Figure 1.
Expression of inactive GSK3β is upregulated following environmental enrichment in the brains of nontransgenic but not APPswe/PS1ΔE9 mice.
(A) Expression levels of GSK3β and its inactive form pGSK3β ser 9 are comparable in the cortex and hippocampus of nontransgenic and APPswe/PS1ΔE9 mice at 2 months of age as detected by Western blot analysis and densitometric quantification (P = 0.2419 cortex, P = 0.2161 hippocampus, N = 4 for nontransgenic, N = 4 for APPswe/PS1ΔE9, Student's t test). (B) Levels of pGSK3β ser 9 decrease in the hippocampus of 6 month-old APPswe/PS1ΔE9 (N = 4) compared to levels at 2 months of age (N = 4), suggesting an increased activity of GSK3β at 6 months (*P<0.05, Student's t test) as total GSK3β levels are preserved. (C,D) Levels of pGSK3β ser 9 increase in the hippocampus (C) and cortex (D) of nontransgenic but not of APPswe/PS1ΔE9 mice following experience in an enriched environment. Values are means ± SE (arbitrary units) [*P<0.05 nontransgenic hippocampus (N = 4 for SH, N = 4 for EE), P = 0.4685 APPswe/PS1ΔE9 hippocampus (N = 4 for SH, N = 4 for EE), ***P<0.0001 nontransgenic cortex (N = 4 for SH, N = 4 for EE), P = 0.9302 APPswe/PS1ΔE9 cortex (N = 5 for SH, N = 5 for EE), Student's t test].
Figure 2.
Akt expression and activity are upregulated following environmental enrichment in the brains of nontransgenic but not APPswe/PS1ΔE9 mice.
(A) Expression levels of the active form of Akt kinase, pAkt ser 437, is comparable in the cortex and hippocampus of nontransgenic and APPswe/PS1ΔE9 mice at 2 months of age as detected by Western blot analysis and densitometric quantification (P = 0.4331 cortex, P = 0.4740 hippocampus, N = 4 for nontransgenic, N = 4 for APPswe/PS1ΔE9, Student's t test). (B) Levels of pAkt ser 437 decrease in the hippocampus of 6 month-old APPswe/PS1ΔE9 mice (N = 4) compared to levels at 2 months of age (N = 4), suggesting decreased activity at 6 months (*P<0.05, Student's t test). (C,D) Levels of pAkt ser 437 increase in the hippocampus (C) and cortex (D) of nontransgenic but not of APPswe/PS1ΔE9 mice following experience in an enriched environment, again with little or no change in total Akt levels. Values are means ± SE [(arbitrary units) *P<0.05 nontransgenic hippocampus (N = 4 for SH, N = 4 for EE), P = 0.9856 APPswe/PS1ΔE9 hippocampus (N = 4 for SH, N = 4 for EE), *P<0.05 nontransgenic cortex (N = 4 for SH, N = 4 for EE), P = 0.8602 APPswe/PS1ΔE9 cortex (N = 5 for SH, N = 5 for EE), Student's t test].
Figure 3.
Hippocampus-specific upregulation of BDNF following environmental enrichment in nontransgenic and APPswe/PS1ΔE9 is accompanied by increased levels of TrkB receptors and tPA.
(A) Real time RT-PCR of RNA extract of the hippocampus of nontransgenic and APPswe/PS1ΔE9 mice revealed comparable levels of BDNF in the hippocampus of nontransgenic and APPswe/PS1ΔE9 at 2 months of age (P = 0.5517, N = 5 for nontransgenic SH, N = 5 for APPswe/PS1ΔE9 SH, Student's t test). (B) BDNF levels increase in the hippocampus of nontransgenic and APPswe/PS1ΔE9 mice following environmental enrichment as detected by real time RT-PCR [*P<0.05 for nontrangenic SH (N = 5) vs. nontransgenic EE (N = 5), **P<0.01 for APPswe/PS1ΔE9 SH (N = 5) vs. APPswe/PS1ΔE9 EE (N = 5), Student's t test]. (C) Upregulation of TrkB receptors was also observed in the hippocampus of nontransgenic and APPswe/PS1ΔE9 mice following environmental enrichment as detected by real time RT-PCR [*P<0.05 for nontrangenic SH (N = 5) vs. nontransgenic EE (N = 5), **P<0.01, for APPswe/PS1ΔE9 SH (N = 5) vs. APPswe/PS1ΔE9 EE (N = 7), Student's t test]. (D,E) BDNF levels increase in the hippocampus [***P<0.001 for nontransgenic SH (N = 6) vs. nontransgenic EE (N = 6), ***P<0.001 for APPswe/PS1ΔE9 SH (N = 6) vs. APPswe/PS1ΔE9 EE (N = 6), one-way ANOVA], but not in the cortex [P = 0.8356 for nontransgenic SH (N = 6) vs. nontransgenic EE (N = 6), P = 0.0842 for APPswe/PS1ΔE9 SH (N = 6) vs. APPswe/PS1ΔE9 EE (N = 6), one-way ANOVA] of nontransgenic and APPswe/PS1ΔE9 following environmental enrichment as determined by ELISA. (F) Upregulation of tPA mRNA level in the hippocampus of nontransgenic and APPswe/PS1ΔE9 following environmental enrichment as determined by real time RT-PCR [*P<0.05 for nontrangenic SH (N = 6) vs. nontransgenic EE (N = 6), *P<0.05, for APPswe/PS1ΔE9 SH (N = 6) vs. APPswe/PS1ΔE9 EE (N = 7)].
Figure 4.
No changes in levels of Akt-induced ERK or PKC signaling following environmental enrichment.
(A,B) Protein expression levels of pERK were comparable in the cortex of nontransgenic and APPswe/PS1ΔE9 mice following experience in an enriched environment as detected by Western blot analysis (A) and densitometric quantification (B), [P = 0.5874 for nontransgenic (N = 4 per group), P = 0.1031 for APPswe/PS1ΔE9 (N = 4 per group), Student's t test]. (C) No statistically significant change in the level of PKC following environmental enrichment as detected by real-time RT-PCR [P = 0.6504 for nontransgenic SH (N = 6) vs. nontransgenic EE (N = 6), P = 0.1364 for APPswe/PS1ΔE9 SH (N = 6) vs. APPswe/PS1ΔE9 EE (N = 6), Student's t test].
Figure 5.
Differential regulation of neurotrophins following environmental enrichment in the brains of nontransgenic and APPswe/PS1ΔE9 mice.
(A,C,E) mRNA levels of NGF (A), NT-3 (B) and IGF-1 (C) are comparable in the hippocampus of nontransgenic and APPswe/PS1ΔE9 mice at 2 months of age, as determined by real time-RT-PCR (NGF, P = 0.3945; NT-3, P = 0.0914 and IGF-1, P = 0.6949, N = 6 for nontransgenic, N = 6 for APPswe/PS1ΔE9, Student's t test). (B) NGF levels increased in the hippocampus of nontransgenic and APPswe/PS1ΔE9 mice following environmental enrichment [*P<0.05 for nontransgenic SH (N = 6) vs. nontransgenic EE (N = 6), *P<0.05 for APPswe/PS1ΔE9 SH (N = 6) vs. APPswe/PS1ΔE9 EE (N = 6), Student's t test]. (D) Levels of NT-3 were upregulated in the hippocampus of nontransgenic but not APPswe/PS1ΔE9 following environmental enrichment [*P<0.05 for nontransgenic SH (N = 6) vs. nontransgenic EE (N = 6), P = 0.0640 for APPswe/PS1ΔE9 SH (N = 6) vs. APPswe/PS1ΔE9 EE (N = 7), Student's t test]. (E) Levels of IGF-1 increased following environmental enrichment in the hippocampus of APPswe/PS1ΔE9 but not nontransgenic mice [P = 0.4077 for nontransgenic SH (N = 7) vs. nontransgenic EE (N = 7) *P<0.05 for APPswe/PS1ΔE9 SH (N = 6) vs. APPswe/PS1ΔE9 EE (N = 6), Student's t test].
Figure 6.
Upregulation of CREB and NMDAR transcription in the hippocampus of nontransgenic and APPswe/PS1ΔE9 mice following environmental enrichment.
Real time RT-PCR suggests that levels of (A) CREB were significantly upregulated in the hippocampus of nontransgenic and APPswe/PS1ΔE9 mice following environmental enrichment [*P<0.05 for nontransgenic SH (N = 7) vs. nontransgenic EE (N = 7), **P<0.01 for APPswe/PS1ΔE9 SH (N = 6) vs. APPswe/PS1ΔE9 EE (N = 6), Student's t test], but not levels of (B) CaMKIV [P = 0.2326 for nontransgenic SH (N = 6) vs. nontransgenic EE (N = 6), P = 0.5772 for APPswe/PS1ΔE9 SH (N = 6) vs. APPswe/PS1ΔE9 EE (N = 7)], (C) CaMKII [P = 0.0642 for nontransgenic SH (N = 7) vs. nontransgenic EE (N = 7), P = 0.3524 for APPswe/PS1ΔE9 SH (N = 6) vs. APPswe/PS1ΔE9 EE (N = 7)] or (D) PSD-95 [P = 0.4337 for nontransgenic SH (N = 7) vs. nontransgenic EE (N = 7), P = 0.4236 for APPswe/PS1ΔE9 SH (N = 7) vs. APPswe/PS1ΔE9 EE (N = 7)]. (E,F) Levels of NMDAR1 [*P<0.05 for nontransgenic SH (N = 6) vs. nontransgenic EE (N = 6), **P<0.01 for APPswe/PS1ΔE9 SH (N = 6) vs. APPswe/PS1ΔE9 EE (N = 6)], but not GluR [P = 0.1256 for nontransgenic SH (N = 6) vs. nontransgenic EE (N = 6), P = 0.3937 for APPswe/PS1ΔE9 SH (N = 6) vs. APPswe/PS1ΔE9 EE (N = 6)], are upregulated in the hippocampus following environmental enrichment.
Figure 7.
Environmental enrichment upregulates CREB phosphorylation in the hippocampus of wild type but not APPswe/PS1ΔE9 mice.
(A) Western blot analysis of expression level of total CREB and phosphorylated CREB (pCREB) shows an increase in CREB phosphorylation in the hippocampus of wild type but not APPswe/PS1ΔE9 mice. Levels of total CREB were comparable in mice maintained in standard group- housing (GH) or EE in both genotypes. (B,C) Quantification of total CREB and pCREB levels in the hippocampus of (B) nontransgenic (*P<0.05 pCREB/actin, *P<0.05 pCREB/total CREB, P = 0.9161 total CREB/actin, N = 3 per group) and (C) APPswe/PS1ΔE9 mice (P = 0.8451 pCREB/actin, P = 0.9827 pCREB/total CREB, P = 0.8295 total CREB/actin, N = 4 per group).
Figure 8.
Proposed signaling network upregulated in the hippocampus of wild type and FAD-linked APPswe/PS1ΔE9 transgenic mice following environmental enrichment.
Experience in environmental enrichment upregulates the expression of several neurotrophic factors, i.e., brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF). Upregulation of insulin growth factor 1 (IGF-1) is observed in the hippocampus of APPswe/PS1ΔE9 but not wild type mice, while upregulation of neurotrophin-3 (NT-3) is observed in the hippocampi of wild type but not APPswe/PS1ΔE9 mice. Upregulation of tissue plasminogen activator (tPA) suggests that upregulation of BDNF signaling may be enhanced by the conversion of immature BDNF into the mature form by plasmin. Based on the observed upregulation of the BDNF receptor tyrosine kinase B (TrkB), we hypothesize that neurotrophin binding to their receptors activates intracellular signaling cascades. Intracellularly, several pathways are activated: Protein kinase B (Akt) is phosphorylated, and may downregulate glycogen synthase kinase β (GSK3β) activity that in turn, results in downregulation of tau phosphorylation. This pathway is blocked in the APPswe/PS1ΔE9 mice. Alternatively, a Mitogen-activated protein kinase pathway Ras/Raf/MEK/MAPK, 40S ribosomal protein S6 kinase and mitogen- and stress-activated protein kinase (RSK2/MSK) signaling might be activated following neurotrophin activation. In addition, neurotransmitter (i.e., NMDAR)-induced Ca2+/calmodulin-dependent protein kinase cascade is activated. These pathways lead to upregulation of CREB phosphorylation in wild type mice, which in turn, regulates gene expression necessary for the formation of long-term memory, including BDNF and IGF-2.