Neuronal Regulation of Vascular Development and Maturation in the Retina

视网膜血管发育和成熟的神经元调节

• 批准号: 10869255
• 负责人: Dritan Agalliu
• 金额: $ 2.15万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10869255
• 关键词: Affect; Angiogenic Factor; Astrocytes; Blood Vessels; Blood-Retinal Barrier; Cell Count; Data; Development; Endothelial Cells; Enzymes; Functional disorder; GLAST Protein; Genetic; Glutamate Transporter; Glutamate-Ammonia Ligase; Glutamates; Glutamine; Knowledge; Mediating; Metabolic; Molecular; Mouse Strains; Muller' s cell; Mus; Nerve Degeneration; Neuroglia; Neurons; Neurotransmitters; Pathway interactions; Photoreceptors; Phototransduction; Process; Regulation; Retina; Retinal Diseases; Retinal Ganglion Cells; Role; Signal Transduction; Synapses; Synaptic Cleft; Testing; Transducers; angiogenesis; beta catenin; cholinergic; excitotoxicity; extracellular; imaging approach; neural; novel therapeutic intervention; pharmacologic; photoactivation; postnatal; response; retinal angiogenesis; retinal rods; vascular factor

PROJECT SUMMARY Reciprocal interactions among neuroglial and vascular components in the developing postnatal retina are critical for proper vascular development (angiogenesis) and maturation [establishment of the blood-retina barrier (BRB)]. Although neuronal or glial cell-derived signals that promote angiogenesis and BRB maturation are being elucidated, we still do not understand: a) how neuronal activity and b) which specific neurotransmitter(s) influence these processes; and c) whether neurotransmitters act on endothelial cells (ECs) directly or indirectly, via astrocytes or Müller cells. The superficial plexus vasculature develops during the spontaneous cholinergic wave of neural activity and photoactivation of Opn4+ retinal ganglion cells (RGC) (P1- P9). In contrast, the deep plexus vascular development and BRB maturation span both cholinergic and glutamatergic waves of neuronal activity (P10-P14), glutamatergic synaptic activity of photoreceptors as well as photoactivation of Opn4+ RGCs. Transient pharmacological blockade of cholinergic waves delays deep plexus angiogenesis and BRB maturation; however, the role of extracellular glutamate in these processes is unknown. In preliminary studies, we have used two mouse strains to assess the effects of glutamate release on retinal angiogenesis and BRB maturation: a) Vglut1 -/- mice that lack glutamate release in the synaptic cleft and b) Gnat1-/- mice, in which rod photoreceptors lack the phototransduction machinery and remain depolarized, thus releasing excess glutamate. We have found that neuronal activity-dependent glutamate release is a positive regulator of deep plexus angiogenesis and BRB maturation. These effects are mediated by induction of Norrin expression in Müller glia and Norrin/b-catenin pathway activation in ECs. Based on these preliminary data, we hypothesize that extracellular glutamate levels are sensed by Müller cells, which in turn operate as transducers to induce expression of angiogenic and BRB-forming factors and promote deep plexus angiogenesis and BRB maturation. We will test this hypothesis through three aims. First, we will examine how modulation of extracellular glutamate levels [Vglut1-/- mice (no glutamate release) and Gnat1-/- mice (high glutamate release)] regulates retinal angiogenesis and structural and functional BRB integrity in the developing retina. Next, we will test whether Müller cells sense extracellular glutamate levels and respond by inducing expression of angiogenic and barriergenic factors. We will examine Müller cell responses [cell number, expression of GLAST (Slc1a3; a major glutamate transporter), glutamine synthetase (an enzyme responsible for converting glutamate to glutamine) and Norrin (an angiogenesis factor expressed by Müller cells)] in Vglut1- /- and Gnat1-/- mice. Finally, we will test if activation of Norrin/b-catenin signalling, that promotes angiogenesis and BRB maturation, in ECs can rescue deficits of Vglut1-/- mice. These studies will provide a mechanistic understanding of how glutamatergic synaptic activity regulates vascular development and maturation in the retina and elucidate how glutamate excitotoxicity affects blood vessels in neurodegenerative retinal diseases.

项目摘要 发育中的产后视网膜中神经和血管成分之间的相互作用是 对于适当的血管发育至关重要（血管生成）和成熟[建立血液retina 障碍物（BRB）。 正在阐明，我们仍然不明白：a）神经元活动和b）哪些特定 神经递质影响这些过程； c）神经递质是否对内皮细胞作用（EC） 直接或间接通过星形胶质细胞或müller细胞。浅表丛脉管系统在 神经活性的赞成胆碱能波和Opn4+视网膜神经节细胞（RGC）的光活化（P1-- P9）。相比之下，深层血管发育和BRB成熟涵盖胆碱能和 神经元活性的谷氨酸能波（P10-P14），感光体的谷氨酸能突触活性 作为OPN4+ RGC的光活化。胆碱能波的瞬态药物阻断延迟了 丛血管生成和BRB成熟；但是，细胞外谷氨酸在这些过程中的作用是 未知。在初步研究中，我们使用了两种小鼠菌株来评估谷氨酸释放的作用 关于视网膜血管生成和BRB成熟：a）vglut1 - / - 小鼠在合成裂裂中缺乏谷氨酸 b）gnat1 - / - 小鼠，其中杆光感受器缺乏光转导机械并保持 去极化，从而释放过多的谷氨酸。我们发现神经元活性依赖性谷氨酸 释放是深层血管生成和BRB成熟的正调节剂。这些效果是介导的 通过在Müller神经胶质中诱导Norrin表达和EC中的Norrin/B-catenin途径激活。基于这些 初步数据，我们假设Müller细胞感知细胞外谷氨酸水平 作为换能器诱导血管生成和BRB形成因子的表达并促进深丛 血管生成和BRB成熟。我们将通过三个目标检验这一假设。首先，我们将研究如何 调节细胞外谷氨酸水平[VGLUT1 - / - 小鼠（无谷氨酸释放）和GNAT1 - / - 小鼠（高 谷氨酸释放）]调节发育中的视网膜血管生成以及结构和功能性BRB完整性 视网膜。接下来，我们将测试müller细胞是否感知细胞外谷氨酸水平并通过诱导的反应 血管生成和肺泡因子的表达。我们将检查müller细胞反应[细胞数， Glast的表达（SLC1A3；主要谷氨酸转运蛋白），谷氨酰胺合成酶（负责的酶 用于将谷氨酸转化为谷氨酰胺）和Norrin（Müller细胞表达的血管生成因子）] / - 和GNAT1 - / - 小鼠。最后，我们将测试是否激活诺林/b-catenin信号传导，从而促进血管生成 和BRB成熟，在EC中可以救出VGLUT1 - / - 小鼠的定义。这些研究将提供机械 了解谷氨酸能突触活动如何调节血管发育和成熟 视网膜和阐明谷氨酸兴奋性如何影响神经退行性残留疾病的血管。