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）调控生理与行为领域诸多约24小时周期的自维持节律。在诸多生物中，氧化应激与衰老会对昼夜节律系统及睡眠产生负面影响。反之，生物钟功能丧失会降低机体对氧化应激的抵抗能力，还可能缩短寿命、加速大脑衰老与神经退行性病变。本研究以果蝇（Drosophila）为模型，探究了生物钟紊乱对运动衰老与寿命的影响。研究发现，三种节律紊乱突变体（ClkAR、cyc0与tim0）以及处于阻断生物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。与之形成对比的是，ClkAR突变体的年龄相关性运动功能缺陷（通过振动诱导攀爬实验评估）进展速度，显著快于cyc0、tim0突变体，以及持续光照环境下的对照野生型果蝇。ClkAR突变体大脑内的活性氧（reactive oxygen species, ROS）随衰老积累程度更高，但这似乎并非该突变体运动功能加速衰退的诱因。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, RNAi）抑制Clk基因（而非Cyc基因）的表达，会导致与ClkAR突变体相似的攀爬能力丧失。反之，仅在上述神经元中恢复Clk基因功能，即可挽救ClkAR突变体的运动功能表型，且这一效应不依赖于行为节律性。ClkAR突变体运动功能加速衰退的现象，依赖于PDF受体（PDF receptor）的表达，且与后脑外侧后1区（posterior protocerebral lateral 1, PPL1）簇内多巴胺能神经元（dopaminergic neurons）的明显丢失相关。当ClkAR突变处于凋亡缺陷背景中时，该神经元丢失现象可被挽救。在支配蕈形体（mushroom bodies）的一对PPL1神经元中损伤多巴胺合成过程，会使正常野生型果蝇出现运动功能加速衰退。综上，本研究揭示了果蝇大脑昼夜节律神经元中Clk基因一项全新的、不依赖于昼夜节律的功能：维持特定亚群的多巴胺能神经元，从而避免运动系统过早衰老。