Stimuli promoting the survival of spiral ganglion neurons

描述（由申请人提供）：毛细胞丧失后，去传入螺旋神经节神经元（SGN）丧失其外周突起并逐渐死亡。SGN变性降低了人工耳蜗的疗效，人工耳蜗是目前唯一的感音神经性耳聋治疗方法。电刺激促进了体内去传入SGN的存活，提高了使用电刺激维持耳聋个体SGN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，并将这些知识应用于预防体内SGN变性。我们发现，SGN死亡的大鼠与JNK-Jun通路中增加的促凋亡信号相关。在耳聋后早期，SGN中的促生存信号也减少，这明显表现为CREB磷酸化减少。目的1使用JNK抑制剂和JNK 3-/-小鼠的脑内输注来确定JNK活性是否是SGN体内死亡所必需的，如果是，则何时是必需的。我们还询问JNK抑制或JNK 3缺失促进外周过程变性的程度。由于Jun磷酸化和SGN死亡发生在毛细胞死亡后很长时间，我们在目标2中询问耳蜗中的其他耳聋后退行性变化是否可以解释SGN死亡，重点是神经胶质细胞的死亡，外周突变性和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，以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。使用这些，我们发现CaMKII将去极化与抑制促凋亡JNK信号传导联系起来。我们进一步表明，CaMKII通过募集非受体蛋白酪氨酸激酶FAK和Pyk 2以及蛋白激酶B（PKB）来实现这一点。这让人想起肽类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。在目标3中，我们通过测试Rac/Cdc 42小GTP酶在通过去极化-CaMKII-Pyk 2/FAK途径抑制JNK信号传导中的作用，进一步开发了这种新的信号传导途径，并与神经营养因子平行。与我们以前的研究一样，实验方法使用转染到培养的SGN中的抑制性和功能获得性构建体靶向所提出的途径中的特定步骤。人工耳蜗植入的生理活动和刺激由各种频率的脉冲组成。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传导的研究扩展到模式化的电活动。我们问是否图案ES招聘的新的信号通路，我们已经确定在去极化SGN。我们还在Aim 4中询问，在体内去传入SGN中抑制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.Sensorineural hearing loss影响约20，000，000美国人，并且目前替代丧失的感觉细胞的功能的唯一手段是耳蜗植入物，其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，以提高人工耳蜗植入的长期疗效，目前有超过40，000名美国人使用。

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.