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

描述（由申请人提供）：青光眼是全球最常见的致盲原因之一，虽然有许多不同形式的青光眼在临床表现和疾病进展方面存在显着差异，但它们都有一个共同的终点，即视网膜神经节细胞的丧失（RGC）。需要新的治疗方法，因为目前的治疗方法可以延迟，但不能阻止疾病的进展。开发新疗法的主要障碍之一是对疾病发病机制的不完全理解。最近的研究表明RGCs的缺失是由于细胞凋亡引起的，然而，触发细胞凋亡的分子机制仍然存在争议。Fas配体（FasL）是一种促凋亡蛋白，其在视网膜中组成性表达，在视网膜中其被认为通过诱导浸润性炎性细胞的凋亡或通过防止新生血管生成来维持免疫赦免以保护组织免受破坏。然而，有两种形式的FasL，膜结合形式（mFasL）和通过金属蛋白酶切割膜结合蛋白产生的可溶性形式（sFasL）。我们以前对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品系，其中FasL金属蛋白酶切割位点发生突变，以防止膜结合蛋白的切割。在这些小鼠中，FasL表达和调节正常，但它们不能切割FasL，因此，只能表达mFasL（称为？CS小鼠）。这是第一次，这些小鼠使我们能够研究在体内功能的膜FasL的可溶性FasL的情况下。为了确定mFasL在青光眼发生发展中的作用，我们将mFasL基因与青光眼发生发展相关基因进行回交。将CS敲入突变导入DBA/2 J菌株（D2. ? CS小鼠），其发展自发性眼内压（IOP）升高和RGC损失。我们的初步研究结果表明，mFasL是一个关键的中介RGC凋亡过程中的青光眼的发展。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中的其他视网膜神经元。我们推测，在响应于由IOP升高引起的损伤，视网膜内Fas和FasL的表达增加。然而，视网膜细胞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更高水平的mFasL，而神经保护性小胶质细胞表达更高水平的sFasL。将在三个特定目的中检验该假设：（目的1）证明D2中IOP升高。?不能切割FasL的CS小鼠触发增加的视网膜神经毒性。(Aim 2）确定在D2中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缺乏。(Aim 3）使用与来自D2的永生化视网膜小胶质细胞的共培养系统确定小胶质细胞介导的体外神经毒性的机制。? CS小鼠和原代RGC。 公众健康相关性：青光眼是一种慢性疾病，最终可导致视力丧失和失明。当视网膜中的某种细胞死亡（视网膜神经节细胞）时，就会发生视力丧失。科学家们还不清楚这些细胞死亡的确切原因。我们至少发现了这些细胞死亡的一个原因。此外，我们还发现了一种阻止这些细胞死亡的方法。这项研究将为青光眼的治疗干预带来新的靶点。

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.