Stimuli promoting the survival of spiral ganglion neurons

描述(申请人提供)：毛细胞丢失后，去传入的螺旋神经节神经元(SGN)失去其外周突起并逐渐死亡。SGN变性降低了人工耳蜗术的疗效，而人工耳蜗术是目前治疗感音神经性耳聋的唯一方法。电刺激促进了体内去传入SGN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预防体内SGN变性。我们发现，耳聋大鼠SGN的死亡与JNK-Jun通路中促凋亡信号的增加有关。在耳聋后早期，SGN中的生存信号也减少，明显表现为CREB磷酸化减少。目的1通过卵泡内注射JNK抑制剂和JNK3-/-小鼠来确定JNK活性是否是体内SGN死亡所必需的，如果是，何时是必需的。我们还询问了JNK抑制或JNK3缺失在多大程度上促进了外周突起的退化。由于Jun磷酸化和SGN死亡发生在毛细胞死亡很久之后，在目标2中，我们询问耳蜗声后的其他退行性变化是否可以解释SGN的死亡，重点是神经胶质细胞的死亡、外周突起变性和NT-3表达的丧失。接下来，我们转向膜电活动如何促进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间隔中的单个细胞内信号通路。利用这些，我们证明了CaMKII将去极化与抑制促凋亡的JNK信号联系在一起。我们进一步证明，CaMKII通过招募非受体蛋白酪氨酸激酶、FAK和PYK2以及蛋白激酶B(PKB)来做到这一点。这让人想起了多肽神经营养因子通过其受体蛋白酪氨酸激酶和PKB抑制JNK信号的机制。在目标3中，我们通过测试RAC/CDC42小GTP酶在去极化-CaMKII-Pyk2/FAK通路抑制JNK信号中的作用，进一步开发了这一新的信号通路，并与神经营养素平行。与我们以前的研究一样，通过将抑制和功能获得的基因导入培养的SGN的实验方法，构建了针对所提出的途径中的特定步骤的结构。人工耳蜗的生理、生理活动和刺激由不同频率的脉冲组成。在目标4中，我们使用体外电刺激(ES)系统将我们对细胞内信号的研究扩展到图案化的电活动。我们问模式化的ES是否招募了我们在去极化SGN中发现的新的信号通路。我们还在Aim 4中询问在体内抑制去传入SGN中促凋亡信号的最佳频率是多少，以及在体内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大约2000万美国人，目前唯一替代失去的感觉细胞功能的手段是人工耳蜗术，它直接刺激耳蜗神经元。我们的研究重点是改善存活神经元的存活和功能，以提高目前超过40,000美国人使用的人工耳蜗术的长期疗效。

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.