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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-β (Aβ) 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 Aβ-dependent neuronal and behavioral alterations, and memory deficits were reduced by irreversible blockade of μ-opioid receptors with the antagonist β-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.
Alzheimer’s disease (AD) causes progressive memory loss and behavioral abnormalities that may reflect alterations in neurotransmitter and neuromodulatory systems. This notion has led to several treatments (Bales et al., 2006; Lleó et al., 2006). Acetylcholinesterase inhibitors and a glutamate receptor antagonist are widely used, while others are in clinical trials, including inhibitors of calcium channels, GABA receptors, or serotonin receptors (Roberson and Mucke, 2006). Current treatments provide limited benefit, and the prevalence of AD is rising at an alarming pace (Ferri et al., 2005). Additional therapeutic strategies are urgently needed. To address this need, we investigated the role of the enkephalin signaling pathway in the pathogenesis of AD.
Enkephalins are part of the endogenous opioid system, which modulates many functions affected by AD, including learning and memory (Messing et al., 1979; Gallagher, 1982; Gallagher et al., 1983; Bodnar and Klein, 2005), synaptic plasticity (Derrick et al., 1992; Do et al., 2002), and emotional behaviors (Nieto et al., 2005). Preproenkephalin (Penk) is proteolytically cleaved to produce met- and leu-enkephalins in the regulated secretory pathway. Enkephalins are stored in dense-core vesicles and co-released with fast-acting neurotransmitters, such as glutamate, after high-frequency synaptic stimulation (Caudle et al., 1991).
Enkephalin peptides and Penk mRNA are expressed by diverse cells in the brain and periphery (Mansour et al., 1995). In the hippocampus, enkephalin levels are particularly high in the lateral perforant pathway, which arises in the entorhinal cortex, and in mossy fibers, which arise in the dentate gyrus (Gall et al., 1981; Stengaard-Pedersen, 1983). Both pathways are severely affected by AD (Gomez-Isla et al., 1996; Blennow et al., 2006).
Enkephalins bind and activate μ- and δ-opioid receptors (Duka et al., 1981; Mansour et al., 1995). Enkephalins modulate learning and memory (Messing et al., 1979; Gallagher, 1982; Gallagher et al., 1983) and synaptic plasticity (Derrick et al., 1992; Williams and Johnston, 1996; Do et al., 2002) by activating μ-opioid receptors, and emotional behaviors by activating δ-opioid receptors (Nieto et al., 2005). Hippocampal μ-opioid receptors are found on cell bodies, dendrites, and axon terminals of inhibitory interneurons (Kalyuzhny and Wessendorf, 1998; Drake and Milner, 1999; Stumm et al., 2004). Consequently, enkephalins can have complex pre- and postsynaptic effects (Arvidsson et al., 1995; Ding et al., 1996). For example, activation of μ-opioid receptors hyperpolarizes GABAergic interneurons (Madison and Nicoll, 1988; Svoboda and Lupica, 1998), disinhibiting principal cells and increasing neuronal excitability (Nicoll et al., 1980; Simmons and Chavkin, 1996).
Most studies of the enkephalin system in AD have focused on changes in opioid receptor binding (Rinne et al., 1993; Mathieu-Kia et al., 2001). AD cases showed significant reductions in μ- and δ-opioid receptor binding in the molecular layer of the dentate gyrus (DG) and in the entorhinal cortex (Jansen et al., 1990), possibly reflecting compensatory adjustments to changes in enkephalin levels. Consistent with this possibility, elevated hippocampal enkephalin levels have been identified in aged transgenic mice overexpressing human amyloid precursor protein (hAPP) carrying familial AD (FAD)-linked mutations (Diez et al., 2000; Diez et al., 2003). It was unknown whether alterations in enkephalins and opioid receptors contribute to cognitive and behavioral deficits in AD or in hAPP mice.
To elucidate the role of met-enkephalin (ENK) in Aβ-induced neuronal and behavioral deficits, we studied hAPP mice from lines J20 and J9, which have a range of AD-related synaptic, neuronal, and behavioral deficits (Hsia et al., 1999; Mucke et al., 2000; Palop et al., 2003; Chin et al., 2005; Palop et al., 2005; Palop et al., 2007; Roberson et al., 2007).
hAPP mice and NTG controls were from lines J9 and J20, which express hAPP with the Swedish (K670N, M671L) and Indiana (V717F) FAD mutations, and from line I5, which expresses wildtype hAPP (Rockenstein et al., 1995; Mucke et al., 2000). Mice with neuronal overexpression of Fyn kinase (line N8) were crossed with mice from line J9 to establish FYN/hAPP doubly transgenic mice as described (Chin et al., 2004; Chin et al., 2005). hAPP mice from the J20 line were crossed with Tau-deficient mice (Dawson et al., 2001) as described (Roberson et al., 2007) to produce offspring with two (Tau+/+), one (Tau+/−), or no (Tau−/−) functional Tau alleles in the presence or absence of hAPP. All lines had been crossed for >10 generations with C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME). For tissue isolation, mice were deeply anesthetized and flush-perfused transcardially with 0.9% saline. The left hemibrain was snap frozen on dry ice and stored at −70°C for ELISA quantitation of Aβ1-x and Aβ1-42 (Mucke et al., 2000). The right hemibrain was drop fixed in 4% paraformaldehyde for 48 h at 4°C, washed in phosphate buffered saline (PBS), and placed in a 30% sucrose solution for 24 h at 4°C. All experiments were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco.
Formalin-fixed hippocampal tissues from AD cases (five women and three men; age: 71-90 years; mean ± S.D: 83.1±7.2) and non-demented controls (three women and four men, age: 71-102 years; mean ± S.D: 84.7±12.8) were obtained from the Alzheimer’s Disease Research Center at the University of California at San Diego and processed as described (Palop et al., 2003). Formalin-fixed tissue was postfixed in 4% phosphate-buffered paraformaldehyde for 72 h before sectioning.
Snap-frozen brain tissues were homogenized with guanidine buffer followed by ELISA measurements of human Aβ peptides (Cheng et al., 2007). The ELISA for Aβ1-42 detects this specific peptide. The ELISA for Aβ1-x (approximates total Aβ) recognizes N-terminal fragments of Aβ containing the first 28 amino acids.
Vibratome sections (40 μm, humans) and sliding microtome sections (30 μm, mice) were immunostained with the standard avidin-biotin/peroxidase method as described (Palop et al., 2003). Briefly, after reducing endogenous peroxidase activity and blocking nonspecific binding, floating sections were incubated for 15-16 h with anti-ENK (1:5,000 for mouse tissue; 1:500 for human tissue; Chemicon), anti-calbindin (1:30,000; Swant, Bellinzona, Switzerland), or biotinylated anti-Aβ (3D6, 1:400; Elan Pharmaceuticals, South San Francisco, CA) at 4°C. Primary antibodies (except 3D6) were detected with biotinylated goat anti-rabbit secondary antibodies (1:200, Vector Laboratories, Burlingame, CA). Diaminobenzidine was used as a chromagen. Some sections from human tissue were counterstained with Hematoxylin 560 (Surgipath Medical Ind., Richmond, IL) for 30 sec. Images were acquired on a BX-60 microscope (Olympus, Melville, NY) with a DEI-470 digital camera (Optronics, Goleta, CA). ENK immunostaining was quantitated in two sections (300 μm apart) per mouse. Integrated optical density was determined with a Bioquant Image Analysis System (R&M Biometrics, Nashville, TN) and averaged in two areas (0.04 mm2 each) in each of the following regions: molecular layer of the dentate gyrus, hilar region of the dentate gyrus, stratum lucidum of CA3, stratum lacunosum-moleculare of CA3, and stratum radiatum of CA1. ENK levels were calculated from the integrated optical density (IOD) in target regions divided by the IOD in stratum radiatum of CA1 of the same section (as an internal control region which did not differ between NTG and hAPPFAD mice). For correlation analyses and when only one region is displayed in a graph, ENK immunoreactivity levels are expressed relative to the average value in NTG mice. Calbindin levels and Aβ plaques were measured as described (Palop et al., 2003).
Total RNA from individual hippocampi or dissected entorhinal cortex from 450-μm-thick horizontal mouse brain sections was isolated with RNeasy mini columns (Qiagen). Reverse transcription (RT) reactions (Applied Biosystems, Foster City, CA) contained 300 ng of DNase-treated total RNA and random hexamer and oligo-d(T) primers (2.5 μM each). Diluted reactions (6 ng of cDNA) were analyzed with SYBR green PCR reagents in duplicate on an ABI Prism 7700 sequence detector (Applied Biosystems). Penk and hAPP mRNA levels were determined relative to standard curves made with serial dilutions of pooled cDNAs (30, 6, and 1.2 ng) from all samples. Values were normalized to individual housekeeping gene expression (mouse GAPDH) and are reported relative to NTG or hAPPWT controls. The slopes of standard curves, control reactions without RT, and dissociation curves of products indicated adequate PCR quality. The following primers were used. Penk: forward, 5 ′- GCCTTGTCAATGATGTTTCTTGTC-3 ′ ; reverse , 5 ′- CAACATAGCCATAAGAGACCAATACTG-3′. mGAPDH: forward, 5′-GGGAAGCCCATCACCATCTT-3′; reverse, 5′-GCCTTCTCCATGGTGGTGAA-3′. hAPP: forward, 5′-GAGGAGGATGCTCGGATGTCT-3′, reverse 5′-AGCCACTTCTTCCTCCTCTGCTA-3′.
Mice were treated with the irreversible μ-opioid receptor antagonist β-funaltrexamine (β-FNA) (Takemori et al., 1981) (Tocris Bioscience, Ellisville, MO) or vehicle. β-FNA was dissolved in water to a concentration of 0.5 mg/ml and injected subcutaneously (s.c.) at 5 mg/kg once a week for 3 weeks. To minimize stress effects from the injections, behavioral testing began 24-48 h after the injections. During weeks 2 and 3 of treatment, mice were tested in the water maze. Mice were sacrificed 1 week after the fourth injection.
Open field. Spontaneous locomotor activity was measured with an automated Flex-Field/Open Field Photobeam Activity System (San Diego Instruments, San Diego, CA). Mice were placed individually in a clear plastic chamber (41 × 41 × 30 cm), and their horizontal and vertical movements were monitored for 15 min with two 16 × 16 photobeam arrays. Total ambulations in the periphery of the open field were analyzed as the main measure. The apparatus was thoroughly cleaned with 70% ethanol between trials.
The Y-maze consisted of three compartments (10 cm × 10 cm × 10 cm) connected with 4 cm × 5 cm × 10 cm passages. Each mouse was placed in one of the compartments and allowed to explore for 6 min. The total number of arm entries was recorded. The apparatus was thoroughly cleaned with 70% ethanol between trials.
The water maze consisted of a pool (122 cm diameter) filled with water (18 ± 1°C) made opaque with non-toxic white tempera paint powder, and was placed in a room surrounded by distinct extramaze cues. Two identical pools were used in two different rooms with distinct cues. Pretraining and visible platform training were performed in one room, whereas hidden platform training was performed in the second room. Mice were first given four pretraining trials in which they had to swim down a channel (15 cm × 122 cm) and mount a platform hidden 1.5 cm below the water surface at the end of the channel. The following day, mice were trained in the actual water maze. For cued training, the platform (14 cm × 14 cm) was submerged (1.5 cm) but visibly cued with a black and white striped mast (15 cm in height). On three consecutive days, mice received two training sessions per day separated by 3-4 hours. Each session consisted of two training trials with an intertrial interval of 10 min. For each session, the platform was moved to a new location and the drop location was changed semi-randomly between trials. Mice were then moved to the second room for hidden platform training during which a similar platform was submerged (1.5 cm) and not visibly cued. On five consecutive days, mice received two training sessions separated by 3-4 hours. Each session consisted of three trials (10 min between trials). The platform location remained the same throughout the hidden platform training, but the drop location was changed semi-randomly between trials. Mice had 60 sec to locate the platform during training. Mice that did not find the platform were guided to it and placed on it for 10 sec. A spatial probe trial was carried out 16-18 h after the third day of training (beginning of session 7) and 24 and 72 hours after the hidden platform training. The platform was removed, and mice were allowed to swim in the pool for 60 sec. Escape latencies, swim paths, swim speeds, percent time spent in each quadrant, and platform crossings were recorded with an EthoVision video tracking system (Noldus, Netherlands).
Mice were treated with β-FNA (5 mg/kg, s.c.) or vehicle weekly for 2 weeks. Forty-eight hours after the last β-FNA injection, baseline nociceptive responses were tested on a hot plate analgesia meter (IITC, Woodland Hills, CA) set at 55 ± 0.1°C. Mice were placed in a Plexiglas cylinder (diameter, 4.5 inches) on the hot plate, and the time it took for the mice to lick a hindpaw or jump was recorded. Mice were then immediately removed from the hot plate and injected with morphine (10 or 20 mg/kg, i.p., Sigma, St. Louis, MO) dissolved in 0.9% saline to a concentration of 1mg/ml or 2 mg/ml. Thirty minutes after the morphine injections, mice were placed on the hotplate again and response latencies were recorded. Morphine-induced analgesia was expressed as the percent maximum possible effect (%MPE), calculated as follows: (test latency – baseline latency)/(60-s cut-off baseline latency) × 100. Approximately 1 h after the morphine injection, mice were placed in an open field and morphine-induced locomotor activity was monitored for 5 min.
Statistical analyses were performed with Statview 5.0 (SAS Institute, Cary, NC). Differences between means were assessed by Student’s t test and differences among means by one-, two, or three-way ANOVA (with or without repeated measures), followed by Fisher’s PLSD post-hoc test or Dunnett’s t test. Correlations were assessed by linear regression analysis.
hAPPFAD mice from line J20, which have high levels of Aβ in the hippocampus and neocortex (Mucke et al., 2000), had much higher levels of ENK immunoreactivity in the mossy fiber and lateral perforant pathways than NTG controls (Fig. 1A-D). At 2-4 months of age, hAPPFAD mice showed increased ENK immunostaining primarily in the hilar region of the dentate gyrus and in mossy fiber terminals in the stratum lucidum of CA3 (Figs. 1A,B and and2A).2A). By 6 months, hAPPFAD mice had increased ENK levels also in the terminal zones of the lateral perforant path: the outer molecular layer of the dentate gyrus and the stratum lacunosum-moleculare of CA1/3 (Figs. 1C,D and and2B).2B). Consistent with these findings, 6-month-old hAPPFAD mice had higher levels of ENK in the entorhinal cortex than NTG mice, particularly in the superficial layer I and the pyramidal cell layers II/III (data not shown), from which the perforant path emanates. In both hAPPFAD mice and NTG controls, ENK expression in the outer molecular layer was confined to the outer two-thirds of this layer and was more prominent in the ventral than the dorsal region of the dentate gyrus (Fig. 1A-D and data not shown), which is typical for mice (Gall, 1988).
Compared with non-demented controls, human AD cases also had increased ENK levels in the hilus, outer molecular layer, and stratum lacunosum-moleculare (Fig. 1E-J), underlining the relevance of the alterations observed in hAPPFAD mice to the human condition. Moreover, the highest levels of ENK were observed in the cases with the highest (worst) scores on the Blessed Dementia Scale (Fig. 1G-J).
To determine if the increased ENK immunoreactivity in hAPPFAD mice was caused by changes in gene expression, we measured Penk mRNA levels in the hippocampus and entorhinal cortex by quantitative RT-PCR. In both brain regions, Penk mRNA levels were 2-3-fold higher in hAPPFAD mice than in NTG controls (Fig. 2C). In addition, hippocampal levels of ENK immunoreactivity and Penk mRNA were strongly correlated in hAPPFAD mice (Fig. 2D).
To exclude the possibility that ENK elevations in hAPPFAD mice were caused by hAPP overexpression per se, we measured the levels of Penk mRNA and ENK immunoreactivity in 2–3-month-old transgenic mice from line I5. Mice from this line express wildtype hAPP (hAPPWT) at levels comparable to those of hAPPFAD in the J20 line (Fig. 3B). Because of the FAD mutations, total Aβ1-x and Aβ1-42 levels were much higher in the hippocampus of hAPPFAD mice than in hAPPWT mice (Fig. 3A). Hippocampal levels of Penk mRNA were also higher in hAPPFAD mice than in hAPPWT mice (Fig. 3B), and not significantly different between hAPPWT mice and NTG controls (data not shown). Compared with NTG controls, only hAPPFAD mice, but not hAPPWT mice, showed marked increases in ENK immunostaining in the dentate gyrus (Fig. 3C). Densitometric analysis confirmed that there was no significant difference in ENK immunostaining in the hilus, mossy fibers and stratum lacunosum-moleculare between hAPPWT mice and NTG controls (Fig. 3D), although there was a small increase in ENK immunostaining in the outer molecular layer of the dentate gyrus. These data suggest that increased ENK expression in hAPPFAD mice is dependent on Aβ levels or on another effect of the FAD mutations.
ENK elevation in the dentate gyrus of aged hAPPFAD mice has previously been attributed to amyloid deposition (Diez et al., 2000; Diez et al., 2003). However, increased ENK expression was detectable in hAPPFAD mice at 2-3 months of age (Figs. 1B, ,2A2A and and3B),3B), before Aβ deposits form in line J20 (Mucke et al., 2000). To assess if Aβ deposition worsens ENK increases in older mice, we examined the relationship between ENK elevation and plaque formation in 4-6-month-old hAPPFAD mice (Fig 3E). We found no correlation between hippocampal ENK expression and Aβ deposition at this age. Thus, the ENK elevations in hAPPFAD mice are not dependent on plaque formation.
hAPPFAD mice with high levels of Aβ have depletions of the calcium-binding protein calbindin-D28K in granule cells of the dentate gyrus that correlate well with other Aβ-dependent alterations, including deficits in the Morris water maze task (Palop et al., 2003; Chin et al., 2004; Chin et al., 2005; Palop et al., 2005; Palop et al., 2007). Such reductions in calbindin also occur in humans with AD and likely reflect a compensatory response to Aβ-induced alterations in neuronal activity and calcium influx (Palop et al., 2003)(Palop et al., 2007). To determine if the dysregulation of calbindin and ENK expression have related underlying mechanisms, we compared the level of hippocampal immunostaining of calbindin and ENK by densitometry. Calbindin depletions in the OML of the dentate gyrus correlated with ENK elevations in the hilus of 2-4-month-old and 6-month-old hAPPFAD mice (Fig. 4).
To test whether ENK elevations also correlate with behavioral alterations, 3-5-month-old hAPPFAD mice and NTG controls were trained in a Morris water maze (Fig. 5). Consistent with previous reports (Palop et al., 2003; Kobayashi and Chen, 2005; Cheng et al., 2007; Roberson et al., 2007), hAPPFAD mice showed learning deficits in this test (Fig. 5A). Spatial memory was assessed in a probe trial (hidden platform removed) at the beginning of the fourth day of hidden platform training (before training session 7). hAPPFAD mice crossed over the target location less often (Fig. 5B) and spent less time searching in the target quadrant (data not shown) than NTG controls. Deficits in water maze performance correlated with increased ENK levels (Fig. 5C,D) and with calbindin depletion (Palop et al., 2003; Supplemental Fig. 1A,B). hAPPFAD mice also showed hyperactivity in an open field (2258 ± 184 total movements for hAPPFAD vs. 1505 ± 53 for NTG, p < 0.001) and a Y-maze (33.8 ± 3.5 arm entries for hAPPFAD vs. 23.9 ±1.3 for NTG, p < 0.01). Hyperactivity is a behavioral phenotype displayed by several hAPP lines (Kobayashi and Chen, 2005) and may relate to impaired hippocampal encoding of contextual information (Godsil et al., 2005). Indeed, hyperactivity in hAPPFAD mice correlated with spatial learning and memory impairments in the water maze (Supplemental Fig. 2). Notably, hyperactivity also correlated with elevations in hippocampal ENK levels (Fig. 5E,F) and depletion of calbindin (Supplemental Fig. 1C,D) in hAPPFAD mice.
Together, these results suggest that similar mechanisms may be involved in the Aβ-induced alterations in ENK and calbindin levels, both of which correlated tightly with behavioral abnormalities in hAPPFAD mice.
Increasing neuronal levels of the tyrosine kinase Fyn sensitizes neurons to aberrant overexcitation and to other Aβ-induced abnormalities (Chin et al., 2004; Chin et al., 2005; Palop et al., 2007). Overexpression of Fyn in low-expresser hAPP mice (line J9, hAPPlow) elicits memory deficits as well as prominent calbindin reductions in granule cells of the dentate gyrus (Chin et al., 2005). In contrast, reduction of the microtubule-associated protein Tau prevents cognitive impairments and neuronal depletions of calbindin in hAPPFAD mice with high levels of Aβ (line J20), most likely through a mechanism that protects against aberrant neuronal overexcitation (Palop et al., 2007; Roberson et al., 2007). By experimentally modulating the levels of Fyn and Tau, we tested whether the ENK alterations may be related to the same pathogenic cascade that leads from increased Aβ levels to aberrant neuronal overexcitation and cognitive decline.
First, we compared hippocampal ENK expression in four groups of 7-9-month-old mice: FYN transgenic mice with neuronal overexpression of mouse Fyn (Kojima et al., 1997; Kojima et al., 1998), hAPP mice from line J9 (hAPPlow) that express hAPPFAD and Aβ at roughly half the levels found in hAPP mice from line J20 (Mucke et al., 2000), FYN/hAPPlow doubly transgenic mice, and NTG mice. Compared with NTG mice, only FYN/hAPPlow mice had significant increases in ENK in the dentate gyrus (Fig. 6), demonstrating ENK elevation in an independent hAPPFAD line and implicating Fyn-related pathways in the pathogenesis of this alteration.
Next, to determine if Tau reduction prevents Aβ-dependent increases in ENK levels, we compared hippocampal ENK levels in the following groups of mice at 5-7 months: hAPPFAD mice from line J20 with two (hAPP / +/+ FAD Tau ), one (hAPP /Tau+/−), or no (hAPP -/- FAD FAD/Tau ) functional Tau alleles, and Tau+/+, Tau+/−, or Tau−/− mice without hAPPFAD (Fig. 7). Tau reduction dose-dependently prevented ENK elevations in the hilus (Fig. 7A,B) and mossy fibers (Fig. 7A,C) of hAPPFAD mice, but did not alter ENK expression in the absence of hAPPFAD (Fig. 7).
The association of increased ENK levels with behavioral deficits suggests that ENK either mediates or counteracts Aβ-induced neuronal dysfunction. To differentiate between these possibilities, we assessed whether inhibiting ENK signaling improves or worsens Aβ-induced behavioral alterations. We treated 9-10-month-old hAPPFAD and NTG mice with the long-lasting, irreversible μ-opioid receptor antagonist β-funaltrexamine (β-FNA) or with vehicle once a week for three weeks. During the second and third week of treatments, mice were trained in the Morris water maze (Fig. 8).
On the first training trial (T1), mice swam a similar distance (Fig. 8A) and required a similar amount of time (data not shown) to find the visible platform, independent of treatment or genotype. Subsequently, it took vehicle-treated hAPPFAD mice longer to learn the visible platform task than vehicle- or β-FNA-treated NTG mice, although by the end of the training vehicle-treated hAPPFAD mice performed as well as the NTG control groups (Fig. 8A). Notably, β-FNA treatment significantly improved the performance of hAPPFAD mice in this task (Fig. 8A).
hAPPFAD mice took significantly longer than NTG mice to learn the hidden platform component of the water maze task independent of treatment (Fig. 8B). β-FNA treatment had no effect on the performance of NTG mice (Fig. 8A,B) or on swim speeds in either genotype (data not shown).
Spatial memory was assessed in probe trials carried out at the beginning of the fourth day of training (before session 7), and 24 h and 72 h after the completion of hidden platform training (Fig. 8C-F and Supplemental Fig. 3). In the last probe trial, β-FNA treatment significantly increased the amount of time hAPPFAD mice spent searching in the target quadrant (Fig. 8C,D) and the number of times they crossed over the target location (Fig. 8E), compared with vehicle-treated hAPPFAD mice. β-FNA treatment also reduced the time it took hAPPFAD mice to reach the target location (Fig. 8F). In NTG mice, the dose of β-FNA used here only increased the percent time they spent in the target quadrant on the first probe trial but had no effects on target crossings or time required to reach the target location (Supplemental Fig. 3). Independent repetition of the above experiment in two additional cohorts of mice with each cohort including 7-11 mice per genotype and treatment yielded similar results (data not shown).
To confirm that the dose of β-FNA used in the behavioral studies was effective in blocking μ-opioid receptor function in hAPP mice and NTG mice, we pretreated mice once a week for two weeks with either β-FNA (5 mg/kg, s.c., per week) or vehicle. We then tested whether the β-FNA pretreatment antagonized morphine-induced analgesia and hyperactivity 48 h after the last drug injection (Supplemental Fig. 4). β-FNA pretreatment significantly antagonized the antinociceptive actions of morphine in the hot plate test (Supplemental Fig. 4A). β-FNA pretreatment also antagonized morphine-induced hyperactivity (10 mg/kg morphine) in an open field (Supplemental Fig. 4B). Since there was no significant interaction between genotype and the effects of β-FNA on morphine-induced analgesia or hyperactivity by ANOVA, the data obtained in different genotypes were combined. Together, these results confirm that β-FNA effectively antagonized μ-opioid receptor activity in our mice, consistent with previous findings (Takemori et al., 1981; Frischknecht et al., 1983; Broadbear et al., 2000).
This study shows that high levels of Aβ increase the expression of Penk and ENK in the hippocampus and entorhinal cortex independent of plaque formation. Lower levels of Aβ had similar effects when neurons were sensitized to Aβ by increased expression of Fyn kinase. Lowering endogenous Tau, which increases resistance to aberrant neuronal overexcitation (Palop et al., 2007; Roberson et al., 2007), reduced the Aβ-induced increase in ENK levels. Because ENK levels correlated with behavioral alterations and neuronal calbindin depletion, we hypothesized that overstimulation of μ-opioid receptors by ENK mediates some Aβ-induced neurological impairments. Indeed, treatment of hAPP mice with a μ-opioid receptor antagonist reduced Aβ-dependent water-maze deficits. We also found that ENK elevations in the hippocampus of AD cases increased with the severity of the disease. Because excessive stimulation functionally uncouples opioid receptors from G-proteins and causes internalization (Williams et al., 2001; von Zastrow et al., 2003), reductions in μ-opioid receptor binding in hippocampus and dentate gyrus of AD patients (Jansen et al., 1990; Rinne et al., 1993; Mathieu-Kia et al., 2001) may be compensatory responses to ENK elevation.
Increased ENK levels in the hippocampus and entorhinal cortex of 21-27-month-old hAPP mice from two other transgenic lines have been postulated to represent a response to plaque-induced neuronal injury (Diez et al., 2000; Diez et al., 2003). However, ENK elevations were prominent in hAPP mice at 2-4 months of age, when plaques have not yet formed in line J20 mice (Mucke et al., 2000). Moreover, ENK levels did not correlate with the extent of Aβ deposits in older mice. These results are consistent with our recent demonstration that other neuronal alterations and memory deficits in 3-4-month-old hAPP mice from line J20 correlate with forebrain levels of the soluble Aβ oligomer Aβ*56, but not with plaque loads (Cheng et al., 2007).
To assess whether ENK elevations in hAPP mice may be caused by hAPP rather than by Aβ, we compared hAPPFAD mice from line J20 with hAPPWT mice from line I5, which have similar hAPP levels but different Aβ levels. Hippocampal levels of Penk mRNA and ENK immunoreactivity were significantly greater in hAPPFAD mice than in hAPPWT mice and NTG controls, which had comparable levels. These results are consistent with the hypothesis that Penk and ENK elevations in hAPP mice are caused by nonfibrillar Aβ, although we cannot exclude contributions by Aβ-independent effects of FAD mutations (De Jonghe et al., 2001; Hecimovic et al., 2004; Wiley et al., 2005). hAPPFAD mice from the lower-expresser J9 line (hAPPlow) had normal ENK levels, suggesting that relatively high levels of Aβ are required to increase ENK expression. Consistent with reports that Fyn sensitizes neurons to Aβ-induced molecular and functional alterations in hAPPlow mice (Chin et al., 2004; Chin et al., 2005; Palop et al., 2007), increased expression of Fyn elicited prominent increases in ENK expression in hAPPlow/FYN mice. Thus, it is extremely unlikely that ENK elevation in line J20 was caused by a line-specific insertional mutation.
Increased ENK immunoreactivity in mossy fibers and perforant pathway could reflect increased neuronal ENK production, decreased ENK degradation, or accumulation of ENK due to reduced synaptic release. Although these possibilities are not mutually exclusive, the significant increases in Penk mRNA levels in the hippocampus and entorhinal cortex of hAPPFAD mice suggest that increased ENK production plays a major role. The tight correlation between increases in ENK immunoreactivity and Penk mRNA across different hAPPFAD mice further supports this conclusion.
Although the precise mechanisms underlying the Aβ-induced increase in Penk and ENK expression remain to be determined, it is likely that aberrant increases in neuronal activity are involved. hAPPFAD mice have increased susceptibility to seizures induced by kainate or pentylenetetrazole (PTZ) (Del Vecchio et al., 2004; Palop et al., 2007; Roberson et al., 2007), which may relate to the increased incidence of seizures in AD patients (Amatniek et al., 2006; Palop et al., 2006). Seizures increase hippocampal ENK levels (Gall, 1988; Morris et al., 1988; Moneta and Höllt, 1990), which may facilitate recurrent limbic seizure activity by disinhibiting neuronal networks via activation of μ-opioid receptors (Kanamatsu et al., 1986; Hong et al., 1988). Intraventricular injection of μ-opioid receptor agonists induced nonconvulsive seizures in rats (Snead and Bearden, 1980; Tortella et al., 1987), whereas blocking μ-opioid receptors with β-FNA attenuated seizure susceptibility in rats treated with ENK (Tortella et al., 1987) or kainate (Liu et al., 2004). Thus, Aβ-induced increases in neuronal excitability may increase neuronal ENK expression, which could promote recurrent seizure activity. Consistent with this possibility, hAPPFAD mice have spontaneous nonconvulsive seizure activity (Palop et al., 2007). Furthermore, ENK elevations in hAPPFAD mice were prevented by Tau reduction and exacerbated by overexpression of Fyn, manipulations that are known to prevent or exacerbate aberrant neuronal overexcitation in hAPPFAD mice, respectively (Palop et al., 2007; Roberson et al., 2007).
Increased ENK expression may contribute to neural network dysfunction in hAPPFAD mice and AD. ENK elevations correlated tightly with calbindin depletions in the dentate gyrus, which correlate with reductions in other synaptic activity-dependent proteins and with learning and memory deficits (Palop et al., 2003; Palop et al., 2005). In NTG mice and rats, peripheral administration of ENK, leu-enkephalin, or morphine caused dose-dependent impairments in acquisition and retention of active and passive avoidance conditioning (Izquierdo et al., 1980; Izquierdo and Dias, 1981; Martinez et al., 1985). In rats, morphine also impaired spatial learning and memory in a water maze, an effect that was blocked with the opioid receptor antagonist naloxone (McNamara and Skelton, 1991). Even in untreated mice and rats, peripheral injection of naloxone or selective μ-opioid receptor antagonists after training enhanced retention of active and passive avoidance conditioning (Izquierdo, 1979; Messing et al., 1979; Izquierdo and Graudenz, 1980; Gallagher, 1982, 1985). Opioid antagonists also improved spatial memory in a water maze (Decker et al., 1989) and working memory in a radial arm maze (Gallagher et al., 1983; Canli et al., 1990). These results suggest that activation of μ-opioid receptors by exogenous or endogenous opioids inhibits memory formation or retention. Indeed, water maze deficits, and hyperactivity in an open field and a Y-maze correlated with hippocampal ENK elevations in hAPPFAD mice, suggesting a strong association between Aβ-induced increases in ENK expression and behavioral abnormalities.
To test more directly whether ENK elevations play a causal role in Aβ-induced behavioral deficits, we treated hAPPFAD mice with β-FNA. In hAPPFAD mice, this irreversible μ-opioid receptor antagonist improved cued platform learning but not hidden platform learning, possibly because the former task is easier. Although the cued platform task can be learned in the absence of distal spatial cues, NTG mice and rats do use such cues to advantage in solving this task, a strategy that requires normal hippocampal function (Whishaw et al., 1995; Hamilton et al., 2004). Partial improvement in hippocampal function may have helped hAPPFAD mice to solve the cued platform task, but may have been insufficient to solve the hidden platform task, which depends more heavily on spatial learning.
β-FNA may also have affected other brain regions and behavioral domains. Although hAPPFAD mice have normal visual acuity, they have difficulty associating a visual cue with escape from water (deIpolyi et al., 2007), an impairment that might relate to visual processing deficits (O’Brien et al., 2001; Kergoat et al., 2002; Mapstone et al., 2003) or topographic agnosia (Kiyosawa et al., 1989; Mendez and Cherrier, 2003) in AD. While the precise neural substrates of these behavioral deficits are unknown, aberrant activation of μ-opioid receptors might be involved. β-FNA might also improve motivation, since morphine appears to impair water maze acquisition, in part, by reducing the impetus to escape onto the platform, an effect that can be blocked with naloxone (McNamara and Skelton, 1991).
β-FNA did not affect hidden platform learning but improved spatial memory retention in hAPPFAD mice, as revealed by their performance in the probe trial. We speculate that β-FNA improves memory retention in hAPPFAD mice by counteracting Aβ-induced increases in ENK signaling through μ-opioid receptor stimulation in the hippocampus and entorhinal cortex.
In a double-blind, placebo-controlled clinical study, naloxone improved cognitive and behavioral performance of AD patients (Reisberg et al., 1983). Although subsequent studies found no significant improvements (Pomara et al., 1985; Serby et al., 1986; Tariot et al., 1986), the opioid receptor antagonist naltrexone markedly improved cognitive performance in specific tests in one of nine (Serby et al., 1986) and one of ten (Pomara et al., 1985) AD patients. Overstimulation of μ-opioid receptors might contribute more critically to cognitive failure in some AD patients than others. It is also possible that a more specific μ-opioid receptor antagonist might enhance cognitive performance in a larger proportion of AD patients. Additional studies are warranted to further explore the therapeutic potential of reducing ENK production or, specifically, μ-opioid receptor activity in the context of AD-related cognitive impairments.
We thank H. Solanoy, X. Wang, and D. Kim for technical support, J. Harris and M. Cisse for comments on the manuscript, G. Howard and S. Ordway for editorial review, J. Carroll for preparation of graphics, and D. McPherson and T. Hamto for administrative support. The study was supported by NIH Grants AG022074 and NS041787 to L.M. and RR018928 to the J. David Gladstone Institutes.