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: 8237645
• 负责人: MEREDITH GREGORY-KSANDER
• 金额: $ 48.5万
• 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/8237645
• 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. PUBLIC HEALTH RELEVANCE: Glaucoma is a chronic disease that can ultimately lead to loss of vision and blindness. The loss of vision occurs when a certain type of cell in your retina dies (retinal ganglion cells). It is unclear to scientists exactly why these cells die. We have discovered at least one reason why these cell die. In addition, we have also found a way to stop these cells from dying. This research will lead to novel targets for therapeutic intervention in glaucoma.

描述（由申请人提供）：青光眼是全球失明的最常见原因之一，尽管有许多不同形式的青光眼在临床表现和疾病进展中有很大差异，但它们都共享一个常见的终点，这是视网膜神经节细胞的丧失（RGC）。需要新的治疗方法，因为当前的疗法可能会延迟，但不能阻止疾病进展。新疗法发展的主要障碍之一是对疾病发病机理的不完全理解。最近的证据表明，RGC的丧失是由于细胞凋亡引起的，但是，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，在视网膜中构成型表达，在该蛋白质中，人们认为它通过维持免疫特权，通过诱导浸润性炎性细胞的凋亡或防止新生天的发生来保护组织免受破坏。但是，有两种形式的FASL，一种结合膜形式（MFASL）和可溶性形式（SFASL），这些形式是由金属蛋白酶裂解的膜结合蛋白的裂解产生的。我们先前关于MFASL和SFASL功能的研究表明，MFASL是促凋亡的，而SFASL是抗凋亡的。我们最近开发了一种独特的敲入小鼠菌株，其中将FASL金属蛋白酶裂解位点突变以防止膜结合蛋白的裂解。在这些小鼠中，FASL得到正常表达和调节，但它们无法裂解FASL，因此，只能表达MFASL（称为“ CS小鼠）”。这些小鼠首次使我们能够在没有可溶性FASL的情况下研究膜FASL的体内功能。为了确定MFASL在青光眼发展中的功能，我们回联系？ CS敲入突变进入DBA/2J菌株（D2。？CS小鼠），该菌株会自发地升高眼内压（IOP）和RGC丢失。我们的初步结果表明，MFASL是青光眼发展过程中RGC凋亡的关键介体。此外，SFASL具有重要的神经保护作用，不仅可以防止RGC损失，而且还可以保护高血压眼睛中的其他视网膜神经元。我们假设，为了响应IOP升高引起的损伤，视网膜内FA和FASL的表达增加。然而，视网膜凋亡的程度取决于小胶质细胞表达的膜 /可溶性FASL的比率。神经毒性小胶质细胞表达更高水平的MFASL，而神经保护小胶质细胞表达更高水平的SFASL。该假设将以三个特定的目的进行检验：（目标1）证明D2中的IOP升高。 ？无法裂解FASL触发的CS小鼠增加了视网膜神经毒性。 （AIM 2）确定D2中是否增加了视网膜神经毒性。 ？ CS小鼠是由于MFASL表达增加和/或缺乏SFASL所致。 （AIM 3）使用与D2永生化的视网膜小胶质细胞的共培养系统在体外确定小胶质细胞介导的神经毒性的机制。 ？ CS小鼠和初级RGC。 公共卫生相关性：青光眼是一种慢性疾病，最终可能导致视力和失明丧失。当视网膜死亡（视网膜神经节细胞）中的某种类型的细胞（视网膜神经节细胞）中的某种类型的细胞时，视力丧失。对于这些细胞而言，科学家尚不清楚为什么这些细胞死亡。我们发现了这些细胞死亡的至少一个原因。此外，我们还找到了一种阻止这些细胞死亡的方法。这项研究将导致对青光眼治疗干预的新靶标。