Mediators of Lithium Action in Olfactory Epithelium

描述(由申请人提供)：双相情感障碍(BD)是一种慢性衰弱疾病，具有巨大的社会负担。我们开发治疗BD的改进疗法的能力的一个主要障碍是我们对其病理生理学的了解不完整。锂，用于治疗BD，改变了大量基因的稳态mRNA水平，特别是GSK-32、肌醇单磷酸酶(IMPase)、MIP合成酶、Bcl-2、Akt、蛋白激酶C、Marcks、胰岛素样生长因子结合蛋白-2(IGFBP-2)和ERK。但目前还不清楚，这些分子变化是如何带来临床反应的，如果有的话。到目前为止，研究锂在活体BD患者中枢神经(CNS)组织中的细胞和分子效应一直是不可行的。尽管死后大脑对生物标志物的研究很有用，但它们不足以捕捉到BD的状态依赖的分子特征。血细胞可能不能充分反映神经细胞中的分子分布。在这项研究中，我们建议使用鼻活检的嗅觉上皮(OE)，结合新的激光捕获显微解剖来阐明前瞻性确定的锂临床反应的分子和细胞标记。随后将确定这些分子模式在来自相同受试者的淋巴母细胞中的平行配置，以可能用于血液标记物的临床和预后用途。OE是中枢神经系统中的一个独特部分，在整个生命过程中不断再生和分化为成熟的嗅觉感受器神经元(ON)。这为检验BD的神经发育假说提供了一条途径，通过检查增殖(P75NGRF)神经元前体和成熟(嗅觉标记蛋白)神经元(OE中神经发生和神经元存活的替代标记物)密度的变化。同样令人特别感兴趣的是，与锂作用相关的基因调节失调，因此BD可能反映在嗅觉功能的缺陷上。因此，结合嗅觉组织中情绪稳定的分子机制和嗅觉功能的检测，可以促进我们对BD的病理生理学的认识，并为BD的其他发病机制的研究提供新的数据来源。此外，经确认的蛋白质研究证实的淋巴母细胞(LB)中验证基因的平行研究(来自OE研究)可能会抢占血液标记物未来的临床和预后用途。在本项目的具体目标1中，我们建议使用我们成功的BD受试者招募方法，确定40名患有BD急性发作的非药物、不吸烟的受试者，以及20名性别、年龄和种族匹配的非吸烟正常对照(CTL)。在特定的目标2中，我们将：a)比较BD和CTL通过RT-PCR测量的(上述)9个锂作用候选基因的mRNA水平，以及b)检测与锂相关的mRNA水平的变化对BD受试者临床反应(LR)的影响。为了进一步阐明特定目标3中情绪稳定的细胞机制，我们将采用免疫组织化学技术检测治疗前后所有参与者OE样本中针对增殖低亲和力神经生长因子受体(P75NGFR)、有丝分裂后未成熟神经元(生长相关蛋白43[GAP43])和嗅觉标记蛋白(OMP)的细胞类型特异性抗体的密度，以确定增殖神经元前体和成熟神经元(分别是神经发生和神经元存活的替代标记物)密度增加是否是锂相关分子改变对LR影响的中介。在其他研究中，我们将：1)检查BD和CTL之间嗅觉的差异；2)确定基线基因活性和嗅觉功能之间的关联，以及性别对这些关联的影响；3)确定基线嗅觉功能和锂相关的嗅觉变化作为BD严重程度和情绪稳定的替代标记物的有效性；以及4)确定OE和LB在锂作用的分子标记物上的平行处置。这些目标的实现将促进我们对BD的病理生理学的理解，促进具有特定期望的情绪稳定效果的新的和改进的治疗方法的发展，并为科学家团队进一步研究BD的其他发病机制提供新的数据资源。 公共卫生相关性：我们早就应该更好地了解双相情感障碍(BD)的病理生理学，并开发改进的治疗方法，这种疾病会给患者造成广泛的疾病负担，给社会带来巨大的经济负担。我们现在已经开发了新的技术来前瞻性地研究锂治疗期间情绪稳定的细胞和分子决定因素，使用的是急性疾病BD患者的中枢神经系统组织--嗅觉上皮(OE)。这项建议的目标是：1)招募自动患病的BD患者，在单一锂治疗前后评估临床症状并获得重复的OE活组织检查；2)确定其变化预测临床反应的分子标志物；3)确定锂诱导的分子改变的效果是否通过促进神经发生和神经元存活而介导；以及4)检测嗅觉功能和血细胞中的基因活性，以作为疾病相关的、锂可改变的BD分子失调的标志物。本研究可促进BD靶向治疗的发展。

DESCRIPTION (provided by applicant): Bipolar Disorder (BD) is a chronic debilitating illness with substantial societal burden. A major impediment in our ability to develop improved therapeutics for BD is the fact that our understanding of its pathophysiology is incomplete. Lithium, used for treatment of BD, alters steady-state mRNA levels of a large number of genes; notably GSK-32, inositol monophosphatase (IMPase), MIP Synthase, Bcl-2, Akt, Protein Kinase C, MARCKS, insulin-like growth factor binding protein-2 (IGFBP-2), and ERK. But it remains unknown, how any of these molecular changes bring about clinical response, if, at all. Hitherto, studying cellular and molecular effects of lithium in the central nervous (CNS) tissues of living BD patients has been infeasible. Although postmortem brains have been useful for studies of biomarkers, they are insufficient to capture state-dependent molecular signature of BD. Blood cells may not reflect adequately, molecular disposition in neuronal cells. In this study, we propose use of olfactory epithelium (OE) via nasal biopsy, combined with novel laser-captured microdissection to elucidate molecular and cellular markers of prospectively-determined clinical response to lithium. This will be followed by determination of parallel dispositions of these molecular patterns in lymphoblasts obtained from same subjects, for possible clinical and prognostic utility of blood markers. OE is a unique part of the CNS that continually regenerates and differentiates into mature olfactory receptor neurons (ORNs) throughout life. This presents an avenue to examine the neurodevelopmental hypotheses of BD through examination of changes in the densities of proliferating (p75NGRF+) neuronal precursors and mature (olfactory marker protein+) neurons (surrogate markers of neurogenesis and neuronal survival in OE). Also of particular interest, dysregulation of genes relevant to lithium action, and therefore BD may be reflected in deficits in olfactory functions. Therefore, combined study of molecular mechanisms underlying mood stabilization in olfactory tissue and tests of olfactory function could advance our understanding of the pathophysiology of BD and generate new data resource to study other pathoetiologies of BD. Furthermore, parallel studies of validated genes (from OE studies) in lymphoblasts (LB), confirmed by confirmed protein studies, may preempt future clinical and prognostic utility of blood markers. In specific aim 1 of this project, we propose to use our successful recruitment approach of BD subjects, to ascertain 40 non-medicated, non-smoking subjects with acute episodes of BD and 20 gender-, age-, and race- matched non-smoking normal controls (CTL) for this study. In specific aim 2, we will: a) compare BD and CTL on mRNA levels of (above-stated) 9 candidate genes of lithium action measured via RT-PCR, and b) examine the effect of lithium-associated changes in mRNA levels on clinical response (LR) in the BD subjects. To further illuminate cellular mechanisms of mood stabilization in specific aim 3, we will employ immunohistochemical techniques to examine densities of cell type-specific antibodies directed at the proliferating low-affinity nerve growth factor receptor (p75NGFR), postmitotic immature neurons (growth- associated protein 43 [GAP43]), and olfactory marker protein (OMP) in OE samples of all participants pre- and post-treatment, to determine if increased density of proliferating neuronal precursors and mature neurons (surrogate markers of neurogenesis and neuronal survival, respectively) are mediators of the effect of lithium- associated molecular alterations on LR. In other studies, we will: 1) examine differences in olfaction between BD and CTLs; 2) determine the association between baseline gene activity and olfactory function, and the impact of gender on these associations; 3) determine the utility of baseline olfactory function and lithium- associated changes in olfaction as surrogate markers of BD severity and mood stabilization; and 4) determine parallel dispositions between OE and LB on molecular markers of lithium action. Accomplishment of these aims will advance our understanding of the pathophysiology of BD, facilitate development of novel and improved therapeutics with specific desired effect on mood stabilization, and generate new data resource for team of scientists to further investigate other pathoetiologies of BD. PUBLIC HEALTH RELEVANCE: We are well overdue for better understanding of the pathophysiology of, and for development of improved therapeutics of Bipolar disorder (BD), a condition that causes extensive illness burden to sufferers and substantial economic burden to the society. We have now developed novel techniques to prospectively study cellular and molecular determinants of mood stabilization during lithium treatment, using a central nervous system tissue, olfactory epithelium (OE) of acutely ill BD patients. The goals of this proposal are: 1) recruit autely ill BD patients to assess clinical symptoms and obtain repeated OE biopsies, before and after lithium monotherapy; 2) identify molecular markers whose alterations predict clinical response; 3) determine if the effect of lithium-induced molecular alterations is mediated through enhanced neurogenesis and neuronal survival; and 4) examine olfactory function and gene activity in blood cells for utility as markers of disease-associated, lithium-modifiable molecular dysregulation of BD. This study could enhance development of targeted therapeutics for BD.