Role of mechanosensation in retinal function and dysfunction

青光眼是一种毁灭性致盲疾病，它们共同代表了美国不可逆失明的主要原因。有大量证据表明，由眼压升高介导的病理性机械刺激在青光眼的病因学中起着因果作用。目前的建议是表征这种疾病的分子机制，这种机制可能是细胞反应的基础，但也可能在其他涉及视网膜机械应力的疾病中发挥关键作用，如糖尿病视网膜病变、缺血和黄斑水肿。我们发现一种机械敏感阳离子通道TRPV4选择性定位于视网膜神经节细胞（RGCs）和Muller胶质细胞。由于这是青光眼中特异性靶向的两种细胞类型，我们假设机械敏感通道介导病理性IOP升高的影响。该建议的中心焦点是通过结合生物物理，细胞和翻译方法来表征RGCs中的这种转导机制。在Aim 1中提出的研究将建立机械敏感通道激活和脱敏的分子机制，以及它们在钙转运、细胞生理和RGC存活中的作用。我们将测试在“低张力”病理下模拟RGC损伤的条件，并测试一些机械敏感通道可能导致兴奋性毒性RGC损伤的模型。在体外和体内青光眼模型中，无毒小分子拮抗剂在阻断压力刺激的RGCs损失方面具有显著的有效性，拟议的研究也将利用初步工作。本研究的目的二是建立在目的一的压敏通道特征的基础上，研究这些机制如何调节RGCs和视网膜星形胶质细胞的肿胀反应。虽然细胞通常会对正常的光诱发神经元活动做出反应，但在缺血和糖尿病等病理情况下，肿胀会加剧，并且可能具有高度的神经毒性。拟开展的研究将探索包括肿胀激活钙通道、水通道、钙波和调节体积减少机制在内的分子复合物。因此，我们提出的研究目标是建立一个直观的概念和实验框架，以帮助我们统一对视网膜IOP转导、细胞肿胀、体积传感和视网膜细胞钙稳态的理解。通过这样做，它将有助于预测通过直接流体静压细胞膜而起作用的机械力的影响，以及确定由拉伸、拉伸和膨胀激活的分子机制。然后，我们将使用诱导型和慢性青光眼的小鼠模型来测试这些预测。这可能有助于完善我们对青光眼、糖尿病视网膜病变和缺血等视力障碍的机械损伤的理解，并有助于开发有效的神经保护治疗方法。

DESCRIPTION (provided by applicant): Glaucoma is complex of devastating blinding diseases that together represent the primary cause of irreversible blindness in the U.S. There is substantial evidence that pathological mechanical stimulation mediated by an increase in intraocular pressure (IOP) plays a causal role in the etiology of glaucoma. The present proposal is to characterize the molecular mechanisms which might underlie the cellular response in this disease but could also play a key function in other diseases that involve mechanical stress in the retina, such as diabetic retinopathy, ischemia and macular edema. We found that a mechanosensitive cation channel, TRPV4, is selectively localized to retinal ganglion cells (RGCs) and in Muller glial cells. Because these are the two cell types that are specifically targeted in glaucoma, we hypothesize that mechanosensitive channels mediate the effects of pathological increases in IOP. The central focus of the proposal is to characterize this transduction mechanism in RGCs by combining biophysical, cellular and translational approaches. Studies proposed in Aim 1 will establish the molecular mechanism of mechanosensitive channel activation and desensitization, their role in calcium transport, cellular physiology and RGC survival. We will test conditions that mimic RGC injury in "low-tension" pathologies and test a number of models under which mechanosensitive channels might contribute to excitotoxic RGC injury. The proposed studies will also capitalize on preliminary work which shows remarkable effectiveness of non toxic small molecule antagonists in blocking pressure-stimulated loss of RGCs in vitro and in vivo glaucoma models. Aim 2 of the proposed research builds on the characterization of pressure-sensitive channels in Aim 1 to study how these mechanisms regulate the swelling response of RGCs and retinal astroglia. Although cells typically swell in response to normal light-evoked neuronal activity, swelling is exacerbated in pathological conditions such as ischemia and diabetes, and can be highly neurotoxic. The proposed studies will explore the molecular complexes that involve swelling-activated calcium channels, water channels, calcium waves and regulatory volume decrease mechanisms. Thus, the goal of proposed research is to establish an intuitive conceptual and experimental framework that helps unify our understanding of retinal IOP transduction, cell swelling, and volume sensing and calcium homeostasis in retinal cells. By doing so, it will help predict the effects of mechanical forces that act through direct hydrostatic compression of cellular membranes as well as determine molecular mechanisms that are activated by tensile stretching, pulling and swelling. We will then test these predictions using mouse models of inducible and chronic glaucoma. This may help to refine our understanding of mechanical injury in vision disorders such as glaucoma, diabetic retinopathy and ischemia and contribute to developing effective neuroprotective treatments.