Lacritin Regulated Ocular Surface Homeostasis

描述(申请人提供)：‘乳酸菌素’是人类泪液中一种眼部特异性的前分泌和生存因子。据报道，人类干眼症患者的泪液中存在选择性催乳素缺乏症。在这里，我们验证了这一假设，即泪蛋白是角膜感觉神经活动的关键调节因子，是基础撕裂和眼表新陈代谢的关键调节因子，是抵抗压力生存的关键。重要 这一假说的含义是，活性催乳素缺乏可能引发干眼病，局部应用催乳素可能具有治疗作用。我们最近报道了治疗缺水干眼症的小鼠。自身免疫调节(AIRE)缺陷[AIRE-/-]小鼠服用Lacritin后，泪液流量增加，眼表健康。Lacritin还可以减轻泪腺炎症。在正常的兔眼上，我们发现单一剂量的催乳素可以持续数小时。我们在大鼠身上进行的新的电生理学研究表明，局部应用lacritin可以增强角膜感觉神经的基础泪液刺激性‘干性反应’。干性反应是由TRPM8阳离子通道调节的，该通道敏锐地感觉到眼睛自然脱水的结果--轻微的角膜降温。从Lacritin的C-末端(‘C-25’)截断25个氨基酸可以消除干反应刺激和撕裂，这意味着细胞靶向的能力存在于这个结构域中。我们最近发现，人工合成的KQFIENGSEFAQKLLKKFS(‘N-94/C-6’)结构域与Lacritin一样有效，并与细胞表面硫酸乙酰肝素蛋白多糖‘syndecan-1’以及肾上腺素能2C受体特异性地相互作用。这一发现提示了一种神经调节机制，因为肾上腺素能2C和/或2A受体调节TRPM8的活动。我们刚刚报道了在炎性细胞因子应激下，泪囊素如何恢复角膜上皮细胞的眼表功能。通过依赖于长寿因子‘Forkhead Box 03’(FOX03)和鲜为人知的‘ATG101’的信号通路，lacritin刺激线粒体氧化磷酸化和自噬。自噬是细胞中常见的质量控制机制。ATG101的结合伙伴提示了lacritin加速自噬和恢复线粒体功能的机制(分别通过自噬介体ATG13和线粒体ETFA)。我们的工作假设是，催乳素对正常的泪水、眼表和腺体健康是必不可少的。我们的近期目标是阐明催泪素刺激的泪液和眼表健康的统一机制。我们的具体目标是(1)稳定N-94/C-6或更小的合成形式的Lacritin，(2)表征局部Lacritin如何促进基底部撕裂，以及(3)解决Lacritin如何恢复眼表健康。弗吉尼亚大学夏洛茨维尔分校 弗吉尼亚詹姆斯·麦迪逊大学哈里森堡弗吉尼亚大学旧金山旧金山加利福尼亚州托马斯·杰斐逊大学费城宾夕法尼亚州

DESCRIPTION (provided by applicant): 'Lacritin' is an ocular specific prosecretory and prosurvival factor in human tears. Selective lacritin deficiency has been reported in human dry eye tears. Here we test the hypothesis that lacritin is a key regulator of corneal sensory nerve activity for basal tearing, and of ocular surface metabolism for survival against stress. Important implications of this hypothesis are that deficiency of active lacritin may initiate dry eye disease and that topical lacritin may be therapeutic. We recently reported on treating mice suffering from aqueous deficient dry eye disease. Autoimmune regulator (Aire)-deficient [Aire-/-] mice dosed with lacritin displayed enhanced tear flow and ocular surface health. Lacritin also reduced lacrimal gland inflammation. On normal rabbit eyes, we found that a single lacritin dose was effective for hours. Our new electrophysiological studies in rats reveal that topical lacritin enhances the basal tear stimulatory 'dry response' of corneal sensory nerves. The dry response is regulated by TRPM8 cation channels that exquisitely sense slight corneal cooling as a consequence of the natural tendency of the eye to dessicate. Truncating 25 amino acids from lacritin's C- terminus ('C-25') abrogated dry response stimulation and tearing, implying that the capacity for cell targeting resides within this domain. We recently found that the domain generated synthetically as KQFIENGSEFAQKLLKKFS ('N-94/C-6') is as potent as lacritin, and interacts specifically with the cell surface heparan sulfate proteoglycan 'syndecan-1', and with the adrenergic ¿2C receptor. This discovery suggests a neural regulatory mechanism since adrenergic ¿2C and/or ¿2A receptors modulate TRPM8 activity. We just reported how lacritin restores ocular surface function in corneal epithelial cells stressed with inflammatory cytokines. Through a signaling pathway dependent of the longevity factor 'Forkhead Box 03' (FOXO3) and on the little known 'ATG101', lacritin stimulates mitochondrial oxidative phosphorylation and autophagy. Autophagy is a common quality control mechanism in cells. Binding partners of ATG101 suggest mechanisms by which lacritin accelerates autophagy and restores mitochondrial function (respectively through autophagy mediator ATG13, and mitochondrial ETFA). Our working hypothesis is that lacritin is essential for normal tearing, and ocular surface and glandular health. Our immediate goal is to elucidate unifying mechanisms of lacritin stimulated tearing and ocular surface health. Our specific aims are to (1) stabilize N-94/C-6 or smaller synthetic form of lacritin, (2) characterize how topical lacritin promotes basal tearing, and (3) resolve how lacritin restores ocular surface health. University of Virginia Charlottesville Virginia James Madison University Harrisonburg Virginia University of California, San Francisco San Francisco California Thomas Jefferson University Philadelphia Pennsylvania