Targeting intracellular calcium leak as novel therapy for diabetes

 描述(由申请人提供)：拟议的研究性研究将有助于改善首席调查员(PI)的教育和职业目标。此外，候选人将研究2型糖尿病(T2 DM)胰岛素分泌受损的机制。候选人已经为拟议研究的头两年(K99阶段)制定了一个重要的教育计划。该计划还包括课程。此外，候选人还将接受导师和其他专家的科学和专业研究技能培训。候选人将进行实验，以研究细胞内钙泄漏在T2 DM病理生理学中的功能作用的分子机制，并将为此类疾病的治疗提出创新的、有针对性的治疗方法。在指导阶段结束后，候选人将建立一个数据、知识和科学技能池，这将增强他成功过渡到独立的资历。在K99阶段，候选人计划参加关于生物统计学、拨款撰写/研究资金、人类主体保护和负责任的研究行为的课程。此外，他还与他的导师和他的顾问/合作者制定了一项具体的培训计划，为他提供关于内质网应激/线粒体健康、β细胞动力学、离子通道的科学知识。这些方面代表着有用的工具，他将能够转移到自己的实验室。除了他将获得的新技术外，拟议中的项目还需要他最近才接触到的领域的知识，如线粒体病理生理学和代谢信号。因此，他在他的顾问委员会中包括了一些在这些特定领域具有杰出职业生涯的科学家，这些科学家将 提供尽可能高级别的指导。他的所有顾问和合作者都是顶尖的学术科学家(见《预算理由》文件中有关人员的完整名单)。候选人还将在几个国际会议上介绍他的工作，以便获得广泛的科学受众的意见，从而扩大他的科学网络。这将促进他向独立研究职位的过渡，并为他申请独立R01级资金提供坚实的科学基础。应用的核心问题是：(1)通过RyR2的细胞内钙泄漏在胰腺β细胞中的作用是什么？(2)含有RyR2通道泄漏的β细胞中葡萄糖刺激的胰岛素分泌受损的机制是什么？(3)如何防止T2 DM供体或糖尿病动物模型的人胰岛发生这种泄漏？(4)预防细胞内钙泄漏如何影响T2 DM的病理生理？为了解决这些问题，候选人设计了一个有两个主要分支的实验计划。第一个阶段为K99阶段，以目前提交的初步数据为基础。这组实验旨在确定β细胞中钙离子通过RyR2通道泄漏的机制，RyR2通道是迄今为止未披露的葡萄糖诱导的胰岛素释放的关键角色。申请人将使用携带RyR2基因突变的敲入小鼠在体内和体外表征泄漏RyR2的功能方面，该基因突变使通道泄漏。同样的突变是一种罕见的综合征的特征，称为儿茶酚胺能多形性室性心动过速(CPVT)；有趣的是，作为支持本应用的材料提供的研究表明，这些患者中的大多数都表现出葡萄糖耐量异常。候选人将验证体外泄漏诱导对胰岛素释放的影响。此外，他将在CPVT小鼠模型中测试一种口服可用小分子(Rycal)，看看这种能够防止细胞内钙泄漏的药物干预是否可以影响β细胞的动力学。该计划的第二个分支进入R00阶段，候选人将在此阶段确定通过RyR2抑制细胞内钙泄漏是否改善了已建立的T2 DM模型的葡萄糖稳态。这些模型将以高通量分析方法为特征，并可能出现新的目标。同样重要的是，阐明将在K99阶段研究的机制(内质网应激、线粒体、离子通道)也可能识别新的靶点，并可能为T2 DM的治疗提供新的方法。最终，这些策略和干预措施的应用，以及针对新因素的分析，集中在内质网应激和钙离子通量上，很可能将为治疗糖稳态受损的情况指明新的方向。而且，凭借申请者在K99阶段获得的科学和专业技能，向独立职位的过渡肯定会更容易。总体而言，目前的申请将使应聘者具备新的知识和有用的工具，以继续在分子代谢和糖尿病研究方面的独立职业生涯。他的培训计划经过精心设计，以促进在哥伦比亚大学突出的环境中完美地产生数据，以及PI对现代生物医学研究工具的熟悉，他将把这些工具转移到他的独立实验室。他的K99/R00奖申请的批准将导致对影响β-细胞病理生理学的新机制的准确定义，挑战现有的范式，从而为糖尿病的治疗提供潜在的新疗法。

 DESCRIPTION (provided by applicant): The proposed research study will facilitate the improvement of the education and career goals of the principal investigator (PI). Moreover, the candidate will investigate the mechanisms that underlie impaired insulin secretion in type 2 diabetes mellitus (T2DM). The candidate has set up an important educative plan for the first two years (K99 phase) of the proposed study. This plan includes also courses. In addition, the candidate will be trained in scientific and professional research skills by his mentors and other experts. The candidate will perform experiments to investigate the molecular mechanisms underlying the functional role of intracellular calcium leak in the pathophysiology of T2DM and will suggest innovative, targeted therapeutic approaches for the treatment of such a disease. Upon conclusion of the mentored phase the candidate will have established a pool of data, knowledge and scientific skills that will enhance his credentials for successful transition to independence. During the K99 phase the candidate has planned to attend courses on biostatistics, grant writing/research funding, human subject protection, and responsible conduct of research. In addition, he has set up a specific training plan with his mentors and his advisors/collaborators that will provide him with scientific knowledge on ER stress/mitochondrial fitness, β-cell dynamics, ion channels. These aspects represent useful tools that he will be able to transfer to his own laboratory. Besides the new techniques that he will acquire, the proposed project requires knowledge in areas that he has only recently been associated with, such as mitochondrial pathophysiology and metabolic signaling. Therefore, he has included in his advisory board a number of scientists with outstanding careers in these particular fields that will provide guidance of the highest possible level. All his advisors and collaborators are top-notch academic scientists (see a complete list in Personnel, in the `Budget justification' document). The candidate will also present his work at several international meetings, in order to receive input by a broad scientific audience, thereby expanding his scientific network. This will promote his transition to an independent research position and provide him a robust scientific foundation from which to apply for independent R01-level funding. The application core questions are: (1) Which is the functional role of intracellular Ca2+ leak via RyR2 in pancreatic β-cells? (2) What i the mechanism underlying impaired glucose-stimulated insulin secretion in β-cells harboring leaky RyR2 channels? (3) How can the leak be prevented in human islets from T2DM donors or animal models of diabetes? (4) How the prevention of intracellular Ca2+ leak can impact the pathophysiology of T2DM? To address these questions the candidate has designed an experimental plan with two main branches. The first falls into the K99 phase and is based on preliminary data of the current submission. This set of experiments aims to identify the mechanism that makes the Ca2+ leak via RyR2 channels in β-cells a heretofore undisclosed key player in glucose-induced insulin release. The applicant will characterize both in vivo and ex vivo the functional aspects of leaky RyR2 using knock-in mice harboring genetic mutations in RyR2 that renders the channel leaky. The same mutations characterize a rare syndrome, known as catecholaminergic polymorphic ventricular tachycardia (CPVT); intriguingly, studies provided as material supporting the present application have demonstrated that most of these patients display glucose intolerance. The candidate will verify the effects of the induction of the leak in vitro on insulin release. Moreover, he will test an orally available small molecule (Rycal) in the CPVT mouse models, to see whether such pharmacological intervention, able to prevent intracellular Ca2+ leak, can affect β-cell dynamics. The second branch of the plan falls into the R00 phase, where the candidate will determine whether the inhibition of intracellular Ca2+ leak via RyR2 improves glucose homeostasis in established models of T2DM. These models will be characterized with high throughput analysis methods and new targets may come up. Equally important, the elucidation of the mechanisms that will be investigated in the K99 phase (ER stress, mitochondria, ion channels) might also identify new targets and may suggest novel approaches for the treatment of T2DM. Ultimately, application of these strategies and interventions, as well as targeting of new factors that the analysis focused on ER Stress and Ca2+ fluxes is most likely going to indicate new directions to treat conditions of impaired glucose homeostasis. Moreover, with the scientific and professional skills acquired by the applicant during the K99 phase, the transition to independent position will be certainly facilitate. Overall, the current application will equip the candidate with novel knowledge and useful tools to continue for independent career in molecular metabolism and diabetes research. His training plan has been meticulously designed to facilitate flawless production of data in the prominent environment of Columbia University, as well as the PI's acquaintance with modern tools of biomedical research that he will transfer to his independent laboratory. Approval of his application for the K99/R00 award will result in the precise definition of novel mechanisms affecting β-cell pathophysiology, challenging current paradigms and will thereby provide potential new therapeutics for the treatment of diabetes mellitus.