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Early Alzheimer's disease (AD) is marked by cholinergic hypofunction, neuronal marker loss, and decreased nicotinic acetylcholine receptor (nAChR) density from the cortex and hippocampus. α7 nAChRs expressed on cholinergic projection neurons and target regions have been implicated in neuroprotection against Aβ toxicity and maintenance of the septo-hippocampal phenotype. We tested the role that α7 nAChRs perform in the etiology of early AD by genetically deleting the α7 nAChR subunit from the Tg2576 mouse model for AD and assessing animals for cognitive function and septo-hippocampal integrity. Thus, Tg2576 mice transgenic for mutant human amyloid precursor protein (APP) were crossed with α7 nAChR null mice (A7KO) in order to render an animal with elevated Aβ in the absence of α7 nAChRs (A7KO-APP). We found that learning and memory deficits seen in 5-month-old APP mice are more severe in the A7KO-APP animals. Analyses of animals in early stage pre-plaque cognitive decline revealed signs of neurodegeneration in A7KO-APP hippocampus as well as loss of cholinergic functionality in the basal forebrain and hippocampus. These changes occurred concomitant with the appearance of a dodecameric oligomer of Aβ that was absent from all other genotypic groups; generating the hypothesis that increased soluble oligomeric Aβ may underlie further impairment of A7KO-APP cognitive function. Thus, α7 nAChRs in a mouse model for early stage AD appear to serve a neuroprotective role through maintenance of the septo-hippocampal cholinergic phenotype and preservation of hippocampal integrity possibly through influences on Aβ accumulation and oligomerization.
In early AD, there is impairment in hippocampus-based episodic memory that is improved through enhancement of cholinergic transmission, indicating that hypoactivity of septo-hippocampal projections underlie the earliest AD symptomatology (Riekkinen et al., 1987), which appear to be via reductions in choline acetyltransferase (ChAT) activity as well as depletion of nicotinic acetylcholine receptors (nAChRs)(Perry et al., 1978; Banerjee et al., 2000; Pappas et al., 2000).
The α7 subtype of nAChR is expressed by basal forebrain cholinergic projection neurons and within the hippocampus (Hunt and Schmidt, 1978; Fabian-Fine et al., 2001; Azam et al., 2003). Several studies have demonstrated that α7 nAChR activation attenuates Aβ toxicity; in vivo, α7 nAChRs have been shown to maintain the septo-hippocampal cholinergic phenotype suggesting that α7 nAChR activation represents a useful therapeutic strategy for AD (Jonnala and Buccafusco, 2001; Kihara et al., 2001; Kihara et al., 2004; Li et al., 2005; Ren et al., 2007). However, this view is confounded by the fact that α7 nAChRs and Aβ peptides have been shown to interact in vitro and in vivo, including human post-mortem AD brain (Dineley, 2007; Wang et al., 2009). Furthermore, recent work suggests that α7 nAChR–Aβ interaction in aged AD mouse models may exacerbate their pathological features (Ren et al., 2007; Soderman et al., 2008; Dziewczapolski et al., 2009). The role of α7 nAChRs in the pathology of pre-plaque AD when early cognitive decline is underway has yet to be directly tested.
In this study, we tested the hypothesis that α7 nAChRs are neuroprotective during early stage AD by investigating the effects of α7 nAChR gene deletion on cognitive function and septo-hippocampal integrity in the Tg2576 (APP) transgenic animal model for AD. In addition to Aβ over-production and accumulation, this animal model exhibits early onset (5-months-of-age) and progressive memory deficits, impaired synaptic plasticity, and alterations of several biochemical markers (Hsiao et al., 1996; Dineley et al., 2001; Apelt et al., 2002; Dineley et al., 2002a; Westerman et al., 2002; Spires et al., 2005; Jacobsen et al., 2006). Yet, cholinergic hypofunction and marked hippocampal neurodegeneration are noticeably absent (Apelt et al., 2002; Stein and Johnson, 2002; Luth et al., 2003; Wenk et al., 2004; Watanabe et al., 2009).
Whereas α7 nAChR knock-out (A7KO) mice neither show cognitive deficits nor exhibit morphological CNS abnormalities, we found that cognitive deficits seen in 5-month-old APP transgenic mice are more severe when α7 nAChR receptors are absent (A7KO-APP) (Orr-Urtreger et al., 1997; Paylor et al., 1998). Biochemical analyses on 5-month-old A7KO-APP revealed significant reduction in hippocampal and basal forebrain ChAT activity and loss of hippocampal neurons and markers; stereological analyses indicated more pronounced loss of hippocampal pyramidal neurons and volume loss compared to APP mice. These observations were concomitant with the appearance of ~56 kD soluble Aβ oligomers in the hippocampus of A7KO-APP mice which may underlie the further impairment of cognitive function in these mice. Thus, α7 nAChRs in a model of early stage AD appear to serve a neuroprotective role through maintenance of the septo-hippocampal cholinergic phenotype and preservation of hippocampal integrity possibly through influences on Aβ accumulation and oligomerization.
Unless otherwise stated, all reagents were purchased from Sigma Aldrich.
Synaptophysin (1:1,000 Synaptic Systems#101002); VAChT (1:200 Santa Cruz Biotechnology#sc-7716); ChAT (1:200 Santa Cruz#sc-20672); β-actin (1:10,000 Sigma Aldrich#A5441); MAP2 (1:2,000 Millipore#AB5622, Millipore); ERK and phospho-ERK (1:5,000 Cell Signaling Technologies #9102 and #9101, respectively), 4G8 (1,5000 Covance #SIG-39200). Secondary antibodies were purchased from Cell Signaling Technologies (#7074 anti-rabbit, #7076 anti-mouse) and Santa Cruz (sc-2350 anti-goat). APP C-terminal antibodies were a kind gift from Drs. Christopher Eckman (Mayo Clinic, FL) and Kumar Sambamurti (Medical University of South Carolina, SC). OC antibody (1:5,000) was a kind gift from Dr. Rakez Kayed.
We studied mice that overproduce Aβ (APP) and lack α7 nAChRs (A7KO). The A7KO-APP genotype was generated by first crossing Tg2576 (50% C57Bl6, 50% SJL) with mice heterozygous for the null mutation in α7 (congenic C57Bl6, n=10), then crossing the appropriate heterozygous F1 mice to each other. Genotype of each animal was determined by PCR of genomic DNA isolated from tail biopsy material obtained at weaning (Orr-Urtreger et al., 1995; Hsiao et al., 1996; Orr-Urtreger et al., 1997). The nomenclature for the mouse models generated in this study is designated WT (wild-type), A7KO, APP, and A7KO-APP.
Behavioral analysis was performed on 5-month-old littermate mice: WT n=15; A7KO n=6; APP n=15; APP-A7KO n=9. 3-month-old mice: WT n= 4; A7KO-APP n=3. For biochemistry, animals 5- or 12-month-old were analyzed; 3-6 animals of each genotype were used. For histological and stereological analyses, both 3-5 and 16-19 -month-old mice were analyzed; 6 animals of each genotype were used.
Mice were housed 3-5 per cage in a room with a 12-hour light/dark cycle (lights on at 06:00) with access to food and water ad libidum. Male and female mice were used, all bred in-house. he University of Texas Medical Branch operates to comply with the USDA Animal Welfare Act (Public Law 89-544) as amended by PL91-579 (1970) PL94-279 (1976) and 45 CRF37618 (6-30-80); Health Research Extension Act of 1985 (Public Law 99-158); follows the Public Health Service Policy on Humane Care and Use of Laboratory Animals; and the Guide for Care and Use of Laboratory Animals DHEW (NIH) 85-23 revised 1985.
Animals were evaluated for general health, sensory perceptiveness, activity level, as well as object recognition and associative learning performance at 5-months-of-age according to our previously published methods (Dineley et al. 2002b; Dineley et al. 2007; Taglialatela et al., 2009) with some modifications as described. The experimenter was blind to the genotype of the subjects. See Supplemental Material for detailed methods of additional behavioral testing.
Each mouse was habituated to an empty novel object recognition open field box for two 10-minute test sessions 24 hours apart. Twenty-four hours after the last habituation session, mice were subjected to training in a 10minute session of exposure to two identical, non-toxic objects (metal or hard plastic items) in the open field box. The time spent exploring each object was recorded using ObjectScan (Clever Sys. Inc.); an area 2 cm2 surrounding the object is defined such that nose entries within 2-cm of the object was recorded as time exploring the object. After the training session, the animal was returned to its home cage. After a retention interval of 24 hours, the animal was returned to the arena in which two objects, one identical to the familiar object but previously unused (to prevent olfactory cues and prevent the necessity to wash objects during experimentation) and one novel object. The animal was allowed to explore for 10 minutes, during which the amount of time exploring each object was recorded. Objects were randomized and counterbalanced across animals. Animals that spent <7 seconds exploring the objects during the 10-minute test session were omitted from analysis (de Bruin and Pouzet, 2006). Objects and arenas were thoroughly cleaned with 70% isopropanol between trials.
The two-pairing paradigm for fear conditioning training consists of placing the animal in the fear conditioning apparatus (Med Associates) for a total of 7 minutes. Animals were left free to explore for 3 minutes, and then a 30-second acoustic-conditioned stimulus (CS; white noise, 80 dB) was delivered. At the end of the CS, a 2-second shock unconditioned stimulus (US; 1.0 mA) was applied to the grid floor. The CS-US pairing was delivered again at the 5-minute mark. Fear learning testing was performed 24 hours post-training. To evaluate contextual fear learning, the animals were returned to the training context, and freezing behavior was scored for 5 minutes. To evaluate cued fear learning, the animals were placed in a different context (novel odor, lighting, cage floor, and visual cues) at least 4 hours following contextual testing. Baseline behavior was scored for 3 minutes, and then the CS was presented for a period of 3 minutes. Freezing behavior was scored with Actimetrics FreezeFrame software captured with real time digital video. Data are expressed as percent freezing in each 60-second epoch or a mean of these 5 epochs.
Upon completion of behavioral analyses, mice were euthanized by decapitation or transcardial perfusion under anesthesia. Brains were removed, immediately placed into ice-cold 0.9% NaCl then stored at −70°C. For transcardial perfusion, mice were anesthetized with avertin and transcardially perfused with phosphate-buffered saline (PBS) (pH 7.4), followed by 4% (w/v) paraformaldehyde fixative in PBS (pH 7.4). Whole brains were removed and post-fixed overnight at 4°C. For young mice (3-5 months), whole brains were stored in 70% ethanol, paraffin-embedded and horizontally sectioned at 10 microns. For aged (16+ months-old) mice, whole brains were sucrose-protected, cut into 1 mm blocks and coronally-sectioned from the fornix through the entire hippocampus at 25 microns. Sections were deparaffinized (if needed) and rehydrated, then stained with 0.5% cresyl violet or immunostained with anti-neuronal nuclear antigen (NeuN); in the case of aged samples, sections were also stained with 0.2% Congo Red (Paxinos and Watson, 2003; Wilcock et al., 2006). For anti-NeuN immunostaining, all sections underwent heated antigen retrieval with 0.01M sodium citrate (pH 6.0), blocking with 5% (v/v) normal goat serum in PBS and overnight incubation with anti-NeuN (1:2000, Millipore# MAB377). Anti-NeuN was detected using the ABC method (Universal ABC kit, Vector Laboratories) and streptavidin-Cy3 (1:400, Jackson Immunoresearch #016-160-084).
Within the hippocampal formation, the counting of pyramidal neurons and granule cells was performed in the pyramidal cell layer of the CA1 and CA3 subfields and granule cell layer of the dentate gyrus (DG). The hippocampal subdivisions and layer boundaries were delineated in cresyl violet-stained sections (see supplementary material Figure 1 for illustrated technique). A semiautomatic stereology system was used by a trained individual blind to the genotype of each mouse to make unbiased estimates of layer-specific neuronal numbers (i.e. cells with visible nuclei/nucleolus) and volumes on cresyl violet-stained sections. The optical fractionator technique was utilized to estimate neuronal numbers and the Cavalieri principal to determine hippocampal subfield layer volumes as described by Bonthius et al. (Bonthius et al., 2004).
For both the pyramidal and granule cell layers, the section sampling fraction (ssf) was 1/15, the area sampling fraction (asf) was 0.01 microns and the thickness sampling fraction (tsf) was 0.7 microns. The total number of neurons (N) in each layer brain region was estimated as N =(ΣQ-)(1 /tsf)(1/asf)(1/ssf). The determination of whole hippocampus and layer volumes was performed using point counting (with a 50 micron × 50 micron superimposed grid) and the Cavalieri principle. Starting at the dorsal surface, both neuron counting and volume-related measurements were performed at the same time in serial order. For all neuronal counting and volumetric analyses, a 10x objective was utilized to define the boundaries of the hippocampal subfield layers. Using a 100x oil-immersion objective, cresyl violet staining was utilized to localize cell nuclei, then neurons based on cell morphology; only nuclei within clear focus of the dissector probe were counted.
Sixteen to 19 -month-old APP and A7KO-APP mice were processed for histochemistry as described. Whole hippocampus and septum boundaries were outlined, electronically measured, and recorded using StereoInvestigator 8.0 software (MicroBrightField. City, VT, USA) on 5 randomly-selected serial sections (spaced 140 microns apart). Using the Cavalieri principle to determine reference volumes (Bonthius et al., 2004) a 25 micron by 25 micron grid was superimposed on each section and the total number of points was recorded for each of these sections that contained the hippocampus and intersected with Congo Red staining. The mean percentage of amyloid plaque load was determined by dividing the total volume of Congo Red staining by the total volume of hippocampus (x 100%).
Frozen brain tissue was homogenized then sonicated in detergent-free RIPA-based buffer plus protease and phosphatase inhibitors followed by centrifugation at 100,000 × g for 1 hour at 4°C. The supernatant was removed, and the pellet was sonicated in RIPA buffer containing protease and phosphatase inhibitors, 0.5% Triton X-100, 0.5% deoxycholoate, 0.1% SDS
For detection of oligomers by immunoblot, see Kayed et al. (Kayed et al., 2007) for more detail. Briefly, frozen hippocampi were homogenized in 100 μL PBS containing protease and phosphatase inhibitors, centrifuged at 10,000 rpm for 5 minutes to pellet insoluble material. Supernatant was collected.
For the detection of all other proteins, see Dineley et al. (Dineley et al., 2001) for more detail. Briefly, frozen tissue was sonicated in a HEPES-based buffer containing protease and phosphatase inhibitors. Protein concentrations were determined with the Pierce Microplate BCA Protein Assay Kit (Thermoscientific). Samples were mixed with sample buffer, resolved by SDS-PAGE, transferred to PVDF, and then probed with the appropriate primary and secondary antibodies. Antibodies were visualized using chemiluminescence (ECL) and densitometric analysis was performed using the AlphaEaseFC software (Alpha Innotech) from film exposures in the linear range for each antibody and normalized to control (WT littermate) samples. Normalized control values were determined from each film by averaging control values, dividing each control and transgenic sample density by the average of the control set, and then determining the average and SEM for control and transgenic samples. By loading a fixed, equal amount of protein, several experiments may be compared to each other. Loading equivalency is verified by probing for an abundant ubiquitous protein such as actin.
ChAT activity assays were performed according to the Fonnum method (Fonnum, 1975). Briefly, brain tissue was sonicated in homogenization buffer (0.5% Triton X-100, 10 mM EDTA pH7.4, plus protease inhibitors). ChAT activity was measured by incubating samples with 14C-acetylCoA in the presence of excess cold substrate and AChE inhibitor for 30 minutes at 37°C. The reaction was stopped with the addition of ice-cold H2O. 14C labeled product was separated from the reaction mixture by organic extraction and counted with liquid scintillant for one minute. Blank samples did not contain any tissue. Olfactory bulb homogenate served as positive control.
ChAT activity was calculated as follows:
Signal Select (BioSource) according to manufacturer's instructions. Aβ or APP content was determined by comparison of unknown sample intensity to a standard curve.
Data were analyzed using GraphPad Prism software.
For novel object recognition tests, the time exploring the novel object (familiar versus novel) is reported as the recognition index and is calculated using the following formula:
Statistical analysis of NOR data is done by first performing a one-sample t-test to determine if the mean percent exploration time is significantly different from a theoretical mean of 0.5000. This is followed by one-way ANOVA to determine group differences.
Statistical analysis of biochemistry and behavior data was done by one-way ANOVA, followed by Tukey or Neuman-Keuls post hoc tests. For paired analyses, two-way Student's t-test was performed. p≤0.05 is considered significant. Graphs were plotted with mean and standard error of the mean (SEM).
We first determined the behavioral and cognitive effects of deleting the α7 nAChR gene product in APP mice. We considered this especially important to pursue because α7 nAChRs have been implicated in contradictory roles in the etiology of AD: activation of α7 nAChRs by conventional nicotinic ligands has been shown to be protective against Aβ toxicity, however, α7 nAChR − Aβ interaction has also been suspected in mediating Aβ toxicity (Dineley, 2007; Ren et al., 2007; Clifford et al., 2008; Soderman et al., 2008; Wang et al., 2009). Understanding which phenotype predominates during early-stage cognitive decline will provide great insight into the etiology of AD. Towards this end, A7KO, APP, A7KO-APP, and WT littermates were subjected to general health and behavior assessment followed by an evaluation of associative learning performance at 5-months-of-age; the age at which cognitive deficits are first apparent (Dineley et al., 2002b; Dineley et al., 2007; Taglialatela et al., 2009). Our standard behavioral screen included, in addition to assessment of such parameters as weight, temperature, general reflexes, etc., evaluation of: open-field behavior, rotating rod testing, pre-pulse inhibition, object recognition, and cued and contextual Pavlovian fear conditioning. This battery of tests allows evaluation of a variety of sensory responses, including hearing and vision, general activity, reflexes, motor coordination, motor learning, and associative learning.
We did not detect statistically significant group differences by genotype regarding weight, temperature, general reflexes, or sensory responses (data not shown). We also did not observe any group differences in the acquisition or performance of the rotating rod test or in pre-pulse inhibition indicating that motor learning, coordination and sensorimotor gating of the startle reflex are normal (supplementary material, Figure 2-rotarod and Figure 3-prepulse inhibition). These results are consistent with previous studies on APP and A7KO mice (Paylor et al., 1998; Dineley et al., 2002b; Dineley et al., 2007).
The time spent exploring a novel object versus a familiar object is a measure of attention and non-spatial declarative memory (Broadbent et al., 2004). We have previously demonstrated that 5-month-old APP mice (Tg2576) are impaired in novel object recognition (NOR) when the retention interval is manipulated to model intermediate and long-term memory (Taglialatela et al., 2009). The retention interval is the amount of time the animals must retain the memory of the identical 3D objects presented during the training session prior to the testing session when one of the familiar objects is replaced with a novel one. NOR training was performed on 5-month-old WT, A7KO, APP, and A7KO-APP mice (Figure 1). Utilizing a 24-hour retention interval to model long term memory, WT and A7KO scored recognition indices during the testing phase that were statistically higher than a theoretical mean of 0.5 to represent a ‘chance’ recognition index. However, neither APP nor A7KO-APP mice exhibited any preference for object exploration. One-way ANOVA analysis found that APP and A7KO-APP performance was statistically different from WT littermates (F(3,72)=11.13) but not each other. Therefore, when a 24-hour retention interval is utilized to model long-term memory, APP and A7KO-APP mice are impaired in object recognition. Since NOR cannot distinguish degrees of cognitive impairment, our next experiments were designed to address whether A7KO-APP have a more severe cognitive deficit than APP.
Next, 5-month-old WT, A7KO, APP, and A7KO-APP were subjected to 2- and 5-pair training for fear conditioning (Figure 2a). As was previously measured in Tg2576 mice, freezing to the context 24 hours following 2-pair training for fear conditioning was impaired in the APP mice (Figure 2b; Corcoran et al., 2002; Dineley et al., 2002b; Comery et al., 2005; Dineley et al., 2007). This deficit was statistically significant in comparison to both WT and A7KO littermates. Likewise, as was previously reported by Paylor et al. (Paylor et al., 1998), α7 null mice did not exhibit any deficits in contextual learning. All groups of mice froze to a similar extent in the cued test for fear learning as previously reported for A7KO and APP mice (supplementary material Figure 4; Paylor et al., 1998; Dineley et al., 2002b; Dineley et al., 2007). In summary, A7KO-APP mice exhibited a contextual fear conditioning deficit that was of similar magnitude to the APP littermates. Hence, following 2-pair training for fear conditioning, APP and A7KO-APP mice are impaired in contextual but not cued memory. Cued conditioning is an index of associative learning that is amygdala-dependent and hippocampus-independent. Thus, impairment of contextual but not cued fear learning indicates that a selective hippocampus deficit underlies this phenotype.
We have previously reported that the contextual fear learning deficit exhibited by 5-month-old Tg2576 mice following 2-pair training is rescued if the animals are put through a more rigorous 5-pair training paradigm (Dineley et al., 2002b). This was also the case for the APP mice evaluated in this study. One-week after 2-pair training, each group of mice was subjected to a 5-pair training paradigm. As was the case in our previous study, APP froze in contextual testing to a similar extent as WT littermates (Figure 2c). As expected, A7KO also froze to the same extent as WT and APP mice (Figure 2c). In contrast, A7KO-APP mice froze significantly less than all other groups of mice in the contextual test for fear learning (Figure 2c). All genotypic groups of mice froze to a similar extent in the cued test (data not shown). Statistical analysis of total freezing behavior in the contextual test following 2- and 5-pairing training shows that APP mice significantly improved in the contextual test following 5-pair training whereas APP-A7KO mice did not (Figure 2d,e,f).
In order to verify that the fear conditioning deficit in A7KO-APP mice is an age-dependent and not purely genetic phenomenon, we put 3 month old A7KO-APP and WT control littermates through the 2-pair fear conditioning paradigm and both groups froze to the same extent in the contextual and cued (supplementary material Figure 5). Therefore, the deficit detected in these animals at 5-months-of-age represents an age-dependent decline in hippocampus-dependent cognitive function. Furthermore, loss of α7 nAChRs appears to accelerate the cognitive decline in APP mice since, at 5-months-of-age, APP fear conditioning deficit is rescued with a more rigorous training paradigm and A7KO-APP is not.
Hyperactivity or an inability to perceive the shock stimulus might confound our results, especially if hyperactivity results in an inability to freeze; the fact that these animals freeze in the cued test argues against this but we also evaluated these mice for spontaneous activity in the open field test. We also measured their threshold to flinch, jump, and vocalize to increasing electrical shock intensities as an index of their sensitivity to the shock stimulus and found no differences. There also were no statistically significant differences by genotype in the distance traveled, rearing, center to distance ratio, or time spent in the center of the open field box indicating that the failure of APP and A7KO-APP mice to freeze in the contextual test is not due to hyperactivity or hypo-anxiety (supplementary material Figure 6). In addition, these animals exhibited similar thresholds to flinch, jump, and vocalize to increasing shock intensities as the other genotypes in this study suggesting normal sensitivity to foot shock (supplementary material Figure 6).
Quantification of Aβ in the hippocampus of young and aged APP and A7KO-APP mice was measured. Brain tissue was harvested from mice 5- and 12-months-of-age then subjected to quantitative Aβ ELISA specific for human Aβ1-40 and 1-42. Five-months-old A7KO-APP mice had an approximately 50% reduction in hippocampal Aβ1-40 load compared to APP (Figure 3a), but Aβ1-42 was not significantly altered. By 12-months-of-age, both Aβ1-40 and Aβ1-42 levels in A7KO-APP hippocampus were about 70% lower than that measured in APP (Figure 3b). Thus, A7KO-APP mice have a selective reduction in hippocampal Aβ compared to APP. Although significantly lower than APP, the A7KO-APP Aβ load is still abnormally high. At 5-months-of-age, A7KO-APP hippocampus contains ~60 pmoles Aβ/g; this is about 6-fold higher than reported for wildtype mice (Kawarabayashi et al., 2001).
Further analysis of these results by way of calculating the ratio of Aβ42:Aβ40 demonstrates that although total Aβ is lower in A7KO-APP hippocampus, the relative proportion of the more toxic peptide, Aβ1–42, is significantly increased (Figure 3c). This raises the possibility that the more severely compromised cognitive status of A7KO-APP mice compared to APP may result from selective enrichment for Aβ1-42.
Brain sections from APP and A7KO-APP animals 16-19 months-of-age were processed for amyloid staining with Congo Red then assessed for percent area of the hippocampus occupied by plaques. Consistent with an overall reduction in Aβ level in A7KO-APP hippocampus, plaque load in these animals was significantly lower than APP. Percent area values were 0.74% ± 0.12 and 0.53% ± 0.06, for APP and A7KO-APP, respectively (Figure 3d).
Utilizing a combination of antibodies directed against different epitopes of the amyloid precursor protein and probing homogenates fractionated into membrane-associated and soluble material we evaluated amyloid precursor protein processing in APP and A7KO-APP hippocampus (Figure 4a). Using 6E10 antibody that recognizes the N-terminal region of human Aβ to probe membrane preparations indicates that transgene expression is unchanged (Figure 4b); however C-terminal fragments (CTFs) generated from β-secretase cleavage of the holoprotein (CTF-β) are reduced in A7KO-APP samples compared to APP (Figure 4b). Soluble hippocampal material probed with 6E10 antibody indicated that cleavage of the amyloid precursor protein by α-secretase (sAPP-α) is enhanced (Figure 4b). We verified enhanced α-secretase processing of the holoprotein by probing membrane proteins from APP and A7KO-APP hippocampus with an antibody directed against the C-terminal portion of the 695 splice-variant of the amyloid precursor protein. Membrane-associated CTFs generated through α-secretase cleavage of the APP were increased and those generated through β-secretase cleavage were reduced in A7KO-APP hippocampus compared to APP (Figure 4c).
We verified that amyloid precursor protein expression was unchanged by evaluating extracts of hippocampus from 5-month-old APP and A7KO-APP mice. Amyloid precursor protein was detected in membrane preparations by sandwich ELISA that selectively detects the human holoprotein or quantitative immunoblotting using antibody 22C11 that will detect both mouse and human amyloid precursor protein. With both methods, we found no significant change in amyloid precursor protein level between 5-month-old APP and A7KO-APP in hippocampus (Figure 4d). Considered together, these results indicate that the amyloid precursor protein in A7KO-APP hippocampus is preferentially processed toward the α-secretase pathway and suggests that this mechanism underlies the reduced Aβ load in the hippocampus of these animals. However, it does not account for the shift toward a higher proportion of Aβ1-42 compared to Aβ1-40. We next determined whether the shift toward Aβ1–42 in A7KO-APP hippocampus resulted in alterations in oligomeric assemblies of Aβ peptides.
Many lines of evidence suggest a role for soluble oligomeric Aβ species in the pathology of AD (Walsh et al., 2002; Lesne et al., 2006; Kayed et al., 2007; Lacor et al., 2007; Venkitaramani et al., 2007; Glabe, 2008; Poling et al., 2008). Previous reports have identified at least two conformationally distinct types of soluble Aβ oligomers: prefibrillar oligomers and fibrillar oligomers (Kayed et al., 2007; Glabe, 2008). The fibrillar oligomers, as opposed to the prefibrillar oligomers, have recently been shown to correlate with cognitive decline (MMSE scores) and the neuropathological hallmarks of AD (Tomic et al., 2009).
Soluble oligomeric assemblies of Aβ were analyzed in hippocampal tissue from WT, A7KO, APP, and A7KO-APP animals using immunoblot with OC antibody, selective for fibrillar oligomeric amyloid structures (Kayed et al., 2007; Glabe, 2008). Samples from 5-month-old animals revealed that A7KO-APP hippocampus exhibits two species of fibrillar oligomeric (OC-positive) assemblies with approximate molecular weights of 27 kD and 56 kD (Figure 4e). These soluble oligomeric species were essentially absent from WT, A7KO, and APP hippocampus under identical exposure conditions. However, upon over-exposure, the 27kD band, but not 56 kD band, was present in APP hippocampal samples. Therefore, only 5-month-old A7KO-APP hippocampi contain a soluble, fibrillar, oligomeric Aβ dodecamer at this age. The 56 kD soluble oligomer does not appear until 6-months-of-age in Tg2576 brain, suggesting that loss of α7 nAChRs both influences APP processing and Aβ oligomer accumulation in this mouse model (Lesne et al., 2006).
The observation that 5-month-old A7KO-APP mice are more severely cognitively impaired than APP littermates, concomitant with enhanced accumulation of soluble Aβ oligomers, prompted us to consider the possibility that this would be accompanied by signs of neurodegeneration. As such, we performed quantitative stereology and biochemical assays in order to evaluate neuron number, somato-dendritic and pre-synaptic integrity in the hippocampus of 5-month-old WT, A7KO, APP, and A7KO-APP littermates.
We found initial evidence of hippocampal neurodegeneration when we evaluated the levels of the presynaptic marker synaptophysin and the somato-dendritic marker with quantitative immunoblot. Previous studies on APP mice have failed to detect robust deficits in synaptophysin at any age and MAP2 decline has only been reported for aged APP animals (9-24 -month-old; Savage et al., 2002; Fonseca et al., 2004). These and other studies suggest that postsynaptic structures are more vulnerable than presynaptic ones in APP mice (Spires et al., 2005). Consistent with this, we found no alteration of hippocampal synaptophysin in any of our four genotypic groups of mice (p=0.26; one-way ANOVA F(3,22)=1.46; data not shown). On the other hand, MAP2 levels in 5-month-old A7KO and A7KO-APP hippocampus were significantly lower compared to WT and APP littermates indicating that post-synaptic integrity may be affected by loss of α7 nAChRs (Figure 5). However, since MAP2 is not specifically localized to dendritic structures, the role of α7 nAChRs in post-synaptic integrity and whether this is accompanied by structural changes in synapses such as dendritic spine loss cannot be addressed with the current methodology.
Next we used quantitative stereological techniques on cresyl violet-stained sections to estimate layer-specific alterations in neuronal number and volume in the hippocampal CA1 and CA3 pyramidal layers and DG granule cell layer of littermate WT, A7KO, APP, and A7KO-APP mice. An example of our cresyl violet staining results compared to NeuN immunoreactivity are shown (Figure 6) and an illustration of the CA1 and CA3 pyramidal neuron layer boundaries as well as DG granule cell layer boundary used in this study can be found in supplemental material. We discovered alterations in the hippocampal formation of both APP and A7KO-APP mice, yet A7KO-APP were more severely affected (Table I). A7KO-APP hippocampus showed significant reduction in neuron number and volumes in a region and layer specific manner. For example, compared to WT, A7KO, and APP littermates, A7KO-APP showed significant decrease in: 1) DG granule cell layer volume, 2) CA3 pyramidal neuron number, 3) CA3 pyramidal neuron layer volume (see *, Table 1). The APP group also exhibited shrinkage of DG granule cell region volume and loss of pyramidal neurons from CA1 (see *, Table 1). Loss of CA1 pyramidal neurons without a corresponding loss of CA1 volume suggests that this reduction was not sufficient to influence volume. Since there was not a corresponding loss of granule cells from DG in APP or A7KO-APP hippocampus, this suggests that the volume shrinkage observed is possibly due to loss of somatic volume; an idea consistent with the observed reduction in MAP2 with quantitative immunoblot. However, A7KO-APP CA3 pyramidal neuron layer exhibited significant loss of both pyramidal neuron number and layer volume. Future studies will evaluate additional cell populations in hippocampus that are enriched for α7 nAChRs such as interneurons (e.g., GAD67-positive), and glia (e.g., GFAP- and S100β- positive) (Teaktong et al., 2003; Khiroug et al., 2003).
Similar volumetric measurements in the septum found no significant volume difference between the four groups of mice (p=0.98, one-way ANOVA, F(3,20)=0.07; data not shown). These results suggest that neuron and volume loss in A7KO-APP occurs first in the hippocampus, a cholinergic target region, rather than in the source nuclei, and that the role of α7 nAChRs in maintaining hippocampal integrity is evident at a young age (prior to the onset of plaques). Our next step was to evaluate if α7 nAChRs are also important in the maintenance of septo-hippocampal cholinergic integrity.
We next evaluated the status of the presynaptically-located cholinergic marker, ChAT, in the hippocampus of 5-month-old WT, A7KO, APP and A7KO-APP mice. Quantitative immunoblot for hippocampal ChAT protein revealed a 50% reduction in A7KO-APP (Figure 7a). Reduced ChAT protein in A7KO-APP hippocampus was paralleled by a similar reduction in ChAT enzyme activity (Figure 7b). The protein signal for the vesicular acetylcholine transporter (VAChT) was also assessed in A7KO-APP samples, but this signal did not reach statistical significance (p=0.41, one-way ANOVA F(3,17)=1.02; data not shown). Thus, loss of pre- and post-synaptic proteins as well as loss of neurons/volume from hippocampus of A7KO-APP mice reveals more severe neurodegeneration in A7KO-APP hippocampus compared to APP, A7KO, and WT groups. These observations indicate that in the absence of α7 nAChRs and in the presence of excess Aβ, hippocampal cholinergic function and neuron integrity is compromised.
The major source of cholinergic input to the hippocampus and neocortex is from the basal forebrain, including the medial septum, diagonal band nuclei, and basal nucleus. We therefore investigated the relative level of ChAT in this region of WT, A7KO, APP, and A7KO-APP mice. Immunoblot quantification suggested a trend for decreased ChAT protein in the basal forebrain region of A7KO and A7KO-APP mice was evident (Figure 7c). When ChAT activity was assayed, we found a statistically significant reduction in enzyme activity in both A7KO and A7KO-APP basal forebrain samples (Figure 7d). This suggests that, in vivo and regardless of Aβ load, α7 nAChRs are important for maintaining cholinergic function in the basal forebrain.
We tested whether the loss of hippocampal and basal forebrain ChAT activity in 5-month-old A7KO-APP animals is an age-dependent phenomenon by evaluating ChAT activity in 3 -month-old WT, A7KO, APP and A7KO-APP mice. At this age, all genotypic groups exhibited statistically equivalent ChAT activity in hippocampus and basal forebrain (supplementary material Figure 7), however, ChAT activity in genotypes lacking the α7 nAChR showed a definite trend for up-regulation. This observation is similar to those made in human post mortem brain in which ChAT activity is up-regulated in samples from patients with early AD (DeKosky et al., 2002; Ikonomovic et al., 2003; Counts et al., 2007). This suggests that the loss of ChAT protein and activity is both age- and α7 nAChR-dependent.
In addition, to its well-known role in neuroprotection, the ERK MAPK cascade is important for certain hippocampus-dependent learning and memory tasks including contextual fear conditioning (Atkins et al., 1998). We have previously shown that soluble oligomeric Aβ, comprised mainly of trimers and hexamers, activates ERK MAPK via α7 nAChRs (Dineley et al., 2001; Bell et al., 2004). Given the enhancement of hexameric (and higher) Aβ assemblies in the A7KO-APP brain, we wanted to assess the status of ERK protein and its activation state in A7KO-APP hippocampus. Using quantitative immunoblot technique, we probed for total ERK MAPK as well as the phosphorylated, and therefore activated, form of ERK MAPK (phospho-ERK). While total ERK MAPK was unaltered, we found a significant reduction in phospho-ERK in the A7KO-APP samples (Figure 8). There were no alterations in the other groups of mice. The fact that A7KO hippocampal ERK MAPK activity was unaffected suggests that under normo-Aβ conditions, baseline ERK MAPK activity is not primarily dependent upon functional α7 nAChRs. APP ERK MAPK was also unaffected; we have previously reported that hippocampal ERK MAPK activity is elevated in dentate gyrus, but not CA1, in similarly aged APP (Dineley et al., 2001). The fact that we analyzed the entire hippocampus in this experiment may have masked regional differences detected earlier. Nonetheless, these observations show that a known downstream target of hippocampal α7 nAChR activation, ERK MAPK, is negatively affected when this receptor is absent.
Experimental destruction of septo-hippocampal cholinergic neurons universally results in reduced cholinergic markers in hippocampus. Most notably this is expressed as reduced ChAT activity within hippocampus (Kuo et al., 1996; Tapiola et al., 2000; Andreasen et al., 2003) Consistent with lesion studies and observations in AD brain, compromise of basal forebrain cholinergic function leads to similar concessions within the hippocampus of 5-month-old A7KO-APP mice supporting the notion that compromise of basal forebrain cholinergic neurons is the underlying mechanism for ChAT reduction in A7KO-APP hippocampus. However, the fact that A7KO mice exhibit loss of ChAT activity in basal forebrain with no effect on hippocampal ChAT indicates that the added stress of excess Aβ in conjunction with loss of α7 nAChRs is necessary to induce such changes in 5-month-old animals.
Previous work on an APP mouse model found impaired cholinergic neurotransmission in the cortex and hippocampus but not the basal forebrain of 19-21 -month-old animals (Apelt et al., 2002; Luth et al., 2003). Thus, the A7KO-APP mouse model for AD is the first to exhibit significant cholinergic hypofunction within the basal forebrain and a critical cholinergic projection area: the hippocampus.
We found that hippocampus-dependent memory deficits previously detected in APP mice are more severe when α7 nAChRs are absent. A7KO mice are cognitively normal, thus cognitive deficits in A7KO-APP cannot be directly attributed to loss of α7 nAChRs (Paylor et al., 1998). In contrast, hippocampal ACh neurotransmission is an essential component during the acquisition and retrieval phases of context-dependent learning such as fear conditioning and NOR (Scali et al., 1997; Nail-Boucherie et al., 2000; Sanders et al., 2003; Rogers and Kesner, 2004; Winters et al., 2008). Therefore, the combined loss of cholinergic activity and hippocampal integrity likely contributes to the accelerated decline of cognitive function in 5-month-old A7KO-APP mice compared to their APP littermates.
Similar to the human disease, the underlying mechanisms for cognitive impairment in older APP animals are likely distinct from those underlying cognitive deficits in 5-month old animals. In fact, α7 nAChRs may perform a deleterious role in later stage AD as exemplified by 13-22 month-old PDAPP in which α7 nAChR deletion improved cognitive performance (Dziewczapolski et al., 2009). Such an antithetical outcome may reflect a dynamic relationship between α7 nAChRs and age-dependent Aβ accumulation and aggregation, discussed below.
Although hippocampal Aβ load was significantly reduced compared to APP mice, the amount of Aβ in the hippocampus of A7KO-APP mice is still abnormally high when compared to wild-type values (Kawarabayashi et al., 2001). Reductions in Aβ load in A7KO-APP hippocampus were not due to altered expression of the human APPSWE transgene suggesting that reduction in A7KO-APP Aβ load is likely the result of enhanced α-secretase processing of the APP holoprotein. This latter fact was demonstrated with immunoblot analyses of membrane-associated and soluble APP metabolites; reductions in hippocampal CTF-β were concomitant with elevations of CTF-α and sAPP-α.
Although total Aβ is reduced in A7KO-APP hippocampus, the Aβ species considered to be more prone to aggregation, and therefore more toxic, Aβ1–42 is enriched along with the enhanced accumulation of soluble fibrillar oligomers. Soluble, fibrillar, oligomeric Aβ has recently been implicated in correlating most strongly with cognitive status in MCI and early AD (Tomic et al., 2009). These observations on Aβ species and assemblies in A7KO-APP hippocampus may be related to effects on hippocampal Aβ accumulation in the absence of α7 nAChRs. In addition, α7 nAChR influence on APP processing and Aβ accumulation may also depend on the APP sequence itself since Aβ accumulation in the PDAPP mouse model expressing a different APP mutant (containing both the ‘Swedish’ and ‘Indiana’ mutations) did not exhibit any differences in APP processing or Aβ levels (Dziewczapolski et al., 2009). Nonetheless, greater understanding of the relationship between α7 nAChRs, APP processing, and Aβ accumulation in the hippocampus may allow positive modulation of these processes and lead to useful therapeutic strategies in early AD.
Several studies have demonstrated that nAChR activation attenuates Aβ toxicity and promotes the integrity of the cholinergic phenotype; protection from Aβ toxicity is proportional to the number of α7 nAChRs expressed by cells (Jonnala and Buccafusco, 2001) (Kihara et al., 1997). APP mice continue to up-regulate α7 nAChRs as Aβ accumulates, possibly why this AD model does not exhibit significant loss of cholinergic function and neurodegeneration (Dineley et al., 2001; Dineley et al., 2002b). We have previously shown that pM to nM Aβ42 oligomer preparations activate α7 nAChRs, leading to Ca2+ influx and ERK MAPK activation, a second messenger system not only important for learning and memory but also important for resistance to Aβ toxicity (Dineley et al., 2001; Dineley et al., 2002a; Bell et al., 2004). Thus the observed loss of basal ERK activity in A7KO-APP hippocampus may contribute to one or all of the following observations in these mice: learning and memory deficits, loss of cholinergic functionality, and hippocampal neurodegeneration (Sweatt, 2001; Thatcher et al., 2006; Williams et al., 2007).
We propose a model for early AD in which α7 nAChRs are neuroprotective through a two-pronged mechanism (Figure 9). The first prong of our model proposes that oligomeric Aβ is capable of transiently activating α7 nAChRs with subsequent activation of its downstream messengers that provide a crucial neurotrophic signal for the maintenance of septo-hippocampal integrity (Williams et al., 2007). The second prong of our model posits that α7 nAChRs also counterbalance the toxic effects of Aβ oligomer accumulation. This is based on our observation that hexameric and dodecameric Aβ assemblies are enhanced in A7KO-APP hippocampus. Aβ-α7 nAChR interaction may sequester Aβ during early AD as well as retard the progression of oligomerization. Thus, in early AD, α7 nAChRs serve a neuroprotective role both through receptor activation as well as through harboring oligomeric Aβ away from additional toxic interactions (Walsh et al., 2002; Walsh et al., 2005).
As AD progresses and Aβ oligomers accumulate, we envision that Aβ-α7 nAChR interaction eventually leads to functional blockade of α7 nAChRs through essentially irreversible association with Aβ. In fact, α7 nAChR-selective agonists are unable to activate these receptors in APP transgenic mice and recent work on human AD postmortem tissue and indicate that much of the receptor protein is functionally inactivated due to association with Aβ peptide (Ren et al., 2007; Soderman et al., 2008; Wang et al., 2009); (Perry et al., 1978; Banerjee et al., 2000; Martin-Ruiz et al., 2000; Dineley et al., 2001; Dineley et al., 2002b; Teaktong et al., 2003; Counts et al., 2007; Ikonomovic et al., 2009; Yu et al., 2009). In contrast to its role in early AD, α7 nAChRs may actually contribute to the pathology of late-stage AD through deleterious effects on synaptic integrity, cholinergic functionality, and cognition. In support of this notion is a recent study in which deletion of α7 nAChRs from the PDAPP mouse model, led to an improvement in cognition in aged (13-22 months) animals (Dziewczapolski et al., 2009).
In summary, this study has shown that at an age when APP mice first exhibit cognitive deficits, A7KO-APP mice exhibit more pronounced AD-like pathology: enhanced accumulation of soluble oligomeric Aβ, more severe cognitive impairment, loss of hippocampal markers, loss of cholinergic functionality, and loss of hippocampal neuron number and volume. The results presented here suggest that the hippocampus is particularly vulnerable to the absence of α7 nAChRs during early-stage cognitive decline induced by elevated Aβ and other APP metabolites.
Future work on this animal model will strive to reconcile: 1) the influence of neuronal versus glial cell populations on the A7KO-APP phenotype since α7 nAChRs are expressed by both these cell types (Teaktong et al., 2003); 2) how loss of α7 nAChRs on interneurons contributes to hippocampal dysfunction in A7KO-APP mice since α7 nAChRs are particularly enriched on this hippocampal neuron population (Khiroug et al., 2003); 3) whether presynaptic versus somato-dendritic α7 nAChRs differentially contribute to neuroprotection or neurotoxicity since there is evidence that Aβ activates presynaptic α7 nAChRs whereas much evidence suggests that Aβ blocks somato-dendritic α7 nAChRs (Liu et al., 2001; Pettit et al., 2001; Dougherty et al., 2003). Perhaps, the neuroprotective role for α7 nAChRs resides largely within the somato-dendritic receptors, whereas the toxicity of Aβ may be primarily through activation of those residing on pre-synaptic boutons. These last investigations must be accompanied by higher resolution quantitative assessments of synaptic integrity, e.g., spine density, in order to further the initial evaluation of hippocampal structural integrity performed herein.
The authors wish to thank Duy Bui, Angela Chang, Dale Hogan, Kenneth J. O'Riordan, Urmi Sengupta, and Wei Song, for their expert technical assistance. This work was supported by funds from the Alzheimer's Association, the John Sealy Memorial Endowment for Biomedical Research, and a kind gift from Jerry and Winkie Mohn to KTD; funds from the NINDS, NIMH, American Health Assistance Foundation, and Evelyn F. McKnight Foundation to JDS; RO1 NS40039 and RO1 AG20670 to HZ, and APA/NIMH to CH.