Notch signaling pathways in auditory support cell differentiation and maintenance

描述（由申请人提供）：在人类和其他哺乳动物物种中，机械感觉毛细胞的损伤是不可逆的，导致耳聋和平衡障碍。值得注意的是，非哺乳动物的脊椎动物可以再生失去的毛细胞。在鸟类中，支持细胞已被证明可以通过细胞分裂或直接反分化的机制取代丢失的毛细胞。在哺乳动物的听觉感觉器官中，支持细胞不能再生丢失的毛细胞。引人注目的是，我们的研究结果表明，哺乳动物支持细胞保留了作为毛细胞祖细胞的内在能力，但它们再生毛细胞的可塑性受到外部抑制信号的积极抑制。我们的长期目标是利用支持细胞的潜在可塑性来开发基于支持细胞的毛细胞替代策略。为了实现这一目标，提高我们对发育和成熟支持细胞中活跃的分子程序的认识是至关重要的。我们认为，为了“重编程”并诱导支持细胞的去分化，进而诱导支持细胞向毛细胞的反分化，我们首先必须了解支持细胞谱系中控制分化和维持的信号。本研究的目的是确定Notch信号通路（一种进化古老且高度保守的细胞间通讯机制）是否在支持细胞分化和细胞维持中起作用。在我们提案的Specific Aim 1中，我们将测试Notch信号是否在支持细胞分化中起指导作用。我们将测试诱导激活形式的Notch1受体是否足以诱导支持细胞的命运，以及3-分泌酶抑制剂抑制Notch信号是否会影响支持细胞分化的发生和进展。在我们提案的Specific Aim 2中，我们将在支持细胞中切除Notch信号通路的核心成分Rbpj，以确定Notch信号是否需要支持完整耳蜗和毛细胞受损耳蜗的细胞维持。为此，我们将Rbpj条件小鼠突变体与他莫昔芬诱导的CreERT系结合使用。我们期望这一分析将为驱动支持细胞分化的分子机制提供有价值的见解，并阐明Notch信号在成人耳蜗中支持细胞维持的功能。与此同时，我们将解决一个长期存在的问题——“哺乳动物毛细胞受损耳蜗中Notch信号的持续存在是否导致了毛细胞再生的缺乏？”解决这个问题与人类健康有关，因为在成熟耳蜗中使用3-分泌酶抑制剂抑制Notch信号可以为恢复毛细胞再生的潜在能力提供一种手段。

DESCRIPTION (provided by applicant): In humans and other mammalian species, damage to mechano-sensory hair cells is irreversible, leading to deafness and balance disorders. Remarkably, non-mammalian vertebrates regenerate lost hair cells. In birds, supporting cells have been shown to replace lost hair cells by mechanisms involving either cell division or direct trans-differentiation. In the mammalian auditory sensory organ, supporting cells do not regenerate lost hair cells. Strikingly, our findings suggest that mammalian supporting cells retain the intrinsic ability to function as hair cell progenitors, but their plasticity to regenerate hair cells is actively suppressed by external inhibitory cues. Our long-term goal is to utilize the latent plasticity of supporting cells to develop supporting cell based hair cell replacement strategies. To attain this goal, it is vital to improve our knowledge of the molecular programs active in developing and in mature supporting cells. We reason that to "reprogram" and induce de- differentiation of supporting cells and consequently trans-differentiation of supporting cells into hair cells, we first have to understand the signals that control differentiation and maintenance in the supporting cell lineage. The goal of this proposal is to determine if the Notch signaling pathway, an evolutionary ancient and highly conserved cell-to-cell communication mechanism, functions in supporting cell differentiation and cell maintenance. In Specific Aim 1 of our proposal, we will test if Notch signaling plays an instructive role in supporting cell differentiation. We will test if induction of an activated form of the Notch1 receptor is sufficient to induce supporting cell fate and whether inhibition of Notch signaling with 3-secretase inhibitors effects the onset and progression of supporting cell differentiation. In Specific Aim 2 of our proposal, we will ablate Rbpj, a core component of the canonical Notch signaling pathway, in supporting cells to address if Notch signaling is required for supporting cell maintenance in the intact and hair cell damaged cochlea. To do so we will employ Rbpj conditional mouse mutants in combination with tamoxifen inducible CreERT lines. We anticipate that this analysis will provide valuable insight into the molecular mechanisms that drive supporting cell differentiation and elucidate the function of Notch signaling in supporting cell maintenance in the adult cochlea. In parallel, we will address a longstanding question-"does persistence of Notch signaling in the hair cell damaged cochlea underlie the lack of hair cell regeneration in mammals?" Addressing this question is relevant to human health as inhibiting Notch signaling using 3-secretase inhibitors in mature cochlea could provide a means for restoring a latent capacity to regenerate hair cells.