Mechanisms of retrograde signaling between muscle and motor neurons

描述（由申请人提供）：在运动神经元（MN）通过轴突切断术失去与肌肉的功能连接后，MN特性发生各种变化，包括转换为再生模式。当运动轴突重新与肌肉建立突触联系时，这些变化恢复正常。这一证据表明，突触接触介导肌肉和MN之间的重要相互作用，通常能够表达正常的MN特性并抑制再生，但基本机制知之甚少。我们已经表明，运动终板乙酰胆碱受体（ACHR）的封锁产生轴突切断样的变化，在MN电流阈值的尖峰激活（基电流），一个重要的指标MN兴奋性。我们获得的其他证据表明，肌纤维动作电位或机械活动不参与这种信号传导。这些观察结果表明，肌肉和MN之间的逆行信号可能是通过ACHR激活完成的。一个重要的问题是ACHR介导的信号转导的丢失是否会引起更广泛的轴突切断后效应。如果是这样的话，那么轴突切断的影响可能是基于ACHR介导的肌肉逆行信号的丢失，而不是损伤本身。这一新颖的想法将在具体目标1中得到检验。其他现有证据表明，一个显着的Ca2+内流是由终板ACHR的激活启动。这种电流可以用于激活肌肉内的下游机制，从而将ACHR激活与最终产生向MN的逆行信号联系起来。在位于运动终板的几种Ca 2+敏感分子中包括神经元型一氧化氮合酶（nNOS）。Ca2+激活nNOS产生一氧化氮（NO），其可以通过扩散直接向运动终末或激活运动终板处的进一步下游级联而向MN发出信号，最终提供逆行信号。在具体目标2中，我们将通过确定外源性NO是否可以防止ACHR阻断后MN性质的轴突切断样变化来测试nNOS参与MN的逆行信号传导。这些研究的结果将增加新的洞察力的因素，触发轴突切断反应在MN和开始识别的分子机制，在肌肉的基础信号，控制在最低MN兴奋性。 公共卫生相关性：这项工作的重点是了解肌肉和运动神经元之间逆行信号传导的机制。基于我们收集的数据，我们已经发展了这样的假设，即这种信号是由运动终板乙酰胆碱受体激活。拟议的研究的目的是测试这种信号的丢失是否是运动神经元对轴突切断的反应的基础，并开始鉴定肌肉中介导逆行信号的分子机制。

DESCRIPTION (provided by applicant): After motoneurons (MNs) lose functional connectivity with muscle by axotomy, a variety of changes occur in MN properties including a switch to a regenerative mode. These changes return to normal when motor axons re-establish synaptic contact with muscle. This evidence indicates that synaptic contact mediates important interactions between muscle and MNs that normally enable expression of normal MN properties and inhibit regeneration, but underlying mechanisms are poorly understood. We have shown that blockade of motor endplate acetylcholine receptors (ACHRs) produces axotomy-like changes in MN current threshold for spike activation (rheobase current), an important metric of MN excitability. Additional evidence we have obtained shows that muscle fiber action potential or mechanical activity is not involved in this signaling. These observations suggest that retrograde signaling between muscle and MNs may be accomplished via ACHR activation. An important question is whether loss of ACHR-mediated signaling can provoke a wider range of post-axotomy effects. If so, then the effects of axotomy may be based on the loss of ACHR-mediated retrograde signaling from muscle rather than injury itself. This novel idea will be tested in Specific Aim 1. Other available evidence indicates that a significant Ca2+ influx is initiated by activation of endplate ACHRs. Such currents could serve to activate downstream mechanisms within muscle and thus link ACHR activation to eventual production of retrograde signals to MNs. Included among several Ca2+-sensitive molecules located at the motor endplate is neuronal nitric oxide synthase (nNOS). Ca2+ activation of nNOS produces nitric oxide (NO) which may signal MNs directly via diffusion to motor terminals or activate further downstream cascades at the motor endplate that ultimately provide retrograde signaling. In Specific Aim 2, we will test the involvement of nNOS in retrograde signaling to MNs by determining whether exogenous NO can prevent axotomy-like changes in MN properties after ACHR blockade. The results of these studies will add new insight into factors that trigger the axotomy response in MNs and begin the identification of molecular mechanisms in muscle which underlie signaling that controls at a minimum MN excitability. PUBLIC HEALTH RELEVANCE: This work is focused on understanding mechanisms underlying retrograde signaling between muscle and motor neurons. Based on data we collected, we have developed the hypothesis that this signaling is initiated by motor endplate acetylcholine receptor activation. The purpose of the proposed studies is to test whether loss of this signaling underlies the motor neuron response to axotomy and to begin identification of molecular mechanisms in muscle that mediate retrograde signaling.