Glucagon-like peptide-1 cleavage product GLP-1(9-36) amide rescues synaptic plasticity and memory deficits in Alzheimer's disease model mice

Abstract

Glucagon-like peptide-1 (GLP-1) is an endogenous intestinal peptide that enhances glucose-stimulated insulin secretion. Its natural cleavage product GLP-1(9-36)(amide) possesses distinct properties and does not affect insulin secretion. Here we report that pretreatment of hippocampal slices with GLP-1(9-36)(amide) prevented impaired long-term potentiation (LTP) and enhanced long-term depression induced by exogenous amyloid β peptide Aβ((1-42)). Similarly, hippocampal LTP impairments in amyloid precursor protein/presenilin 1 (APP/PS1) mutant mice that model Alzheimer's disease (AD) were prevented by GLP-1(9-36)(amide). In addition, treatment of APP/PS1 mice with GLP-1(9-36)(amide) at an age at which they display impaired spatial and contextual fear memory resulted in a reversal of their memory defects. At the molecular level, GLP-1(9-36)(amide) reduced elevated levels of mitochondrial-derived reactive oxygen species and restored dysregulated Akt-glycogen synthase kinase-3β signaling in the hippocampus of APP/PS1 mice. Our findings suggest that GLP-1(9-36)(amide) treatment may have therapeutic potential for AD and other diseases associated with cognitive dysfunction.

Figure 1.

AD-associated aberrant synaptic plasticity is prevented by GLP-1(9-36)^amide. A, HFS induced LTP in vehicle-treated hippocampal slices (white squares; n = 5) but not in slices treated with 500 nm Aβ_(1–42) (red circles; n = 5). In contrast, 100 pm GLP-1(9-36)^amide enabled normal LTP induction in the presence of Aβ_(1–42) (blue triangles; n = 6). GLP-1(9-36)^amide alone did not alter LTP induction (gray diamonds; n = 5). The dashed line represents the normalized baseline value of 100%. B, Cumulative data showing mean fEPSP slopes 80 min after HFS from the LTP experiments in A. C, Aβ_(1–42) facilitated induction of LTD (black triangles; n = 6) by a weak LFS protocol (1 Hz, 300 pulses), which otherwise did not induce LTD (gray circles; n = 5). D, Aβ_(1–42) failed to facilitate LTD induction by weak LFS in the presence of either GLP-1(9-36)^amide (gray triangles; n = 8) or 500 nm MitoQ (black triangles; n = 7). E, Treatment of slices with GLP-1(9-36)^amide did not affect baseline fEPSPs (n = 8). Shown on the right are traces of representative fEPSPs during the baseline recording before and after GLP-1(9-36)^amide application. F, HFS-induced LTP was impaired in slices from APP/PS1 mice (white squares; n = 9) and was rescued by GLP-1(9-36)^amide treatment (black circles; n = 6). Shown on the right are traces of representative fEPSPs before and after HFS. GLP-1(9-36)^amide, Aβ_(1–42), and MitoQ were added 30 min before HFS or LFS and left throughout the recording.

Figure 2.

Impaired spatial and conditioned fear memory in APP/PS1 mice is rescued by GLP-1(9-36)^amide. A, Escape latency in the Morris water maze plotted against the training days. APP/PS1 mice (red circles; n = 7) were slower to learn than wild-type (WT) mice (white squares; n = 7). In contrast, GLP-1(9-36)^amide-treated APP/PS1 mice (blue squares; n = 8) exhibited learning curves similar to wild-type mice. Repeated-measures ANOVA were used, p = 0.0001. B, Percentage of time spent in the target quadrant during a 60 s probe trial. APP/PS1 mice spent less time in the target quadrant compared with wild-type mice. GLP-1(9-36)^amide-treated APP/PS1 mice demonstrated occupancy time similar to wild-type mice. ANOVA and post hoc Tukey's multiple comparison tests were used with p < 0.05 as significance criteria. C, During the visible platform test, no difference was observed for escape latency among the different groups. Repeated-measures ANOVA test was used, p = 0.24. D, Mean freezing behavior in a contextual fear conditioning test 24 h after training. APP/PS1 mice (n = 7) displayed a significant reduction in freezing compared with wild-type mice (n = 7). In contrast, GLP-1(9-36)^amide-treated APP/PS1 mice (n = 7) displayed significantly more freezing behavior than APP/PS1 mice. ANOVA and post hoc Tukey's multiple comparison tests were used with p < 0.05 as significance criteria. E, Mean freezing behavior in cued fear conditioning test 24 h after training. APP/PS1 mice (n = 7) displayed a significant reduction in freezing compared with wild-type mice (n = 7). In contrast, GLP-1(9-36)^amide-treated APP/PS1 mice (n = 7) displayed significantly more freezing behavior than APP/PS1 mice. ANOVA and post hoc Tukey's multiple comparison tests were used with p < 0.05 as significance criteria.

Figure 3.

GLP-1(9-36)^amide application does not change levels of either APP or Aβ in APP/PS1 mice. A, B, Western blotting was performed on brain extracts of mice from behavioral experiments. No change in the levels of either APP or Aβ was detected between APP/PS1 mice and GLP-1(9-36)^amide-treated APP/PS1 mice. n = 5. C, Immunofluorescence confocal microscopy for Aβ in the CA1 region of hippocampal slices harvested from APP/PS1 mice treated with GLP-1(9-36)^amide and control group. Scale bar, 50 μm.

Figure 4.

GLP-1(9-36)^amide normalizes increased mitochondrial superoxide levels associated with AD. A, Treatment of hippocampal slices with 500 nm Aβ_(1–42) increased the MitoSOX fluorescent signal (red) in area CA1 compared with control slices. DAPI staining is shown as blue. Pretreatment of slices with 100 pm GLP-1(9-36)^amide prevented the Aβ-induced elevation in the MitoSOX fluorescent signal. B, The MitoSOX fluorescent signal was increased in slices from APP/PS1 mice compared with wild-type (WT) slices. Treatment of slices with GLP-1(9-36)^amide normalized the increased fluorescent signal. Results are representative of three independent experiments. Scale bar, 50 μm.

Figure 5.

GLP-1(9-36)^amide treatment reverses activating dephosphorylation of GSK3β in APP/PS1 mice. Immunofluorescence for phosphorylated GSK3β on serine 9 (p-GSK3β) in area CA1 of hippocampal slices was reduced in APP/PS1 mice but was restored to wild-type (WT) levels by GLP-1(9-36)^amide treatment. Scale bar, 25 μm.

Figure 6.

Activating dephosphorylation of GSK3β and inactivating dephosphorylation of Akt in hippocampal slices of APP/PS1 mice is reversed by GLP-1(9-36)^amide. A, In slices from wild-type (WT) mice, GLP-1(9-36)^amide treatment did not change phosphorylated GSK3β levels on serine 9 (p-GSK3β). n = 6. B, Reduced level of phospho-GSK3β in APP/PS1 mice was reversed by GLP-1(9-36)^amide. n = 6. C, In slices from wild-type mice, GLP-1(9-36) treatment did not change phosphorylated Akt levels on serine 473 (p-Akt). n = 6. D, Reduced levels of phospho-Akt in APP/PS1 mice was reversed by GLP-1(9-36)^amide. n = 6. veh, Vehicle.