Development of Novel Cell Free Therapies to Prevent Age-related Motor Neuron loss

解决老龄化的不利影响不仅是BBSRC的一个优先研究领域，也是工业部门的一个优先研究领域。该项目旨在通过建立学术界（雷丁大学）和工业界（自然生物科学有限公司）之间的联盟，利用雷丁大学的一项新发现，开发新的无细胞试剂，以防止年龄引起的运动神经元损失。大量文献表明，随着年龄的增长超过60岁，由于MN的丧失导致生活质量下降，甚至经常死亡，人体肌肉会发生去神经支配。永久性失神经支配的肌肉最终会消失，取而代之的是脂肪和纤维组织。本项目将利用我们及时的发现，小鼠中肌生长抑制素基因的缺失导致了MN的前所未有的存活，而MN通常会在生长过程中通过程序性细胞死亡被消除（Elashry et al . 2010 Journal of Anatomy, submitted）。由于肌肉生长抑制素突变只影响骨骼肌，我们已经提出了“骨骼肌产生扩散营养因子作为神经保护剂，防止与年龄相关的运动神经元死亡”的中心假设。这个假设将由学生来检验。这项研究将利用雷丁大学的帕特尔教授和自然生物科学有限公司的史蒂夫·雷博士之间的战略联盟，后者为研究领域带来了广泛的（包括促进神经再生的无细胞疗法的专业知识开发）和研究试剂（显示MN过早死亡的小鼠系，包括SODG93A小鼠系）。项目目的：(1)使用体外平台确定在缺乏肌肉生长抑制素的情况下发育的MN是否对神经毒素具有抗性。(2)确定体内肌生长抑制素缺失时形成的抗衰老MN是否表现出年轻MN的生存特征。(3)通过体外实验确定肌生成抑制素组织生成的细胞游离试剂（条件培养基、微泡、RNA）是否具有神经保护作用。(4)确定目标(3)中开发的无细胞试剂是否在体内具有神经保护特性。时间：0-9个月。通过标准方法从野生型和肌肉生长抑制素缺失小鼠中分离和培养胎儿MN （Kume etal . (2005) Neuroscience Letters 383:199-202）。培训：细胞培养，使用转基因动物。地点：英国雷丁大学，10-18个月。MN从肌生长抑制素无效到暴露于谷氨酸或一氧化氮激动剂的神经保护特性将被建立。培训：细胞培养，数据统计分析。地点：NATURAL BIOSCIENCES LTD . 12-18个月。建立肌肉生长抑制素/SODG93A细胞系。培训：动物处理，分子生物学。地点：雷丁大学19-30个月。肌生长抑制素/ SODG93A系运动神经的组织学检查。培训：共焦显微镜，电子显微镜。地点：雷丁大学28-33个月。从肌肉生长抑制素无效的肌肉中分离无细胞生物活性试剂（条件培养基、微泡、RNA）。培训：分子生物学，生物化学。地点：NATURAL BIOSCIENCES LTD . 28-36个月。证实从肌肉生长抑制素缺失小鼠中提取的细胞游离试剂的神经保护特性，以防止谷氨酸或一氧化氮激动剂的毒性作用。培训：毒理学，统计学。地点：NATURAL BIOSCIENCES LTD . 36-48个月。量化SODG93A小鼠肌生成抑制素无源细胞游离试剂的神经保护证据。培训:组织学。学生将每24个月在Patel教授和NATURAL BIOSCIENCES LTD.的实验室中度过。Natural Biosciences SA每年将提供15000英镑用于动物、组织培养、试剂和差旅费。这个项目的产出将用于未来的项目拨款申请-详情将在后面的章节中介绍。

Addressing the adverse effects of ageing is a priority research area not only of the BBSRC but also of the Industrial sector. This project aims to develop new and novel cell free reagents to prevent age-induced motor neuron loss by forging an alliance between academia (University of Reading) and industry (NATURAL BIOSCIENCES LTD) to exploit a new finding made at the University of Reading. An extensive body of literature exists showing that as age increases beyond 60 years, human muscle undergoes denervation, due to a loss of MN that leads to a poor quality of life and often death. Permanently denervated muscle is ultimately lost and replaced by fat and fibrous tissue. This project will exploit our timely finding that deletion of the gene Myostatin in the mouse results in an unprecedented survival of MN, which are normally eliminated by programmed cell death during (Elashry et al 2010 Journal of Anatomy, submitted). Since the myostatin mutation only affects skeletal muscle, we have developed the central hypothesis 'that the skeletal muscle produces diffusible trophic factors that act as neuro-protective agents that prevent age related motor neuron death'. The hypothesis will be tested by the student. This study will take advantage of a strategic alliance between the groups of Professor Patel, who at the University of Reading have made this key discovery and Dr Steve Ray of NATURAL BIOSCIENCES LTD who brings to the research arena extensive (including expertise development of cell free therapies to promote neural regeneration) and research reagents (mouse lines displaying premature MN death, including SODG93A mouse line. Project aims: (1) To determine using in-vitro platforms whether MN that develop in the absence of myostatin are resistant to neuro-toxins. (2) To determine in-vivo whether the ageing resistant MN that form in the absence of myostatin display the survival characteristics of young MN. (3) To conduct in-vitro experiments to determine whether cell free reagents (conditioned media, micro vesicles, RNA) generated from myostatin null tissue harbour neuro-protective properties. (4) To determine whether cell free reagents developed in aim (3) offer neuro-protective properties in an in-vivo context. Timeline: 0-9 months. Isolation and culturing foetal MN from wild type and myostatin null mice by standard methods (Kume et al (2005) Neuroscience Letters 383:199-202). Training: cell culture, working with transgenic animals. Venue: University of Reading 10-18 months. Neuroprotective properties of MN from myostatin nulls to exposure to glutamate or nitric oxide agonists will be established. Training: cell culture, statistical analysis of data. Venue: NATURAL BIOSCIENCES LTD 12-18 months. Establish myostatin/SODG93A line. Training: animal handling, molecular biology. Venue: University of Reading 19-30 months. Histological examination of motor nerves in myostatin/ SODG93A line. Training: con-focal microscopy, electron microscopy. Venue: University of Reading 28-33 months. Isolation of cell free biologically active reagents (conditioned media, micro vesicles, RNA) from myostatin null muscle. Training: molecular biology, biochemistry. Venue: NATURAL BIOSCIENCES LTD 28-36 months. Confirm the neuroprotective properties of cell free reagents derived from myostatin null mice to prevent the toxic effects of glutamate or nitric oxide agonists. Training: toxicology, statistics. Venue: NATURAL BIOSCIENCES LTD 36-48 months. Quantify evidence of neuroprotection of cell free reagents from myostatin nulls in SODG93A mice. Training: histology. Venue: University of Reading The student will spend about each 24 months in the laboratory of Professor Patel and NATURAL BIOSCIENCES LTD. Natural Biosciences SA will provide £15,000 per year for animal, tissue culture, reagent and travel costs. Outputs from this project will be used for a future project grant application- details in later sections.