Cortico-Spinal Repopulation from Precursors for Repair

描述（由申请人提供）：所提出的实验的长期目标是通过i）从内源性前体（“干细胞”）诱导新的皮质脊髓运动神经元（CSMN）的神经发生; ii）支持其存活，以及iii）募集到具有下运动神经元群体的功能性突触回路中，来修复受损或退化的皮质脊髓回路。最近，我们已经操纵内源性前体原位在成年小鼠进行神经发生（新神经元的诞生）从头在成年小鼠新皮层，在那里它通常不会发生。 在合作工作中，我们诱导行为功能的神经元替代原位鸣禽同源鸟类内源性前体。这些实验表明，存在一系列分子信号，通过这些信号可以诱导新神经元的诞生，即使在神经发生通常不会发生的成人新皮层中也是如此。更直接相关的是我们最近的发现：1）内源性前体细胞也可以被特异性诱导原位分化为皮质脊髓运动神经元（CSMN），并向脊髓投射; 2）我们可以分离，流式细胞仪纯化（至>99.5%纯度），并在不同的发育阶段培养CSMN以分析对存活和分化的谱系特异性控制，特别是轴突伸长的速率/程度。我们假设，我们可以通过提高新生神经元的存活率和/或其轴突生长到支持性脊髓靶点的速率和程度来大幅增加新招募的CSMN的数量。我们还假设，新生神经元可以精确地分化成新的功能CSMN，接受传入突触，并成为突触整合。我们的五个目标将在体外和体内直接测试这些假设和相关假设。拟议的研究将：目的1）对诱导的成年CSMN的谱系特异性分化进行更完整的分析，并对诱导的新生CSMN的神经发生、神经元分化、连接和长期存活的时间过程进行更详细的表征;目标2，3）在体外研究一组选择的候选生长和神经营养因子，其能够潜在地提高阶段特异性存活和/或或在不同发育阶段采用流式细胞仪纯化CSMN，以及水浴与局部因子应用，增强CSMN的生长和回路连接性;目的4）通过脑室内输注来自体外实验和目的5）的高度选择的候选生长因子来操纵和增加诱导的成人CSMN神经发生和脊髓投射。使用ICC和逆转录病毒GFP-大麦凝集素表达研究CSMN分化和潜在传入和传出突触形成的精确性。总之，这项工作的最终目标是修复皮质脊髓运动神经元回路的内源性神经前体原位操作，而无需移植。

DESCRIPTION (provided by applicant): The long-term goal of the proposed experiments is the repair of damaged or degenerated cortico-spinal circuitry via i) induction of neurogenesis of new cortico-spinal motor neurons (CSMN) from endogenous precursors ("stem cells"); ii) support of their survival, and iii) recruitment into functional synaptic circuitry with the lower motor neuron population. Recently, we have manipulated endogenous precursors in situ in the adult mouse to undergo neurogenesis (birth of new neurons) de novo in adult mouse neocortex, where it does not normally occur. In collaborative work, we induced behaviorally functional neuronal replacement in situ in songbirds by homologous avian endogenous precursors. These experiments demonstrated that there exists a sequence and combination of molecular signals by which the birth of new neurons can be induced, even in the adult neocortex where neurogenesis does not normally occur. Even more directly relevant to this proposal are our recent findings that 1) endogenous precursors can also be specifically induced to differentiate in situ into cortico-spinal motor neurons (CSMN) with projections to the spinal cord; and 2) that we can isolate, FACS purify (to >99.5% purity), and culture CSMN at distinct developmental stages for analysis of lineage-specific controls over survival and differentiation, particularly rate / extent of axon elongation. We hypothesize that we can substantially increase the number of newly recruited CSMN by enhancing the survival of the newborn neurons and/or their rate and extent of axonal outgrowth to supportive spinal cord targets. We also hypothesize that newborn neurons can differentiate precisely into new functional CSMN, receive afferent synapses, and become synaptically integrated. Our five Aims will test these and related hypotheses directly, in vitro and in vivo. The proposed research will: Aim 1) undertake a more complete analysis of lineage-specific differentiation of the induced adult-born CSMN, and a more detailed characterization of the time course of induced neurogenesis, neuronal differentiation, connectivity, and long-term survival of newborn CSMN); Aims 2,3) investigate in vitro a select set of candidate growth and neurotrophic factors enabling potentially stage-specific enhanced survival and/or enhanced outgrowth and circuit connectivity of CSMN, employing FACS purification of CSMN at distinct developmental stages, and bath vs. localized factor application; Aim 4) manipulate and increase the induced adult CSMN neurogenesis and spinal projections by intraventricular infusion of highly selected candidate growth factors from the in vitro experiments and Aim 5) investigate the precision of CSMN differentiation and potential afferent and efferent synapse formation using ICC and retroviral GFP-barley lectin expression. Together, this work aims toward the ultimate goal of repair of cortico-spinal motor neuron circuitry by manipulation of endogenous neural precursors in situ, without transplantation.