RETINAL/SENSORY GROWTH CONE INHIBITION BY PROTEOGLYCANS

蛋白聚糖对视网膜/感觉锥生长的抑制

• 批准号: 2545870
• 负责人: Diane M Snow
• 金额: $ 10.3万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-30 至 1999-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2545870
• 关键词: abstracting; calcium flux; cell adhesion molecules; chick embryo; developmental neurobiology; dorsal root; fluorescent dye /probe; growth cones; growth inhibitors; immunocytochemistry; laminin; neuronal guidance; proteoglycan; retina; retinal ganglion; tissue /cell culture; tubulin

Proteoglycans (PGs) are a structurally diverse class of molecules that interact with many extracellular matrix (ECM) and cell surface components. PGs play a role in many cellular processes, including inhibition of retinal ganglion and dorsal root ganglion cell outgrowth. The mechanisms by which PGs inhibit neurite outgrowth are unknown. Therefore, the goal of this proposal is to investigate the hypothesis that PGs may inhibit growth cone migration by two fundamental mechanisms: 1) limiting access to growth promoting adhesive molecules, and 2) triggering the reorganization of the growth cone cytoskeleton through transient rises in [Ca2+]. First, we will use 35S-CSPG and 3H-laminin to determine the amounts of these molecules bound to the substrata in DRG cultures. We will then do behavioral assays to determine whether PGs limit cell surface access to the growth-promoting adhesive molecule laminin, and to determine whether such masking can lead to growth cone turning at a PG border. Second, we will determine if there are changes in the cytoskeletal proteins tubulin and actin, or in related proteins, as growth cones contact and are inhibited by PGs. We will use injection of fluorescent actin and tubulin, or immunocytochemistry of fixed cultures, and treatment with cytochalasin to inactivate growth cone filopodia. Third, contact with PGs induces a large rise in [Ca2+]i within growth cones. Therefore, we will determine the role of the transient elevation of [Ca2+]i in growth cones that contact CSPG, and test whether changes in [Ca2+]i can induce inhibition of growth cone migration. Fluorescence imaging of the calcium indicator fura-2/AM will be used in combination with pharmacological reagents to block the influx or release of Ca2+. Elevation of [Ca2+]i may affect the morphology and trajectory of the elongating growth cone, 1) through direct action on cytoskeletal components of the growth cone, or 2) through second messenger systems that regulate the dynamic motile apparatus of growth cones. These experiments are important because: 1) they will lead to an understanding of the mechanisms by which PGs regulate growth cone guidance, and 2) they may provide molecular methods by which to manipulate PGs in adult tissue to enable CNS regeneration, since PGs are expressed and are inhibitory to regenerating nerve cells, following CNS injury.

蛋白聚糖（PGS）是一种结构上多样化的分子， 与许多细胞外基质（ECM）和细胞表面相互作用 成分。 PGS在许多蜂窝过程中起作用，包括 抑制视网膜神经节和背根神经节细胞的生长。 PG抑制神经突生长的机制尚不清楚。 因此，该提议的目的是研究假设 PG可以通过两种基本机制抑制生长锥迁移： 1）限制促进生长促进粘合分子的获取，2） 通过触发生长锥细胞骨架的重组 瞬态在[Ca2+]中上升。 首先，我们将使用35s-CSPG和3H氯胺敏来确定 这些分子与DRG培养物中的基质结合。 然后我们会做 行为分析以确定PGS是否限制了细胞表面的访问 促进生长的粘合分子层粘连蛋白，并确定是否 这种掩盖会导致在PG边界的生长锥转弯。 第二，我们 将确定细胞骨架蛋白微管蛋白是否有变化 和肌动蛋白，或相关蛋白作为生长锥接触，并且是 被PG抑制。 我们将使用荧光肌动蛋白的注射和 固定培养物的微管蛋白或免疫细胞化学，并与 细胞切拉斯蛋白会使生长锥丝状疾病灭活。 第三，接触 PGS在生长锥内[Ca2+] i的[Ca2+] i都大幅上升。 因此，我们 将确定[Ca2+] I在生长中的瞬时升高的作用 接触CSPG的锥体，并测试[Ca2+]中是否可以诱导变化 抑制生长锥迁移。 钙的荧光成像 指标fura-2/am将与药理结合使用 阻断Ca2+涌入或释放的试剂。 [Ca2+] i的高程 可能会影响延长生长锥的形态和轨迹， 1）通过直接作用生长锥的细胞骨架成分， 或2）通过调节动态运动的第二个使者系统 生长锥的设备。 这些实验很重要，因为：1）它们将导致 了解PGS调节生长锥的机制 指导和2）它们可以提供分子方法 操纵成人组织中的PG使CNS再生，因为PG是 表达并抑制了CNS的再生神经细胞 受伤。