The role of neuronal activity on retinal angiogenesis and blood-retina barrier (BRB) maturation

神经元活动对视网膜血管生成和血视网膜屏障（BRB）成熟的作用

• 批准号: 10592014
• 负责人: Saptarshi Biswas
• 金额: $ 13.4万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10592014
• 关键词: Address; Affect; Astrocytes; Basement membrane; Blood Vessels; Blood-Retinal Barrier; Caveolae; Cell Count; Cell Death; Cell Proliferation; Cells; Data; Defect; Degenerative Disorder; Development; Endothelial Cells; Endothelium; Enzymes; Eye; GLAST Protein; Genetic Models; Glutamate Receptor; Glutamate Transporter; Glutamate-Ammonia Ligase; Glutamates; Glutamine; Growth; Impairment; Ischemia; Knowledge; Ligands; Measures; Mediating; Membrane Proteins; Metabolic; Mouse Strains; Muller' s cell; Mus; Mutant Strains Mice; Nerve Degeneration; Neurodegenerative Disorders; Neuroglia; Neurons; Neurotransmitters; PECAM1 gene; Pathway interactions; Permeability; Phase; Phenotype; Photoreceptors; Process; Retina; Retinal Diseases; Retinal Ganglion Cells; Role; Signal Pathway; Signal Transduction; Synapses; Testing; Tight Junctions; Tracer; Transcript; Transducers; Transforming Growth Factor beta; VEGFA gene; angiogenesis; beta catenin; cholinergic; cholinergic neuron; early onset; excitotoxicity; extracellular; genetic approach; glutamatergic signaling; insight; neural; neurovascular unit; notch protein; novel; novel therapeutic intervention; pharmacologic; photoactivation; postnatal; response; retinal angiogenesis; retinal rods; sensor; single-cell RNA sequencing; transcriptomics; uptake; vascular factor; Address; Affect; Astrocytes; Basement membrane; Blood Vessels; Blood-Retinal Barrier; Caveolae; Cell Count; Cell Death; Cell Proliferation; Cells; Data; Defect; Degenerative Disorder; Development; Endothelial Cells; Endothelium; Enzymes; Eye; GLAST Protein; Genetic Models; Glutamate Receptor; Glutamate Transporter; Glutamate-Ammonia Ligase; Glutamates; Glutamine; Growth; Impairment; Ischemia; Knowledge; Ligands; Measures; Mediating; Membrane Proteins; Metabolic; Mouse Strains; Muller' s cell; Mus; Mutant Strains Mice; Nerve Degeneration; Neurodegenerative Disorders; Neuroglia; Neurons; Neurotransmitters; PECAM1 gene; Pathway interactions; Permeability; Phase; Phenotype; Photoreceptors; Process; Retina; Retinal Diseases; Retinal Ganglion Cells; Role; Signal Pathway; Signal Transduction; Synapses; Testing; Tight Junctions; Tracer; Transcript; Transducers; Transforming Growth Factor beta; VEGFA gene; angiogenesis; beta catenin; cholinergic; cholinergic neuron; early onset; excitotoxicity; extracellular; genetic approach; glutamatergic signaling; insight; neural; neurovascular unit; notch protein; novel; novel therapeutic intervention; pharmacologic; photoactivation; postnatal; response; retinal angiogenesis; retinal rods; sensor; single-cell RNA sequencing; transcriptomics; uptake; vascular factor

PROJECT SUMMARY Reciprocal interactions among neuroglial and vascular components of the postnatal retina are critical for proper angiogenesis and the establishment of the blood-retinal barrier (BRB). Although neuronal or glial cells-derived signals that promote angiogenesis and BRB maturation are being elucidated, we still do not understand: a) how neuronal synaptic activity in the retina influences these processes; b) which neurotransmitter(s) contribute to these processes; and c) whether neurotransmitters act on endothelial cells (ECs) directly or indirectly, via astrocytes or Müller cells. The superficial plexus vasculature develops (P1-P8) during the spontaneous cholinergic wave of neural activity (P0-P10) and photoactivation of Opn4+ retinal ganglion cells (RGC). In contrast, the deep plexus vasculature and BRB development span cholinergic and glutamatergic waves of neural activity (P10-P14), glutamatergic synaptic activity of photoreceptors (P10 onwards) as well as photoactivation of Opn4+ RGCs. Transient pharmacological blockade of cholinergic wave impairs deep plexus angiogenesis and BRB maturation; however, the role of extracellular glutamate in these processes is unknown. In preliminary findings, we have primarily used three mouse strains to assess the effects of synaptic glutamate release on retinal angiogenesis and the BRB: a) Vglut1 -/-, that lack glutamate release, b) Gnat1-/-, in which rod photoreceptors remain depolarized, thus release excess glutamate, and c) Chrnb2-/-, where cholinergic wave is abolished and glutamatergic wave begins early (P8). We have found that synaptic glutamate release is a positive regulator of deep plexus angiogenesis and BRB maturation. This effect is mediated by inducing expression of Norrin in Müller glia and Norrin/b-catenin pathway activation in ECs. Based on these preliminary data, we hypothesize that released 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 glutamate levels [Vglut1-/- mice (no glutamate release), Gnat1-/- mice (high glutamate release), and Chrnb2-/- mice (early onset of glutamatergic wave)] regulates retinal angiogenesis and structural and functional BRB integrity. Next, we will determine whether Müller cells respond to extracellular glutamate levels by inducing expression of angiogenic and barriergenic factors. We will examine Müller cell response [cell number, expression of GLAST (Slc1a3; a major glutamate transporter), glutamine synthetase (enzyme responsible for converting glutamate to glutamine) and Norrin (the major Wnt ligand expressed by Müller cells)] in Vglut1-/-, Gnat1-/- and Chrnb2-/- mice. Finally, we will test if activation of endothelial Norrin/b-catenin signalling that promotes angiogenesis and BRB maturation can rescue deficits in Vglut1-/- mice. These studies will provide a mechanistic understanding of how glutamatergic synaptic activity regulates angiogenesis and BRB in the developing retina and provide new insights into effects of excitotoxicity on neurodegenerative diseases in the retina.

项目摘要 产后视网膜的神经和血管成分之间的相互作用对适当至关重要 血管生成和血红视网膜屏障（BRB）的建立。尽管神经元或神经胶质细胞衍生 阐明了促进血管生成和BRB成熟的信号，我们仍然不明白：a） 视网膜中的神经元突触活动会影响这些过程。 b）哪些神经递质有助于 这些过程； c）神经递质是否直接或间接地对内皮细胞（EC）作用 星形胶质细胞或müller细胞。浅表丛脉管系统的发展（P1-P8） 神经活性的胆碱能波（P0-P10）和OPN4+视网膜神经节细胞（RGC）的光活化。在 对比，深丛脉管系统和BRB发育跨越胆碱能和谷氨酸能的神经元波 活性（P10-P14），光感受器的谷氨酸能突触活性（P10）以及光激活 OPN4+ RGC。胆碱能波的瞬时药物阻断损害了深丛血管生成和 BRB成熟；但是，细胞外谷氨酸在这些过程中的作用尚不清楚。在初步 调查结果，我们使用了主要的三种小鼠菌株来评估突触谷氨酸释放的影响 视网膜血管生成和BRB：a）vglut1 - / - ，缺乏谷氨酸释放，b）gnat1 - / - ，其中杆 光感受器保持定义，因此释放超过谷氨酸，c）chrnb2 - / - ，其中胆碱能波为 废除和谷氨酸能波早期开始（P8）。我们发现合成谷氨酸能释放是正 深层血管生成和BRB成熟的调节剂。这种效应是通过诱导的表达来介导的 ECS中的Müller神经胶质中的Norrin和Norrin/B-catenin途径激活。基于这些初步数据，我们 假设释放的谷氨酸水平是由müller细胞感知的，而穆勒细胞又作为传感器作用 诱导血管生成和BRB形成因子的表达，并促进深层血管生成和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谷氨酸 水平[VGLUT1 - / - 小鼠（无谷氨酸释放），GNAT1 - / - 小鼠（高谷氨酸释放）和Chrnb2 - / - 小鼠（早期 谷氨酸能波的发作）调节视网膜血管生成以及结构和功能性BRB完整性。下一个， 我们将通过诱导表达的表达来确定müller细胞是否对细胞外谷氨酸水平做出反应 血管生成和肺泡因子。我们将检查müller细胞响应[细胞数，胶状的表达 （SLC1A3；主要谷氨酸转运蛋白），谷氨酰胺合成酶（负责将谷氨酸转化为的酶 谷氨酰胺）和诺林（Müller细胞表达的主要Wnt配体）]在vglut1 - / - ，gnat1 - / - 和chrnb2 - / - 小鼠中。 最后，我们将测试是否激活促进血管生成和BRB的内皮诺林/b-catenin信号传导 成熟可以营救在vglut1 - / - 小鼠中定义。这些研究将对如何进行机械理解 谷氨酸能突触活动调节发育中的视网膜中的血管生成和BRB，并提供新的见解 兴奋性毒性对视网膜中神经退行性疾病的影响。