Stimuli promoting survival of spiral ganglion neurons

螺旋神经节神经元（SGN）随着毛细胞的凋亡而逐渐死亡。 电刺激促进了这种去传入的SGN在体内的存活，提高了使用电刺激来维持SGN在耳聋个体中的存活的可能性-实际上允许耳蜗植入物取代毛细胞的营养和感觉功能。 我们的第一个目标是提供一个详细的时间过程中的关键分子事件特征的细胞凋亡死亡的SGNs后耳聋，并描述这些事件是如何受到电刺激。将使用耳毒素破坏毛细胞对大鼠进行麻醉，在SGN死亡的约100天期间，将通过化学方法和生物化学方法测定关键代表性凋亡调节剂和介质的水平、磷酸化状态和亚细胞定位。 这将揭示在毛细胞损失后SGN存在于细胞凋亡过程中的哪个阶段，从而确定预防SGN死亡的潜在疗法将最成功应用的时间。 第二个目标是检验我们以前的研究所暗示的假设：去极化招募了三种不同的激酶系统，它们独立地和相加地促进SGN存活，作用于不同的细胞区室和不同的底物。环AMP依赖性蛋白激酶和Ca 2 +/钙调蛋白依赖性蛋白激酶（CaMK）II似乎在细胞质中起作用，并磷酸化线粒体凋亡调节因子，CaMK II通过涉及酪氨酸激酶的间接途径这样做。 CaMKIV在细胞核中起作用，磷酸化转录因子CREB。 为了检验这一假设，将使用慢病毒载体将编码活化蛋白激酶突变体或激酶抑制剂蛋白的基因引入SGN中，这些蛋白质限于特定的亚细胞位置，例如，线粒体或细胞核，通过生理靶向序列。 对于这些研究，我们使用我们开发的系统，其中SGN在体外被电刺激或去极化以维持其存活。 与此同时，我们将这些激酶系统的药理学抑制剂或激活剂注入听力或听力受损大鼠或植入刺激电极的听力受损大鼠的耳蜗。 这些实验将确定这些细胞内信号在体内促进存活的程度，并验证它们在体内介导电刺激的存活促进作用，就像它们在体外一样。

Spiral ganglion neurons (SGNs) die gradually following the loss of hair cells by the process of apoptosis. Electrical stimulation promotes the survival of such deafferented SGNs in vivo, raising the possibility of using electrical stimulation to maintain survival of SGNs in deaf individuals - in effect allowing cochlear implants to replace the trophic as well as the sensory function of hair cells. Our first goal is to provide a detailed timecourse of the key molecular events characteristic of apoptosis during the death of SGNs following deafening and describe how these events are affected by electrical stimulation. Rats will be deafened using ototoxin to destroy the hair cells and, during the approximately 100 day period over which SGNs die, the levels, the phosphorylation state and the subcellular localization of key representative apoptosis regulators and mediators will be determined immunohistochemically and biochemically. This will reveal at what stage(s) in the apoptotic process the SGNs exist following loss of hair cells, thereby identifying the time during which potential therapies for preventing SGN death would be most successfully applied. The second goal is to test the hypothesis implied by our previous studies: depolarization recruits three distinct kinase systems that independently and additively promote SGN survival, acting in distinct cellular compartments and on distinct substrates. Cyclic AMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase (CaMK) II, appear to function in the cytoplasm and phosphorylate mitochondrial apoptotic regulators, with CaMKII doing so by an indirect route involving tyrosine kinases. CaMKIV functions in the nucleus, phosphorylating the transcription factor CREB. To test this hypothesis, a lentiviral vector will be used to introduce genes encoding activated protein kinase mutants or kinase inhibitor proteins into SGNs, with these proteins restricted to specific subcellular locations, e.g., mitochondria or nucleus, by means of physiological targeting sequences. For these studies, we use the system we have developed, in which SGNs are electrically stimulated or depolarized in vitro to maintain their survival. In parallel, we will infuse pharmacological inhibitors or activators of these kinase systems into the cochleae of hearing or deafened rats, or deafened rats with implanted stimulating electrodes. These experiments will determine the extent to which these intracellular signals promote survival in vivo and verify that they mediate the survival-promoting effect of electrical stimulation in vivo as they do in vitro.