Figure 1.
Heat acclimation (HA) induces improved motor recovery after TBI.
Mice were subjected to CHI and motor ability was evaluated by the neurological severity score (NSS) at 1 h post injury for initial disability assessment and 1, 3 and 7 days thereafter. ΔNSS represent the recovery of injured mice as measured between 1 h post injury and any later time point, n = 9–12 per group (A). Heat Acclimation (HA) reduces depressive like behavior after CHI. Mice were subjected to CHI or sham operation and 24 h or 7 days thereafter to the forced swimming test. Immobility time was determined out of 240 seconds of record (B). Values represent the mean ± SEM. n = 5–6 per group. *p<0.05 vs. normothermic (NT) mice, **p<0.05 vs. sham mice within the same group.
Figure 2.
HA induces inhibition of glycogen synthase kinase 3β (GSK3β) and c-Jun N terminal kinase (JNK).
Mice were subjected to CHI or sham operation, and injured cortexes were removed at 6, 12 or 24β showing significant inhibition of GSK3β by 24 h post injury in heat acclimated mice (HA) as compared with normothermic mice (NT) (A). Representative western blots of JNK phosphorylation, that is related to enhanced kinase activity, showing reduced JNK activity in HA mice after CHI (B). Values represent the mean ± SEM. n = 5–6 per group. *p<0.05 vs. NT mice, **p<0.05 vs. sham HA mice.
Figure 3.
Heat acclimation (HA) enhances ser33/37thr/41 phosphorylation of β catenin without change in its nuclear/cytosolic levels.
Mice were subjected to CHI or sham operation, and injured cortexes were removed at 6, 12 or 24/37thr/41 phosphorylation of β catenin is induced in injured HA mice (A). Total cytosolic fool length (FL) β catenin remain unchanged (B). Total nuclear β catenin remain unchanged. Anti α tubulin (1∶2000) was used to verify the absence of cytosolic proteins (C). 70–80 kD cytosolic β catenin fragment is significantly elevated after CHI in normothermic (NT) mice (D). Values represent the mean ± SEM. n = 5–6 per group. *p<0.05 vs. NT mice, **p<0.05 vs. sham HA mice within the same group.
Figure 4.
Heat acclimation induces tyrosine 654 phosphorylation of β catenin.
Mice were subjected to CHI or sham operation, and injured cortexes were removed at 6 or 24β catenin, and Y654 β catenin was detected using western blots. Total β catenin was used as loading control (A). Immunostaining of the injured brain for Y654 β catenin (green) and DAPI (blue) shows induced Y654 β catenin phosphorylation restricted to injury area (B). Values represent the mean ± SEM. n = 5–6 per group. *p<0.05 vs. NT mice, **p<0.05 vs. sham mice, within the same group.
Figure 5.
Heat acclimation induces basal N-cadherin and post injury detachment of the cadherin-catenin complex.
Representative western blots of N cadherin, showing significant basal induction of N cadherin which lasts up to 6(HA) as compared with normothermic mice (NT) (A). β-catenin was immunoprecipitated (IP) with protein A beads and separated on SDS-PAGE gels. Immunoblotting (IB) of N cadherin indicates disassociation of cadherin-catenin complex in injured HA mice. (−) Immunoprecipitation of non β-catenin related protein (Bcl-xL) with the same beads. (+) input from non immunoprecipitation blot of 60 µg protein from injured HA brain (B). Values represent the mean ± SEM. n = 5–6 per group. *p<0.05 vs. NT mice, **p<0.05 vs. sham mice, within the same group.
Figure 6.
Heat acclimation induces transient reduction in membrane β-catenin and synaptophysin.
Membrane cellular fraction extracts were separated on SDS-PAGE gels and analyzed using western blotting. After the injury a significant reduction in membrane bound β-catenin were seen at 72 h post injury in heat acclimated (HA) mice and after 7 days in normothermic (NT) mice. Membrane bound β-catenin levels were recovered by 7 days after injury (A) Blots of membrane extracts show significant reduction in synaptophysin levels in both groups at 72 h after injury with a recovery seen only in HA mice by 7 days post injury (B). The absence of synaptophysin in the cytosol verifies membrane protein enrichment. Values represent the mean ± SEM. n = 5–6 per group. *p<0.05 vs. NT mice, **p<0.05 vs. sham mice,within the same group.
Figure 7.
Schematic representation of suggested sequent of events determining β catenin fate in heat acclimated mice after closed head injury (CHI).
In the first hours after CHI, basal high levels of brain derived neurotrophic factor (BDNF) and N-cadherin lead to abrupt increase of Akt phosphorylation upon CHI, resulting in inhibition of c-Jun N-terminal kinase (JNK) and glycogen kinase 3β (GSK3β). Inhibited GSK3β may lead to reduced ser33/37thr41 phosphorylation of β catenin later on. Increased ser33/37thr41 phosphorylation of β catenin implies on involvement of another player, potentially another kinase or hypoxia inducible factor 1α (HIF1α). Simultaneously, high levels of BDNF consequence in induced tyrosine 654 phosphorylation of β catenin, superior to the ser33/37thr41 phosphorylation which allows β catenin to escape degradation or nuclear translocation. Those events lead to weakening the catenin-cadherin complex in cell membrane, enabling synaptic changes at 1 day post injury. 1 week after the injury, β catenin translocates back to newly created synapses to form cadherin-catenin complex and establish cell-cell adhesion.