Data from: Drosophila clock is required in brain pacemaker neurons to prevent premature locomotor aging independently of its circadian function

Circadian clocks control many self-sustained rhythms in physiology and behavior with approximately 24-hour periodicity. In many organisms, oxidative stress and aging negatively impact the circadian system and sleep. Conversely, loss of the clock decreases resistance to oxidative stress, and may reduce lifespan and speed up brain aging and neurodegeneration. Here we examined the effects of clock disruptions on locomotor aging and longevity in Drosophila. We found that lifespan was similarly reduced in three arrhythmic mutants (ClkAR, cyc0 and tim0) and in wild-type flies under constant light, which stops the clock. In contrast, ClkAR mutants showed significantly faster age-related locomotor deficits (as monitored by startle-induced climbing) than cyc0 and tim0, or than control flies under constant light. Reactive oxygen species accumulated more with age in ClkAR mutant brains, but this did not appear to contribute to the accelerated locomotor decline of the mutant. Clk, but not Cyc, inactivation by RNA interference in the pigment-dispersing factor (PDF)-expressing central pacemaker neurons led to similar loss of climbing performance as ClkAR. Conversely, restoring Clk function in these cells was sufficient to rescue the ClkAR locomotor phenotype, independently of behavioral rhythmicity. Accelerated locomotor decline of the ClkAR mutant required expression of the PDF receptor and correlated to an apparent loss of dopaminergic neurons in the posterior protocerebral lateral 1 (PPL1) clusters. This neuronal loss was rescued when the ClkAR mutation was placed in an apoptosis-deficient background. Impairing dopamine synthesis in a single pair of PPL1 neurons that innervate the mushroom bodies accelerated locomotor decline in otherwise wild-type flies. Our results therefore reveal a novel circadian-independent requirement for Clk in brain circadian neurons to maintain a subset of dopaminergic cells and avoid premature locomotor aging in Drosophila.

昼夜节律钟（Circadian clock）调控生理与行为中诸多以约24小时为周期的自维持节律。在诸多生物体中，氧化应激（oxidative stress）与衰老会对节律系统及睡眠产生负面影响。反之，节律钟功能丧失会降低机体对氧化应激的抗性，或缩短寿命、加速脑衰老与神经退行性变。本研究以果蝇（Drosophila）为模型，探究了节律钟紊乱对运动衰老与寿命的影响。我们发现，3株节律紊乱突变体（ClkAR、cyc0与tim0）以及处于阻断节律钟功能的持续光照环境下的野生型果蝇，其寿命均出现了相似程度的缩短。与之相反，ClkAR突变体的衰老相关运动功能缺陷——以震动诱导攀爬实验监测的结果为准——相较于cyc0、tim0突变体，或持续光照下的对照果蝇，出现得显著更早、进展得更快。随衰老进程，ClkAR突变体脑内的活性氧（Reactive oxygen species）积累量更高，但这似乎并未参与该突变体运动能力的加速衰退。在表达色素分散因子（pigment-dispersing factor, PDF）的中枢起搏神经元中，通过RNA干扰（RNA interference）使Clk而非Cyc失活，会导致与ClkAR突变体相似的攀爬能力下降。反之，仅需在这些细胞中恢复Clk功能，即可挽救ClkAR突变体的运动表型，且该挽救效应与行为节律性无关。ClkAR突变体运动能力的加速衰退，依赖于PDF受体的表达，且与后脑外侧后1群（posterior protocerebral lateral 1, PPL1）中的多巴胺能神经元明显丢失存在相关性。当ClkAR突变置于凋亡缺陷背景中时，这种神经元丢失现象可被逆转。在一对仅支配蕈形体（mushroom bodies）的PPL1神经元中损伤多巴胺合成，会加速野生型果蝇的运动衰老。综上，本研究揭示了果蝇脑节律神经元中Clk的一种非依赖昼夜节律的全新功能：维持特定亚群的多巴胺能神经元，从而避免果蝇出现过早的运动衰老。