Role of Circadian Clock Components in Apoptosis and Tauopathy in Drosophila

项目总结 在人类中，很明显，生物钟功能的降低与糖尿病发病率的增加有关。 神经退行性变(如阿尔茨海默病；阿尔茨海默病)。虽然有假设认为昼夜节律失调 和神经退行性变可能存在联系，但这些拟议联系的机制仍不确定。我的实验室有 有证据表明，果蝇的生物钟成分调节对细胞死亡的敏感性和 长时间暴露在光下的神经退化途径。双倍体蛋白激酶(DBT)，它 磷酸化关键的生物钟蛋白来控制它们的日常积累，是共同的环节。在各种情况下 DBT激酶活性降低的基因型，DRONC caspase，它将各种靶蛋白裂解为 启动细胞死亡途径，激活并裂解tau蛋白，这与增强 直立性眼病的苍蝇眼模型中的变性。DBT最近与另外两种昼夜节律蛋白相互作用 由我的实验室(SPAG和BDBT)发现，以维持对DRONC激活的抑制。反过来，SPAG可能会 与热休克蛋白90(HSP90)和tau相互作用，以减少tau病。此外，激活 DRONC caspase更广泛地出现在不表达转基因的细胞中，从而降低了DBT活性 并由昼夜节律神经肽PDF发出信号。这些发现导致我们提出了一种机制，在这种机制中 昼夜节律成分DBT激酶磷酸化DRONC半胱氨酸天冬氨酸氨基转移酶抑制其激活和DRONC- 依赖tau裂解，这可能会导致tau病。抑制细胞死亡似乎需要 DBT的磷酸化。拟议中的工作将测试这一机制，并绘制出生物学后果 在苍蝇模型中。目的1.确定DBT依赖的caspase激活和tau的生化途径 互动。具体地说，我的实验室将调查DBT激酶是否直接磷酸化DRONC 半胱氨酸天冬氨酸氨基转移酶抑制其裂解，以及细胞死亡诱导刺激，如紫外线刺激 DBT的自动磷酸化、SPAG辅伴蛋白和BDBT FKBP的解离及裂解 DRONC.BDBT辅伴侣调节DRONC激酶激活的能力将在 新的bdbt crispr突变体，以及spag辅伴蛋白与HSP90和tau相互作用的性质将是 评估过了。目的2.确定果蝇眼中tau的裂解是否对DBT活性降低作出反应 导致典型的肌萎缩侧索硬化症特征，有助于增强神经退行性变，或者 DBT降低的作用是通过细胞死亡(caspase导向的)途径独立于tau介导的。 我们将试图通过只在成虫体内而不是在整个果蝇中表达这些蛋白质来诱导眼睛退化 为了更准确地模拟年龄相关的神经退行性变，眼睛的发育。我们将确定 在苍蝇眼中，DBT激酶的减少在多大程度上诱导了细胞死亡途径，以及在多大程度上 肌萎缩侧索硬化症的各个方面都是下游的后果(例如，tau磷酸化、NFT形成和 线粒体和细胞周期功能障碍)。此外，被切割的tau在下游产生tautation的能力 将测试DBT激酶的还原，以及DRONC caspase激活在神经退行性变中的作用 通过用RNAi拮抗DRONC或用非磷酸化形式的 DRONC.目的3.确定DBT介导的caspase相互作用的机制 激活和衰老、光照和昼夜节律的PDF信号通路。我们已经找到了那道光 在暴露7小时后，在我们的果蝇中诱导视叶中的半胱氨酸天冬氨酸酶，与昼夜时间无关 降低了大脑昼夜节律细胞中的DBT活性。这种影响包括昼夜节律PDF发出的信号 神经肽，并可出现在不表达转基因的细胞中，降低DBT激酶的活性。我们会 确定昼夜节律和视觉光感受器在这一过程中的重要性，以及野生型苍蝇 由于衰老而产生紧张症。发生DRONC caspase激活的细胞将是 以及PDF神经肽受体是否会增加DBT激酶的磷酸化 以及细胞内DBT水平的下降。最后，将确定caspase的激活是否 观察会影响先前观察到的视叶中依赖光的神经元重塑过程。

PROJECT SUMMARY In humans, it is clear that reduced function of the circadian clock is associated with increased incidence of neurodegeneration (e.g., Alzheimer’s Disease; AD). While it has been hypothesized that circadian dysfunction and neurodegeneration may be linked, the mechanism for these proposed links remains uncertain. My lab has evidence that the circadian clock components in Drosophila regulate sensitivity to cell death and neurodegeneration pathways after prolonged exposure to light. The doubletime protein kinase (DBT), which phosphorylates key circadian clock proteins to control their daily accumulation, is the common link. In various genotypes with reduced DBT kinase activity, the DRONC caspase, which cleaves various target proteins to initiate cell death pathways, becomes activated and cleaves tau protein, which is associated with enhanced degeneration in a fly eye model for tauopathy. DBT interacts with two other circadian proteins recently discovered by my lab (SPAG and BDBT) to maintain inhibition of DRONC activation. In turn, SPAG may interact with heat shock protein 90 (HSP90) and tau to reduce tauopathies. Furthermore, activation of the DRONC caspase occurs more broadly in cells that do not express the transgenes knocking down DBT activity and is signaled by the circadian neuropeptide PDF. These findings lead us to propose a mechanism in which the circadian component DBT kinase phosphorylates DRONC caspase to inhibit its activation and DRONC- dependent tau cleavage, which can lead to tauopathy. The inhibition of cell death seems to require phosphorylation of DBT. The work proposed will test this mechanism and map out the biological consequences in the fly model. Aim 1. Determine the biochemical pathway of DBT-dependent caspase activation and tau interactions. Specifically, my lab will investigate whether DBT kinase directly phosphorylates the DRONC caspase to inhibit its cleavage, and whether cell cell-death inducing stimuli like UV stimulate autophosphorylation of DBT, dissociation of SPAG cochaperone and BDBT FKBP from DBT and cleavage of DRONC. The capacity of BDBT cochaperone to regulate activation of DRONC kinase will be investigated in novel bdbt CRISPR mutants, and the nature of SPAG cochaperone interactions with HSP90 and tau will be assessed. Aim 2. Determine whether tau cleavage in the fly eye in response to reduced DBT activity leads to typical tauopathy features that contribute to enhanced neurodegeneration, or whether the effect of reduced DBT is mediated independently of tau via cell death (caspase-directed) pathways. We will try to induce eye degeneration by expressing these proteins only in the adult fly instead of throughout eye development in order to more accurately mimic age-dependent neurodegeneration. We will determine the extent to which cell death pathways are induced in the fly eye by DBT kinase reductions and the extent to which various aspects of tauopathy are downstream consequences (e.g., tau phosphorylation, NFT formation, and mitochondrial and cell cycle dysfunction). Also, the capacity of cleaved tau to produce tauopathy downstream of DBT kinase reductions will be tested, and the role of DRONC caspase activation in the neurodegeneration will be assessed by antagonizing DRONC with RNAi or potentiating it with nonphosphorylatable forms of DRONC. Aim 3. Determine the mechanisms for the interaction between DBT-mediated caspase activation and aging, light and the circadian rhythm PDF signaling pathway. We have found that light induces caspases in the optic lobes after 7 hrs of exposure, independent of circadian time, in our flies with reduced DBT activity in the circadian cells of the brain. The effect involves signaling by the circadian PDF neuropeptide and can occur in cells that do not express the transgenes reducing DBT kinase activity. We will determine the importance of circadian and visual photoreceptors for this process, and whether wild type flies produce tauopathy as a consequence of aging. The cells in which the DRONC caspase activation occurs will be determined, and whether the PDF neuropeptide receptor produces an increase in DBT kinase phosphorylation and a decrease in DBT levels in the cells. Finally, it will be determined whether the caspase activation we observe affects a previously observed light-dependent neuronal remodeling process in the optic lobes.