Toxoplasma gondii alters NMDAR signaling and induces signs of Alzheimer’s disease in wild-type, C57BL/6 mice

Eight- to 12-week-old C57BL/6 (wild-type) male and female mice were purchased from Jackson Laboratories. Mice were bred and housed under specific pathogen-free conditions in the animal facility at Cornell University.

Mice were randomly assigned to mock-infected and T. gondii-infected groups. T. gondii was orally infected following the infection protocol from Mahamed et al. [6]. Briefly, wild-type mice were infected with 10 T. gondii ME49 cysts which were maintained in Swiss Webster mice and isolated before infection. Survival rates and weight loss were monitored daily. For our experiments, groups of mice were euthanized at 15, 30, 60, and 90 days post infection and tissues (brain, spleen, spinal cord) were collected for further processing.

Mice were terminally anesthetized using a ketamine-xylazine cocktail and perfused with ice-cold 4% paraformaldehyde (PFA), and tissues were collected and embedded with OCT. Samples were also flash frozen in liquid nitrogen for further analysis. For immunohistochemistry, sections were cut at 5–8-um thickness and post-fixed with 4% PFA.

For thioflavin S plaque staining, frozen sections were fixed with 4% PFA, stained with primary antibody, anti-T. gondii antibody (1:1000). Samples were washed twice with PBS and incubated with fluochrome conjugated secondary antibody for 1 h. Samples were washed twice and incubated with 1% ThS (Sigma Aldrich) before differentiating in 70% ethanol. Slides were washed in distilled water mounted and coverslipped with DAPI. Sections were imaged and quantified for ThS⁺ immunoreactivity in infected and non-infected mice brains.

Mouse embryonic cortical neurons were purchased from Life Technologies (Cat# A15586) and cultured for 6 days before infection as per manufacturer’s instructions. mCherry pru strain of T. gondii (kind gift of Dr. Denkers, Cornell University, Ithaca) was propagated in HS27 cells. Media of neuronal cells were changed every 3 days and cells were infected with 1 multiplicity of infection (MOI) of mCherry pru strain of T. gondii for up to 3 days. Cells were washed with ice-cold PBS twice and fixed with 4% PFA for 20 min and blocked with 5% goat serum for 1 h. Cells were then incubated overnight with anti-MAP-2 (MAB3418, Millipore, 1:200) and anti-NMDAR (Abcam, Cat# ab17345, 1:200) and washed twice with PBS. Cells were then incubated with fluorochrome conjugated secondary antibodies, washed, and coverslipped with prolonged gold DAPI for imaging.

Sociability in mock-infected and T gondii-infected mice was measured once during the animal’s light cycle at day 60 post infection. We used a modified version of the Crawley’s sociability and preference for social novelty test that has been described previously [21]. The same open field arena (18 × 18 × 18 in.) used for the open field test was used to test social behavior. Two identical wired cups were placed in opposite sides of the arena. The test was divided into two 10-min sessions. In session 1, a mouse (stranger mouse 1) was placed under one of the wired cups, while the second wired cup located in the opposite side was left empty. The duration of the active contacts between the test mouse and both the empty cup and the cup containing stranger mouse 1 was recorded. An active contact was defined as any instance in which the mouse touched the wired cup with its snout or paws. In session 2, a second (novel) mouse was placed under the cup that had been empty during session 1. The duration of active contacts between the test mouse and both the familiar mouse and the novel mouse was recorded. The stranger mice and the subject mice were the same genotype, sex, weight, and age and were not littermates of each other. Both sessions were videotaped and manually analyzed by an observer blinded to the treatment conditions.

Animals were habituated by placing each mouse in a clean, empty cage as described previously [22]. After 15 min, the mouse was moved to a new empty cage and allowed to habituate for 15 min. This process was repeated once more before the mouse was placed in the final cage for another 15 min prior to testing. The mouse was then presented with a square piece (2 × 2 in.) of filter paper that was impregnated with either an attractive scent (peanut butter, in concentrations ranging from 2.5 to 10%) or an aversive scent (10% trichloroacetic acid (TCA)). A piece of filter paper impregnated with water was used as control. The mouse was exposed to each scent for 3 min and the session was videotaped. The amount of time the mouse spent sniffing the filter paper was then recorded. The scents were presented in the following order: water, 2.5% peanut butter solution, 5% peanut butter solution, 10% peanut butter solution, and 10% TCA. After being exposed to a particular scent, each mouse was returned to a clean, empty cage for another round of habituation. Both the test and the analysis were carried out by an experimenter blinded to the treatment conditions.

All statistics were performed using GraphPad Prism 6 for Windows. All quantifications and analyses were performed by an observer blinded to the treatment conditions. Intensity quantification of NMDAR, Neurotracer, VGLUT1/2, and GAD67 were performed using Zen Digital Imaging software and were analyzed by one-way ANOVA followed by Bonferroni’s post hoc test. Quantification of 6E10⁺ signal, Thioflavine S⁺ signal, TUNEL⁺ neurons, TUNEL⁺ T. gondii cysts, and BAG1⁺ cysts was manually performed and analyzed using one-way ANOVA followed by Bonferroni’s post hoc test. Densitometry analysis of AT8, NMDA, VGLUT1/2, and GAD67 was performed using ImageJ and analyzed by one-way ANOVA followed by Bonferroni’s post hoc test. Open field data were analyzed by two-tailed Student’s t tests with Welch’s correction for samples having possibly unequal variances. Data from the social behavior test and the olfactory sensitivity test were analyzed by two-way ANOVA followed by Bonferroni’s post hoc test to correct for multiple comparisons. Barnes maze data were analyzed using two-way repeated measures ANOVA followed by Bonferroni’s post hoc test. The number of animals of each sex used in each experiment is specified in the figure legends. Data were presented as mean ± SEM. Data were considered statistically significant when p < 0.05.

The high prevalence of T. gondii-infected individuals and its association with neurodegenerative diseases led us to investigate whether the parasite had the capability to induce any of the major pathological signs of AD such as Aβ deposition or pTau in C57BL/6 wild-type mice. We infected wild-type mice with T. gondii and collected brains from control (mock-infected) and infected mice on days 15, 30, 60, and 90 post infection. Day 15 post infection is the pre-chronic infection stage when few parasites are found in the brain, while days 30, 60, and 90 represent the chronic (cyst) stage [23]. By day 15 post infection, we observed a few T. gondii-stained cysts associated with neurons (labeled with neurotracer) in the cortex and in the hippocampus of infected mice (Fig. 1a). There was a marked decrease in neurotracer intensity in the infected mice versus mock-infected controls. Some areas of the cortex showed no neurotracer signal, indicating a loss of neurons. Magnified images of the cortex and hippocampus showed an even more pronounced difference in neurotracer intensity. However, in control mice, those areas showed a more dense and compact neuronal staining (Fig. 1a). This suggests that T. gondii is associated with neuronal cells and may contribute to neuronal loss. Others have shown that T. gondii is not only associated with neurons [24, 25] but also with microglia and astrocytes [26‐28]. To determine the distribution of T. gondii cysts throughout the brain, we scanned the entire brain of T. gondii-infected and mock-infected controls from days 15 to 90 post infection and quantified numbers of T. gondii cysts. Representative images of BAG1⁺ cysts in the cortex at days 30 and 60 are shown (Fig. 1b). We observed increasing frequency of T. gondii cysts stained with BAG1 antibody and/or T. gondii antigen at days 15, 30, 60, and 90 post infection (Fig. 1b). There were significantly more BAG1⁺cysts at day 90 compared to days 15 and 30 (F(3,12) = 8.487, p = 0.0027, one-way ANOVA). Moreover, T. gondii cysts were prominently clustered in specific brain regions (Fig. 1b), including the pre-frontal cortex and the olfactory bulb, two areas where others have observed cyst formation [29, 30]. These brain areas are also prominently associated with cognitive function and chemical signal response [31].

We next determined whether T. gondii infection induced signs of AD in wild-type mice. We therefore analyzed the brains of T. gondii-infected mice for signs of Aβ plaques, which is one of the major hallmarks of AD (Fig. 2). We stained brains from T. gondii-infected mice and mock-infected controls with 6E10, an antibody that is reactive to amino acid residue 1–16 of human beta amyloid [32]. Intriguingly, we observed that the brains of T. gondii-infected mice stained positive for 6E10, whereas no sign of 6E10 staining was observed in mock-infected controls (Fig. 2a and Additional file 1: Figure S1). Moreover, there was co-localization of 6E10 and BAG1-positive T. gondii cysts as early as day 15 post infection (Fig. 2a). We next determined whether Aβ staining co-localized only with T. gondii cysts or whether widespread Aβ immunoreactivity was found in other areas of the brain where T. gondii cysts were not present. Indeed, we observed Aβ immunoreactivity in other areas of the brain where they did not co-localize with cysts at days 60 and 90 post infection (Fig. 2a, d). One-way ANOVA did not reveal a significant difference in 6E10⁺ signal (F(2,6) = 3.064. p = 0.1211). However, direct group comparisons between 6E10⁺ signal at days 30 and 90 showed that 6E10⁺ signal in the cortex increased significantly (p = 0.0450, two-tailed student’s t test, Fig. 2b). 6E10⁺ signal also increased significantly in the hippocampus from day 30 to day 90 (F(2,7) = 10.22, p = 0.0084, one-way ANOVA. Fig. 2c). By 90 days post infection, quintessential plaque morphology was seen with 6E10 staining (Fig. 2a) surrounding cysts and also near cysts. We stained day 60 brain sections with normal mouse IgG and normal rabbit IgG to confirm antibody specificity of the 6E10 and BAG1 antibodies, respectively. We observed no 6E10 or BAG-1 signal in the brains at day 60 post infection, indicating that the antibodies were specific (Additional file 1: Figure S1). To further confirm the presence of Aβ immunoreactivity, we next stained brains of T. gondii-infected and mock-infected mice with Thioflavin S, a dye that fluoresces an apple green color when specifically bound to Aβ (Fig. 2d). We stained day 60 infected mice brains with Thioflavin S and observed strong Thioflavin S immunoreactivity surrounding T. gondii cysts, confirming that T. gondii induces Aβ immunoreactivity in infected mice but not in mock-infected controls (Fig. 2d).

Since the 6E10 antibody we used is reactive to amino acid residues 1–16 of human beta amyloid, we wanted to test whether we could see Aβ plaques in our mouse samples when using an antibody reactive to mouse beta amyloid (anti-beta amyloid antibody, Abcam (Cat#2539)). We observed anti-beta amyloid signal in the brain cortex at days 30 and 60 post infection and found BAG-1 signal in comparable regions of the cortex. (Additional file 2: Figure S2A). We also observed 6E10 signal in tissue sections from the same animals we used to look for mouse Aβ but did not find any 6E10 signal when using isotype controls (Additional file 2: Figure S2B). Together, these findings show that T. gondii infection was capable of inducing signs of neurodegeneration, as well as, Aβ immunoreactivity, which is one of the major hallmarks of AD.

We investigated whether T. gondii infection induced Tau hyperphosphorylation (pTau), another major hallmark of AD [33]. Tau is a microtubule-associated protein that is involved in the transport of neurotransmitters through axons [33]. Phosphorylation of Tau protein forms neurofibrillary tangles in neuronal cells causing collapse of the cytoskeleton, thereby hindering neurotransmission. To determine whether T. gondii induced pTau expression, we performed immunohistochemistry using the AT8 antibody which specifically stains pTau [34]. We observed pTau staining in the brains of T. gondii-infected mice beginning at day 15, with increased staining intensity by day 60 post infection (Fig. 3a, b). The intensity of pTau signal increased as the infection progressed, thus, day 60 infected mice had significantly stronger pTau staining compared to the brains at day 15 or 30 post infection (Fig. 3a, b). This was most pronounced in the cortex and hippocampus, which are areas that are critically involved in memory and cognition [35, 36]. We also saw strong pTau staining in the choroid plexus of infected animals at day 30 (Fig. 3b). We confirmed these results by Western blot using protein samples from the brains of mock-infected and T. gondii-infected mice at days 30 and 60. We found a significant increase in protein levels of AT8 in the brains of infected mice at day 60 post infection (F(2,9) = 5.895, p = 0.0231, one-way ANOVA. Fig. 3c–d). Based on these cumulative data, we conclude that infection by T. gondii directly induces major pathological hallmarks of AD.

Glutamate is the major excitatory neurotransmitter in the CNS and it is released at a high rate at synapses in the brain. Glutamate may be endocytosed or released by glutamatergic NMDARs that are widely expressed on neuronal cells and functions in maintaining CNS homeostasis [37, 38]. NMDAR plays a predominant role in controlling synaptic plasticity including learning and memory function; and NMDAR dysfunction is widely implicated in the pathophysiology of AD [39]. Given the strong evidence showing that Aβ causes neuronal pathology and dysfunction that can be abrogated in the presence of NMDAR antagonism [40], we hypothesized that the Aβ immunoreactivity found in infected mice may alter NMDAR expression. We examined the brains of infected and mock-infected wild-type mice on days 15, 30, and 60 post infection to evaluate NMDAR expression with an antibody against the NMDAR subunit 1 (NR1) and co-stained with neurotracer. Intriguingly, at days 15 and 30, we observed a significant decrease in NMDAR expression intensity especially in the cortex and hippocampus, when compared to controls (Fig. 4a–c). This effect was even more pronounced at day 60, which showed a dramatic decrease in NMDAR expression (Fig. 4a–c). Loss of NMDAR signal was also consistent with a significant decrease in neuronal signal suggesting that NMDAR loss was related to neuronal loss (Fig. 4a, d, e). We also observed that NMDARs co-localized directly with T. gondii cysts, further supporting the notion that T. gondii is responsible for NMDAR loss (Fig. 4h). We tested the effect of T. gondii infection on protein levels of NMDAR by Western blot using protein samples from the brains of mock-infected and T. gondii-infected mice. We found significantly reduced protein levels of NMDAR in the brains of infected mice at day 60 post infection (F(2,12) = 4.906, p = 0.0277, one-way ANOVA, Fig. 4f, g). We also observed that NMDARs co-localized directly with T. gondii cysts, further supporting the notion that T. gondii is responsible for NMDAR loss (Fig. 4h). To further confirm whether T. gondii infection directly caused loss of NMDAR on neurons, we infected neuronal cells in vitro with 1 MOI of T. gondii (mCherry pru strain). Mock-infected cells were used as controls. We observed a similar decrease in NMDAR expression on infected neurons for up to 72 h post infection (Fig. 4i). Intriguingly, any remaining NMDAR expression was tightly clustered around T. gondii cysts both in vivo and in vitro, and by 72 h, there was almost complete loss of NMDAR signal (Fig. 4i). This decrease in NMDAR signal may be similar to NMDAR antagonism that may lead to receptor under-excitation and hypofunction resulting in cognitive impairment, similar to what is observed in AD [15]. Since T. gondii cysts were in close contact with NMDAR (Fig. 4i), it is reasonable to hypothesize that the parasite interaction with NMDAR hindered normal neurotransmission, although this hypothesis needs further investigation. The potent downregulation or loss of NMDAR is strongly indicative of the Aβ-induced synaptic depression and impaired synaptic plasticity that is seen in AD [41, 42].

Glutamate uptake and transport are mediated by vesicular glutamate transporters (VGLUTs). VGLUT1 functions in neurotransmission and VGLUT2 functions in synaptic plasticity. In AD patients, VGLUT expression and function are impaired in the pre-frontal cortex [43], and olfactory dysfunction and atrophy are one of the early signs of AD [20]. Olfactory dysfunction causes a loss of sensory input in the entorhinal cortex and hippocampus [20]. The excitatory VGLUT2 is a molecular marker for glutamatergic olfactory sensory neurons, which transmit information from the nose to the brain [44, 45]. Since the olfactory bulb was heavily infected with T. gondii cysts, and olfactory dysfunction is one of the early indicators of AD, we examined VGLUT2 expression in the olfactory bulb on days 30 and 60 post infection. Compared to control mice that expressed their full complement of VGLUT2 in the olfactory glomeruli, we observed that more than two thirds of olfactory VGLUT2 expression were lost in T. gondii-infected mice on days 30 and 60 (Fig. 5a, b). This loss of olfactory VGLUT2 is indicative of extensive loss of olfactory sensory neurons in infected mice. Interestingly, there was also a notable decrease in VGLUT2 expression in the hippocampus where T. gondii cysts were also present (data not shown). This dramatic loss of VGLUT2 expression in the olfactory glomeruli is indicative of severe glutamatergic dysfunction and disturbance of synaptic plasticity that usually leads to cognitive impairment. On the other hand, VGLUT1 expression significantly increased in areas of the brain where VGLUT2 expression was decreased, suggesting that VGLUT1 may be compensating for the loss of VGLUT2 in these areas (F(2,27) = 15.78, p < 0.0001, one-way ANOVA, Fig. 5a, c). Western blots using whole-brain samples from control and infected mice at day 60 revealed unchanged levels of VGLUT2 protein (F(2,14) = 1.422, p = 0.2739, one-way ANOVA, Fig. 5g, i) and significantly increased levels of VGLUT1 in the infected brains compared to controls (F(2,14) = 4.327, p = 0.0344, one-way ANOVA, Fig. 5g, h).

GAD67 (l-glutamic acid decarboxylase67) is one of two enzymes that generates γ-amino butyric acid (GABA). GAD67 is responsible for synthesizing more than 90% of GABA in the brain and its activity is rate limiting [46]. Alteration in GABAergic neurotransmission is associated with AD, as an increase in the expression of GAD67 was found in the brains of AD patients and in AD transgenic mice [47]. To determine whether GABAergic signaling was altered in infected mice, we examined the expression level of GAD67 in the brains of mock-infected and T. gondii-infected mice on days 30 and 60 post infection. GAD67 was upregulated in discrete regions of the infected mouse brain including the olfactory bulb (Fig. 5d–e). The overall expression of GAD67 was significantly increased over control at day 60 post infection (F(2,24) = 10.45, p = 0.0005, one-way ANOVA). Protein levels of GAD67 were also moderately increased at days 30 and 60 post infection (F(2,3) = 2.002, p = 0.2803, one-way ANOVA, Fig. 4g, j). In a recent report, GAD67 expression was displaced from the synaptic termini and redistributed throughout the neuropil in T. gondii-infected mice [48]. Thus, it is plausible that GAD67 increase is an attempt to re-establish GABAergic nerve terminals in areas where neurotransmission is most deficient. Interestingly, the increased GAD67 expression occurred at sites where T. gondii cysts were most prevalent (Fig. 5f), suggesting that T. gondii cyst formation caused alterations in intracellular GABA production. Furthermore, this is consistent with previous reports that showed that T. gondii uses GABA as its carbon source for its metabolism [49].

To determine whether T. gondii infection led to cell death in infected mice, we pinpointed areas in the prefrontal cortex that specifically contained T. gondii cysts and double stained with antibodies to both TUNEL and NeuN. We observed co-localization of both TUNEL and NeuN in the areas where T. gondii cysts were present (Fig. 6a). This indicates that T. gondii induced neuronal cell death. In addition, the number of NeuN⁺ TUNEL⁺ cells increased significantly as infection progressed (F(2,22) = 17.20, p < 0.0001, one-way ANOVA, Fig. 6c). We also observed areas in the olfactory bulb that were positive for TUNEL but not for NeuN, suggesting that other cell types were also undergoing apoptosis (Fig. 6b). However, the percentage of NeuN⁺ TUNEL⁺ cells was significant after infection compared to the control. We surmise these other cell types are astrocytes and/or microglia as previous reports have shown significant damage to these cells post infection [36]. We next asked whether similar cell death occurred in the olfactory bulb, which was heavily infected with T. gondii cysts. Indeed, we observed strong TUNEL⁺ staining in the entire olfactory bulb (Fig. 6b), suggesting that neuronal cells in the olfactory bulb were undergoing cell death, which is consistent with the loss of olfactory VGLUT2 staining in the olfactory glomeruli (Fig. 5a). Moreover, we observed that some areas of the olfactory bulb were devoid of cells noted by the loss of DAPI staining (data not shown), indicating that cell death had already occurred in those areas. Control sections show the presence of cells in these olfactory regions.

With disruption of NMDAR, alterations of GAD67 and extensive damage to neuronal cells, we set out to determine if there were any behavioral changes affecting mice infected with T. gondii cysts. We observed T. gondii clusters in both the prefrontal cortex and the hippocampus, two brain regions that have been linked to regulation of social behavior [50, 51]. To determine whether the effects of T. gondii infection extended to changes in social behavior, we used a modified version of the Crawley’s sociability and preference for social novelty test previously described [21]. Mock-infected mice displayed a preference for the cup containing stranger mouse 1 over the empty cup, which is indicative of normal social behavior (Fig. 7a). Two-way ANOVA revealed that T. gondii-infected mice spent significantly more time with the cup containing stranger mouse 1 over the empty container, also indicative of normal behavior (F(1,40) = 7.553, p = 0.0089, Fig. 7a). Specific two-group comparisons revealed that control mice preferred the container with the novel mouse (stranger mouse 2) over the familiar mouse (stranger mouse 1) in the second phase of the experiment, and this difference was significant (p = 0.0229, two-tailed Student’s t test). However, T. gondii-infected animals did not show a preference for either container, indicating an alteration in recognition of social novelty (p = 0.5461, two-tailed Student’s t test, Fig. 7b).

To understand whether the observed effect was mediated by hyperactivity or motor impairments in the infected animals, we assessed the activity of the same cohort of animals in an open field test environment. T. gondii infection did not affect the distance traveled (Fig. 7c, p = 0.9669, two-tailed Student’s t test) or average velocity (Fig. 7d, p = 0.9866, two-tailed Student’s t test) in the open field. However, T. gondii-infected animals spent more time in the corners of the open field (Fig. 7e, p = 0.0896, two-tailed Student’s t test) and significantly less time in the center compared to mock-infected controls (Fig. 7f, p = 0.0365, two-tailed Student’s t test). This behavioral outcome has been associated with increased anxiety [52] and is also common among AD patients [53]. Taken together, these results indicate that T. gondii-infected mice had normal motor activity but displayed anxiety-like behavior and failed to recognize social novelty.

Since VGLUT2 expression was reduced in T. gondii-infected mice and since olfactory dysfunction is one of the early indicators of AD, we examined the effects of T. gondii infection on olfactory sensitivity on the same group of animals in which social behavior was measured using a protocol that has been described previously [22]. The effect of T. gondii infection on olfactory sensitivity varied depending on sex. In contrast to infected male animals, mock-infected males showed a clear concentration-dependent preference for the peanut butter solution and a clear dislike for the aversive scent, trichloroacetic acid (TCA), indicating a loss of olfactory sensitivity in male mice infected with T. gondii ((F(1,75) = 6.800, p = 0.0110, two-way ANOVA, Fig. 8a). Overall, control female mice spent less time sniffing each scent compared to control male mice, although they did show a preference for the 10% peanut butter solution over both water and the aversive scent, TCA (Fig. 8b). Infected female mice displayed similar behavior compared to their respective controls and there was no significant main effect of experimental condition (F(1,105) = 0.0005111, p = 0.9820, two way-ANOVA, Fig. 8b), indicating that olfactory sensitivity in infected female mice was not significantly affected. Thus, olfactory sensitivity was reduced in male mice but it is intact in female mice.

The brains of T. gondii-infected mice showed a significant reduction in the expression of NMDAR, which is heavily involved in the formation of spatial and contextual memories. We tested the effect of T. gondii infection on spatial learning and exploration in the Barnes maze using a new cohort of mice. Control and infected animals were tested twice per day for five consecutive days. Testing sessions were separated by 15 min and data from both sessions were averaged for analysis. A main effect of time revealed that performance of the control mice improved over the 5-day testing period (F(4,64)  =  7.045, p  <  0.0001, two-way repeated measures ANOVA), while infected mice showed only a mild improvement in remembering the escape box location by day 5 of testing (Fig. 9a). Control mice were able to find and enter the escape box in significantly less time compared to infected animals at days 2, 3, 4, and 5 of testing (F(1,16) = 51.32, p  <  0.0001, two-way repeated measures ANOVA), indicating that spatial learning and memory in T. gondii-infected mice were severely impaired (Fig. 9a, b). These same animals moved a similar distance (p = 0.5222, two-tailed Student’s t test, Fig. 9c) and moved at a similar velocity (p = 0.5477, two-tailed Student’s t test, Fig. 9d) in the open field compared to the control mice, indicating that motor deficits cannot explain the impairment in spatial memory we observed. As with previous experiments, infected mice spent significantly more time in the corners of the open field (p = 0.0297, two-tailed Student’s t test, Fig. 9e) and less time in the center (p = 0.1028, two-tailed Student’s t test, Fig. 9f), indicating anxiety-like behavior.