Figure 5. Expression of catalytically inactive PDE4A5 in hippocampal neurons prevents memory deficits and alterations in the cAMP-PKA-LIMK-cofilin signaling pathway associated with sleep deprivation.

(A) Mice were injected with pAAV₉-CaMKIIα0.4-eGFP or pAAV₉-CaMKIIα0.4-PDE4A5^catnull-VSV into the hippocampus to drive neuronal expression of eGFP or catalytically inactive full-length PDE4A5 (PDE4A5^catnull). (B) Robust PDE4A5^catnull expression was observed at the expected molecular weight, 108 kDa, in hippocampal lysates. (C–E) PDE4A5^catnullexpression was observed in all 3 subregions of the hippocampus. (F–H) PDE4A5^catnullwas not expressed in astrocytes reflected by a lack of co-labeling between PDE4A5^catnull and GFAP expression. (I) Sleep deprivation causes a reduction in LIMK serine 596 phosphorylation in the hippocampus that is prevented by PDE4A5^catnull expression (n = 7–8; two-way ANOVA, effect of virus F_1,27 = 3.299, p=0.08; effect of sleep deprivation F_1,27 = 6.124, p=0.02; interaction effect F_1,27 = 11.336, p=0.002; eGFP SD group versus other groups p<0.05). (J) Sleep deprivation causes a reduction in cofilin phosphorylation in the hippocampus that is prevented by PDE4A5^catnull expression (n = 9–10; two-way ANOVA, effect of virus F_1,35 = 4.122, p=0.05; effect of sleep deprivation F_1,35 = 2.885, p=0.1; interaction effect F_1,35 = 9.416, p=0.004; eGFP SD group versus other groups p<0.05). (K, L) Three hours of recovery sleep after five hours of sleep deprivation restores hippocampal LIMK phosphorylation at serine 596 and cofilin phosphorylation at serine 3 to those observed in non-sleep deprived controls (p>0.45 for both comparisons). (M) Mice expressing eGFP or PDE4A5^catnull were trained in the hippocampus-dependent object-place recognition task and immediately sleep deprived for 5 hr after training (SD) or left undisturbed (NSD). Hippocampal PDE4A5^catnull expression prevents memory deficits caused by sleep deprivation (n = 8–10; two-way ANOVA, effect of virus F_1,33 = 2.626, p=0.115; effect of sleep deprivation F_1,33 = 2.311, p=0.138; interaction effect F_1,33 = 7.485, p=0.01; posthoc Dunnet test eGFP SD group versus other groups p<0.05). In all blots, each lane represents one individual animal. NSD: non-sleep deprived, SD: sleep deprived, SD+RS: sleep deprived plus recovery sleep. Scale bar, 100 µm. Values represent the mean ± SEM. *p<0.05 by posthoc Dunnet’s posthoc test. See also Figure 5—figure supplement 1.

DOI: http://dx.doi.org/10.7554/eLife.13424.018

Figure 5—figure supplement 1. Expression of catalytically null PDE4A5 in the hippocampus: Catalytically inactive PDE4A5 without the unique N-terminal localization domain fails to prevent memory deficits associated with sleep loss.

(A) PDE4A5^catnull expression in hippocampal neurons did not significantly affect PDE4 activity in the hippocampus (n = 7, Student’s t test p=0.097). (B) PDE4 activity in the prefrontal cortex was not altered by expression of the catalytically inactive PDE4A5^catnull in the hippocampus (n = 7–8, Student’s t test p=0.162). (C) PDE4 activity in the cerebellum was not changed by expression of the catalytically inactive PDE4A5^catnull in the hippocampus (n = 7–8, Student’s t test p=0.293). (D) Expression of the catalytically inactive PDE4A5^catnull in hippocampal neurons did not alter the total time spent exploring objects during training in the object-place recognition task (n = 8–10, two-way ANOVA, effect of virus F_1,33 = 0.043, p=0.873). All groups show a decrease in the total object exploration time during consecutive training sessions (two-way ANOVA effect of session F_2,66 = 32.777, p=0.0001). Mice expressing PDE4A5^catnull had a slightly but non-significantly lower object exploration time during the first training session, and a slightly but non-significantly higher object exploration time during the last training session (interaction effect F_2,66 = 4.875, p=0.011, one way ANOVAs per session, p>0.05). (E) Mice expressing PDE4A5^catnull spend a similar time in the periphery of the open field as mice expressing eGFP in hippocampal neurons (n = 8, Student’s t test, p=0.292). (F) Mice were injected with pAAV₉-CaMKIIα0.4-eGFP or pAAV₉-CaMKIIα0.4-PDE4A5^catnullΔ4-VSV into the hippocampus to drive neuronal expression of eGFP or catalytically inactive full-length PDE4A5 which lacked the N-terminal domain unique for PDE4A5 (PDE4A5^catnullΔ4). A VSV-tag was included to discriminate between endogenous PDE4A5 and the truncated PDE4A5^catnullΔ4. (G) PDE4A5^catnullΔ4 protein levels in the hippocampus 4 weeks after viral injection. A sample blot probed with an isoform-nonspecific PDE4A antibody revealed the presence of both wild-type PDE4A5 protein and truncated PDE4A5^catnullΔ4 protein. Probing the blot with an antibody for the HA-tag confirmed that the truncated protein is indeed the N-terminal lacking catalytically inactive PDE4A5^catnullΔ4. Each band represents an individual animal (H) Expression of the catalytically inactive PDE4A5^catnullΔ4 lacking the N-terminal domain in hippocampal neurons did not affect total object exploration time during training in the object-place recognition task (n = 7–9, two-way ANOVA effect of virus F_1,29 = 0.470, p=0.498). All groups show decreased total object exploration times during consecutive training sessions (two-way ANOVA effect of session F_2,58 = 13.597, p=0.0001; interaction effect F_2,58 = 0.555, p=0.557). (I) Mice expressing eGFP or the N-terminal domain lacking inactive form of PDE4A5^catnullΔ4 were trained in the hippocampus-dependent object-place recognition task. Sleep deprivation causes memory deficits in both eGFP and PDE4A5^catnullΔ4 mice (n = 7–9; two-way ANOVA effect of sleep deprivation F_1,29 = 18.131, p=0.0001; effect of virus F_1,29 = 1.064, p=0.311; interaction effect F_1,29 = 0.001, p=0.986; eGFP NSD versus EGFP SD, posthoc Tukey’ t test p=0.0054; PDE4A5^catnullΔ4 NSD versus PDE4A5^catnullΔ4 SD, posthoc Tukey’ t test p=0.0037). Dotted line indicates chance level performance. NSD: non-sleep deprived, SD: sleep deprived. Values represent the mean ± SEM. ^#p<0.01 by Tukeys t test.

DOI: http://dx.doi.org/10.7554/eLife.13424.020