Cellular therapy for the inner ear

 描述(申请人提供)：神经回路的退化，无论是通过突触丢失或神经细胞死亡，都是神经系统功能障碍的重要原因，而导致耳聋的传入听神经丢失是这些过程在耳蜗处的表现。我们以前的工作表明，来自移植干细胞的神经元在化学去传入后可以重新支配Corti器官的毛细胞，这表明这种神经回路可以重建。在这些研究中，我们提出了两种类型神经变性的修复问题：首先，我们在体外和体内突触损伤模型中，研究了通过操纵轴突引导和神经营养因子是否可以增加毛细胞的再神经和突触发生；其次，我们在完全传入神经元丧失的模型中研究了前体细胞转分化为神经元的作用和这一回路的再生。突触和神经修复的测量包括体外对新突触的电生理和组织学分析，以及体内听功能的外周、中枢和行为评估。了解成人耳朵毛细胞与神经连接重新形成的机制对于治疗导致听力损失的神经功能障碍的方法很重要。具体目标包括三个相互关联的实验，以探索可能影响听觉系统外周和中枢神经重新支配成功的关键变量。在目标1中，我们评估了轴突导向抑制剂和轴突生长促进剂对传入突触丢失但神经节细胞保留的耳蜗毛细胞再支配的影响。在体外系统中(由于给予红藻氨酸导致传入突触丢失) 使用电生理测量来评估突触功能：刺激毛细胞后神经元的动作电位和兴奋性突触后电流的激发，以及抑制 谷氨酸能突触的药物阻滞剂对这种突触功能的影响。在活体系统中(噪声损伤导致传入突触丢失)指标包括突触分析以及听觉脑干反应和失真产物耳声发射。评估是基于免疫组织化学测定的与内、外毛细胞形成的突触的形态和数量。在目标2中，我们定义了Sox2和Ngn1在雪旺细胞向神经元转化过程中的作用和最佳表达水平。我们已经分离出可以从螺旋神经节转化为神经元的前体细胞，我们已经证明这些细胞来自雪旺细胞。我们测试了Sox2和Ngn1在这种转换中的作用。我们使用哇巴因在体内评估雪旺细胞向神经元的转化，并通过调节相同基因的表达来刺激内源性细胞向螺旋神经节细胞的转化。在目标3中，我们记录了听觉中脑中单个神经元对声音刺激的放电活动，这些刺激被传递到重新神经支配和未治疗的耳朵。我们将神经恢复的标记物与改善声音辨别能力的行为指标进行比较。

 DESCRIPTION (provided by applicant): Degeneration of neural circuitry, either through synaptic loss or neural cell death, is an important cause of nervous system dysfunction, and afferent auditory nerve loss leading to deafness is a manifestation of these processes occurring in the cochlea. Our previous work has shown that neurons from transplanted stem cells can reinnervate hair cells of the organ of Corti after chemical de-afferentation, suggesting that this neural circuit can be reconstituted. In these studies we ask questions about repair of both types of neural degeneration: first, we investigate whether reinnervation and synaptogenesis with hair cells can be increased by manipulating axonal guidance and neurotrophic factors in in vitro and in vivo models of synapse damage; second, we study the effect of progenitor cell transdifferentiation to neurons and the regeneration of this circuit in a model of complete afferent neuron loss. Measurements of synaptic and neural repair include electrophysiological and histological analysis of new synapses in vitro and immunohistochemical as well as peripheral, central, and behavioral assessments of auditory function in vivo. Understanding the mechanisms underlying re-formation of neural connections to hair cells in the adult ear is important to therapeutic approaches for the treatment of neural dysfunction that causes hearing loss. The Specific Aims comprise three inter-related experiments to probe key variables likely to influence the success of peripheral and central reinnervation of the auditory system. In Aim 1 we assess the effect of inhibitors of axonal guidance as well as promoters of axonal growth on the reinnervation of hair cells in cochlear models with loss of the afferent synapse but preservation of ganglion cells. In the in vitro system (afferent synapses lost due to kainate administration) we use electrophysiological measurements to assess synaptic function: firing of action potentials and excitatory postsynaptic currents by the neurons after stimulation of hair cells, and inhibition of this synaptic function by pharmacological blockers of glutamatergic synapses. In the in vivo system (noise damage causing afferent synapse loss) metrics include synaptic analysis as well as auditory brainstem response and distortion product otoacoustic emissions. Assessments are based on the morphology and number of synapses formed with inner and outer hair cells determined immunohistochemically. In Aim 2 we define the roles and optimal expression levels of Sox2 as well as Ngn1 for Schwann cell to neuronal conversion. We have isolated progenitor cells that can be converted to neurons from spiral ganglion, and we have shown that these cells arise from Schwann cells. We test the role of Sox2 and Ngn1 in this conversion. We assess conversion of Schwann cells to neurons in vivo using ouabain to produce a model of auditory nerve damage, and we stimulate the conversion of endogenous cells to spiral ganglion cells by modulating expression of the same genes. In Aim 3 we record the spiking activity from single neurons in the auditory midbrain to sound stimuli delivered to the reinnervated and untreated ears. We compare markers of neural recovery to behavioral measures of improved sound discrimination.