Stimuli promoting survival of spiral ganglion neurons

螺旋神经节神经元（sgn）随着毛细胞的丧失而逐渐死亡。电刺激促进了这种失传入sgn在体内的存活，提高了使用电刺激维持聋人sgn存活的可能性——实际上允许人工耳蜗取代毛细胞的营养和感觉功能。我们的第一个目标是提供耳聋后sgn死亡过程中凋亡的关键分子事件的详细时间过程，并描述这些事件如何受到电刺激的影响。用耳毒素破坏毛细胞使大鼠耳聋，在大约100天的sgn死亡期间，通过免疫组织化学和生物化学方法确定关键的代表性凋亡调节因子和介质的水平、磷酸化状态和亚细胞定位。这将揭示毛细胞丢失后SGN在凋亡过程中的哪个阶段存在，从而确定防止SGN死亡的潜在疗法最成功应用的时间。第二个目标是验证我们之前的研究所暗示的假设：去极化招募三种不同的激酶系统，它们独立地和加性地促进SGN的存活，作用于不同的细胞室和不同的底物。环amp依赖性蛋白激酶和Ca2+/钙调素依赖性蛋白激酶（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，CaMKII通过涉及酪氨酸激酶的间接途径起作用。CaMKIV在细胞核中起作用，使转录因子CREB磷酸化。为了验证这一假设，将使用慢病毒载体将编码活化的蛋白激酶突变体或激酶抑制剂蛋白的基因引入sgn，通过生理靶向序列将这些蛋白限制在特定的亚细胞位置，例如线粒体或细胞核。在这些研究中，我们使用了我们开发的系统，其中sgn在体外受到电刺激或去极化以维持其存活。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耳聋大鼠的耳蜗，或植入刺激电极的耳聋大鼠。这些实验将确定这些细胞内信号在体内促进生存的程度，并验证它们在体内介导电刺激促进生存的作用，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.