Fig 1.
Age-related locomotor impairment (ARLI) in cyc0, tim0 and ClkAR arrhythmic mutants.
(A) ARLI of cyc0 and tim0 mutants is accelerated relative to wild-type flies (ctrl) housed in LD. (B) ARLI of wild-type Canton-S flies (ctrl) is accelerated under constant light (LL) that stops the clock. (C) The effect of ClkAR mutation on ARLI is stronger and occurs earlier than the effect of cyc0, tim0 or LL. (D) ClkAR/+, but not cyc0/+, heterozygote mutants also exhibit an accelerated locomotor decline as compared to controls. (E) No effect on ARLI is observed for cyc0 and tim0 mutants in constant light (LL). (F) In contrast, ClkAR mutants exhibit an accelerated ARLI under constant light when compared to wild-type flies (ctrl) kept in LL as well. Graphs display the means ± SEM from 2–3 independent experiments.
Fig 2.
Clk inactivation in the s-LNv induces faster ARLI in aging flies.
(A) Down-regulating Clk expression selectively in all PDF-expressing neurons (pdf>ClkRNAi) results in a premature locomotor decline. (B) Flies with reduced cyc expression in the PDF neurons (pdf>cycRNAi) showed no ARLI defect. (C) Expressing ClkRNAi in the s-LNv selectively (R6-Gal4 driver) had the same effect on climbing abilities as pdf>ClkRNAi. (D) Ablation of PDF neurons by expressing the pro-apoptic gene hid in these cells did not alter ARLI. Graphs display the means ± SEM from 2 independent experiments.
Fig 3.
Rescue of ClkAR ARLI phenotype by Clk expression in the PDF-expressing pacemaker neurons.
(A) Reintroducing Clk expression in the PDF neurons of ClkAR flies (pdf>Clk;ClkAR) significantly rescued the premature locomotor decline of the ClkAR mutation. (B) Clk overexpression in PDF neurons had no effect on SING decline in a wild-type background. Graphs display the means ± SEM from 2 independent experiments.
Fig 4.
Clk regulates brain ROS levels independently from ARLI.
(A, B) Brain ROS levels are higher in ClkAR mutants at both 10 and 31 days of age as compared to ctrl and cyc0 flies. Scale bars in A: 100μm. (C) Downregulating Clk expression in the PDF neurons, which accelerated ARLI (see Fig 2A) had no effect on brain ROS levels as compared to controls. (D) Brain ROS levels of ClkAR flies in which Clk expression was restored in the PDF neurons only, resulting in largely rescued ARLI (see Fig 3A), are unaltered in comparison to respective controls in the same ClkAR background. Histograms display the mean ± SEM of brain ROS levels from 2–3 independent experiments with 6–8 brains per genotype in each experiment.
Fig 5.
Specific loss of dopaminergic PPL1 neurons in Clk-deficient flies.
TH-IR cells were counted in confocal stacks of brains dissected from flies of the indicated ages and genotypes. In all panels, histograms display the mean ± SEM of TH-IR cell numbers in the PAL, PPL1, PPL2, PPM1/2 and PPM 3 clusters, from 2 independent experiments with 8–10 brains per genotype in each experiment. (A) At 10 days of age, the number of TH-IR neurons in the different dopaminergic neuronal clusters of Canton-S (ctrl), ClkAR and cyc0 flies was similar. (B) ClkAR mutants exhibited a selective loss of TH-IR cells in the PPL1 neuronal cluster at 31 days post-eclosion when compared to either controls or cyc0. (C) Loss of TH-IR cells in the PPL1 neuronal cluster was also observed at 31 days post-eclosion in ClkAR/+ heterozygote flies. (D) Reducing Clk levels in the PDF neurons specifically decreased the number of PPL1 TH-IR cells. PAL, protocerebral anterior lateral; PPM1-3, protocerebral posterior median 1–3; PPL1-2, protocerebral posterior lateral 1–2.
Fig 6.
ClkAR locomotor and neuronal phenotypes require the PDF receptor.
(A) ARLI was assayed in the ClkAR and han5304 mutants, and in the han5304;;ClkAR double mutant. The latter did not show the accelerated ARLI characteristic of ClkAR flies. The graph displays the means ± SEM from 2 independent experiments. (B) Histograms display the mean ± SEM of TH-IR cell numbers in the PAL, PPL1, PPL2, PPM1/2 and PPM 3 clusters, from 2 independent experiments with 7–8 brains per genotype in each experiment. At 31 days of age, no TH-IR cell loss was observed in either han5304 or han5304;;ClkAR, contrary to the loss of TH-IR cells in the PPL1 cluster characteristic of aged ClkAR flies.
Fig 7.
PPL1 dopaminergic neuron disruption accelerates ARLI.
(A,B) Expression of the pro-apoptotic gene hid in dopaminergic neurons of the PPL1, PPL2 and PPM3 clusters with TH-D1-Gal4 and TH-D’-Gal4 drivers accelerates ARLI in aging flies. (C) THRNAi expression using MZ840-Gal4 that targets one pair of dopaminergic neurons (the MB-V1 in each PPL1 cluster) as well as non-dopaminergic brain cells similarly speeded up SING decline with age. Data in panels A and B are from 2 separate experiments, with 5 groups of flies tested at each age for each genotype, the graphs showing means and sem for these 5 groups. Panel C is an average of 2 independent experiments with the indicated genotypes.
Fig 8.
Proposed link between PDF-expressing and dopaminergic neurons in the Drosophila brain.
Reducing Clk expression in the s-LNv neurons (bright red with blue outline) accelerates ARLI, and perturbates the dopaminergic PPL1 cluster (somas shown in blue). The PDF receptor is involved, possibly overactivated by PDF released from the s-LNvs. In the ClkAR mutant, this could in particular negatively affect the MB-V1 dopaminergic neuron, either directly or indirectly, leading to accelerated ARLI. The projections of the l-LNv neurons (brown red) onto the right brain hemisphere were omitted for clarity.