Stimuli promoting the survival of spiral ganglion neurons

描述（由申请人提供）：毛细胞丢失后，失传入螺旋神经节神经元（sgn）失去其外周进程并逐渐死亡。SGN变性降低了人工耳蜗的疗效，而人工耳蜗是目前治疗感音神经性耳聋的唯一方法。电刺激促进失传入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。我们使用体外和体内方法来确定电活动如何防止SGN死亡，并将这一知识应用于体内SGN变性的预防。我们发现耳聋大鼠的SGN死亡与JNK-Jun通路中促凋亡信号的增加有关。在耳聋后早期，sgn中促生存信号也减少，表现为CREB磷酸化减少。Aim 1使用耳蜗内灌注JNK抑制剂和JNK3-/-小鼠来确定体内SGN死亡是否需要JNK活性，如果需要，则何时需要。我们还询问JNK抑制或JNK3缺失在多大程度上促进了周围过程的退化。由于Jun磷酸化和SGN死亡发生在毛细胞死亡后很长一段时间，我们在Aim 2中询问耳蜗的其他退行性变化是否可以解释SGN死亡，重点关注胶质细胞的死亡、外周过程变性和NT-3表达的丧失。接下来我们将讨论膜电活动如何促进SGN存活的问题。我们已经开发了分子试剂来选择性地激活或沉默特定亚细胞区室中的单个细胞内信号通路。利用这些，我们发现CaMKII将去极化与促凋亡JNK信号的抑制联系起来。我们进一步表明，CaMKII通过募集非受体蛋白酪氨酸激酶FAK和Pyk2以及蛋白激酶B （PKB）来实现这一功能。这让人想起肽神经营养因子通过其受体蛋白-酪氨酸激酶和PKB抑制JNK信号传导的机制。在Aim 3中，我们通过测试Rac/Cdc42小gtpase通过去极化- camkii - pyk2 /FAK途径抑制JNK信号通路的作用，进一步开发了这种新的信号通路，并与神经营养因子并行。正如我们之前的研究一样，实验方法是将抑制和功能获得构建物转染到培养的sgn中，靶向所提出途径中的特定步骤。人工耳蜗的生理、听觉活动和刺激由不同频率的脉冲组成。在Aim 4中，我们使用体外电刺激（ES）系统将细胞内信号传导的研究扩展到模式电活动。我们想知道模式ES是否招募了我们在去极化sgn中发现的新的信号通路。在Aim 4中，我们还询问了在体内抑制失传入sgn中促凋亡信号传导的最佳频率是多少，以及体内ES是否也以camkii依赖的方式招募FAK/Pyk2。感觉神经性听力损失影响了大约2000万美国人，目前唯一替代失去的感觉细胞功能的方法是人工耳蜗，它直接刺激耳蜗神经元。我们的研究重点是提高存活神经元的存活率和功能，以提高人工耳蜗的长期疗效，目前有超过40,000名美国人使用人工耳蜗。

DESCRIPTION (provided by applicant): After loss of hair cells, the deafferented spiral ganglion neurons (SGNs) lose their peripheral process and gradually die. SGN degeneration reduces the efficacy of cochlear implants, currently the only treatment for sensorineural deafness. Electrical stimulation promotes survival of 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. We use in vitro and in vivo approaches to determine how electrical activity prevents SGN death and apply this knowledge to prevention of SGN degeneration in vivo. We showed that SGN death in deafened rats is correlated with increased proapoptotic signaling in the JNK-Jun pathway. Early in the post-deafening period there is also decreased prosurvival signaling in SGNs, evident as decreased CREB phosphorylation. Aim 1 uses intracochlear infusion of a JNK inhibitor and JNK3-/- mice to determine whether JNK activity is necessary for SGN death in vivo and, if so, when is it necessary. We also ask the extent to which JNK inhibition or JNK3 deletion promotes degeneration of peripheral processes. Because Jun phosphorylation and SGN death occur long after hair cells have died, we ask, in Aim 2, whether other post-deafening degenerative changes in the cochlea can account for SGN death, focusing on the death of glial cells, peripheral process degeneration, and loss of NT-3 expression. We next turn to the question of how membrane electrical activity promotes SGN survival. We have developed molecular reagents to selectively activate or silence individual intracellular signaling pathways in specific subcellular compartments. Using these, we showed that CaMKII links depolarization to suppression of proapoptotic JNK signaling. We further show that CaMKII does so by recruiting nonreceptor protein-tyrosine kinases, FAK and Pyk2, and protein kinase B (PKB). This is reminiscent of the mechanism by which peptide neurotrophic factors suppress JNK signaling via their receptor protein-trosine kinases and PKB. In Aim 3, we further develop this novel signaling pathway, and parallelism with neurotrophins, by testing the role of Rac/Cdc42 small GTPases in suppression of JNK signaling by the depolarization-CaMKII-Pyk2/FAK pathway. As in our previous studies, the experimental approach using transfection into cultured SGNs of inhibitory and gain-of-function constructs targeting specific steps in the proposed pathway. Physiologi,cal activity and stimulation by cochlear implants consists of impulses of various frequencies. In Aim 4, we extend our studies of intracellular signaling to patterned electrical activity using a system for in vitro electrical stimulation (ES). We ask whether patterned ES recruits the novel signaling pathways we have identified in depolarized SGNs. We also ask in Aim 4 what is the optimal frequency for suppression of proapoptotic signaling in deafferented SGNs in vivo and whether in vivo ES also recruits FAK/Pyk2 in a CaMKII-dependent manner.Sensorineural hearing loss affects about 20,000,000 Americans and the only current means to replace the function of the lost sensory cells is the cochlear implant, which directly stimulates cochlear neurons. Our research focuses on improving the survival and function of surviving neurons in order to improve the long-term efficacy of cochlear implants, currently used by over 40,000 Americans.