Mechanisms of retrograde signaling between muscle and motor neurons

描述（由申请人提供）：在运动神经元（MNs）通过轴切术失去与肌肉的功能连接后，MN属性发生各种变化，包括切换到再生模式。当运动轴突与肌肉重新建立突触接触时，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MN特性并抑制再生，但潜在的机制尚不清楚。我们已经证明，运动终板乙酰胆碱受体（achr）的阻断会导致神经元尖刺激活的电流阈值（流变基电流）发生类似闭锁术的变化，这是神经元兴奋性的一个重要指标。我们获得的其他证据表明，肌纤维动作电位或机械活动与这种信号传导无关。这些观察结果表明，肌肉和MNs之间的逆行信号可能通过ACHR激活来完成。一个重要的问题是，achr介导的信号丢失是否会引起更广泛的轴切开术后效应。如果是这样，那么肛门切开术的效果可能是基于achr介导的肌肉逆行信号的丧失，而不是损伤本身。这个新颖的想法将在具体目标1中进行测试。其他可用的证据表明，Ca2+内流是由终板achr的激活引起的。这种电流可以激活肌肉内的下游机制，从而将ACHR的激活与最终向MNs产生逆行信号联系起来。位于运动终板的几种Ca2+敏感分子中包括神经元一氧化氮合酶（nNOS）。Ca2+激活nNOS产生一氧化氮（NO），一氧化氮可能通过扩散到运动终端直接向MNs发出信号，或在运动终板激活进一步的下游级联，最终提供逆行信号。在Specific Aim 2中，我们将通过确定外源性NO是否可以阻止ACHR阻断后MN性质的axocut样变化，来测试nNOS在向MN逆行信号传导中的参与。这些研究的结果将增加对触发肌神经网络中轴切开术反应的因素的新见解，并开始识别肌肉中的分子机制，这些机制是控制肌神经网络兴奋性最低的信号的基础。

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.