Shedding light on glaucoma: optogenetics regulation of ciliary phosphoinositides

揭示青光眼：睫状磷酸肌醇的光遗传学调控

• 批准号: 9378737
• 负责人: Yang Sun
• 金额: --
• 依托单位: VETERANS ADMIN PALO ALTO HEALTH CARE SYS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-10-01 至 2020-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9378737
• 关键词: Affect; Age; Apical; Aqueous Humor; Biological Assay; Blindness; Cadaver; Cartilage; Cations; Cell membrane; Cells; Cilia; Cleaved cell; Clinic; Cues; Data; Development; Disease; Exhibits; Eye; Genes; Genetic Transcription; Glaucoma; Goals; Inositol; Kidney; Knowledge; Laboratories; Lead; Light; Lipids; Mammals; Membrane; Methods; Molecular; Mus; Mutate; Nitric Oxide; Open-Angle Glaucoma; Operative Surgical Procedures; Optics; Organelles; Pathogenesis; Patients; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiologic Intraocular Pressure; Play; Population; Primary Open Angle Glaucoma; Process; Production; Protein Dephosphorylation; Protein translocation; Regulation; Regulatory Element; Relaxation; Research; Risk Factors; Rodent Model; Role; Signal Pathway; Signal Transduction; Steroids; Surface; Techniques; Therapeutic; Tissues; Trabecular meshwork structure; Trabeculectomy; Translating; Veterans; Wild Type Mouse; aged; anterior chamber; aqueous; bone; fluid flow; genetic regulatory protein; glaucoma surgery; in vivo; inositol-1,4,5-trisphosphate 5-phosphatase; lipid mediator; modifiable risk; mouse model; novel; novel strategies; novel therapeutic intervention; novel therapeutics; optogenetics; polarized cell; pressure; protein activation; public health relevance; recruit; small molecule

 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.

 描述（由申请人提供）： 青光眼是世界上不可逆转的失明的主要原因，它影响了超过285，000名美国退伍军人。眼内压（IOP）升高与原发性开角型青光眼的发生和进展相关，IOP是该疾病唯一可改变的危险因素。然而，引起IOP升高的机制仍然知之甚少。小梁网细胞对于维持正常的房水流动和眼内压至关重要。在其他组织如肾脏、骨骼和软骨中，初级纤毛在流体流动中发挥机械感觉作用。在这里，我们进一步探讨了潜在的小梁网细胞的纤毛在眼睛中发挥机械感觉作用。事实上，我们以前的研究结果表明，初级纤毛内的磷脂酰肌醇脂质是眼压的重要调节因子。在初级纤毛中，肌醇磷酸酶OCRL与TRPV 4相互作用，TRPV 4是一种机械感觉通道，当TRPV 4受到刺激时，可降低小鼠的IOP。先前的研究已经确定磷酸肌醇调节TRPV 4活性。我们推测，磷酸肌醇信号在纤毛的小梁网细胞调节水流出通过TRPV 4通道。在本申请中，我们提出采用新颖的光遗传学技术来选择性地指导蛋白质易位和睫状肌醇激酶/磷酸酶的活化以调节TRPV 4活性和IOP。在目的1中，我们将通过肌醇磷酸酶OCRL的光遗传学刺激来确定经由OCRL的PIP 2去磷酸化的改变是否影响小鼠的睫状体流出功能和IOP。然后，我们将评估光学刺激异位表达的OCRL是否可以挽救类固醇诱导的青光眼（SIG）小鼠模型。我们预期通过小鼠眼前房中的光响应OCRL激活TRPV 4通道将降低IOP。在目标2中，我们将确定通过PI 3 K激活的PIP 2磷酸化是否会影响野生型小鼠的睫状体流出功能和IOP。 我们预期PI 3 K信号传导的光遗传学刺激将增加IOP并降低流出功能。然后，我们将确定小分子抑制PI 3 K是否降低类固醇诱导的青光眼小鼠模型中的IOP。在目标3中，我们将确定类固醇诱导的青光眼患者是否表现出小梁细胞膜磷酸肌醇含量的变化。我们还将比较接受小梁切除术的患者与正常年龄匹配的尸体对照的培养的小梁网细胞中的磷酸肌醇水平。这项拟议中的研究预计将对哺乳动物眼压机械感觉的理解产生重大影响，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有助于开发新的青光眼治疗策略，使我们的退伍军人受益。