APP cognitive deficits worsen in the absence of α7 nAChRs
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.
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 (). 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.
Figure 1 Using a 24-hour retention interval, APP and A7KO-APP exhibit a novel object recognition deficit compared to WT and A7KO. WT and A7KO mice showed a preference for the novel object following a 24-hour delay between identical object exploration during training (more ...)
Next, 5-month-old WT, A7KO, APP, and A7KO-APP were subjected to 2- and 5-pair training for fear conditioning (). 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 (; 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.
Figure 2 APP performance in the contextual test for fear learning is rescued with 5-pair training, APP-A7KO is not. a, Schematic depicting regimen for training 5-month-old mice for fear conditioning either with 2-pairs or 5-pairs of CS-US. Data expressed as mean (more ...)
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 (). As expected, A7KO also froze to the same extent as WT and APP mice (). In contrast, A7KO-APP mice froze significantly less than all other groups of mice in the contextual test for fear learning (). 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 ().
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
Total Hippocampal Aβ Load is decreased yet Aβ1-42 is enriched in A7KO-APP
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 (), 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 (). 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
Figure 3 Compared to APP, A7KO-APP have higher proportion of Aβ42 in hippocampus despite reduced total amyloid load that results in reduced plaque load in A7KO-APP hippocampus. Extracts of hippocampus were subjected to Aβ sandwich ELISA, a, 5-month-old, (more ...)
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 (). 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.
Reduced hippocampal Aβ is accompanied by reduced plaque load in A7KO-APP
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 ().
APP transgene expression unchanged; CTFs indicate altered APP processing in A7KO-APP hippocampus
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 (). Using 6E10 antibody that recognizes the N-terminal region of human Aβ to probe membrane preparations indicates that transgene expression is unchanged (); however C-terminal fragments (CTFs) generated from β-secretase cleavage of the holoprotein (CTF-β) are reduced in A7KO-APP samples compared to APP (). Soluble hippocampal material probed with 6E10 antibody indicated that cleavage of the amyloid precursor protein by α-secretase (sAPP-α) is enhanced (). 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 4 Enhanced accumulation of soluble, fibrillar, oligomeric Aβ and altered APP processing in A7KO-APP hippocampus. a, Schematic depicting the APP within the cell membrane. Antibody epitopes and secretase cleavage sites are designated. b, Immunoblot (more ...)
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 (). 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.
Enhanced Aβ oligomer accumulation in A7KO-APP hippocampus
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 (). 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
Signs of hippocampal neurodegeneration in A7KO-APP mice
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 (). 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.
Figure 5 Signs of hippocampal neurodegeneration in A7KO-APP hippocampus. Quantitative analysis of MAP2 immunoblots demonstrates reduced MAP2 in 5-month-old A7KO and A7KO-APP hippocampus. Data reported as mean ± SEM band density normalized to WT values. (more ...)
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 () 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 (). 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 *, ). The APP group also exhibited shrinkage of DG granule cell region volume and loss of pyramidal neurons from CA1 (see *, ). 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
Figure 6 Histological images of the hippocampal formation from young (a1, a2) WT, (b1, b2) A7KO, (c1, c2) APP and (d1, d2) A7KO-APP mice. Sections were treated with (1) cresyl-violet (CV) or immunostained with (2) anti-NeuN. Images demonstrate that CV staining (more ...)
Quantitative stereological analyses of hippocampal neuron counts and volume for WT, A7KO, APP, A7KO-APP
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.
Loss of Cholinergic markers and functionality in A7KO-APP mice
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 (). Reduced ChAT protein in A7KO-APP hippocampus was paralleled by a similar reduction in ChAT enzyme activity (). 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.
Figure 7 5-month-old A7KO-APP mice have reduced ChAT protein and activity in hippocampus and basal forebrain. a, Quantification of band intensity following immunoblot reveals a 50% reduction in ChAT protein in the hippocampus of A7KO-APP mice. *p<0.02, (more ...)
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 (). When ChAT activity was assayed, we found a statistically significant reduction in enzyme activity in both A7KO and A7KO-APP basal forebrain samples (). 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.
Loss of ERK activity in A7KO-APP hippocampus
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 (). 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.
Figure 8 5-month-old A7KO-APP mice have reduced hippocampal ERK activity with no alteration in total ERK. Quantification of band intensity following immunoblot for a, phospho-ERK, reveals significant reduction in ERK activity in A7KO-APP hippocampus. *indicates (more ...)