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. 2009 Jan 16:1249:191-201.
doi: 10.1016/j.brainres.2008.10.054. Epub 2008 Nov 5.

Exercise can increase small heat shock proteins (sHSP) and pre- and post-synaptic proteins in the hippocampus

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Exercise can increase small heat shock proteins (sHSP) and pre- and post-synaptic proteins in the hippocampus

Shuxin Hu et al. Brain Res. .

Abstract

The molecular events mediating the complex interaction between exercise and cognition are not well-understood. Although many aspects of the signal transduction pathways mediate exercise induced improvement in cognition are elucidated, little is known about the molecular events interrelating physiological stress with synaptic proteins, following physical exercise. Small heat shock proteins (sHSP), HSP27 and alpha-B-crystallin are co-localized to synapses and astrocytes, but their role in the brain is not well-understood. We investigated whether their levels in the hippocampus were modulated by exercise, using a well characterized voluntary exercise paradigm. Since sHSP are known to be regulated by many intracellular signaling molecules in other cells types outside the brain, we investigated whether similar regulation may serve a role in the brain by measuring protein kinase B (PKB/Akt), pGSK3 and the mitogen activated protein (MAP) kinases, p38, phospho-extracellular signal-regulated kinase (pERK) and phospho-c-Jun kinase (pJNK). Results demonstrated exercise-dependent increases in HSP27 and alpha-B-crystallin levels. We observed that increases in sHSP coincided with robust elevations in the presynaptic protein, SNAP25 and the post-synaptic proteins NR2b and PSD95. Exercise had a differential impact on kinases, significantly reducing pAkt and pERK, while increasing p38 MAPK. In conclusion, we demonstrate four early novel hippocampal responses to exercise that have not been identified previously: the induction of (1) sHSPs (2) the synaptic proteins SNAP-25, NR2b, and PSD-95, (3) the MAP kinase p38 and (4) the immediate early gene product MKP1. We speculate that sHSP may play a role in synaptic plasticity in response to exercise.

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Figures

Fig.1
Fig.1. Impact of exercise on heat shock proteins (HSP) in the hippocampus
(A) A representative Western immunoblot, showing levels of heat shock proteins and β-actin in sedentary rats (D0) or after 3 days (D3) or 7 days (D7) of voluntary exercise. The relative optical density (OD) of bands was calculated to determine the impact of exercise on brain levels of HSP27 (B), α-B-crystallin (C) and HSP70 (D). β–actin levels were measured to show equal loading and for normalization. Error bars indicate S.E.M. Treatment effects were analyzed by 1 ×1 ANOVA. For HSP27 and α-B-crystallin, values were log transformed to establish homogeneity of variance. Fisher’s LSD analysis was used for post-hoc test of planned comparisons. Asterisks indicate significant difference from D0 (p < 0.05).
Fig. 2
Fig. 2. Exercise-induced modulation of Heat Shock Factor-1 (HSF1) aggregates in the hippocampus
Representative lanes of a Western immunoblot of HSF1 using a 3-15% non-reducing conditions, demonstrate multiple bands in sedentary (D0) rats or rats after 3 (D3) or 7 (D7) days of voluntary exercise. The top three graphs show the treatment means for the relative optical density of each set of bands, representing trimer, dimer and monomer. The bottom right graph shows the ratio of trimer to monomer as an index of aggregation. The ‘monomer’ data were log-transformed, and the ‘ratio’ data were square root-transformed data for homogeneity of variance prior to 1 × 1 ANOVA, and Fisher’s post-hoc analysis was used to determine difference between planned comparisons. Error bars represent S.E.M. Asterisks indicate significant difference from D0 (p < 0.05).
Fig. 3
Fig. 3. Exercise-induced changes in mitogen activatied protein (MAP) kinases
(A) Representative lanes of a Western immunoblot for phosphorylated P38 (pP38), total P38, phosphorylated c-Jun kinase (pJNK). Bands of appropriate molecular weight are shown from hippocampi of sedentary (D0) rats or rats after 3 (D3) or 7 (D7) days of voluntary exercise. pP38 was stripped and re-probed with an antibody to total p38 for normalization. Treatment means for optical density (OD) of bands were calculated, and treatment effects were analyzed by 1×1 ANOVA. Graphs show (B) the ratio of pP38: P38 to reflect enzymatic activity (C) and pJNK levels. Equal loading was confirmed by Coomassie Blue (not shown). Error bars represent S.E.M. Asterisks indicate significant difference from D0 (p < 0.05).
Fig. 4
Fig. 4. Exercise effects on extracellular related kinase (ERK) and its phosphatase mitogen activated kinase phosphatase 1 (MKP1)
(A) Representative lanes of a Western immunoblot for pERK, tERK, and MKP1 from hippocampi of sedentary (D0) rats or rats after 3 (D3) or 7 (D7) days of voluntary exercise. pERK was stripped and re-probed with an antibody to total ERK. MKP1 was run on separate blot. The relative optical density (OD) of bands was measured. Results were evaluated by 1 × 1 ANOVA for (B) phosphophorylated ERK (pERK), (C) total ERK (tERK), (D) the ratio of pERK/tERK as the index of enzyme activity and (E) MAP kinase phosphatase MKP1. Equal loading was confirmed by Coomassie Blue (not shown). Error bars represent S.E.M. Asterisks indicate significant difference from D0 (p < 0.05).
Fig. 5
Fig. 5. Exercise effects on Akt and its down stream target GSK3
(A) Representative lanes of a Western immunoblot for phosphorylated Akt (pAkt), total Akt (Akt), and active phosphorlated GSK3 α and β, and inactive phosphorylated GSK (pGSK3β-ser9) from hippocampi of sedentary (D0) rats or 7 (D7) days of voluntary exercise. pAkt was stripped and re-probed with an antibody to total Akt. GSK3 was run on separate blot. The relative optical density (OD) of bands was measured. Graphs show (B) treatment means for pAkt, (C) total Akt (tAkt) and (D) the ratio of pAkt to Akt as an index of enzymatic activity. (E-G) treatment means of active GSK3α/β tyr279/216 and inactive GSK3ser9. Treatment groups were evaluated by 1 × 1 ANOVA. Equal loading of protein was confirmed by Coomassie Blue (not shown). Error bars represent S.E.M. Asterisks indicate significant difference from D0 (p < 0.05).
Fig. 6
Fig. 6. Exercise induced modulation of synaptic proteins and glial fibriallary acidic protein (GFAP)
(A) Representative lanes of a Western immunoblot for synaptic proteins and GFAP from hippocampi of sedentary (D0) rats or rats after 3 (D3) or 7 (D7) days of voluntary exercise. The relative optical density of bands was measured. Graphs show treatment means for (B) NR2b (C) post-synaptic density protein-95 (PSD95), (D) synaptophysin (SY38), (E) synaptosomal-associated protein-25 (SNAP25) and (F) GFAP, and treatment effects were evaluated by 1×1 ANOVA. Equal loading was confirmed by β-actin and for normalization. Error bars represent S.E.M. Asterisks indicate significant difference from D0 (*p < 0.05, **p = 0.01).
Fig. 7
Fig. 7. Correlation between sHSP with selective synaptic proteins in the hippocampus of sedentary and exercising rats
(A) α-B-crystallin levels correlated with SNAP25 levels (p < 0.0001), even when the outlier was removed (B) p < 0.01.
Fig. 8
Fig. 8. Schematic diagram of possible effects of sHSP on the synapse in the hippocampus after exercise
Molecular factors and pathways that are boxed with red letters and red arrows show our observed effects that we speculate are an early response to exercise modulating synaptic proteins; Letters in blue show our observed effects that are down-regulated; Letters in black are generally accepted pathways for synaptic plasticity; and letters in red show exercise-dependent upregulation. It is unclear whether the sHSP pathway precedes the induction of pERK, pAkt or BDNF. The circled “X” indicates the unknown factor triggering the heat shock cascade (eg. glucocorticoid receptor or temperature change or oxidative free radical). Even rats that ran minimum distances, still showed induction sHSP and synaptic proteins (not shown). Pre-synaptic proteins SNAP25 and synaptophysin were increased as well as the post-synaptic proteins PSD-95 and NR2b, but the localization of the sHSP is not known. The schematic diagram of the molecular pathway is illustrated in the post-synaptic density. However, sHSP can be induced in the astrocytes after hyperthermia and play a critical role in synaptogenesis. L-VGCC; L-Voltage-gated channels

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