Shedding light on glaucoma: optogenetics regulation of ciliary phosphoinositides

 描述(由申请人提供)： 青光眼是世界上导致不可逆转失明的主要原因，它影响着超过28.5万名美国退伍军人。眼压升高与原发性开角型青光眼的发生和发展有关，而眼压是该病唯一可改变的危险因素。然而，导致高眼压的机制仍然知之甚少。小梁细胞对于维持正常的房水流量和眼压至关重要。在肾脏、骨骼和软骨等其他组织中，初级纤毛在液体流动中起着机械感觉的作用。在这里，我们进一步探索小梁细胞纤毛在眼睛中起机械感觉作用的可能性。事实上，我们之前的发现表明，初级纤毛中的肌醇磷脂是眼压的重要调节因素。在初级纤毛中，肌醇磷酸酶OCRL与TRPV4相互作用，TRPV4是一种机械感觉通道，当刺激TRPV4时，它能降低小鼠的眼压。先前的研究已经证实，肌醇磷脂调节TRPV4的活性。我们推测，小梁细胞纤毛内的肌醇磷脂信号通过TRPV4通道调节房水流出。在这一应用中，我们建议使用新的光遗传学技术来选择性地指导蛋白转位和睫状肌酸激酶/磷酸酶的激活，以调节TRPV4的活性和眼压。在目标1中，我们将通过光遗传刺激肌醇磷酸酶OCRL来确定通过OCRL引起的PIP2去磷酸化改变是否影响小鼠的睫状体流出能力和眼压。然后，我们将评估光刺激异位表达的OCRL是否可以挽救类固醇诱导的青光眼(SIG)小鼠模型。我们预计，通过光响应性OCRL激活小鼠眼前房中的TRPV4通道将降低眼压。在目标2中，我们将确定通过PI3K激活的PIP2磷酸化是否会影响野生型小鼠的睫状肌流出能力和眼压。 我们预计PI3K信号的光遗传刺激将增加眼压并减少流出设施。然后，我们将确定在类固醇诱导的青光眼小鼠模型中，抑制PI3K的小分子是否会降低眼压。在目标3中，我们将确定类固醇诱导的青光眼患者的小梁细胞膜磷脂酰肌醇含量是否发生变化。我们还将比较接受小梁切除术的患者和年龄匹配的正常身体对照组培养的小梁细胞中的肌醇磷脂水平。这项拟议的研究有望对理解哺乳动物眼压的机械感觉产生重大影响，从而促进开角型青光眼和其他形式青光眼的新疗法的发现。最终，从这项研究中获得的知识可能允许开发新的青光眼治疗策略，这将使我们的退伍军人受益。

 DESCRIPTION (provided by applicant): Glaucoma is a leading cause of irreversible blindness in the world, and it affects more than 285,000 U.S. veterans. Elevated intraocular pressure (IOP) is associated with both development and progression of primary open angle glaucoma, and IOP is the only modifiable risk factor for the disease. However, the mechanisms causing elevated IOP remain poorly understood. Trabecular meshwork cells are critical to maintaining a normal aqueous fluid flow and intraocular pressure. In other tissues such as the kidney, bones and cartilage, the primary cilia play a mechanosensory role in fluid flow. Here, we further explore the potential for cilia of trabecular meshwork cells to serve a mechanosensory role in the eye. Indeed, our previous findings demonstrate that phosphoinositide lipids within primary cilium are an important regulator of eye pressure. In the primary cilium, inositol phosphatase OCRL interacts with TRPV4, a mechanosensory channel that lowers IOP in mice when TRPV4 is stimulated. Previous studies have established that phosphoinositides regulate TRPV4 activity. We hypothesize that phosphoinositide signaling within the cilia of the trabecular meshwork cells regulates aqueous outflow via TRPV4 channels. In this application, we propose to employ novel optogenetic techniques to selectively direct protein translocation and activation of ciliary inositl kinase/phosphatase to modulate TRPV4 activity and IOP. In Aim 1, we will determine whether alterations in PIP2 dephosphorylation via OCRL affect ciliary outflow facility and IOP in mice by optogenetic stimulation of inositol phosphatase OCRL. We will then assess whether optical stimulation of ectopically expressed OCRL can rescue a steroid-induced glaucoma (SIG) mouse model. We expect that the activation of TRPV4 channels via light-responsive OCRL in the anterior chamber of mouse eye will lower IOP. In Aim 2, we will determine whether PIP2 phosphorylation via PI3K activation will affect ciliary outflow facility and IOP in wild-type mice. We anticipate that optogenetic stimulation of PI3K signaling will increase IOP and reduce outflow facility. We will then determine whether small molecule inhibition of PI3K decreases IOP in the steroid-induced glaucoma mouse model. In Aim 3, we will determine whether steroid-induced glaucoma patients exhibit changes in their trabecular meshwork cell membrane phosphoinositide content. We will also compare phosphoinositide levels in cultured trabecular meshwork cells from patients undergoing trabeculectomy surgery to normal aged- matched cadaveric controls. The proposed research is expected to have a significant impact on the understanding of mechanosensation of eye pressure in mammals, thereby facilitating the discovery of new therapies for open angle glaucoma and other forms of glaucoma. Ultimately, the knowledge obtained from this study may allow for the development of new therapeutic strategies in glaucoma that will benefit our veterans.