Synaptic Compensation as a Probable Cause of Prolonged Mild Cognitive Impairment in Alzheimer’s Disease: Implications from a Transgenic Mouse Model of the Disease

Abstract

Alzheimer’s disease (AD) is a slow, progressive neurodegenerative disease in which cognitive decline takes place over a period of several years with a very variable period of mild cognitive impairment (MCI) and, in some cases, relatively long period before progression to dementia. The cognitive deficit during MCI is probably due to neuronal loss, an intermediate level of amyloid-β (Aβ) plaques and neurofibrillary tangles (NFT) and synaptosis, which is interrupted with a transient compensatory increase. We found impairment in reference memory accompanied by a decrease in the expression of synaptophysin, β-III tubulin, and MAP2 and a trend for GluR1, at 12 weeks of age in 3xTg-AD mice (hAPP_Swe, P301L tau, PS1 [M146V] knock-in), a widely used transgenic model of AD. Past 12 weeks, the cross-sectional analysis of different age groups showed a compensatory increase in synaptic markers relative to that in wild type animals in a topographic and time-dependent manner. When studied across time we found that in 3xTg-AD mice, the compensatory phenomenon occurred in parallel in different regions of the brain. However, this attempt of the brain to repair itself was able to only partially rescue cognitive impairment. These findings for the first time raise the intriguing possibility that AD causing mutated transgenes may initially cause an increase in synaptic and dendritic markers as a compensatory mechanism for synaptic deficit, and this phenomenon, though transient, could be the biological basis of the period of MCI seen in AD.

Keywords

Alzheimer’s disease cognitive impairment neuronal plasticity synaptic compensation 3xTg-AD transgenic mouse model

INTRODUCTION

Alzheimer’s disease (AD) is a progressive neurodegenerative disease and the most common form of dementia in elderly people. It is characterized by two histopathological hallmarks: β-amyloidosis and neurofibrillary degeneration, maximal densities of which are seen at moderate to severe stages of dementia. Increasing evidence suggests that the beginning of the pathological cascade of AD starts many years before the appearance of the clinical symptoms of cognitive impairment. The very early stages of AD are thought to start with synaptic dysfunction, followed by neuronal loss, an intermediate level of amyloid-β (Aβ) and tau pathologies and cognitive impairment [1 –4]. Although the severity of dementia has been shown to correlate with the density of neurofibrillary tangles (NFT) or NFT plus senile plaques, neither of the two pathologies is specific to AD. They are also reported in normal aged individuals. For instance, in the oldest old people dementia is associated with very little pathology, while the absence of dementia was reported in the presence of high levels of Aβ and tau pathologies [5, 6]. Furthermore, it has been shown that there is a significant overlap in the Braak stages between demented and non-demented patients [7, 8]. Synaptic loss, on the other hand, has been shown to be a consistent feature that differentiates demented and non-demented people and significantly correlates with the severity of dementia [9 –11]. Synaptic deficit correlates well with other types of dementia and age-associated decrease in cognitive performance, called normal cognitive aging [12].

Postmortem analysis revealed a drastic decrease in presynaptic and postsynaptic markers in very early AD stages, suggesting the degeneration of the whole synaptic element [13]. Synaptophysin immunoreactivity was reported to decrease by 25% in the cortex of mild AD patients compared to normal cases [14, 15]. A decrease in the number of synapses of ∼25–30% was reported in temporal and frontal cortical biopsies and around 15–35% loss in the number of synapses per neuron within 2–4 years of the onset of AD [16]. Synaptic deficit is even more pronounced in the hippocampus, reaching 44% to 55% [17]. Synaptic degeneration is thought to start in the entorhinal cortex, with the dendrites as the first to undergo degeneration, since they make up to 90% of synaptic contact. Dendritic loss is found to occur in parallel with synaptic loss very early in AD [15, 16]; the ratio of synapses to neurons decreases by 48%, suggesting that synaptic loss is not happening only in the degenerated neurons but also in the remaining neurons [18, 19].

In spite of the reduced capacity of synaptic plasticity in the brain, there is still a residual plastic capacity, as shown by the synaptic compensation phenomenon that happens in the early AD stages [20, 21] and in transgenic mice [22, 23]. In the case of AD, the brain may take advantage of such capacity to compensate for the synaptic damage and slow down the progression of the disease, as seen by a relatively prolonged mild cognitive impairment (MCI) period. In a previous study, although synaptophysin was reported to decrease in the frontal cortex of demented people, an increase was found in cognitively impaired non-demented 90+ year old individuals. This suggests that synaptic compensation counteracts synaptic deficit and accounts for preserved cognition in these individuals in the presence of extensive pathology [24]. In the Rush Religious Orders Study, an increase in the level of synaptophysin in the superior frontal cortex of MCI patients was reported compared to normal individuals [25]. Furthermore, an increase in the level of synaptophysin in elderly people with extensive pathology and normal cognition was found compared to patients diagnosed with clinical and pathological AD [26, 27]. However, the increase in synaptophysin expression does not necessarily mean an increase in the number of synapses, since an increase in the synaptic size to keep an intact total synaptic contact area in the very early stages of the disease has been found [21, 28].

The use of transgenic animal models of AD made it convenient to understand the progression of AD pathology and to develop treatments. Herein, we used the 3xTg-AD mouse [29], a widely used model shown to mimic the neuropathological features of AD such as Aβ and tau pathologies. The Aβ pathology starts at around nine months of age, tau pathology at around twelve months of age, and cognitive impairment as early as two to three months of age in this mouse model [30, 31]. This model was generated by the co-injection of two mutated human transgenes, APP_swe (KM670/671NL) and tau P301L, into the PS1/M146V knock-in embryo [29]. The two mutated transgenes are under the control of Thy 1.2 (Thy 1.2 cell surface antigen) promoter, which is activated during the embryonic days 11 and 15 in mice [32].

In the present study, we show that in a manner reminiscent of AD, in 3xTg-AD mice cognitive impairment is associated with a transient synaptic compensation, which is followed by synaptic deficit.

MATERIAL AND METHODS

Animal and housing

The homozygous 3xTg-AD mice express two mutated human transgenes, APP_Swe and tau P301L mutations, and PS1 knock-in. Breeding pairs of these mice were obtained from Dr. Frank LaFerla through the Jackson Laboratory (New Harbor, ME, USA). Wild type (WT) age-matched controls from the same background strain, a hybrid: 129/Sv×C57BL/6, were also obtained from the Jackson Laboratory. Animals were housed and bred according to approved protocols from our Institutional Animal Care and Use Committee (IACUC), according to the PHS Policy on Human Care and Use of Laboratory animals (revised January, 2013). Animals were housed as 5 animals/cage with a 12:12 h light/dark cycle and with free access to food and water. They were given a period of acclimatization of 45 min to 1 h before any behavioral test was performed. Only young adult female 3xTg-AD mice and age-matched WT control mice were used in this study because it was shown previously that female 3xTg-AD have more aggressive and consistent pathology compared to males, and female mice have more Aβ deposition, worse cognitive performance, and a higher deficit in neurogenesis than males [33 –35]. To exclude any effect of behavioral tests on neurogenesis, animals other than those used for behavioral studies were employed for immunohistochemical/biochemical studies. Twenty-six 3xTg-AD and 28 WT control mice were used for immunohistochemistry and western blots. For behavioral studies, 30 3xTg-AD and 20 WT animals were employed.

Behavioral procedures: Morris water maze task

The Morris water maze task is a test of reference memory, which is a measure of hippocampal function. The test was a modification of the original procedure by Morris et al. [36]. A total of 30 3xTg-AD mice and 20 age-matched WT controls were divided into two batches of 15 3xTg-AD and 10 WT in each batch. The first batch of animals was tested for reference memory by the Morris water maze task at 12–13 weeks and the second batch was tested at 13–14 weeks. The procedure was performed in a circular pool tank, 180 cm in diameter. A 13-cm escape platform was submerged 1 cm under opaque water in the northwest quadrant. The water was made opaque by the addition of a white non-toxic chalk to make the escape platform invisible to mice. Spatial cues were set up in the test room to help the animals locate the place of the platform. The temperature of the water was kept at 21±1°C. In each trial, the animal was given 90 s to find the escape platform. If the animal did not find the escape platform, it was gently guided to it. Each animal was left for 20 s on the escape platform and then returned to its cage. In each trial, the mouse was released with its back toward the center of the tank from a different quadrant and the starting point was changed each day so that the animal would not learn the place of the platform without using the spatial cues. The test was comprised of three acquisition trials for four consecutive days. Each animal performed 12 acquisition trials, which is considered a partial learning for the spatial reference memory task. Twenty-four hours after the last acquisition trial, the retention test or probe trial was conducted in which the escape platform was removed and the animal was allowed to swim freely for 60 s.

The measure of acquisition was the time and distance swum to reach the escape platform. The measures of retention were the percent of time spent in the target quadrant and the number of platform crossings. Behavior of the animal in the Morris water maze was recorded using a Samsung digital camera (SDC 4304) mounted on the ceiling and a SMART version 2.0.14 software (Pan Lab/San Diego Instruments) was used for tracking and timing of each trial.

Tissue processing

Right after completion of the Morris water maze task, six groups of animals (10-, 12-, 13-, 14-,15-, and 16-week-old mice) were chosen for immunohistochemical and biochemical studies. Animals were anesthetized using overdose of avertin and transcardially perfused using 0.1 M PBS. The brain was immediately removed from the skull, the right hemisphere was immersion fixed with 4% paraformaldehyde in 0.1 M PBS for at least 24 h at 4°C, then transferred to 30% sucrose; the left hemisphere was dissected into hippocampus, fore-, mid-, and hind-brains, and stored at –80°C for biochemical studies. After fixation and cryoprotection, the right hemisphere was sectioned into 40 um sagittal sections then saved at –20°C in antifreeze solution (ethylene glycol, glycerol, and 0.1 M PBS in 3:3:4 ratio) until further processing.

Immunohistochemistry

For immunohistochemistry, 3–5 animals/group from the six different groups were chosen randomly and 5 to 6 sections/mouse from every tenth section were used for staining intensity scanning analysis. Sections used for a specific marker from the six groups were immunostained and analyzed by confocal microscopy under identical conditions. The following antibodies were used: mouse anti-synaptophysin (1:500; Chemicon, Temecula, CA, USA); mouse monoclonal anti-SMI52 to MAP2 (1:1000; Covance, Emeryville, CA, USA); rabbit polyclonal anti-β-III tubulin (1:1000; Covance); rabbit polyclonal anti-GluR1 (1:300; Millipore, Temecula, CA, USA). The following secondary antibodies were used: Alexa 488-conjugated goat anti-mouse IgG antibody (1:500, Molecular Probes, Carlsbad, CA, USA) and CY3-conjugated goat anti-rabbit antibody (1:500, Jackson Laboratory, Bar Harbor, Maine, USA). Sections were double-stained either with anti-synaptophysin and anti-GluR1 or with anti-MAP2 and anti-βIII-tubulin.

For all the stainings, maximum projection images were generated employing the average of 15 Z-stacks using a 20X objective with a small pinhole of a Nikon 90i fluorescent microscope equipped with Nikon C1 three-laser confocal system and a Nikon DS U1 digital camera and the entire area of DG, CA1 (stratum radiatum and Stratum pyramidae), CA3 (stratum oriens, stratum radiatum and stratum pyramidae), parietal association, and frontal cortices (all six layers) were analyzed. Mean pixel intensity was measured for each brain region using the Image J software (NIH). Each picture to be analyzed was converted to grey scale and each area to be analyzed was selected, the threshold was adjusted and the mean pixel intensity of the region of interest was quantified.

Western blots

Western blot procedures were done using 10% SDS-PAGE as previously described [37, 38]. The blots were developed using mouse monoclonal anti-synaptophysin antibody (1:3,000; Chemicon, Temecula, CA, USA), the rabbit polyclonal anti-GluR1 antibody (1:1000; EMD Millipore, Billerica, MA, USA) and rabbit monoclonal anti-GAPDH (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as a loading control. The corresponding horseradish peroxidase-conjugated affinity-purified goat anti-mouse IgG (H+L) and anti-rabbit IgG (H+L) secondary antibodies were used (1:5,000; Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA). The blots were visualized using the enhanced chemiluminescence (ECL) reagents, (Pierce, Rockford, IL, USA). The ECL films of the blots were scanned and analyzed using Image J software (NH). Each protein’s band intensity was quantified and then analyzed using image J. Each protein’s band intensity quantification was the result of the subtraction of the background intensity from the original intensity of the band. Finally, the intensity of each band minus the background was normalized to the intensity of the GAPDH band minus the background.

Statistical analysis

Statistical analyses were conducted using Stata and GraphPad Prism software package, version 5.0 (GraphPad Software Inc., La Jolla, CA, USA). Tests of scalar predictors were done with regression models and were performed in Stata 13 (StataCorp, College Station, TX, USA). Statistical significance of the changes in indicator levels over age was performed in regression models. Instead of comparing each week with each other week without regard for the natural ordering of the ages (as an ANOVA would), the regression analyses determined the change in each indicator per week of age across all the ages at once. As the rate of change could vary across ages, we computed models including quadratic and cubic terms for age to see if the rate at which indicators varied could best be predicted by them. When these higher-order terms are included, the results show whether, for example, a model in which indicators rise, then fall, then rise with age best fits the data. We compared the adjusted R-squared for the models to determine which model best fit the data. Table 1 and Table 2 list, for the best-fitting models, the overall F score and degrees of freedom (as in an ANOVA), the p-value that shows the overall statistical significance of the model, the adjusted R-squared (which shows how much of the variance in the indicator over age is explained by the model, adjusted for the number of predictors in the model), and the type of model. A cubic model uses age, age-squared, and age-cubed to predict the indicator. A quadratic model uses age and age-squared, and a linear model uses only age itself. We compared the adjusted R-squared for each model to determine best fit. For multiple group comparisons, one-way or two-way ANOVAs followed by post-hoc tests were used. T-tests were used for all other inter-group comparisons. Data are presented as mean±SEM. For all analyses p < 0.05 was considered to be significant.

RESULTS

Since AD is characterized by impairment in episodic (hippocampal) memory at early stages followed by a decline in other cognitive domains (i.e., executive, visuospatial, visuoperceptive, praxis, language, etc.), we investigated if young adult 3xTg-AD mice show any hippocampal based cognitive impairment. Because the hippocampus is the main brain structure implicated in memory formation, storage, consolidation and retrieval we employed the Morris water maze behavioral task, which is hippocampal-dependent, to test the 3xTg-AD mice. In this task, mice are required to learn the position of a hidden platform using distal spatial reference cues. At the end of the learning trials mice have to form a spatial navigation map using the spatial cues to determine the position of the platform.

To study the effect of synaptic deficit and synaptic compensation on cognition in 3xTg-AD mice, we tested by Morris water maze task one batch of 15 3xTg-AD and 10 WT mice at 12-13 weeks and a second batch of same size at 13-14 weeks.

At 12-13 weeks, synaptic deficit was accompanied by impairment in spatial reference memory in 3xTg-AD mice

The analysis of the swim speed revealed no significant difference between the WT and the 3xTg-AD mice (Fig. 1a, p = 0.84). For this reason, the learning performance of the animals was analyzed as the latency time and the distance to reach the escape platform. We found that both 3xTg-AD and WT mice learned the task well, which was evident from the decrease in the latency to escape across training sessions (Fig. 1a, p < 0.0001). However, the regression of the latency to escape across time showed that the 3xTg-AD mice spent more time to find the escape platform compared to WT (Fig. 1a, p = 0.0085). The analysis of the distance traveled to find the escape platform showed that both groups learned the task well (Fig. 1a, p < 0.0001). However, during the training days, the 3xTg-AD mice had to travel longer distance to find the escape platform (Fig. 1a, p = 0.0261) compared to WT. These data revealed that at 12-13 weeks synaptic deficit was accompanied by impairment in spatial reference memory in 3xTg-AD mice.

To evaluate the memory retention in the 3xTg-AD and WT mice, a probe trial 24 h after the last training session was conducted. The 3xTg-AD mice spent significantly less time in the target quadrant (Fig. 1b; p = 0.0004) and visited the target zone less (Fig. 1b; p < 0.007) than WT animals. These results suggest that memory retention/retrieval was compromised in 3xTg-AD mice and further corroborated that these mice were cognitively impaired.

At 13-14 weeks, synaptic compensation was able to improve learning but not retention and memory in 3xTg-AD mice

The analysis of batch 2 (13-14 weeks) animals showed that there was no difference in the swim speed between the WT and 3xTg-AD mice (Fig. 1a, p = 0.95) and the latency to escape went from highly significant in batch 1 to only a trend in batch 2 (Fig. 1a, p = 0.06). Furthermore, the difference in the distance traveled between WT and 3xTg-AD changed from significant in batch 1 (Fig. 1a, p = 0.0261) to non-significant in batch 2 (Fig. 1a, p = 0.297). Both groups learned the task well since the latency to escape and distance decreased for both groups during the training days (Fig. 1a, p < 0.0001 and p < 0.001, respectively). These data show that synaptic compensation ameliorated learning impairment during the training days in the Morris water maze task.

To determine the effect of synaptic compensation on memory retention we analyzed the percent of time in target quadrant and the number of crossings of the previous platform place. We found that the 3xTg-AD mice spent significantly less time in the target quadrant compared to WT (Fig. 1b, p = 0.0002) while there was no difference between both groups in the number of crossings to the previous place of the platform (Fig. 1b, p < 0.238). This shows that memory retention stayed impaired in 3xTg-AD mice even with the presence of synaptic compensation.

When we pooled the two batches together aged 12–14 weeks, we found that the 3xTg-AD mice were impaired compared to WT in spatial reference memory. The analysis of the swim speed revealed no significant difference between the wild type and the 3xTg-AD mice (Supplementary Figure 1a). For this reason, the learning performance of the animals was analyzed as the latency time to reach the escape platform. We found that animals from the two different groups learned the task well as seen by a decrease in the latency to escape across training sessions (Supplementary Figure 1a, p < 0.0001). However, the regression of the latency to escape across time showed that the 3xTg-AD mice learned more slowly than the WT across the training days (Supplementary Figure 1b, p = 0.048).

The evaluation of the memory retention of the two batches combined in the 3xTg-AD and WT mice was conducted 24 h after the last training session. We found that the 3xTg-AD mice spent significantly less time in the target quadrant (Supplementary Figure 1c, p < 0.001) and visited less the previous place of the platform (Supplementary Figure 1d, p < 0.005) than WT animals. These results showed that the 3xTg-AD mice were impaired in memory retention/retrieval and that the 3xTg-AD mice were impaired in spatial reference memory.

We found enhanced motor learning coordination as reported previously [37 , 40] and normal level of anxiety in the 10–14 weeks 3xTg-AD mice (Supplementary Figure 2a-f).

Impairment in spatial reference memory is accompanied by a transient increase in neuronal and synaptic markers in 3xTg-AD mice

Synaptic deficit is an established phenomenon and an early event in AD. Synaptophysin loss in the frontal cortex strongly correlates with dementia and cognitive impairment [15]. To examine synaptic changes at very early stages of the disease, we evaluated immunohistochemical staining of brains from 3xTg-AD mice aged 10, 12, 13, 14, 15, and 16 weeks using four different antibodies: anti-synaptophysin, anti-GluR1, anti-MAP2, and anti-β-III tubulin.

Cornu ammonis 1 (CA1)

Across time changes in mean pixel intensity showed that the synaptophysin expression in CA1 in 3xTg-AD mice from 10–16 weeks of age was best modeled by a cubic regression model. In this model, synaptophysin expression diminished at 12 weeks of age and was then compensated for from 13 to 16 weeks of age (Fig. 2a; Table 1). Similarly, western blots of synaptophysin expression were best fit by a cubic model, with a significant decrease in synaptophysin level in the hippocampus at 12 weeks compared to 10 weeks, followed by an increase by 13-14 weeks and then a decrease by 15-16 weeks in 3xTg-AD mice (Fig. 3a, b; F (3, 18) = 5.47, p = 0.0075).

The examination of GluR1 expression in the CA1 region showed no significant difference across time (Fig. 2b; Table 1). Similarly, western blots showed no change of the expression level in the hippocampus (Fig. 3c, p = 0.64; Supplementary Table 1).

The expression of the dendritic marker MAP2 was best fit by a linear model (Fig. 2c; Table 1).

The expression of β-III tubulin dropped at 12 weeks compared to 10 weeks and then increased from 13–16 weeks (Fig. 2d; Table 1), though again the best fit was obtained with a linear model.

Cornu ammonis 3 (CA3)

The expression of synaptophysin in the CA3 region was best fit by a cubic model (Supplementary Figure 3a; Table 1). As in the CA1 region, the expression of synaptophysin dropped at 12 weeks compared to 10 weeks and increased from 13 to 15 weeks of age. No significant change was detected in the GluR1 and MAP2 expressions in the CA3 region (Supplementary Figure 3b,c; Table 1). β-III tubulin was best modeled by a cubic regression with a decrease in the expression at 12 weeks compared to 10 weeks and increase from 13–16 weeks of age (Supplementary Figure 3d; Table 1).

Dentate gyrus (DG)

In the DG, the expression of synaptophysin dropped at 12 weeks of age compared to 10 weeks and then increased from 13 to 16 weeks. The changes followed a cubic model (Fig. 4a; Table 1). No change was found in the expression of GluR1 (Fig. 4b; Table 1). The expression of MAP2 followed a quadratic model (Fig. 4c, Table 1); β-III tubulin expression was best fit by a quadratic model with a drop at 12 weeks compared to 10 weeks and increase from 13–16 weeks (Fig. 4d; Table 1).

Parietal cortex

Expression of synaptophysin, MAP2, and β-III tubulin showed a drop in expression across time at 12 weeks compared to 10 weeks and an increase from 13–16 weeks. A cubic model best fit the expression of synaptophysin, while β-III tubulin and MAP2 followed a quadratic model (Supplementary Figure 4a, c, d; Table 1). The expression of GluR1 did not change significantly (Supplementary Figure 4b; Table 1).

Frontal cortex

The expressions of synaptophysin and β-III tubulin changed across time. Synaptophysin followed a cubic model (Supplementary Figure 5a; Table 1), while for β-III tubulin, a linear model fit best (Supplementary Figure 5d; Table 1). Both markers decreased at 12 weeks of age compared to 10 weeks and increased from 13–16 weeks. No significant change was detected in the expression of GluR1 (Supplementary Figure 5b; Table 1) or MAP2 (Supplementary Figure 5c; Table 1).

Cross-sectional comparison between 3xTg-AD and control mice confirms synaptic compensation in the former

To understand if the changes in the expression of the synaptic, dendritic, and neuronal markers are relevant to the poor cognitive performance in the 3xTg-AD mice we analyzed these data as a percent of control (WT) for different ages from the age of 10–16 weeks in different brain regions. Figure 2 and Figure 4 and Supplementary Figure 3–Figure 5 show the pattern of expression of synaptophysin, GluR1, MAP2, and β-III tubulin in the 3xTg-AD alone across time. However, the expression patterns of the four markers also fluctuate in the WT across time.

CA1

Cross-sectional comparison of 3xTg-AD as a percent of control (WT) mice revealed a synaptic deficit at 12 weeks followed by compensation in different brain regions at different ages. In the CA1 region, change in synaptophysin expression was not significant (Supplementary Figure 6a; Supplementary Table 2). Western blots showed changes in synaptophysin expression best fit by a quadratic model with synaptic deficit at 12 weeks compensated from 13 weeks (Fig. 3a-b; F (2, 7) = 12.56; p < 0.005).

GluR1 expression showed no difference between 3xTg-AD mice and WT for all time points studied (Supplementary Figure 6b; Supplementary Table 2).

Similarly, MAP2 expression was comparable in 3xTg-AD and WT mice (Supplementary Figure 6c; Supplementary Table 2).

Level of β-III tubulin did not change significantly but the lowest level was at 13 weeks of age with a slight compensation from 14–16 weeks of age (Supplementary Figure 6d; Supplementary Table 2).

CA3

Cross-sectional comparison of 3xTg-AD as a percent of control mice revealed that synaptophysin expression did not differ significantly between 3xTg-AD and WT (Supplementary Figure 7a; Supplementary Table 2).

GluR1 expression did not change significantly between 3xTg-AD and WT in all the time points studied (Supplementary Figure 7b; Supplementary Table 2).

MAP2 showed a trend toward decrease at 12 weeks, compensated for at 15 weeks, then dropped significantly at 16 weeks in 3xTg-AD compared to WT (Supplementary Figure 7c; Supplementary Table 2).

β-III tubulin expression in the CA3 did not change significantly between 3xTg-AD and WT in the different time points studied (Supplementary Figure 7d; Supplementary Table 2).

DG

Cross-sectional comparison of 3xTg-AD as a percent of control mice showed that synaptophysin expression in the DG did not differ significantly between WT and 3xTg-AD across time (Fig. 5a; Supplementary Table 2).

Overall, there was no significant difference in the expression of GluR1 between 3xTg-AD and WT (Fig. 5b; Supplementary Table 2).

MAP2 expression decreased at 12 weeks, increased from 13–15 weeks, and then dropped again at 16 weeks in 3xTg-AD mice compared to WT (Fig. 5c; Supplementary Table 2).

Overall β-III tubulin expression did not change significantly between 3xTg-AD and WT (Fig. 5d; Supplementary Table 2).

Parietal cortex region

Cross-sectional comparison of 3xTg-AD as a percent of control mice showed that synaptophysin expression decreased significantly at 12 weeks in 3xTg-AD compared to WT and increased from 13–16 weeks (Supplementary Figure 8a; Supplementary Table 2).

GluR1 expression did not change significantly between 3xTg-AD and WT (Supplementary Figure 8b; Supplementary Table 2).

Change in MAP2 expression was not significant (Supplementary Figure 8c, Supplementary Table 2).

Change in β-III tubulin expression was not significant (Supplementary Figure 8d. Supplementary Table 2).

Frontal cortex

Cross-sectional comparison of 3xTg-AD as a percent of control mice showed that synaptophysin decreased at 12 weeks and increased at 14 weeks. The compensation for synaptic deficit occurred from 13 to 15 weeks (Supplementary Figure 9a; Supplementary Table 2).

GluR1 expression showed no significant change (Supplementary Figure 9b; Supplementary Table 2).

MAP2 expression did not change significantly between 3xTg-AD mice and WT (Supplementary Figure 9c, Supplementary Table 2).

β-III tubulin expression in 3xTg-AD mice was lowest at 13 weeks and increased non-significantly at 14 weeks of age. Compensation for synaptic deficit happened mainly at 14 weeks (Supplementary Figure 9d; Supplementary Table 2).

Small changes in the expressions of neuronal and synaptic proteins in some brain regions in WT mice

CA1

Synaptophysin and GluR1 did not change significantly in WT mice (Fig. 6a; Table 2). MAP2 and β-III tubulin significantly changed over time in the CA1 region, best modeled with a cubic regression (Fig. 6a; Table 2). MAP2 decreased from 12 to 13 weeks but was a normal level at 14 to 15 weeks (Fig. 6a; Table 2). β-III tubulin decreased at 12 weeks and then increased at 13 weeks (Fig. 6a; Table 2).

CA3

No significant changes were detected in synaptophysin, MAP2, or β-III tubulin (Fig. 6b; Table 2). GluR1 followed a cubic pattern, with the lowest level at 15 weeks and an increase at 16 weeks (Fig. 6b; Table 2).

DG

No significant changes for any of the markers were detected in the DG area (Fig. 6c; Table 2).

Parietal cortex

The only change that was seen in this region was in the expression of GluR1. It followed a quadratic fit with a decrease from 12–15 weeks and then an increase by 16 weeks (Fig. 6d; Table 2). All the other markers showed no differences.

Frontal cortex

No significant change was seen for any markers in this brain region for WT mice (Fig. 6e; Table 2).

Cross-sectional comparison between 3xTg-AD and control mice shows that synaptic compensation happens at different time points in different brain regions

A comparison of changes in the expression of different synaptic and neuronal markers described above in the 3xTg-AD mice as % of WT showed that synaptic compensation happened in different brain regions at different time points (Fig. 7, Supplementary Table 2). Indeed, for the synaptic marker synaptophysin, for example, synaptic compensation in the CA3 region happened at 14 weeks, while in the parietal and frontal cortices, compensation happened at 13 weeks. Supplementary Table 2 shows the significances of regional changes for synaptic and neuronal markers at different ages.

DISCUSSION

Dementia status in AD is preceded by a relatively long and variable period of MCI. In some individuals, the MCI stage does not progress to AD. This variable MCI stage is probably due to the compensatory neuroregenerative response of the AD brain which, because of an insufficient neurotrophic environment, does not succeed completely [41, 42]. Histopathological studies have shown that the compensatory phenomenon does occur in the AD brain [20].

The present study revealed that the 3xTg-AD mice are cognitively impaired at around 12–14 weeks of age or even before, which is in agreement with recent reports [30, 31]. We further show that the spatial reference memory impairment seen in the young adult 3xTg-AD mice is accompanied by a decrease in the presynaptic marker synaptophysin, the dendritic marker MAP2, the neuronal marker β-III tubulin, and a trend for the postsynaptic marker GluR1. Since MAP2 is located preferentially in the somatodendritic compartment, not only in dendritic spines, and β-III-tubulin is expressed preferentially in the somato-axonal compartment, not only in the presynaptic bouton, the changes in synaptophysin may be due to changes (increase and loss) in dendritic branches and the changes in GluR1 levels may be due to changes (increase and loss) of axons. Most importantly the drop in the synaptic and dendritic markers was seen when the 3xTg-AD mice were compared across time from 10–16 weeks of age. Almost all the above synaptic and neuronal markers except GluR1 show decrease in the expression at 12 weeks of age. However, like in the AD brain, the 3xTg-AD mice show a transient increase in the expression of these markers from 13 to 16 weeks of age. This transient increase in the different markers was able to partially reduce impairment in spatial reference memory. We speculate that this increase to compensate for synaptic deficit is similar to the failed attempt for self-repair by increasing neurogenesis described in the AD brain [41]. Beyond the age of 4 months, the 3xTg-AD mice are known to have a decrease in synaptophysin as well as an apparent synaptic dysfunction [29, 37]. We found that different brain regions compensated for the synaptic deficit at different time points. The comparison of the WT mice across time showed changes in these markers from 10–16 weeks of age. The changes seen in WT mice showed different kinetics from those seen in 3xTg-AD mice. The increase in the expression and the level of synaptophysin beyond 12 weeks in cognitively impaired 3xTg-AD mice seen in this study is in agreement with what was reported previously in the APP_K670_N,M671L+ PSl_M146L double transgenic mice [22, 23]. Similarly, an increase in the levels of expression of PSD95 and GluR1 compared to control animals was shown to be associated with spatial reference memory impairment in aged rats [43].

Consistent with the present study, a previous study using human postmortem brains reported an increase in the expressions of synaptophysin, SNAP-23, MAP2, and α-synuclein in AD postmortem brains at Braak stage 3 and 4. A sharp decrease in synaptophysin expression in AD was seen only at Braak stages 5 to 6 with the full spectrum of Aβ and tau pathologies [20]. An increase in the expression of PSD-95 in the entorhinal cortex of AD cases and presynaptic cholinergic bouton density in the midfrontal gyrus of MCI patients compared to age-matched controls was also previously reported [44, 45]. Similarly, an increase in the expression of the synaptic protein debrin in the superior frontal cortex of MCI patients, followed by a decrease of 40–60% when the disease progresses was reported [25]. The agreement between human and animal studies indicates that the upregulation in synaptic, dendritic, and neuronal markers found in the present study is most probably due to a compensatory phenomenon initiated by the diseased brain to save the remaining intact synapses.

In light of what was found previously in human subjects, findings of the present study in 3xTg-AD mice could be mimicking what happens in AD patients at early stages of the disease. For instance, at the age of 12–14 weeks, the 3xTg-AD mice are considered to be in the early stages of AD-like disease. They have only intracellular Aβ and cognitive impairment; Aβ plaque and tau pathologies are seen only at 9–12 months onwards in these animals.

Several mechanisms of synaptic compensation were proposed in the literature. For instance, the diseased brain can compensate for synaptic and neuronal loss by adjusting the level of firing of the individual neuron and the global neuronal network [46 –49]. Compensation also could happen by increasing the size of the surviving synapses so that the total synaptic area per unit volume remains intact [21, 28]. Compensation by activation of the silenced synapses following the death of inhibitory neurons and an increase in neurogenesis in the dentate gyrus was also reported [50]. Neurite outgrowth as a response to synaptosis has been proposed [51] and coupling between astrocytes and neurons also was proposed to contribute to global synaptic homeostasis [52].

There are three major points of view concerning the role of synaptic compensation. One view considers the phenomenon to be beneficial, helping to slow down the progression of the disease [53]. The second view considers it as detrimental because it affects the brain network and drives disease progression [43 , 55]. The third one considers compensation as beneficial at the early stage of the disease because it slows down its progression [56, 57]. However, in the long run it is detrimental because it becomes the driving force to affecting the whole neuronalnetwork.

We speculate that in the present study the decrease in synaptophysin, MAP2, and β-III tubulin at 12 weeks of age in the 3xTg-AD animals compared to WT and the transient increase in these three markers from 13 to 16 weeks of age is due to a compensatory mechanism in which the diseased brain attempts to repair itself. Because the disease is not fully developed in the young 3xTg-AD mice, the brain still has the capacity to compensate for the synaptic loss. This compensatory mechanism may be the biological basis of slowing down the development of the AD-like pathological features in this mouse model. Indeed, it is known that the 3xTg-AD mouse develops Aβ and tau pathologies only at an advanced age: Aβ pathology at ∼9 months and tau pathology at around 12 months. Thus, it takes several months for these pathological features to develop, and this is considered a long enough period for the progression of the pathologies. We speculate that a similar phenomenon may be occurring in asymptomatic AD or MCI patients’ brains. In fact, the mild cognitive impairment period is relatively long, often lasting several years, and this may be due to the compensation or the self-repair attempt of the brain. However, as the disease progresses the brain is overwhelmed and synaptic deficit reaches a certain threshold level beyond which the brain cannot compensate any more. Perhaps this is why the acceleration of cognitive decline and the appearance of clinical features of AD occur at an advanced stage.

We believe that the unsuccessful attempt to compensate for synaptic deficit is probably due to the lack of the neurotrophic environment in the affected brain. For this reason, prevention strategies starting at an early stage of the disease by boosting the neurotrophic environment through providing a neurotrophic support could be more effective in helping the brain to sustain its attempt to self-repair than intervention at a later stage after a drastic synaptic and neuronal loss is reached. Once the brain has undergone a severe insult of synaptic and neuronal loss, repair would be more difficult than prevention at an early stage of the disease.

Footnotes

ACKNOWLEDGMENTS

We are grateful to Dr. George Merz for his help with confocal microscopy, to Dr. Fei Liu for her critical reading of the manuscript and to Ms. Janet Murphy for secretarial assistance. These studies were supported in part by a Graduate Student Fellowship from the Center for Development in Neuroscience Graduate Program, the New York State Office for People with Developmental Disabilities, and Zenith Award ZEN-12-241433 from Alzheimer’s Association, USA.

Authors’ disclosures available online ().

Appendix

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