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. 2008 May 7;28(19):5007-17.
doi: 10.1523/JNEUROSCI.0590-08.2008.

Enkephalin elevations contribute to neuronal and behavioral impairments in a transgenic mouse model of Alzheimer's disease

William J Meilandt  1 , Gui-Qiu Yu, Jeannie Chin, Erik D Roberson, Jorge J Palop, Tiffany Wu, Kimberly Scearce-Levie, Lennart Mucke
Affiliations

Enkephalin elevations contribute to neuronal and behavioral impairments in a transgenic mouse model of Alzheimer's disease

William J Meilandt et al. J Neurosci. .

Abstract

The enkephalin signaling pathway regulates various neural functions and can be altered by neurodegenerative disorders. In Alzheimer's disease (AD), elevated enkephalin levels may reflect compensatory processes or contribute to cognitive impairments. To differentiate between these possibilities, we studied transgenic mice that express human amyloid precursor protein (hAPP) and amyloid-beta (Abeta) peptides in neurons and exhibit key aspects of AD. Met-enkephalin levels in neuronal projections from the entorhinal cortex and dentate gyrus (brain regions important for memory that are affected in early stages of AD) were increased in hAPP mice, as were preproenkephalin mRNA levels. Genetic manipulations that exacerbate or prevent excitotoxicity also exacerbated or prevented the enkephalin alterations. In human AD brains, enkephalin levels in the dentate gyrus were also increased. In hAPP mice, enkephalin elevations correlated with the extent of Abeta-dependent neuronal and behavioral alterations, and memory deficits were reduced by irreversible blockade of mu-opioid receptors with the antagonist beta-funaltrexamine. We conclude that enkephalin elevations may contribute to cognitive impairments in hAPP mice and possibly in humans with AD. The therapeutic potential of reducing enkephalin production or signaling merits further exploration.

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Figures

Figure 1.
Figure 1.
Increased hippocampal ENK immunoreactivity in hAPPFAD mice and humans with AD. A–D, Horizontal brain sections of 2- to 4-month-old (A, B) and 6-month-old (C, D) mice were immunostained for ENK. Compared with NTG controls (A, C), hAPPFAD mice (B, D) had increased ENK immunoreactivity in the outer molecular layer (arrowhead) and hilar region of the dentate gyrus, the stratum lacunosum-moleculare, and the mossy fibers (arrows). E, F, Horizontal sections of human hippocampal tissue depicting differences in ENK immunoreactivity in a 71-year-old nondemented control (E) and a 90-year-old AD patient (F). G–J, Hippocampal sections from different AD cases were immunostained for ENK and counterstained with hematoxylin. Blessed Dementia Scale scores are shown in parentheses. gc, Granular layer; hil, hilus; mf, mossy fibers; oml, outer molecular layer; slm, stratum lacunosum-moleculare; sp, stratum pyramidale; sr, stratum radiatum. Abbreviations are the same in subsequent figures.
Figure 2.
Figure 2.
Quantitative comparison of hippocampal ENK and Penk expression in hAPPFAD mice and NTG controls. A, B, Densitometric quantitation of ENK immunoreactivity in specific hippocampal subregions (normalized to values from the stratum radiatum of CA1) from sections of 2- to 4-month-old (A) and 6-month-old (B) mice. n = 10–14 mice per age and genotype. C, Quantitative RT-PCR analysis of Penk mRNA levels in the hippocampus of 2- to 3-month-old mice and in the entorhinal cortex of 4- to 6-month-old mice relative to NTG controls. n = 6 (hippocampus) or 11–15 (entorhinal cortex) mice per age and genotype. D, In 2- to 3-month-old hAPPFAD mice, ENK immunoreactivity levels in the hilus correlated with hippocampal Penk mRNA levels. R2 and p values refer to hAPPFAD mice only. *p < 0.05, **p < 0.01, ***p < 0.001 versus NTG (Student's t test). Values are mean ± SEM.
Figure 3.
Figure 3.
Comparable expression of wild-type hAPP does not increase hippocampal ENK and Penk expression. A, B, Hippocampal levels of hAPP mRNA (A right), Penk mRNA (A, left), Aβ1–x (B, left), and Aβ1–42 (B, right) in hAPPWT mice from line I5 and hAPPFAD mice from line J20 (n = 7–10 mice per genotype; age, 2–3 months). C, Coronal sections illustrating typical ENK immunostaining in 4- to 6-month-old mice of the indicated genotypes. D, Densitometric quantitation of ENK immunoreactivity revealed no significant differences between hAPPWT mice and age-matched NTG controls in all hippocampal subfields except the oml of the dentate gyrus. E, Levels of ENK immunoreactivity in the hilus did not correlate with hippocampal plaque loads in 6-month-old hAPPFAD mice. *p < 0.05, **p < 0.01, ***p < 0.001 (Student's t test). Values are mean ± SEM.
Figure 4.
Figure 4.
ENK elevations in hAPPFAD mice correlate with calbindin depletions in the dentate gyrus. A, B, Horizontal brain sections of 2- to 4-month-old (A) and 6-month-old (B) hAPPFAD mice (J20 line) were immunostained for ENK and calbindin, and immunoreactivities were quantitated by densitometry. ENK elevations in the hilus correlated with calbindin depletions in the outer molecular layer in hAPPFAD mice. R2 and p values refer to hAPPFAD mice only.
Figure 5.
Figure 5.
ENK elevations correlate with behavioral alterations in hAPPFAD mice. A, B, hAPPFAD mice from line J20 and NTG controls (n = 10 mice per genotype; age, 3–5 months) were trained in a Morris water maze. A, No differences were found on the first training trial (T1). However, hAPPFAD mice showed learning deficits in the cued-platform (p < 0.05) and hidden-platform (p = 0.001) components of this task (repeated-measures ANOVA). B, During a probe trial (platform removed) at the beginning of the fourth day of training (before session 7), hAPPFAD mice crossed over the target location less often than NTG controls. C–F, ENK elevations in the hilus of hAPPFAD mice correlated with increased average distance required to reach the hidden platform (sessions 1–10) in the water maze (C), decreased target crossings during the probe trial (D), and hyperactivity in the open field (E) and Y-maze (F). R2 and p values refer to hAPPFAD mice only. **p < 0.01 (Student's t test). Values are mean ± SEM.
Figure 6.
Figure 6.
Increased neuronal Fyn expression elicits ENK elevations in low-expresser hAPPFAD mice. hAPPFAD mice from the low-expresser J9 line (hAPPlow) were crossed with mice that overexpress murine Fyn in neurons (FYN). The resulting offspring were analyzed at 7–9 months of age. A, Sagittal sections of the hippocampus immunostained for ENK. B, Densitometric quantitation of ENK immunoreactivity in different subfields of the hippocampus (n = 6–8 mice per genotype). Two-way ANOVA revealed a significant APP × FYN interaction (p < 0.01) for both the hilus and mossy fibers. *p < 0.05, **p < 0.01 versus NTG (Dunnett's test). Values are mean ± SEM.
Figure 7.
Figure 7.
Tau reduction prevents ENK elevations in hAPPFAD mice from line J20. Mice were analyzed at 5–7 months of age. A, Coronal sections of the hippocampus immunostained for ENK. B, C, ENK immunoreactivity in the hilus (B) and mossy fibers (C) was quantitated densitometrically in the groups indicated [n = 7–10 mice per genotype, except for hAPPFAD/Tau+/+ mice (n = 3)]. Two-way ANOVA revealed a significant APP effect (p < 0.001), Tau effect (p < 0.05), and APP × Tau interaction (p < 0.01) for both regions. **p < 0.01 versus Tau+/+ mice without hAPP (Dunnett's test); +p < 0.05, ++p < 0.01 versus Tau+/+ mice with hAPP (Fisher's PLSD test). Values are mean ± SEM.
Figure 8.
Figure 8.
μ-Opioid receptor antagonist ameliorates Aβ-induced behavioral deficits in hAPPFAD mice. hAPPFAD mice and NTG controls were treated with the irreversible μ-opioid receptor antagonist β-FNA or vehicle (Veh) and tested in the Morris water maze (n = 7–9 mice per genotype and treatment; age, 9–10 months). A, B, β-FNA improved the ability of hAPPFAD mice to learn the cued-platform location (p < 0.01 for genotype × treatment interaction by ANOVA; A), but not the hidden-platform location (B). The β-FNA regimen used here had no effect on NTG controls. C–F, Results from a probe trial performed 72 h after the last hidden-platform training trial. C, Representative swim paths show increased preference for the target quadrant in β-FNA-treated hAPPFAD mice. D, Percentage of time spent in different quadrants (p < 0.05 for group × quadrant interaction by ANOVA). E, Number of times mice crossed the platform location or corresponding locations in the other quadrants (p < 0.05 for group × quadrant interaction by ANOVA). F, Latency to reach the platform location (p < 0.05 for genotype × treatment interaction by ANOVA). ###p < 0.001, #p < 0.05 versus all nontarget quadrants; *p < 0.05, **p < 0.01 versus all other groups (Fisher's PLSD test); +p < 0.05 versus β-FNA-treated hAPP mice (Student's t test). Values are mean ± SEM.
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