Infrastructure for Translational Neuroscience Center

转化神经科学中心基础设施

• 批准号: 8940123
• 负责人: Avindra Nath
• 金额: $ 265.88万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 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/8940123
• 关键词: Adult; Affinity; Affinity Chromatography; Agonist; Albumins; Antibodies; Antisense Oligonucleotides; Astrocytes; Autistic Disorder; Bioinformatics; Biological Assay; Biological Markers; Blood; CD34 gene; Cardiac Glycosides; Cell Line; Cell Membrane Proteins; Cell membrane; Cells; Cellular Morphology; Cerebrospinal Fluid; Chemistry; Clinical; Collaborations; Collection; Data Set; Digoxin; Disease; Enzyme-Linked Immunosorbent Assay; Experimental Designs; Extramural Activities; Fluorescence; Gagging; Gene Expression; Generations; Genes; Goals; HERVs; HIV; HIV tat Protein; Hippocampus (Brain); Human; Image; Immobilized Enzymes; In Vitro; Individual; Inflammation; Inflammatory; Intervention; Label; Lead; Life; Liquid Chromatography; Luciferases; Manuscripts; Methods; Modality; Motor Neurons; National Institute of Neurological Disorders and Stroke; Nervous system structure; Neurodegenerative Disorders; Neurons; Oligodendroglia; Ontology; Ouabain; Paper; Pathogenesis; Patients; Peptides; Peripheral; Phase; Play; Pluripotent Stem Cells; Poison; Progress Reports; Proteins; Proteomics; Protocols documentation; Publications; Publishing; Rattus; Reaction; Recombinant Proteins; Regenerative Medicine; Relative (related person); Research; Research Infrastructure; Research Personnel; Research Project Grants; Resolution; Role; Sampling; Series; Serum; Small Interfering RNA; Stem Cell Development; Stem cells; Structure; T-Lymphocyte; Techniques; United States National Institutes of Health; Vascular Endothelial Growth Factor Receptor; Vascular Endothelial Growth Factors; Work; base; cellular imaging; dopamine D3 receptor; enzyme reactor; high throughput screening; induced pluripotent stem cell; inhibitor/antagonist; method development; model development; multiple reaction monitoring; nerve stem cell; nervous system disorder; neurite growth; neurogenesis; neurotoxic; new therapeutic target; novel diagnostics; programs; relating to nervous system; research study; screening; self-renewal; stable cell line; stemness; tandem mass spectrometry; tool; translational neuroscience

Progress Report for Neural Differentiation Unit For the past year, the Neural Differentiation Unit has made continuous progress.We also initiated collaborations with other groups within NINDS and NIH. Specific aim 1: In vitro neurogenesis and development modeling using neural cultures derived from human adult peripheral CD34+ cells. We optimized our protocol and we can now derive both neural stem cells (iNS) and iPSC cells simultaneously from 10 ml of blood. We have published a paper on this technique and another video protocol paper is in press. Specific aim 2:Study the effect of inflammation on oligodendrocyte progenitor cells.We found that T cells released VEGF to increase OPC proliferation through VEGF receptor type 2 (VEGF-R2).These findings may have potential impact on treating T cell inflammatory-related neurodegenerative disorders. A manuscript based on these findings has been submitted for publication. Specific aim 3:Study the effect of HERV K on pluripotent stem cell development. We have found that HERV K components, gag, env and pol expression were increased in iPSCs but diminished rapidly after differentiation. We found Herv-K Env interacted with cell membrane protein CD98HC, which plays an important role in maintaining stemness and cell morphology. Inhibition using siRNA or antibody against Env resulted in stem cell morphological changes. These finding suggested an important role HERV-K may have played in stem cell development. A paper is in prepared for publication based on these findings. Specific aim 4: Study the mechanisms of neurodegenerative disorders. We are generating more iPS/iNS from cells of ALS (3 cell line) and PLS (6 cell lines) in collaboration with Dr. Mary Key Floeter. We are differentiating the cells to motoneurons and will compare their gene expressions with normal controls using microarray. We are also providing human neural cells for Dr. Roche to study the possible role of neuroligin-4 in Autism. Our unit also deriving iNS cells from patient samples from Undiagnosed disorders program and differentiate neural cells to study disease pathogenesis. Progress Report Summary for NTDU Within the NTDU group, we have utilized a number of automated, medium throughput assays in the past year, to screen small compound collections against rat mixed cortical cultures, human neural stem cells and human NSC-derived neurons along with some astrocyte cultures.Weve utilized live cell imaging based assays with rat hippocampal/cortical neurons, as well as developed fluorescence and high resolution phase contrast assays to probe high content imaging endpoints. These assays have been employed in multiple screens, working with numerous investigators (Rao, Malik-CRM, Pant, Major and others). We have continued to generate probe or tool compounds in our chemistry labs to facilitate screening and mechanistic studies in our research projects. We produce and purify a number of recombinant proteins, such as the HIV Tat protein for our own studies and provide that to extramural researchers around the world for their studies. With the Section of Insections of the Nervous System (SINS), we have continued to work on three research projects. We have found that the HERVKenv protein is toxic to mixed rat hippocampal cultures, human neural stem cells and human NSC-derived neurons. The HIV Tat antagonist program has progressed with the generation of LTR-luciferase constructs that are more sensitive to Tat in eliciting Tat-dependent selective LTR activation, and upon incorporation into a stable cell line, will facilitate screening of the 14,000 compound Maybridge HitCreator collection for new lead Tat inhibitors. The studies incorporating HIV Tat with A1-40 continue, as we evaluate whether these neurotoxic effects result from cell membrane interactions or from intracellular means. The NTDU has completed a number of studies in collaboration with the Center for Regenerative Medicine, developing a high throughput assay to discover compounds that were toxic to neural stem cells, but not neurons or astrocytes. The screen of 2000 compounds identified more than 50 compounds toxic to human NSCs but not mixed rat cortical neurons. One class of compounds that we identified as being particularly toxic to human but not rat neural cells, was cardiac glycosides, like digoxin, oleandrin, lanatoside C and ouabain. We continue to work with Dr. Harish Pant and his research team to characterize the neuroprotective actions of the Cdk5 modulator TFP-5. In another series of experiments, treatment of neural stem cells with TFP-5 had no significant effect on NSC proliferation and self renewal. However, we observed that treatment of NSCs with TFP-5 resulted in an accelerated neurite growth and branching. We continue to work with Gene Majors lab to develop a hybridization ELISA assay to quantitate the JCV antisense oligonucleotide levels in serum and CSF. Likewise, we are beginning to work with David Sibleys group to characterize the dopamine D3 receptor agonists that they have identified, using our live cell imaging platforms for quantitative analysis. The Clinical Proteomics Unit goal is to initiate discussions with collaborators in order to provide guidance on aspects of experimental design that are critical to producing high quality analyses and statistically meaningful results. We utilize multi-dimensional liquid chromatography as a primary means of reducing sample complexity but also utilize affinity and electrophoretic techniques when indicated. A significant portion of the Clinical Proteomics Unit efforts focus on methods development. We are developing an automated workflow for cerebrospinal fluid (CSF). Our strategy is to use affinity chromatography to deplete albumin and IgGs from CSF prior to hydrolyzing the remaining proteins in an immobilized enzyme reactor and capture the resultant peptide mixture. Individual depleted CSF samples are labeled with an isobaric mass tags, pooled with several other samples and analyzed by tandem mass spectrometry. Bioinformatic analysis then affords both protein identification and relative quantitation. This list of candidate protein biomarkers will be rapidly quantified in large numbers of clinical samples by Parallel Reaction Monitory (PRM), a high resolution form of Multiple Reaction Monitoring (MRM). Proteomic analysis of non-depleted CSF resulted in 887 protein IDs, while our method resulted in 630 proteins IDs of which 341 were unique to the depleted CSF dataset. Importantly, Gene Ontology analysis indicates a majority of the unique IDs were related to neuronal structure and function.

神经分化单元进展报告 在过去的一年里，神经分化单元不断取得进展。我们还与 NINDS 和 NIH 内的其他小组开展了合作。 具体目标 1：使用源自人类成人外周 CD34+ 细胞的神经培养物进行体外神经发生和发育建模。我们优化了方案，现在可以从 10 ml 血液中同时获取神经干细胞 (iNS) 和 iPSC 细胞。我们已经发表了一篇关于该技术的论文，另一篇视频协议论文正在出版。 具体目标2：研究炎症对少突胶质祖细胞的影响。我们发现T细胞释放VEGF，通过VEGF受体2型（VEGF-R2）增加OPC增殖。这些发现可能对治疗T细胞炎症相关的神经退行性疾病具有潜在影响。基于这些发现的手稿已提交出版。 具体目标3：研究HERV K对多能干细胞发育的影响。我们发现 HERV K 成分、gag、env 和 pol 表达在 iPSC 中增加，但在分化后迅速减少。我们发现 Herv-K Env 与细胞膜蛋白 CD98HC 相互作用，在维持干性和细胞形态方面发挥着重要作用。使用 siRNA 或针对 Env 的抗体进行抑制会导致干细胞形态发生变化。这些发现表明 HERV-K 可能在干细胞发育中发挥重要作用。根据这些发现，正在准备发表一篇论文。 具体目标4：研究神经退行性疾病的机制。我们与 Mary Key Floeter 博士合作，从 ALS（3 个细胞系）和 PLS（6 个细胞系）细胞中产生更多的 iPS/iNS。我们正在将细胞分化为运动神经元，并将使用微阵列将它们的基因表达与正常对照进行比较。我们还为 Roche 博士提供人类神经细胞，以研究 Neuroligin-4 在自闭症中的可能作用。我们的部门还从未确诊疾病项目的患者样本中提取 iNS 细胞，并分化神经细胞来研究疾病发病机制。 NTDU 进度报告摘要 在 NTDU 小组内，我们在过去的一年中利用了许多自动化、中等通量的测定，针对大鼠混合皮质培养物、人类神经干细胞和人类 NSC 衍生神经元以及一些星形胶质细胞培养物筛选了小型化合物集合。我们利用基于活细胞成像的大鼠海马/皮质神经元测定，以及开发的荧光和高分辨率相差测定来探测高通量 内容成像端点。 这些测定已与众多研究人员（Rao、Malik-CRM、Pant、Major 等）合作用于多种筛选。我们继续在化学实验室中生产探针或工具化合物，以促进我们研究项目中的筛选和机理研究。 我们生产和纯化许多重组蛋白，例如用于我们自己研究的 HIV Tat 蛋白，并将其提供给世界各地的校外研究人员进行研究。 在神经系统昆虫科 (SINS) 中，我们继续开展三个研究项目。 我们发现 HERVKenv 蛋白对混合大鼠海马培养物、人类神经干细胞和人类 NSC 衍生神经元具有毒性。 HIV Tat 拮抗剂计划随着 LTR 荧光素酶构建体的产生而取得进展，该构建体对 Tat 更敏感，可引发 Tat 依赖性选择性 LTR 激活，并且在纳入稳定细胞系后，将有助于筛选 14,000 种化合物 Maybridge HitCreator 集合中的新先导 Tat 抑制剂。 将 HIV Tat 与 A1-40 结合的研究仍在继续，我们评估这些神经毒性作用是由细胞膜相互作用还是细胞内手段引起的。 NTDU 与再生医学中心合作完成了多项研究，开发了一种高通量测定法，以发现对神经干细胞有毒的化合物，但对神经元或星形胶质细胞无毒。 对 2000 种化合物的筛选鉴定出 50 多种对人类 NSC 有毒的化合物，但对混合的大鼠皮质神经元没有毒性。 我们确定的一类对人类神经细胞特别有毒但对大鼠神经细胞没有毒性的化合物是强心苷，如地高辛、夹竹桃苷、羊毛花苷 C 和哇巴因。我们继续与 Harish Pant 博士及其研究团队合作，研究 Cdk5 调节剂 TFP-5 的神经保护作用。 在另一系列实验中，用TFP-5处理神经干细胞对NSC增殖和自我更新没有显着影响。然而，我们观察到用 TFP-5 处理 NSC 会导致神经突生长和分支加速。 我们继续与 Gene Majors 实验室合作开发杂交 ELISA 测定法，以定量血清和脑脊液中的 JCV 反义寡核苷酸水平。 同样，我们开始与 David Sibleys 团队合作，使用我们的活细胞成像平台进行定量分析，表征他们已识别的多巴胺 D3 受体激动剂。 临床蛋白质组学部门的目标是与合作者发起讨论，以便为实验设计方面提供指导，这对于产生高质量的分析和具有统计意义的结果至关重要。我们利用多维液相色谱作为降低样品复杂性的主要手段，但在有需要时也利用亲和和电泳技术。 临床蛋白质组学部门的很大一部分工作集中在方法开发上。我们正在开发脑脊液 (CSF) 的自动化工作流程。我们的策略是使用亲和层析去除脑脊液中的白蛋白和 IgG，然后在固定化酶反应器中水解剩余的蛋白质并捕获所得的肽混合物。单个耗尽的脑脊液样本用同量异位质量标签标记，与其他几个样本合并并通过串联质谱法进行分析。然后生物信息学分析提供蛋白质鉴定和相对定量。该候选蛋白质生物标志物列表将通过平行反应监测 (PRM)（多重反应监测 (MRM) 的一种高分辨率形式）在大量临床样本中快速定量。 非耗尽 CSF 的蛋白质组学分析得出 887 个蛋白质 ID，而我们的方法得出 630 个蛋白质 ID，其中 341 个是耗尽 CSF 数据集特有的。重要的是，基因本体分析表明大多数独特 ID 与神经元结构和功能有关。