Injury-Evoked Regeneration Mechanism in Olfactory System

描述(申请人提供)：外周嗅觉神经上皮损伤后能够再生；然而，启动复杂的级联反应导致细胞增殖和神经发生的信号知之甚少。我们将研究ATP作为一种重要的局部营养因子在损伤诱导的嗅神经元再生中的作用。我们假设，对嗅觉上皮的伤害性伤害会触发细胞外ATP诱导的信号级联反应，从而启动神经上皮细胞的再生。我们的这一假说基于这样的观察：(1)细胞损伤、溶解以及调节过程将向细胞外环境释放ATP，(2)激活嘌呤能受体诱导神经营养因子的合成和/或释放，有丝分裂和中枢神经系统的分化。因此，急性损伤细胞释放的ATP可以通过激活嘌呤能受体的有丝分裂和促进生长作用来刺激局部再生。生长促进可通过位于支持细胞和基底干细胞上的G蛋白偶联的嘌呤能受体介导，与其他生长因子受体一样，它们可以诱导一系列细胞内事件，从而触发细胞增殖。我们的具体目标是(1)确定伤害性和生理性刺激是否引起OE中ATP的释放。我们将使用生物发光分析和共聚焦显微镜来确定毒理和生理刺激，释放途径和三磷酸腺苷的来源。(2)确定细胞外ATP和嘌呤能受体激活是否引起神经营养因子的释放。我们将使用电生理学、共聚焦显微镜和免疫细胞化学来测量ATP诱导的膜电容的增加，以此作为胞吐作用、ATP囊泡释放和神经营养因子释放的指标。(3)确定细胞外的ATP和激活的嘌呤能受体是否能引起细胞增殖。我们将通过免疫组织化学以及体外和体内实验来确定ATP是否足以启动未损伤的嗅觉上皮细胞的增殖，或协同作用启动未损伤的嗅觉上皮细胞的增殖，和/或促进损伤诱导的增殖。这一数据显然将对增殖的药理学调控产生影响。改变嘌呤类药物和嘌呤能受体作用的药物很容易获得，并可以在治疗上调节嗅觉系统和中枢神经系统中的细胞增殖和分化。

DESCRIPTION (provided by applicant): The peripheral olfactory neuroepithelium is able to regenerate after injury; however the signals that initiate the complex cascade leading to increased cell proliferation and neurogenesis are poorly understood. We will examine ATP as an important local trophic factor in injury-evoked olfactory neuron regeneration. We hypothesize that noxious insult to the olfactory epithelium triggers an extracellular ATP-induced signaling cascade that initiates regeneration of the neuroepithelium. We base this hypothesis on the observation that (1) cell damage, iysis and also regulated processes will release ATP into the extracellular milieu and (2) activation of purinergic receptors induces synthesis and/or release of neurotrophic factors, mitogenesis, and differentiation in the CNS. Thus, ATP released by acutely injured cells could stimulate localized regeneration due to the mitogenic and growth-promoting effects of purinergic receptor activation. Growth promotion can be mediated by G protein coupled purinergic receptors located on sustentacular support cells and basal stem cells, which, like other growth factor receptors, induce a cascade of intracellular events that trigger cell proliferation. Our specific aims are to (1) Determine if noxious and physiological stimuli evoke release of ATP in the OE. We will identify toxicological and physiological stimuli, pathways of release and sources of ATP using bioluminescence assays and confocal microscopy. (2) Determine if extracellular ATP and purinergic receptor activation elicits the release of neurotrophic factors. We will measure ATP-induced increases in membrane capacitance as an indication of exocytosis, vesicular release of ATP, and release of neurotrophic factors using electrophysiology, confocal microscopy and immunocytochemistry. (3) Determine if extracellular ATP and activation of purinergic receptors elicits proliferation. We will determine if ATP is sufficient to initiate proliferation or acts synergistically to initiate proliferation in the uninjured olfactory epithelium, and/or promotes injury-evoked proliferation using immunohistochemistry and in vitro and in vivo assays. This data will clearly have implications for the pharmacological modulation of proliferation. Agents that modify the effects of purinergics and purinergic receptors are readily available and could therapeutically modulate cell proliferation and differentiation, in the olfactory system and in the CNS.