Neurotoxicity and neuroprotection in the DBA/2J spontaneous model of glaucoma

DBA/2J 自发性青光眼模型的神经毒性和神经保护

• 批准号: 8389866
• 负责人: MEREDITH GREGORY-KSANDER
• 金额: $ 46.08万
• 依托单位: SCHEPENS EYE RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-12-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8389866
• 关键词: Apoptosis; Apoptotic; Backcrossings; Binding; Binding Proteins; Blindness; Brain; CD95 Antigens; Cell Death; Cell Surface Proteins; Cells; Cessation of life; Chronic Disease; Cleaved cell; Clinical; Coculture Techniques; Development; Disease; Disease Progression; Eye; Glaucoma; Immune; In Vitro; Inflammatory Infiltrate; Knock-in Mouse; Lead; Mediating; Mediator of activation protein; Membrane; Metalloproteases; Microglia; Modeling; Molecular; Mouse Strains; Mus; Mutate; Mutation; Neuroglia; Pathogenesis; Patients; Physiologic Intraocular Pressure; Predisposition; Primary Open Angle Glaucoma; Problem Solving; Research; Retina; Retinal; Retinal Ganglion Cells; Scientist; Site; System; Testing; Therapeutic Intervention; Time; Tissues; Tumor Necrosis Factor Ligand Superfamily Member 6; Work; base; cell type; in vivo; interest; neuroprotection; neurotoxic; neurotoxicity; novel; pressure; prevent; pro-apoptotic protein; response to injury; retinal apoptosis; retinal neuron; stem

DESCRIPTION (provided by applicant): Glaucoma is one of the most common causes of blindness worldwide and, while there are many different forms of glaucoma that differ significantly in clinical presentation and disease progression, they all share a common endpoint which is the loss of retinal ganglion cells (RGCs). New treatments are needed, since current therapies can delay, but not stop disease progression. One of the major barriers to the development of novel treatments is the incomplete understanding of the disease pathogenesis. Recent evidence indicates the loss of RGCs is due to apoptosis, nevertheless, the actual molecular mechanism that triggers apoptosis is still controversial. Fas Ligand (FasL) is a pro-apoptotic protein that is constitutively expressed in the retina where it is thought to protect tissue from destruction by maintaining immune privilege, either by inducing apoptosis of infiltrating inflammatory cells or by preventing neoangeogenesis. However, there are two forms of FasL, a membrane-bound form (mFasL) and a soluble form (sFasL) that is produced by metalloproteinase cleavage of the membrane-bound protein. Our previous studies on the function of mFasL and sFasL indicate that mFasL is pro-apoptotic, while sFasL is anti-apoptotic. We recently developed a unique knock-in mouse strain in which the FasL metalloproteinase cleavage sites were mutated to prevent cleavage of the membrane-bound protein. In these mice, FasL is expressed and regulated normally, but they are unable to cleave FasL and therefore, can only express mFasL (termed ? CS mice). For the first time, these mice have allowed us to study the in vivo function of membrane FasL in the absence of soluble FasL. In order to determine the function of mFasL in the development of glaucoma, we backcrossed the ? CS knock-in mutation into the DBA/2J strain (D2. ? CS mice) that develops spontaneous elevated intraocular pressure (IOP) and loss of RGCs. Our preliminary results indicate that mFasL is a critical mediator of RGC apoptosis during the development of glaucoma. Moreover, sFasL has an important neuroprotective effect that not only prevents loss of RGCs, but also protects other retinal neurons in hypertensive eyes. We hypothesize that in response to injury caused by elevated IOP, there is increased expression of Fas and FasL within the retina. However, the extent of retinal apoptosis is determined by the ratio of membrane / soluble FasL expressed by microglia. Neurotoxic microglia express higher levels of mFasL, while neuroprotective microglia express higher levels of sFasL. This hypothesis will be tested in three Specific Aims: (Aim 1) Demonstrate that elevated IOP in D2. ? CS mice that are unable to cleave FasL triggers increased retinal neurotoxicity. (Aim 2) Determine whether increased retinal neurotoxicity in D2. ? CS mice is due to increased mFasL expression and/or the absence of sFasL. (Aim 3) Determine the mechanisms of microglia-mediated neurotoxicity in vitro using a co-culture system with immortalized retinal microglia from D2. ? CS mice and primary RGCs.

青光眼是世界范围内最常见的致盲原因之一，虽然青光眼有许多不同的形式，在临床表现和疾病进展方面存在显著差异，但它们都有一个共同的终点，即视网膜神经节细胞（RGCs）的丧失。需要新的治疗方法，因为目前的治疗方法可以延缓，但不能阻止疾病的进展。发展新疗法的主要障碍之一是对疾病发病机制的不完全了解。最近的证据表明RGCs的丢失是由于细胞凋亡引起的，然而，触发细胞凋亡的实际分子机制仍然存在争议。Fas配体（FasL）是一种促凋亡蛋白，在视网膜中组成性表达，被认为通过诱导浸润性炎症细胞凋亡或阻止新生血管生成来维持免疫特权，从而保护组织免受破坏。然而，FasL有两种形式，一种是膜结合形式（mFasL），另一种是由金属蛋白酶裂解膜结合蛋白产生的可溶性形式（sFasL）。我们之前对mFasL和sFasL功能的研究表明，mFasL具有促凋亡作用，而sFasL具有抗凋亡作用。我们最近开发了一种独特的敲入小鼠品系，其中FasL金属蛋白酶裂解位点发生突变，以防止膜结合蛋白的裂解。在这些小鼠中，FasL正常表达和调节，但它们不能切割FasL，因此只能表达mFasL(称为？CS老鼠)。这些小鼠首次使我们能够在缺乏可溶性FasL的情况下研究膜FasL的体内功能。为了确定mFasL在青光眼发生发展中的作用，我们对？CS敲入突变为DBA/2J菌株(D2。？ CS小鼠)发生自发性眼压升高（IOP）和RGCs丢失。我们的初步结果表明mFasL是青光眼发生过程中RGC凋亡的关键介质。此外，sFasL具有重要的神经保护作用，不仅可以防止RGCs的丢失，还可以保护高血压眼的其他视网膜神经元。我们推测，在IOP升高引起的损伤反应中，视网膜内Fas和FasL的表达增加。然而，视网膜凋亡的程度是由小胶质细胞表达的膜/可溶性FasL的比例决定的。神经毒性小胶质细胞表达较高水平的mFasL，而神经保护性小胶质细胞表达较高水平的sFasL。这一假设将在三个特定目标中得到验证：（目标1）证明D2的IOP升高。？ 不能切割FasL的CS小鼠会增加视网膜神经毒性。（目的2）确定D2是否增加视网膜神经毒性。？ CS小鼠是由于mFasL表达增加和/或sFasL缺失所致。（目的3）利用D2永生化视网膜小胶质细胞共培养系统，确定小胶质细胞介导的体外神经毒性机制。？ CS小鼠和原代RGCs。