Glucocorticoid-induced TrkB receptor regulation by ubiquitination

描述（由申请人提供）：压力和糖皮质激素，在压力经历后释放到血液循环中，已被证明对各种精神疾病的表现有显著贡献，包括创伤后应激障碍（PTSD）和抑郁症。脑源性神经营养因子（BDNF）通过其受体TrkB信号传导在应激介导的前额皮质结构和功能可塑性变化中起关键作用。前额皮质TrkB表达和信号的失调以及糖皮质激素水平的升高与精神分裂症、创伤后应激障碍和抑郁症有关。此外，研究表明，与急性基因组功能相比，糖皮质激素对BDNF信号的慢性治疗作用是通过非基因组作用实现的。然而，调控TrkB受体内化和转换的分子机制尚不清楚。本提案的总体目标是确定泛素化在糖皮质激素诱导的TrkB下调中的作用。我们提供的初步证据表明，慢性糖皮质激素诱导泛素蛋白酶体介导的TrkB降解和泛素E3连接酶c-Cbl的上调。我们还提供证据表明，精神分裂症和双相情感障碍受试者的背外侧前额叶皮层中c-Cbl mRNA水平更高。我们发现在精神分裂症受试者的前额叶皮层中TrkB泛素化增加并与c-Cbl相关。我们假设慢性糖皮质激素诱导前额皮质c-Cbl依赖性TrkB下调。我们还将研究Sprouty2在糖皮质激素诱导的TrkB下调中的作用。Sprouty2通过与c-Cbl的相互作用调节生长因子信号。我们最近的研究发现，在精神分裂症、双相情感障碍和自杀受试者的背外侧前额叶皮层中，Sprouty2 mRNA水平显著降低。我们现在提供了初步证据，表明慢性糖皮质激素暴露会下调Sprouty2的表达。我们提出了一系列全面的研究，旨在通过共免疫沉淀研究、小干扰RNA （siRNA）分析和免疫荧光显微镜实验，研究c-Cbl和Sprouty2在糖皮质激素诱导的初级皮质神经元TrkB下调中的作用。我们的目标是确定慢性糖皮质激素暴露后TrkB下调的细胞和分子机制。提出了两个具体目标。特异性目的1将检验慢性糖皮质激素诱导的TrkB下调是通过c-Cbl介导的假设。Specific Aim 2将验证Sprouty2过表达通过与c-Cbl的相互作用减弱糖皮质激素诱导的TrkB表达下调的假设。更好地表征c-Cbl和Sprouty2蛋白的作用可能有助于深入了解TrkB受体调控的机制。此外，这样一个系统的研究还将提供基本的机制信息，这对最终开发有效治疗几种压力相关病理（包括创伤后应激障碍、精神分裂症和抑郁症）至关重要。

DESCRIPTION (provided by applicant): Stress and glucocorticoid hormones, which are released into the circulation following stressful experiences, have been shown to contribute significantly to the manifestation of various psychiatric illnesses including Post- Traumatic Stress Disorder (PTSD) and depression. The brain-derived neurotrophic factor (BDNF) signaling through its receptor TrkB plays a critical role in stress-mediated changes in structural as well as functional plasticity in prefrontal cortex. Deregulation of TrkB expression and signaling in prefrontal cortex, and elevated glucocorticoid levels are linked to schizophrenia, PTSD and depression. In addition, studies have indicated that the chronic treatment effect of glucocorticoids on BDNF signaling is through non-genomic actions as compared to their acute genomic functions. However, the molecular mechanisms that regulate TrkB receptor internalization and turnover remain unknown. The overall goal of this proposal is to determine the role of ubiquitination in glucocorticoid-induced downregulation of the TrkB. We provide initial evidence demonstrating that chronic glucocorticoid induces ubiquitin-proteasome-mediated degradation of TrkB and up-regulation of the ubiquitin E3 ligase c-Cbl. We also provide evidence that the c-Cbl mRNA levels are higher in the dorsolateral prefrontal cortex of schizophrenia and bipolar subjects. We found an increased TrkB ubiquitination and association with c-Cbl in the prefrontal cortex of schizophrenia subjects. We hypothesize that chronic glucocorticoid induces c-Cbl dependent downregulation of TrkB in prefrontal cortex. We will also investigate the role of Sprouty2 in glucocorticoid -induced downregulation of TrkB. Sprouty2 is known to regulate growth factor signaling through its interaction with c-Cbl. Our recent study has found a significant reduction in Sprouty2 mRNA levels in the dorsolateral prefrontal cortex of schizophrenia, bipolar and suicide subjects. We now provide initial evidence that chronic glucocorticoid exposure downregulates Sprouty2 expression. We propose a comprehensive series of studies aimed at examining the role of c-Cbl and Sprouty2 in chronic glucocorticoid -induced TrkB downregulation in primary cortical neurons using co- immunoprecipitation studies, small interfering RNA (siRNA) analysis and immunofluorescence microscopy experiments. Our objective is to define the cellular and molecular mechanisms that underlie the TrkB downregulation following chronic glucocorticoid exposure. Two Specific Aims are proposed. The Specific Aim 1 will test the hypothesis that chronic glucocorticoid-induced TrkB downregulation is mediated through c-Cbl. The Specific Aim 2 will test the hypothesis that overexpression of Sprouty2 attenuates glucocorticoid-induced downregulation of TrkB expression through its interaction with c-Cbl. A better characterization of the role of c-Cbl and Sprouty2 proteins may open up insights into the mechanisms governing TrkB receptor regulation. In addition, such a systematic study will also provide fundamental mechanistic information that is critical to the eventual development of effective therapy for several stress related pathologies, including PTSD, schizophrenia and depression. PUBLIC HEALTH RELEVANCE: This application will explore the mechanism of TrkB receptor regulation under chronic stress. Given the important role of TrkB signaling in brain function, the findings of this exploratory study (R21) may provide avenues to develop newer therapeutic agents for such debilitating disorders PTSD, addiction, autism, depression and schizophrenia.