TNF-alpha in cell death & neuroprotection in glaucoma

描述（由申请人提供）：虽然青光眼是全球失明的主要原因，但导致青光眼视网膜神经节细胞死亡的确切致病机制和有效的神经保护策略均未知。肿瘤坏死因子-α（TNF-α）最近已被确定为青光眼视网膜神经节细胞死亡的介质。体外证据表明，由活化的神经胶质细胞响应于青光眼应激源（例如升高的眼内压和缺血）而分泌的TNF-α可诱导视网膜神经节细胞的凋亡性死亡。神经胶质细胞产生的TNF-α增加，TNF受体-1，死亡受体，上调视网膜神经节细胞和它们的轴突在青光眼的人的眼睛。还有从高压青光眼大鼠模型获得的令人信服的体内证据，其证明了TNF-α在这些眼睛中的致病作用。(1)（2）TNF-α信号转导参与了这些眼睛中的细胞死亡，如通过激活视网膜caspase-8 -TNF受体家族细胞死亡途径中的近端效应蛋白所证明的;（3）抗TNF-α治疗对高眼压大鼠眼有神经保护作用。其他体内证据来自使用TNF受体-1缺陷小鼠的视神经挤压损伤模型的研究。虽然这不是青光眼的模型，但这些研究的结果提供了证据，表明TNF-α信号转导参与视神经损伤后视网膜神经节细胞的死亡。因此，本申请中提供的现有证据共同为进一步评价青光眼中TNF-α介导的细胞死亡提供了强有力的依据。所提出的实验旨在使用体外和体内模型来确定视网膜神经节细胞和神经胶质细胞中TNF-α介导的细胞死亡和神经保护的精确机制。使用原代共培养物，将在暴露于外源性TNF-α的视网膜神经节细胞和神经胶质细胞中分别研究半胱天冬酶和线粒体事件的激活以及天然保护机制和选定激酶途径的激活。使用大鼠青光眼模型，将在体内研究内源性TNF-α的诱导与细胞死亡之间的关系。此外，将在该体内模型中使用功能和解剖结局进一步确定特异性抗TNF-α治疗提供神经保护的有效性。在本申请中提出的研究中，对细胞死亡和保护的精确机制的更好理解应有助于有效操纵青光眼中视网膜神经节细胞的存活。

DESCRIPTION (provided by applicant): Although glaucoma is a leading cause of worldwide blindness, neither the precise pathogenic mechanisms, which lead to retinal ganglion cell death in glaucoma, nor effective strategies for neuroprotection are known. Tumor necrosis factor-alpha (TNF-alpha) has recently been identified to be a mediator of retinal ganglion cell death in glaucoma. In vitro evidence demonstrates that TNF-alpha secreted by activated glial cells in response to glaucomatous stressors, such as elevated intraocular pressure and ischemia, can induce apoptotic death of retinal ganglion cells. Glial production of TNF-alpha is increased, and TNF receptor-1, a death receptor, is upregulated in retinal ganglion cells and their axons in glaucomatous human eyes. There is also compelling in vivo evidence obtained from a rat model of high-pressure glaucoma, which demonstrates the pathogenic role of TNF-a in these eyes. (1) Similar to glaucomatous human eyes, the expression of TNF-alpha and TNF receptor-1 are increased in rat eyes following intraocular pressure elevation; (2) TNF-alpha signaling is involved in cell death in these eyes, as evidenced by the activation of retinal caspase-8 -a proximal effector protein in the TNF receptor family cell death pathway; (3) anti-TNF-alpha treatment in ocular hypertensive rat eyes can provide neuroprotection. Additional in vivo evidence comes from studies using the optic nerve crush injury model in mice deficient for TNF receptor-1. Although this is not a model for glaucoma, findings of these studies provide evidence that TNF-alpha signaling is involved in the death of retinal ganglion cells following optic nerve injury. Thus, current evidence presented in this application collectively provides a strong rationale for further evaluation of the TNF-alpha mediated cell death in glaucoma. The proposed experiments aim to identify the precise mechanisms of TNF-alpha-mediated cell death and neuroprotection in retinal ganglion cells and glial cells, using in vitro and in vivo models. Using primary co-cultures, activation of caspases and mitochondrial events, and activation of native protection mechanisms and selected kinase pathways will be separately studied in retinal ganglion cells and glial cells exposed to exogenous TNF-alpha. Using a rat glaucoma model, the relationship between the induction of endogenous TNF-alpha and cell death will be studied, in vivo. In addition, the effectiveness of a specific anti-TNF-alpha treatment to provide neuroprotection will be further determined in this in vivo model using both the functional and the anatomical outcomes. An improved understanding of the precise mechanisms of cell death and protection in studies proposed in this application should facilitate efforts to effectively manipulate the survival of retinal ganglion cells in glaucoma.