ERK phosphorylation regulates sleep and plasticity in Drosophila

Abstract

Given the relationship between sleep and plasticity, we examined the role of Extracellular signal-regulated kinase (ERK) in regulating baseline sleep, and modulating the response to waking experience. Both sleep deprivation and social enrichment increase ERK phosphorylation in wild-type flies. The effects of both sleep deprivation and social enrichment on structural plasticity in the LNvs can be recapitulated by expressing an active version of ERK (UAS-ERK(SEM)) pan-neuronally in the adult fly using GeneSwitch (Gsw) Gsw-elav-GAL4. Conversely, disrupting ERK reduces sleep and prevents both the behavioral and structural plasticity normally induced by social enrichment. Finally, using transgenic flies carrying a cAMP response Element (CRE)-luciferase reporter we show that activating ERK enhances CRE-Luc activity while disrupting ERK reduces it. These data suggest that ERK phosphorylation is an important mediator in transducing waking experience into sleep.

Figure 1. ERK activation increases sleep.

A, Pan-neuronal expression of activated ERK in adult flies results in an increase in sleep. Sleep is shown in minutes/hour in RU486 (RU+) fed +;UAS-ERK^SEM/+ ;GSw-elav/+ flies and their vehicle fed controls (RU-). A 2(RU+, RU-) X 24(Hour) ANOVA reveals a significant drug by hour interaction F_(23,1488) = 7.42, P = 5.1E-23,(n = 32/ group). B-D, Total sleep time and sleep bout duration during the light period were significantly increased in RU+ fed +;UAS-ERK^SEM/+ ;GSw-elav/+ compared to RU- controls, p = 7.25E-7 and p = 1.77E-6, respectively; student’s t-test, n=31-32/group. RU+ treated flies showed no change in sleep consolidation at night p = 0.103; student’s t-test, n=31-32/group. Data are presented as mean ± SEM, (* = p < 0.05). E, RU fed +;UAS-ERK^SEM/+ ;GSw-elav/+ showed increased activity during waking (ZT1-ZT12) compared to vehicle fed controls; A 2(RU+, RU-) X 12(Hour) ANOVA reveals a significant main effect of drug F _(1,600) = 88.47, P = 1.08 E-19] (n = 26/ group). F, RU+ fed +;UAS-ERK^SEM/+ ;GSw-elav/+ showed an exaggerated homeostatic response following 12 h of sleep deprivation compared to RU- fed siblings. Data are presented as cumulative loss then gained in minutes. A negative slope indicates sleep lost, and a positive slope indicates sleep gained; when the slope is zero, recovery is complete. A 2(RU+, RU-) X 72(Hour) ANOVA reveals a significant drug by hour interaction [F_(71,3888) = 8.08, P = 2.88E-73] (n = 28/ group). G, Pharmacological inhibition of ERK in adult Cs flies reduces sleep. Adult flies were fed 2mM of the Mitogen-activated protein kinase kinase (MEK) inhibitor SL327. Two-way ANOVA reveals a significant interaction of drug (SL3227, control) treatment by time [F(_23,1392) = 9.57, P = 2.4E-31] (n = 30 for each group). Inset shows that SL327 administration reduces total sleep time significantly in wild type Cs flies but did not alter total sleep in the Drosophila rl ¹ mutants p= 0.08 (shown in inset), suggesting specificity of the drug to cause the sleep reduction phenotype. H-J, Total sleep time and sleep consolidation during the light period were significantly decreased in SL327 fed flies (n=30) compared to vehicle fed controls (n=30), p = 0.0008 and p = 0.0014, respectively; student’s t-test. SL327 did not alter sleep consolidation at night, p = 0.379, (* = p < 0.05). K, Activity during waking (ZT1-ZT12) is significantly reduced in adult Cs flies fed SL327 compared to vehicle fed controls; ; A 2(RU+, RU-) X 12(Hour) ANOVA reveals a significant main effect of drug [F _(1,744) = 57.74, P = 9.01E-14 (n = 32 flies)]. L, No change in sleep homeostasis was observed in SL327 fed Cs flies compared to vehicle fed controls; 2(SL327, control) X 72(Hour) ANOVA,[F_(71,3024) = 0.393, P = 0.999 (n = 22/ group)].

Figure 2. Sleep deprivation activates ERK.

A-B, Immunohistochemistry of ppERK in brains from 5 day old Cs flies that experienced either a full night of sleep (A) or 12 hours of sleep deprivation (B). C, Western blot showing an increase in ppERK activation following 12 h of sleep deprivation in 5 day old Cs flies (4 heads/lane). D, Quantification of the Western blot in C revealed a statistically significant increase in ppERK/TotERK levels , p = 0.0059, student’s t-test, n= 4/group. Data are presented as mean ± SEM. E, Quantification of ppERK levels in wild type Cs flies exposed to mechanical perturbation (handled), the oxidative stressor Paraquat or starvation show no statistically significant differences in ppERK/total ERK levels compared to non-manipulated control flies p = 0.402, p = 0.477 and p = 0.209 respectively, student’s t-test, n = 3/group. Data are presented as mean ± SEM (* = p < 0.05). F, Quantification of the change in ppERK levels using Western blot in rl ¹ mutant flies following 12 hours of sleep deprivation revealed no alteration in ppERK levels p = 0.0815, student’s t-test, n = 3/group. Data are presented as mean ± SEM.

Figure 3. Sleep deprivation alters structural plasticity.

A-B, Immunohistochemistry of pigment dispersing factor (PDF) in brains of wild-type Cs flies that experienced either a full night of sleep (A) or 12 hours of sleep deprivation (B). C, Quantification of PDF immunohistochemistry in A-B. 12 h of Sleep deprivation significantly increases PDF-positive terminals, p = 0.0011, student’s t-test, n= 13-14/group. Values normalized to non-sleep deprived controls and presented as mean ± SEM (* = p < 0.05). D-E, Immunohistochemistry of pigment dispersing factor (PDF) in brains of rl ¹ mutant flies that experienced either a full night of sleep (D) or 12 hours of sleep deprivation (E). F, 12 h of sleep deprivation did not alter the number of PDF-positive terminals, p = 0.083, student’s t-test, n= 16-17/group. Values normalized to non-sleep deprived controls and presented as mean ± SEM (* = p < 0.05).

Figure 4. ERK increases structural plasticity.

A, Cs flies exposed to 5 days of social enrichment with ~45 siblings display an increase in sleep compared to isolated controls. A 2(Isolated, Enriched) X 24 (Time (hour)) ANOVA reveals a significant condition x time interaction [F _(23,720) = 5.044, P = 3.6E-13] (n = 16/ group). The inset shows that this increase in sleep following social enrichment is attenuated in rl ¹ mutant flies (shown as the change in sleep from the isolated siblings also known as ∆sleep). B, Representative Western blot showing that ppERK/totERK levels are increased in Cs flies following social enrichment compared to isolated siblings (4 heads per lane). C) Quantification of the Western blot in B for ppERK/totERK reveal a statistically significant increase in ppERK/totERK levels in the socially enriched group compared to isolated siblings p = 0.0002, student’s t-test. Data are presented as mean ± SEM (* = p < 0.05). D-E, Pan-neuronal expression of activated ERK in adult flies results in an increase number of PDF-positive terminals as revealed by immunohistochemistry. A representative image is shown for RU- fed +;UAS-ERK^SEM/+ ;GSw-elav/+ controls (D) and their RU+ fed experimental group (E). F, Quantification of terminal numbers reveals a significant increase in terminal number p = 0.0076, n= 11/group, student’s t-test. Values normalized vehicle fed controls.

Figure 5. rl ¹ and rut ²⁰⁸⁰ have deficits in ERK activation and structural plasticity in response to social enrichment.

A, In contrast to Cs controls, flies mutant for rl ¹ or rut ²⁰⁸⁰ do not exhibit an increase in ERK activation following social enrichment. Representative Western blot for ppERK and totERK in flies exposed to social enrichment and social isolation (4 heads/lane). B, Quantification of the Western blot in (A) for ppERK/totERK levels reveals a statistically significant increase in ppERK/totERK levels in the socially enriched Cs flies (p = 0.0002) with no statistical increase in ppERK/totERK levels in either rl ¹ or rut ²⁰⁸⁰ mutant flies (p = 0.113 and p = 0.579, respectively, student’s t-test, * = p < 0.05). C, Immunohistochemistry for PDF in brains of Cs, rl ¹ and rut ²⁰⁸⁰ flies exposed to social enrichment and social isolation. D, Quantification of PDF-positive terminals from brains in C. PDF terminals are increased following exposure to social enrichment in Cs flies p = 0.043, but not in rl ¹ and rut ²⁰⁸⁰ flies, p = 0.259 and p = 0.666, respectively, student’s t-test; n=11-15/group Values normalized to the isolated siblings.

Figure 6. ERK affects CRE-luciferase reporter activity.

A, Western blot showing an increase in the nuclear localization of ppERK activation following 12 h of sleep deprivation in 5 day old Cs flies (40 heads/lane). B, Quantification of the Western blot in A revealed a statistically significant increase in ppERK/TotERK levels, students t-test, p = 0.032, n=8. Data are presented as mean ± SEM. C, Sleep profile of flies expressing a wild type version of UAS-RSK ^wt displayed a decrease in sleep. D, Total sleep is significantly decreased in RSK over expressing flies. p = 0.0261; student’s t-test, n= 31-32/group. E, elav/Y; CRE-Luc/UAS-ERK ^SEM flies (blue trace) have elevated total reporter activity compared to elav/Y; CRE-Luc/+ controls (red trace); n=19-21/group. F, Sum of luciferase activity shown in E; students t-test, p =0.04). G, Flies that are heterozygous for the MAPK gene rolled (CRE-Luc/Y; rl ^10A /+; blue trace) show significant reductions in reporter activity compared to controls (CRE-Luc/Y; +/CyO; red trace); n=16-18/group. H, Sum of luciferase activity shown in G; students t-test, p = 0.0006). I, SL327-fed elav/Y; CRE-Luc/+ (black trace) show significant reduction in reporter activity compared to vehicle controls (red trace); n=48/group. J, Sum of luciferase activity shown in I; students t-test, p = 0.008).