Persistent short-term memory defects following sleep deprivation in a drosophila model of Parkinson disease

Abstract

Study objectives: Parkinson disease (PD) is the second most common neurodegenerative disorder in the United States. It is associated with motor deficits, sleep disturbances, and cognitive impairment. The pathology associated with PD and the effects of sleep deprivation impinge, in part, upon common molecular pathways suggesting that sleep loss may be particularly deleterious to the degenerating brain. Thus we investigated the long-term consequences of sleep deprivation on shortterm memory using a Drosophila model of Parkinson disease.

Participants: Transgenic strains of Drosophila melanogaster.

Design: Using the GAL4-UAS system, human alpha-synuclein was expressed throughout the nervous system of adult flies. Alpha-synuclein expressing flies (alpha S flies) and the corresponding genetic background controls were sleep deprived for 12 h at age 16 days and allowed to recover undisturbed for at least 3 days. Short-term memory was evaluated using aversive phototaxis suppression. Dopaminergic systems were assessed using mRNA profiling and immunohistochemistry. MEASURMENTS AND RESULTS: When sleep deprived at an intermediate stage of the pathology, alpha S flies showed persistent short-term memory deficits that lasted > or = 3 days. Cognitive deficits were not observed in younger alpha S flies nor in genetic background controls. Long-term impairments were not associated with accelerated loss of dopaminergic neurons. However mRNA expression of the dopamine receptors dDA1 and DAMB were significantly increased in sleep deprived alpha S flies. Blocking D1-like receptors during sleep deprivation prevented persistent shortterm memory deficits. Importantly, feeding flies the polyphenolic compound curcumin blocked long-term learning deficits.

Conclusions: These data emphasize the importance of sleep in a degenerating/reorganizing brain and shows that pathological processes induced by sleep deprivation can be dissected at the molecular and cellular level using Drosophila genetics.

Figure 1

PD-like pathology in flies expressing human α-synuclein. A, Time line for the progression of α-synuclein pathology in flies expressing UAS α-synuclein under the control of the elavGAL4 driver (αS flies). B, Detection of numerous α-synuclein aggregates in the brains of 20-day-old αS flies. Arrow: large Lewy-body like inclusion in a cell body. Immunohistochemistry on brain sections using anti α-synuclein antibodies. Similar results were obtained in 16 day old αS flies (not shown). C, Genes associated with α-synuclein pathology are down-regulated in 20-day-old αS flies compared to elavGAL4/+ and UAS α-syn/+ controls. Transcript levels are presented as the difference in expression between elavGAL4/+ and UAS α-syn/+ or αS divided by the expression value in elavGAL4/+ flies (% Genetic control change). QPCR data obtained with 2 independent sets of 20 whole fly heads for each condition. One of 2 replicates shown. D, No significant impairment in climbing ability is observed in 16-day-old αS flies; 30-day-old αS flies show significant motor impairments compared to age-matched elavGAL4/+ and UAS α-syn/+ controls. A main effect for age is revealed by 3 (Genotype: elavGAL4/+, UAS α-syn/+, αS) × 2 (Age: 16 vs. 30 days) ANOVA (F_2,12 = 18.1, P = 0.001, *P < 0.05, planned comparison with Tukey Correction). E, APS performance in 20- and 45-day-old αS flies compared to wild-type Cs flies (n = 10 for each group).

Figure 2

Long term learning impairments in old α-synuclein expressing flies. A, Sleep deprivation and test schedule: 16-day-old flies were sleep deprived (TSD, Total Sleep Deprivation) for 12 h (ZT12 to ZT0) and tested for learning (Test) either immediately after sleep deprivation or after being allowed to recover 3 days unperturbed before being tested. Control siblings were left untreated during the same period. All flies were put in TriKinetics tubes for sleep monitoring. B, Learning in sleep deprived αS flies (n = 10) is significantly impaired immediately after sleep deprivation (“No recovery,” left) and after 3 days of recovery (right) compared to untreated age-matched control (n = 10 for each group,*t-test P < 0.05). C, Control metrics: time to complete the learning test (TCT, in minutes), phototaxis index (PI), and Quinine sensitivity index (QSI, in seconds) are similar in sleep deprived flies with 3 days of recovery and untreated age-matched control flies. D, Total daily sleep (min), and average sleep bout duration (min) during both the day and night are similar in sleep deprived and untreated control flies. E, Sleep deprivation in 7-day-old (Young) αS flies or in 20-day-old (Old) flies bearing either elavGAL4 or UAS α-syn alone does not produce long-term performance deficits.

Figure 3

Long term changes in DA signaling. A-B, Long term impairments in αS flies are not associated with an accelerated loss of dopaminergic neurons. Untreated age-matched controls (control) are compared to sleep deprived flies allowed 3-5 days of recovery (sleep deprived). A, Representative dopaminergic neuronal clusters (PPM1,2 and PPM3) in old αS flies,whole-mount immunostaining with anti-TH antibodies). B, Number of dopaminergic neurons included in the PPM1,2, PPM3, PPL1 and PPL2 clusters. Data represents the average obtained from 12 independent clusters. No difference is observed between control and sleep deprived flies allowed 5 days of recovery. C, Under baseline conditions old αS flies show reduced DA receptor mRNA levels compared to their genetic background controls elavGAL4/+ and UAS α-syn/+. Gene expression is presented as % change from elavGAL4/+ control levels (% Genetic control change). One of 2 replicates shown, 20 whole heads used for each condition. (*P < 0.05 one-sample t-test). D, Dopamine receptor mRNA levels are increased after 3 days of recovery following total sleep deprivation (TSD) in αS flies. Gene expression is presented as % change from untreated controls (% Baseline change). One of 2 replicates shown. (*P < 0.05 one-sample t-test).

Figure 4

Pharmacological treatments preventing sleep deprivation induced long term impairments in αS flies. A, Sleep deprivation and test schedule (top). αS flies were tested after 3 days of recovery following 12 h of total sleep deprivation (TSD) and compared to untreated age-matched siblings (Control). Flies fed curcumin did not show long-term performance impairments after sleep deprivation (bottom graph, right). N = 10 for each condition. A 2 (Drug: curcumin vs. vehicle) × 2 (Condition: 3 days recovery vs. control) ANOVA show main effect for Drug (F_2,36 = 4.38, P = 0.04, *planned comparison with Tukey correction P < 0.05). B, Feeding αS flies curcumin (curcumin, white) changes DAT and dDA1 mRNA levels compared to flies fed vehicle (vehicle, black). Gene expression represented as % change from elavGAL4/+ controls fed vehicle. One of 2 replicates shown. C, DA receptor mRNA levels are decreased after 3 days of recovery following sleep deprivation in flies maintained on curcumin (white) and are increased in controls maintained on vehicle (black). Gene expression represented as % change from untreated controls (% Baseline change). One of 2 replicates shown. (*P < 0.05 one-sample t-test). D, Blocking D1 receptor activation during sleep deprivation prevents long term performance deficits. Top scheme: sleep deprivation and test schedule. Old αS flies were fed the D1 antagonist SCH-23390 (1 mg/mL) or vehicle during total sleep deprivation (TSD), then transferred to regular food until being tested for learning 3 days later. Performance in flies fed the D1 antagonist during sleep deprivation (TSD+ D1 antagonist, n = 10) was significantly improved compared to flies sleep deprived on vehicle (TSD, n = 12). The score of age-matched control αS flies is shown for comparison (n = 10) (*t-test, P < 0.05).