大学什里夫波特健康中心获得博士学位，并在布朗大学完成了精神病学住院医师培训。 职业目标和发展：我希望获得评估精神分裂症（SZ）相关的专业知识 死后大脑中的分子破坏，产生 SZ 患者来源的诱导多能干细胞 （iPSC）并将其分化为疾病相关细胞类型，以及测量细胞运输和 通过使用荧光标记的蛋白质结构来调节管腔 pH 值。通过获得这些技能并完成 通过本提案中提出的研究，我将处于有利位置并有竞争力获得独立资助。 研究项目：蛋白质翻译后修饰 (PTM) 和运输方面的缺陷报告于 精神分裂症（SZ）大脑，但根本原因尚不清楚。参与 PTM 的细胞器的功能和 pH 值破坏对运输有很大影响，Na+/H+ 交换器 (NHE) 6-9 是 细胞器 pH 值。在癌细胞中，缺氧会导致能量代谢改变以及 NHE6 的重新分配 内体进入质膜。据报道，SZ 脑中也有类似的代谢变化，这表明 NHE6-9 细胞内分布也可能受到影响，这可能导致蛋白质 PTM 和 贩运。到目前为止，我发现SZ背外侧前额皮质中NHE7/8表达减少 (DLPFC) 而 NHE6/9 不变。尽管如此，NHE6/9 在富含 突触表明这些蛋白质的分布发生了变化。在此，我建议更广泛地确定 NHE6-9 首先在 SZ 死后 DLPFC 中表达和分布，然后在兴奋性皮质神经元中表达和分布 以及从患者来源的 iPSC 分化而来的星形胶质细胞。我还将确定如何介绍和 急性应激源（缺氧）的消除会影响这些蛋白质在这些细胞中的分布。最后，我会 用荧光标记的蛋白质构建体转染细胞以测量 NHE6-9 和神经递质受体 活细胞中的运输以及细胞器 pH 值。这些研究有助于确定 SZ 的新治疗靶点 并导致高通量测定来识别逆转 SZ 相关分子破坏的药物。 指导：本提案的主要指导团队由 James Meador-Woodruff 博士组成，他是一位世界 精神分裂症大脑分子破坏和死后脑组织分析领域的著名专家，Dr. Marek Napierala，重复扩张障碍分子机制及其建模专家 使用 iPSC 分化成多种细胞类型（包括皮质神经元）来治疗疾病，Vladimir 博士 Parpura，神经胶质生物学专家，通过使用活细胞中的囊泡运输可视化 荧光标记的蛋白质构建体。