Progress Report for Neurotherapeutics Unit

神经治疗科进展报告

基本信息

批准号：
10916073
负责人：
Avindra Nath
金额：
$ 139.97万
依托单位：
NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10916073
关键词：
ALS patients Alzheimer&apos s Disease Antibodies Astrocytes Attenuated Binding Biological Assay Biological Products Bipolar Disorder Caenorhabditis elegans Cell Culture Techniques Cell Line Cells Chronic Collaborations Collection Development Electrophysiology (science)Endogenous Retroviruses Epilepsy Extramural Activities FRAP1 gene Fluorescence Fluorescence Microscopy GABA-A Receptor Generations Glioblastoma Glioma Goals HIF1A gene HIV HIV tat Protein Hippocampus Human Image In Vitro Induced pluripotent stem cell derived neurons Label Laboratories Lead Ligands Mediating Microelectrodes Modeling Morphology Mutation National Center for Advancing Translational Sciences National Institute of Allergy and Infectious Disease National Institute of Mental Health National Institute of Neurological Disorders and Stroke Nerve Regeneration Neurology Neurons North Dakota Patients Pituitary-dependent Cushing&apos s disease Population Potassium Progress Reports Property Proteins Rattus Recombinant Proteins Recombinants Research Personnel Research Project Grants Role Sampling Schools Scientist Therapeutic Agents Translational Research Virus Replication Work antagonist chemosensitizing agent dopamine D3 receptor env Gene Products gamma-Aminobutyric Acid high throughput screening imaging platform in vivo induced pluripotent stem cell inhibitor live cell imaging neoplastic cell nerve stem cell nervous system disorder nervous system infection neuroprotection neuropsychiatry neurotoxic neurotoxicity novel therapeutics preclinical development programs promoter protein phosphatase inhibitor-2 protein purification response screening stem cell niche stem-like cell stressor tat Protein therapeutic target

项目摘要

Within the NTDU group, we continue to utilize many automated, medium-throughput cell-based assays to screen small compound collections against rat mixed cortical cultures, human neural stem cells (NSC) and human NSC-derived neurons and astrocytes. We've utilized live cell imaging-based assays with human neurons, as well as developed fluorescence-based assays to probe high content imaging endpoints. These cell-based screening assays, in 384 well plate format, have been successfully employed in multiple screens with multiple investigators. We continue to produce and purify a number of recombinant proteins, such as HERV-K Envelope proteins and HIV Tat protein for our own studies and provide that Tat protein to extramural researchers. SINS (PI: Nath) With the Section of Infections of the Nervous System (SINS), we have continued to work on multiple research projects. We continue to characterize the neurotoxicity resulting from the expression of the HERV-K env protein in NSC and human neurons, and have already identified some compounds and proteins which attenuate HERVK env mediated neurotoxicity. Exogenously added recombinant HERV-K Env protein also elicits significant neurotoxicity. Microelectrode array electrophysiologic studies have provided a functional endpoint to these studies. Specific anti-HERV-K envelope antibodies can block the envelope mediated neurotoxicity, as well as neurotoxic envelope protein contained in CSF samples from ALS patients. (Annals Neurology ref). The HIV Tat antagonist program has incorporated direct label-free binding assays, including protein thermal shift assays, as well as Tat-dependent selective LTR activation assays to complete the screening of more than 2000 compounds to identify new lead Tat inhibitors. The resultant "hit" compounds continue to be characterized for potency and biopharmaceutical properties, as well as activity against HIV viral replication. Additional Tat antagonist identification and characterization has been completed by collaboration with NCATS scientists. The NTDU has continued multiple studies in collaboration with other intramural investigators: Sibley Laboratory: We continue to work with the Sibley laboratory to characterize the dopamine D3 receptor modulators that they have identified, using our live cell imaging platforms for quantitative analysis. This work includes working with C elegans models as well as iPSC derived neuronal cultures. Zaghloul laboratory: Utilizing neuronal cell culture models of epilepsy, we found that chronic neuronal activation results in elevated LDH A via AMPK/Hif1a. Gliomas with IDH mutations promoted elipeptogenesis through mTOR. Chittiboina Laboratory: We worked toward purification of protein targets that display elevated expression and activation in Cushings Disease. Subsequent analysis of the interactions of these two proteins are being investigated Yesh Banasavadi Siddegowda laboratory: We characterized a potential chemosensitizer for GBM and have begun screening for compounds that may work synergistically to eliminate the GBM cells. Wei Lu Laboratory: We are collaborating with Dr. Wei Lu laboratory to characterize the activity of GABA A receptors with their newly identified modulator protein Shisa7. Interaction of GABA A Receptors with Shisa changed the binding activity of a number of GABA ligands. Nearly two thousand compounds have been screened and characterized as GABA A receptor modulators. Laboratories outside NINDS: Simeonov NCATS We have been working with their group to screen compounds for inhibition of Tat mediated HIV LTR activation, evaluating and characterizing the active molecules in confirmatory assays. McMahon laboratory (NIMH) to generate fluorescently labeled NSC and neuronal cultures from control and neuropsychiatric patients, to characterize their morphologic changes in response to stressors or toxic challenges. Completed Safavi Laboratory NIAID: Their laboratory generated iPSC lines from patient derived samples and characterized the derived neuronal populations with MEA and fluorescence microscopy. Extramural collaborations: Jonathan Geiger Laboratory (Univ North Dakota School of Med) We are collaborating with the Geiger laboratory to study the role of endolysosomal function in the neurotoxic activities of the HERV-K Env protein. Asish Shah laboratory (Univ Miami School of Med) Glioblastoma cells with stem cell like properties contain elevated levels of HERV-K Env and the HERV-K elements contribute to the stem cell niche of the glioblastoma, suggesting that HERV-K may be a therapeutic target for these tumor cells. Cell line generation NeuroTherapeutics Development Unit (NTDU): As part of its role in facilitating translational research, the NTDU develops cell lines in human neural stem cells (NSCs) for both internal and collaborative use. Lines have been developed to ubiquitously express the tdTomato fluorescent protein to aid in conducting screening assays. Collaborations between the NTDU and intramural and extramural groups has led to the development of more than ten such NSC lines that will make it possible to conduct medium throughput screens aimed at targeting several neurological diseases including bipolar disorder and Alzheimer diseases. Additionally, the NTDU has also generated additional cell specific lines for internal use that are also available to collaborators. These lines use cell specific promoters to express tdTomato in neurons and astrocytes and could have also have broad utility for conducting screens in distinct neural cells.

在NTDU组中，我们继续利用许多自动化的基于中等的细胞测定法，以筛选针对大鼠混合皮质培养物，人类神经干细胞（NSC）以及人类NSC衍生的神经元和星形胶质细胞的小型化合物集合。我们使用人类神经元利用了基于活细胞成像的测定法，并开发了基于荧光的测定方法来探测高内容成像终点。这些基于384个井板格式的基于细胞的筛选测定已成功地用于多个研究人员的多个屏幕中。我们继续生产和净化许多重组蛋白，例如HERV-K包膜蛋白和HIV TAT蛋白，以进行我们自己的研究，并将TAT蛋白提供给壁外研究人员。罪（PI：NATH）在神经系统感染（罪）的一部分中，我们继续从事多个研究项目。我们继续表征由NSC和人类神经元中HERV-K ENV蛋白表达产生的神经毒性，并且已经确定了一些减弱HERVK ENV介导的神经毒性的化合物和蛋白质。外源添加的重组HERV-K ENV蛋白还引起了明显的神经毒性。微电极阵列电生理研究为这些研究提供了功能终点。特定的抗HERV-K包膜抗体可以阻止包膜介导的神经毒性，以及ALS患者CSF样品中包含的神经毒性包膜蛋白。（Annals神经病学参考）。 HIV TAT拮抗剂程序已结合了无标记的无标记结合测定，包括蛋白质热移分析以及依赖TAT的选择性LTR激活测定法，以完成对2000多种化合物的筛选，以识别新的铅TAT抑制剂。所得的“命中”化合物继续以效力和生物药物特性以及针对HIV病毒复制的活性来表征。与NCATS科学家合作完成了其他TAT拮抗剂的识别和表征。 NTDU继续与其他壁内研究人员合作进行了多项研究： Sibley实验室：我们继续与Sibley实验室合作，以使用我们的实时细胞成像平台进行定量分析来表征他们已经鉴定出的多巴胺D3受体调节剂。这项工作包括与C秀丽隐杆线虫模型以及IPSC衍生的神经元文化合作。 Zaghloul实验室：利用癫痫的神经元细胞培养模型，我们发现慢性神经元激活导致LDH A通过AMPK/HIF1A升高。带有IDH突变的神经瘤通过MTOR促进了极生生成。 Chittiboina实验室：我们致力于纯化蛋白质靶标，这些蛋白质靶标表现出升高的表达和激活。随后研究了这两种蛋白质的相互作用 Yesh Banasavadi Siddegowda实验室：我们表征了GBM的潜在化学敏化剂，并开始筛选可以协同起作用以消除GBM细胞的化合物。 Wei Lu实验室：我们正在与Wei Lu实验室合作，以与新鉴定的调节蛋白SHISA7的受体的活动来表征GABA A受体的活性。 GABA A受体与SHISA的相互作用改变了许多GABA配体的结合活性。已经筛选了近2000种化合物并将其描述为GABA A受体调节剂。 Ninds以外的实验室： Simeonov ncats 我们一直在与他们的组合作筛选化合物，以抑制TAT介导的HIV LTR激活，评估和表征确认性测定中的活性分子。 McMahon实验室（NIMH）从对照和神经精神病患者中产生荧光标记NSC和神经元培养物，以表征其响应压力源或有毒挑战的形态变化。完全的 Safavi实验室Niaid：他们的实验室从患者衍生的样品中产生了IPSC线，并用MEA和荧光显微镜表征了衍生的神经元种群。壁外合作：乔纳森·盖格实验室（Univ North Dakota Med School）我们正在与Geiger实验室合作研究内溶性功能在HERV-K-KENV蛋白的神经毒性活性中的作用。 Asish Shah实验室（MED UNIV迈阿密学校）具有干细胞类似特性的胶质母细胞瘤细胞含有升高的HERV-K ENV水平，而HERV-K元素有助于胶质母细胞瘤的干细胞生态位，这表明HERV-K可能是这些肿瘤细胞的治疗靶标。细胞系的产生神经疗法开发单元（NTDU）：作为促进转化研究的作用的一部分，NTDU在人神经干细胞（NSC）中开发了内部和协作使用的细胞系。已经开发了线条以无处不在表达TDTOMATO荧光蛋白，以帮助进行筛选分析。 NTDU与壁内和壁外组之间的合作导致了十多种此类NSC线的发展，这将使进行旨在针对多种神经系统疾病在内的中等吞吐量筛查，包括双相情感障碍和阿尔茨海默氏症疾病。此外，NTDU还生成了其他特定于内部用途的单元线，还可以为协作者使用。这些线使用细胞特异性启动子在神经元和星形胶质细胞中表达TDTOMATO，并且还可能具有广泛的效用，可以在不同的神经细胞中进行筛选。