Stimuli promoting survival of spiral ganglion neurons

随着毛细胞的死亡，螺旋神经节神经元(SGN)逐渐死亡。电刺激促进了这种去传入的SGN在体内的存活，增加了使用电刺激来维持聋人SGN存活的可能性--实际上允许人工耳蜗术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死亡过程中具有细胞凋亡特征的关键分子事件的详细时间过程，并描述这些事件是如何受电刺激影响的。将用耳毒素致大鼠耳聋，破坏毛细胞，在SGN死亡的大约100天期间，将用免疫组织化学和生化方法测定关键代表细胞凋亡调节因子和介导物的水平、磷酸化状态和亚细胞定位。这将揭示在毛细胞丢失后SGN存在于凋亡过程的哪个阶段(S)，从而确定预防SGN死亡的潜在治疗方法将最成功地应用的时间。第二个目标是验证我们之前的研究所暗示的假设：去极化招募了三个不同的激酶系统，它们独立地和相加地促进SGN的存活，作用于不同的细胞间隔和不同的底物。CAMP依赖的蛋白激酶和钙/钙调素依赖的蛋白激酶(CaMK)II似乎在细胞质中发挥作用，并磷酸化线粒体的凋亡调节因子，CaMKII通过涉及酪氨酸激酶的间接途径实现这一功能。CaMKIV在细胞核中发挥作用，使转录因子CREB磷酸化。为了验证这一假设，慢病毒载体将被用来将编码激活的蛋白激酶突变体或激酶抑制蛋白的基因导入SGN，这些蛋白通过生理靶向序列被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，我们使用我们开发的系统，在该系统中，SGN在体外受到电刺激或去极化以维持其存活。同时，我们将把这些激酶系统的药理抑制剂或激活剂注入听力或耳聋大鼠的耳蜗内，或植入刺激电极的耳聋大鼠的耳蜗内。这些实验将确定这些细胞内信号促进体内存活的程度，并验证它们在体内介导电刺激的促进存活效应，就像它们在体外所做的那样。

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.