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Accumulation of β-amyloid (Aβ) and loss of synapses are hallmarks of Alzheimer’s disease (AD). How synaptic activity relates to Aβ accumulation and loss of synapses is a current topic of major interest. Synaptic activation promotes Aβ secretion, and chronic reduction of synaptic activity reduced Aβ plaques in an AD transgenic mouse model. This suggested beneficial effects of reducing synaptic activity in AD. We now show that reduced synaptic activity causes detrimental effects on synapses and memory despite reducing plaques using two different models of chronic synaptic inhibition: deafferentation of the barrel cortex and administration of benzodiazepine. An interval of prolonged synaptic inhibition exacerbated loss of synaptophysin compared to synaptically more active brain in AD transgenic but not wild-type mice. Furthermore, an interval of benzodiazepine treatment, followed by a washout period, exacerbated memory impairment in AD transgenic mice. Exacerbation of synaptic and behavioral abnormalities occurred in the setting of reduced Aβ plaques but elevated intraneuronal Aβ immunoreactivity. These data support beneficial effects of synaptic activation on Aβ-related synaptic and behavioral impairment in AD.
Alzheimer’s disease (AD) is characterized by the aberrant accumulation of β-amyloid peptides (Aβ) and the progressive loss of synapses in the brain (Selkoe, 2002). Synapses are considered the earliest site of pathology, and synaptic loss is the best pathological correlate of cognitive impairment in subjects with AD (Terry et al., 1991; Gouras et al., 2010). The relation between synaptic activity and Aβ in AD pathogenesis is a topic of major interest. Synaptic activity has been shown to increase Aβ secretion in vitro and in vivo (Kamenetz et al., 2003; Cirrito et al., 2005). Since addition of extracellular Aβ alters synapses and impairs synaptic plasticity, chronic synaptic activity could therefore be detrimental. In support of this scenario, reduction of activity by induction of sleep reduced Aβ secretion and plaque burden in brains of AD mice (Kang et al., 2009). Moreover, epilepsy is associated with increased plaque pathology, and cortical hyperexcitability is seen in brains of AD mouse models (Mackenzie and Miller, 1994; Palop et al., 2006; Palop et al., 2007). On the other hand, synaptic activity is essential for synaptic plasticity and memory formation. Higher educational attainment and involvement in intellectually stimulating activities are associated with reduced risk of AD (Stern, 2006). In addition, environmental enrichment and behavioral training reduced amyloid deposition and improved memory in AD transgenic mice (Lazarov et al., 2005; Billings et al., 2007), although these behavioral manipulations are complex and are not necessarily straightforwardly related to synaptic activity. We previously provided mechanistic data demonstrating that synaptic activation reduced levels of intraneuronal Aβ and protected synapses in primary neuron models of AD (Tampellini et al., 2009). Intriguingly, these studies showed that synaptic activity promotes trafficking of the amyloid precursor protein (APP) to synapses, where Aβ is preferentially generated (Goldsbury et al., 2006; Cirrito et al., 2008; Tampellini et al., 2009). In addition, the Aβ-degrading protease neprilysin was critical for the reduction specifically of the intraneuronal pool of Aβ42 with synaptic activation. Although mechanistic biological studies showed quantitative beneficial effects of synaptic activity on synapses in AD transgenic neurons, in vivo studies to determine the role of synaptic activity on synapses and behavior are required to define the relation between synaptic activity and the pathophysiology of the disease. To investigate the role of synaptic activity on Aβ, synapses, and brain function, we chronically reduced activity in mice by two well-established experimental methods used to investigate synaptic plasticity in the brain (Wong-Riley and Welt, 1980; Machin et al., 2006). We now demonstrate that inhibition of synaptic activity reduces plaque burden but increases immunoreactivity of Aβ within neurons and exacerbates loss of synaptophysin in AD transgenic mice. Moreover, despite reducing plaques, chronic treatment with diazepam worsened memory retention.
Three month old Tg19959 mice and wild-type littermates (13 males and 19 females) were divided into 4 groups: wild-type + vehicle (control), wild-type + diazepam, Tg19959 + vehicle, Tg19959 + diazepam. Mice were treated with diazepam (4.5 mg/kg) or vehicle (saline containing 4% propylene glycol and 1% ethanol) by daily intraperitoneal (IP) injection for 6 days a week for 1 month. Mice were given 4 days of washout before testing in the MWM.
Two month old Tg19959 mice and wild-type littermates (5 males and 5 females) were anesthetized with an IP injection of ketamine and xylazine. Surgery was performed as previously described (Tampellini et al., 2009). At 6 months of age, mice were sacrificed with pentobarbital and perfused with 4% paraformaldehyde (PFA) or with 2% PFA and 3.75% acrolein for electron microscopy (EM) studies. Brains were removed and either cut by Vibratome (EM) or incubated at 4°C in increasing percentages of sucrose (10, 20 and 30%) before sectioning by cryostat (immunostaining and immunofluorescence).
Spatial learning and memory were assessed by MWM as described (Dumont et al., 2009). In brief, the hidden platform was located in the middle of the northwest (NW) quadrant, 1 cm beneath water level. Latencies were recorded for 5 days with a video tracking system. Each day mice were placed next to and facing the wall of the basin in 4 different starting positions: north, east, south, and west, corresponding to 4 successive trials. Whenever the mouse failed to reach the platform within the maximally allowed time of 60 seconds, it was placed on the platform for 5 seconds. A probe trial was assessed 24 hours after the acquisition period, removing the platform from the pool. The mice were released on the north side (exactly on the border between NW and NE quadrants) facing the wall. To ensure that any differences were not due to visual deficits, the visible platform version of the water maze was performed after the probe trial.
Ultrastructural quantification of synapses was done using EM as described (Takahashi et al., 2002). Representative images were obtained from deafferented and contralateral barrels, n=5 each. Synapses were identified by the following criteria: presence of a clearly defined synaptic density and evidence of pre- and postsynaptic compartments. Numbers of synapses were counted by an investigator unaware of the origin of the images, and the difference in synapse numbers was expressed as percentage of change from control.
Western blots were performed as described (Dumont et al., 2009). Briefly, snap-frozen brain tissues were homogenized in 6% SDS. Equal protein amounts (25μg) were electrophoresed through 4–12% Bis-Tris NuPage (synaptophysin) or 10–20% Tris-Tricine polyacrylamide (APP, CTFs and Aβ) gels (Invitrogen). After transfer, membranes for Aβ were boiled in PBS for 5 minutes. Membranes were blocked in 5% nonfat dry milk and incubated with primary antibody overnight at 4°C. Horseradish peroxidase-conjugated secondary antibody binding was visualized with enhanced chemiluminescence. Antibodies used: rabbit polyclonal anti-APP 369, mouse monoclonal 6E10 (Covance), mouse monoclonal anti-synaptophysin (Millipore), and mouse monoclonal anti-tubulin (Sigma).
Brains were cut at 40μm thickness with a cryostat and stained for COX (Wong-Riley and Welt, 1980) or immunolabeled for Aβ42 (Millipore) and/or synaptophysin (Millipore) in adjacent sections. The barrel cortices corresponding to the half snouts that did not undergo surgery were used as controls. Immunolabeling for light and fluorescence microscopy was performed as previously described (Li et al., 2004; Takahashi et al., 2010). Brain sections were viewed and images were acquired as described (Li et al., 2004; Tampellini et al., 2009; Takahashi et al., 2010). Metamorph software 7.6 (Universal Imaging Co.) was used for quantitative analysis. Integrated fluorescence intensity per field was measured on thresholded images. Three images per stack were used for each section. Total fluorescence per thresholded picture was automatically quantified.
Statistical comparisons were made using two-tailed t-tests (paired for the deafferentation data, unpaired for the diazepam data) and ANOVA with significance placed at p<0.05. Averages of measurements per section per mouse were considered as an individual measurement (n=1). Data were expressed as mean ± SEM. Statistical analysis was performed using StatView (SAS Institute Inc.) and Excel (Microsoft).
Chronic reduction of synaptic activity in vivo was accomplished using two different approaches: unilateral ablation of whiskers and chronic diazepam treatment. For the former, whisker bulbs were unilaterally and irreversibly removed in 2 to 3 month old Tg19959 mice, which harbor the KM670/671NL and V717F familial AD mutations (Li et al., 2004). These mice develop plaques at approximately 3 months of age. The effects on barrel cortices were examined at 6 months of age. Reduced cytochrome oxidase (COX) staining confirmed reduced neuronal activity in the deafferented compared to the synaptically active control barrel cortices (Figure 1A, upper panels). Barrel cortices with reduced activity demonstrated a 29±5% decrease in the number of Aβ plaques and a 32±8% decrease in the area covered by plaques compared to the control side (Figure 1A, lower panels). At the same time, barrel cortices with reduced activity and reduced plaques showed a 64±30% increase of Aβ42 immunoreactivity in neurons compared to controls (Figure 1B).
To determine the effect of chronic synaptic inhibition on the integrity of synapses, levels of the synaptic marker synaptophysin were examined in the barrel cortices by confocal microscopy. Progressive loss of the pre-synaptic marker synaptophysin is well-established in AD transgenic mice (Mucke et al., 2000) and is considered the best brain correlate of cognitive decline in human AD (Terry et al., 1991). We have previously shown that Tg19959 mice have reduced synaptophysin levels compared to wild-type littermates (Dumont et al., 2009). Remarkably, and despite the reduction in plaques, confocal microscopy demonstrated that levels of synaptophysin in Tg19959 mice were further decreased by 80±3% (Figure 2A, left panels) on the deafferented compared to the synaptically active barrel cortex (control); there also was a 31±14% reduction in levels of PSD-95 on the deafferented compared to control barrels (Supplementary Figure S1). In contrast, wild-type mouse brains with equivalent chronic synaptic inhibition did not show decreases in synaptophysin (Figure 2A, right panels). To evaluate whether the decrease of synaptophysin in the deafferented barrel cortex of Tg19959 mice might reflect a loss of synapses, electron microscopy was used to quantify the number of synapses (Figure 2B). There was a 31±12% reduction in number of synapses in the deafferented barrels compared to control.
Next, diazepam treatment was used to investigate the effect of chronic synaptic inhibition on Aβ, synapses, and cognitive function. Tg19959 mice were treated with diazepam for 1 month beginning at 3 months of age, followed by a 4-day washout prior to behavioral testing. Diazepam treatment reduced plaque burden by 32±12% and 20±9% in cortex and hippocampus respectively, while confocal immunofluorescence of the hippocampus revealed a 41±10% increase in intraneuronal Aβ42 immunoreactivity in CA1 neurons in diazepam-treated compared to vehicle-treated Tg19959 mice (Figure 3A-C). Similar to the deafferentation results, confocal immunofluorescence demonstrated that the already decreased levels of synaptophysin in Tg19959 mice were further decreased by diazepam (Figure 4A). In contrast, immunofluorescence demonstrated an increase in levels of synaptophysin in wild-type mice with synaptic inhibition by diazepam (Supplementary Figure S2). Of note, chronic synaptic inhibition in wild-type barrel cortices was previously reported to lead to an increase in synapses (Machin et al., 2006). Our previous data linked intraneuronal Aβ with reduction in levels of synaptophysin in cultured AD transgenic neurons (Almeida et al., 2005). We now observe a link between Aβ42 accumulation and loss of synaptophysin in brain (Supplementary Figure S3).
We also examined levels of full-length APP, Aβ, CTFs and synaptophysin in Tg19959 mouse brains by Western blotting (Supplementary Figure S4). Diazepam treatment did not significantly alter total brain levels of any of these in either wild-type or Tg19959 mice. This contrasts with the anatomically specific results seen by confocal immunofluorescence, above, likely because homogenization obscures cellular and anatomical structure.
We next tested whether the reduction in plaques from diazepam treatment was associated with behavioral improvement or decline in the Tg19959 mice. As expected, Tg19959 mice performed worse than wild-type littermates during the probe trial of the Morris water maze (MWM; Figure 4B and Supplementary Figure S5A). Remarkably, despite plaque reduction, diazepam treatment further impaired spatial memory performance in Tg19959 mice in the first 15 sec of the probe trial (Figure 4B). Diazepam treatment did not affect performance in the acquisition phase of the MWM in Tg19959 and wild-type mice (Supplementary Figure S5B), and did not significantly affect memory retention in wild-type mice (Supplementary Figure S5C). In addition, there were no differences in non-cognitive components of behavior, such as the visible platform test (Supplementary Figure S5D) and swim speed (not shown), between the Tg19959 and wild type mice or diazepam treated compared to untreated mice.
Thus, we have obtained similar results with two different models of chronic synaptic inhibition, pharmacologic and surgical deafferentation. Although benzodiazepines might have effects other than their well-established effect on GABA receptors, the concordance with the barrel cortex results further supports the primary role of synaptic inhibition in the results presented.
In two different models of chronic synaptic inhibition, there was a reduction in plaque burden but an increase in Aβ42 immunoreactivity within neurons and an exacerbation in synaptophysin and synapse loss. Importantly, despite plaque reduction, chronic inhibition with diazepam exacerbated memory impairment in AD transgenic mice. Although mouse behavior may not necessarily reflect human cognition, the aborted active Aβ42 vaccine clinical trial suggested a similar dissociation between plaques and cognition, with plaque clearance despite continued cognitive decline (Holmes et al., 2008). As another example of dissociation between plaques and cognition, passive immunotherapy with an Aβ antibody improved behavior without plaque reduction in a mouse model of AD (Dodart et al., 2002). In the data reported here, the observed increase of Aβ42 within neurons with synaptic inhibition likely is the primary cause for the reduction of synaptic proteins and impaired memory given our previous mechanistic evidence for this in cultured primary neurons (Tampellini et al., 2009). Evidence increasingly supports that this intraneuronal pool of Aβ42 is critical in AD. Accumulation of intraneuronal Aβ immunoreactivity precedes the appearance of plaques (Gouras et al., 2000; Wirths et al., 2001; Oddo et al., 2003), and the emergence of intraneuronal Aβ correlated with the onset of synaptic, pathological, physiological and behavioral abnormalities in AD transgenic models (Runz et al., 2002; Takahashi et al., 2002; Oddo et al., 2003; Billings et al., 2005; Cruz et al., 2006; Knobloch et al., 2007; Lord et al., 2009; Gandy et al., 2010; Leon et al., 2010; Tomiyama et al., 2010). Of note, a recently described familial AD mutation in APP is associated with intraneuronal Aβ accumulation and oligomerization in the absence of plaques (Tomiyama et al., 2008; Tomiyama et al., 2010). The current work shows the importance of normal synaptic activity in protecting against AD-related pathogenesis, but does not argue against a pathologic role also of synaptic hyperexcitability in AD (Buckner et al., 2005; Palop et al., 2007). Overall, these data have important therapeutic and diagnostic implications for AD, and support the idea that synaptic activity can be protective in AD pathogenesis.
Supported by an Alzheimer’s Association New Investigator Award (DT) and Zenith Award (GKG), and National Institute of Health grants AG020729 (MTL), AG027140, AG028174 and AG09464 (GKG). We thank Dr. George Carlson, McLaughlin Research Institute, for providing the Tg19959 mice. We thank Dr. Teresa Milner and Dr. Costantino Iadecola in our Division of Neurobiology for help with electron microscopy and advice on the barrel cortex experiments.
The authors declare that they have no conflicts of interest.