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. 2022 Nov 16;42(1):e111485. doi: 10.15252/embj.2022111485

Figure 5. Microglial TNFα modulates sleep homeostasis.

Figure 5

  1. Time course of SWA during NREM sleep across a 24 h baseline sleep–wake cycle (left) and in the first 5 h after sleep deprivation (SD; right) in controls CTL (black) and micTNFα‐KO (green) mice. Mean SWA values normalized to the mean SWA during ZT8 to ZT12 of the baseline recording, a period during which sleep need is lowest (see Methods). SWA in baseline sleep: CTL, 14 mice; micTNFα‐KO, 15 mice. Two‐way rANOVA; genotype, F(1,27) = 2.170, P = 0.1523; time: F(3.003,81.09) = 117.8, P < 0.0001; interaction F(8,216) = 2.257, P = 0.0246; SWA after sleep deprivation: CTL, 12 mice; micTNFα‐KO, 15 mice. Two‐way rANOVA; genotype, F(1,25) = 1.902, P = 0.1801; time: F(2.489,62.23) = 415.8, P < 0.0001; interaction F(4,100) = 4.196, P = 0.0035. Post hoc test with Sidak's multiple‐comparisons tests, # P < 0.05 CTL vs. micTNFα‐KO.
  2. Delta change in wake and total sleep amounts between SD and sham in the first 3 h of the recovery phase. Unpaired t‐test (two tailed): wake, # P = 0.0355; total sleep, # P = 0.0359.
  3. Wake, NREM sleep, and REM sleep amounts in the first 3 h of the recovery phase after sleep deprivation (SD, plain) or sham condition (Sham, empty) in CTL and micTNFα‐KO mice. SD: mice were sleep deprived for 6 h starting at ZT0; Sham condition: each mouse was gently awakened at ZT6, 2 days prior to the SD procedure (matched control recording). CTL, 15 mice; micTNFα‐KO, 15 mice. Data are represented as mean ± SEM. Two‐way rANOVA; wake: genotype, F(1,28) = 0.5302, P = 0.4726; SD: F(1,28) = 5.863, P = 0.0222; interaction F(1,28) = 6.105, P = 0.0198; NREM sleep, F(1,28) = 0.7292, P = 0.4004; SD: F(1,28) = 11.40, P = 0.0022; interaction F(1,28) = 2.657, P = 0.1143, REM sleep, F(1,28) = 0.2423, P = 0.6264; SD: F(1,28) = 8.273, P = 0.0076; interaction F(1,28) = 12.91, P = 0.0012. Post hoc test with Sidak's multiple‐comparisons tests, **P < 0.01 and ***P < 0.001 Sham vs. SD and # P < 0.05 CTL vs. micTNFα‐KO.
  4. Quantitative analysis of changes in cortical phosphoproteome between CTL and micTNFα‐KO after sleep deprivation from ZT0 to ZT6 (SD6; yellow).
  5. Identified sleep need phosphoproteins are modulated by microglial TNFα following sleep deprivation. The 994 phosphoproteins that had at least one phosphosite changing in micTNFα‐KO vs. CTL comparison at SD6 (phosphosites with an adjusted P‐value ≤ 0.05 and unique phosphosites; Dataset EV5) comprise all KSPs and 60 of 80 SNIPPS.
  6. Phosphosubstrates of microglial TNFα during sleep deprivation are causally linked to sleep regulation. Venn diagram shows the 200 phosphoproteins with a higher density of TNFα‐modulated phosphosites at SD6. Dark green highlights proteins whose mutation and/or knockdown leads to sleep phenotypes in baseline sleep, NREM SWA, and/or sleep rebound after SD (green tick) (refer to Dataset EV5 for a list of relevant citations). Proteins are listed in descending order of density of TNFα‐modulated phosphosites at SD6.