Toxoplasma gondii alters NMDAR signaling and induces signs of Alzheimer’s disease in wild-type, C57BL/6 mice
- Luisa Torres†1,
- Sudie-Ann Robinson†1,
- Do-Geun Kim†1,
- Angela Yan1,
- Thomas A. Cleland2 and
- Margaret S. Bynoe1Email author
© The Author(s). 2018
Received: 30 January 2018
Accepted: 1 February 2018
Published: 23 February 2018
Alzheimer’s disease (AD) is a progressive neurodegenerative disease associated with cognitive decline and complete loss of basic functions. The ubiquitous apicomplexan parasite Toxoplasma gondii (T. gondii) infects up to one third of the world’s population and is implicated in AD.
We infected C57BL/6 wild-type male and female mice with 10 T. gondii ME49 cysts and assessed whether infection led to behavioral and anatomical effects using immunohistochemistry, immunofluorescence, Western blotting, cell culture assays, as well as an array of mouse behavior tests.
We show that T. gondii infection induced two major hallmarks of AD in the brains of C57BL/6 male and female mice: beta-amyloid (Aβ) immunoreactivity and hyperphosphorylated Tau. Infected mice showed significant neuronal death, loss of N-methyl-d-aspartate receptor (NMDAR) expression, and loss of olfactory sensory neurons. T. gondii infection also caused anxiety-like behavior, altered recognition of social novelty, altered spatial memory, and reduced olfactory sensitivity. This last finding was exclusive to male mice, as infected females showed intact olfactory sensitivity.
These results demonstrate that T. gondii can induce advanced signs of AD in wild-type mice and that it may induce AD in some individuals with underlying health problems.
Alzheimer’s disease (AD) is a progressive neurodegenerative disease associated with decline in cognitive function [1, 2]. There are 5.1 million cases of AD in the USA and almost 2 million people suffering from dementia and cognitive decline . This number will almost triple by the year 2050 due to increased numbers of individuals living beyond 85 years . Major pathological hallmarks of AD include senile amyloid plaques composed of amyloid β (Aβ) protein and intracellular neurofibrillary tangles, with characteristic reactive microgliosis and astrogliosis [1, 2]. AD is also characterized by dystrophic neuritis, neuronal loss, synaptic dysfunction, and cerebral atrophy . Viruses (herpes simplex virus 1), bacteria (Chlamydia pneumoniae), and parasites such as Toxoplasma gondii (T. gondii) are implicated in neurodegenerative diseases including Parkinson’s and AD [3, 4].
T. gondii is an obligate intracellular protozoan pathogen that enters the central nervous system (CNS) following its initial invasion and replication in the gut [5, 6]. T. gondii infects an estimated one third of the adult population worldwide, although seroprevalence varies significantly by country [7, 8] and is highly dependent on place of birth, educational level, living conditions, occupation, race, and ethnicity [9, 10]. Emerging studies implicate T. gondii as a contributor to Parkinson’s disease, schizophrenia, obsessive compulsive disorder, and Tourette syndrome . Little is known about the long-term impact of T. gondii infection on neuronal cells, and their receptors such as the N-methyl-d-aspartate receptor (NMDAR), which is involved in synaptic plasticity and cognition. In response to neural activity, the NMDAR mediates the strength of synaptic transmission . Synaptic loss is one of the major characteristics of AD and AD-related dementias . Studies have shown that NMDARs mediate the downstream pathological effects of Aβ in animal models of AD. For instance, application of Aβ to neurons results in a rapid decrease in NMDAR function and expression . This suggests that NMDAR is critical for maintaining neuronal function.
Glutamate, a major excitatory neurotransmitter, constitutes about 70% of all excitatory synapses in the CNS, and as such, it is tightly regulated . Vesicular glutamate transporters, VGLUT1, 2, and 3, mediate the uptake of glutamate via synaptic vesicles . VGLUT1 functions in neurotransmission while VGLUT2 plays a role in synaptic plasticity and protection from neuronal injury . γ-Aminobutyric acid (GABA) is one of the major inhibitory neurotransmitters found in the adult brain and is synthesized from the conversion of glutamate via the catalytic decarboxylase GAD67. This mainly occurs in the presynaptic area of GABAergic neuron terminals in the brain . Defects in GABAergic signaling are known to cause seizures and have links to AD, temporal lobe epilepsy, and schizophrenia.
We set out to determine whether T. gondii infection induces signs of AD in C57BL/6 (wild-type) mice including Aβ production, which is known to alter NMDAR signaling . Here, we show that T. gondii causes signs of neurodegeneration in wild-type mice. We demonstrate that as infection progresses, T. gondii induces loss of NMDAR signal with concomitant induction of AD pathology including production of hyperphosphorylated Tau and Aβ immunoreactivity in the brain. We also observed neuronal death in the olfactory bulb accompanied by alterations in olfactory sensitivity which is known to occur in patients with AD . The effects on the latter varied depending on sex: While olfactory sensitivity was reduced in male mice, females showed intact olfactory ability. Infected mice also showed alterations in social behavior, anxiety-like symptoms, and alterations in spatial learning. Thus, T. gondii can directly confer neurodegenerative and behavioral signs of AD in infected wild-type 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.
T. gondii infection in mice
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. . 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.
Antibodies and reagents
Anti-APP antibody (6E10) was purchased from Covance (Cat# SIG-39320-1000). Anti-beta amyloid was purchased from Abcam (Cat#2539). Anti-T. gondii antibody was purchased from US biologicals (Cat# T8075-01). BAG1 was a gift from Dr. Dubey. Normal mouse IgG (SC-2025) and normal rabbit IgG (SC-2027) were used as isotype controls to test the specificity of the 6E10 and BAG1 antibodies, respectively. Anti-phospho-Tau antibody (AT8) was purchased from Thermofisher (Cat# MN1020). Anti-NeuN was purchased from Cell Signaling (Cat# 12943S) or Abcam (ab104224). Anti-VGLUT2 (Cat#71555S), anti-GAPDH (Cat# 2118), and anti-Tau (Cat#4019) antibodies were purchased from Cell Signaling. Anti-NMDAR (Cat# ab17345), anti-VGLUT1 (Cat# ab134283), and anti-GAD67 (Cat# ab26116) antibodies were purchased from Abcam. Thioflavin S was purchased from (Sigma Aldrich) and neuro-tracer from Life Technologies.
Immunoblots were performed on whole brain lysates from mock-infected and T. gondii-infected mice. The protein content was quantified by Bradford assay (Bio-Rad). Aliquots of samples were denatured and reduced with sodium dodecyl sulfate (SDS), and β-mercaptoethanol and equivalent concentrations of total protein were separated by SDS-PAGE. The proteins were transferred onto a nitrocellulose membrane. Non-specific protein binding was blocked by incubation with 5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h at room temperature. The membranes were incubated with primary antibodies overnight at 4 °C (anti-NMDAR, anti-VGLUT1, anti-VGLUT2, anti-GAD67, anti-Tau, anti-phospho Tau, and anti-GAPDH), washed, and then probed with either goat anti-rabbit IgG human ads-HRP antibody (Southern Biotech, 1:2000 or goat anti-mouse IgG human ads-HRP antibody (Southern Biotech, 1:2000). Densitometry was performed on images of immunoblots for quantification with image J software.
Preparation of samples for histology
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.
IHC and IFA
In general, frozen samples were washed twice with PBS and blocked with 10% goat serum/casein and incubated with anti-T. gondii primary antibodies, anti-RH strain or anti-BAG1 (1:1000), anti-NMDAR (1:200), anti-VGLUT1 (1:100), anti-VGLUT2 (1:50) or anti-GAD67 (1:100), anti-6E10 (1:200), anti-beta amyloid (1:200), anti-pTau (AT8) (1:1000), and anti-NeuN (1:200) overnight at 4 °C. Samples were washed twice with PBS and incubated with fluorochrome-conjugated secondary antibodies or horseradish peroxidase for 1 h for immunofluorescence assay (IFA) or 20 min for immunohistochemistry (IHC). For neurotracer staining, sections were incubated with neurotracer (1:500) for 30 min at room temperature. For IFA, samples were washed with PBS twice and coverslipped using vectashield with 4′,6-diamidino-2-phenylindole (DAPI) mounting media. For IHC, samples were washed with PBS twice then developed with AEC developing kit (Invitrogen) before counterstaining with hematoxylin. Slides were then coverslipped using fluoromount G mounting media. Images were captured using a Zeiss Axio Imager M1 microscope.
Thioflavin S plaque staining
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.
Neuronal culture and T. gondii infection
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.
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to detect apoptotic cells. The reaction mixture was supplied by Roche’s In Situ Cell Death Detection Kit, AP (Cat# 11684809910). Five- to eight-micrometer frozen sections were fixed with 4% paraformaldehyde for 20 min. Sections were then permeabilized using 0.1% Triton X-100 (Sigma-Aldrich, 9002-93-1) in sodium citrate (Fisher Scientific, 6132-04-3) with PBS for 2 min. Sections were blocked using 10% BSA dissolved in PBS. Primary antibody and TUNEL solution were added to each section. Antibodies included anti-NeuN (1:200) and anti-Toxoplasma gondii cysts (1:1000). Sections were left overnight at 4 °C. Positive and negative controls were prepared as instructed in the protocol. Sections were then incubated with secondary antibody (1:200) antibodies for 1 h. Following, sections were washed and coverslipped with vectashield with DAPI. Images were captured using a Zeiss Axio Imager M1 microscope.
Mobility in an open field was measured once during the animal’s light cycle at day 60 post T. gondii infection. Mock-infected mice were used as control. Each individual mouse was placed in the center of an open field arena (18 × 18 × 18 in.). The movement of the mouse was recorded by a USB webcam (Logitech HD-1820p) and a PC-based video capture software. The recorded video file was further analyzed by video tracking software (Topscan, Clever Systems) to determine the velocity and the total distance traveled by each mouse during the 5-min observation period. The percentage of time spent in the corners and in the center of the open field was also recorded. Both the test and the analysis were performed by an observer blinded to the treatment conditions.
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 . 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.
Olfactory sensitivity test
Animals were habituated by placing each mouse in a clean, empty cage as described previously . 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.
Barnes maze was performed to assess spatial memory 60 days after infection with T. gondii. Mice were placed on the center of a wooden circular platform that was elevated 75 cm off the ground and that contained eight holes spaced 24.5 cm apart. An escape box measuring 10 × 8.5 × 4 cm was placed underneath a randomly selected hole. The platform measured 91 cm in diameter and the escape hole was 5 cm in diameter. The amount of time taken by the mouse to find the escape box (latency to find) and the amount of time taken to enter the escape box (latency to enter) were recorded. The session ended when the mouse entered the escape box or after 5 min. The mouse was left in the escape box for 1 min after entering and before being transferred back to the home cage. If the mouse did not find the escape hole within 5 min, it was placed into the escape box and left there for 1 min. Two trials separated by a 15-min inter-trial interval were performed daily for five consecutive days. The average latencies to find and enter the escape box were used for analysis.
Experimental design and statistical analysis
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.
T. gondii associated with neurons and showed preference for infecting the olfactory bulb and the prefrontal cortex
T. gondii induced Aβ immunoreactivity in infected mice
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.
T. gondii infection induced Tau hyperphosphorylation
T. gondii infection led to loss of NMDAR signal
T. gondii infection led to loss of VGLUT2, which is indicative of loss of olfactory sensory neurons
T. gondii-infected mice showed a discrete spatial increase in GAD67 expression
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 . 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 . 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 . 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 .
T. gondii cysts were associated with neuronal cell death
T. gondii-infected mice had altered social memory and displayed anxiety-like behavior 60 days after infection
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  and is also common among AD patients . 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.
The effects of T. gondii infection on olfactory sensitivity varied depending on sex
We did not observe sex-specific differences in expression or protein levels of VGLUT1/2, NMDAR, or GAD67. We also did not observe sex-specific differences in any of the other behavioral measures we used, indicating that in our model of T. gondii infection sex-dependent differences are limited to olfactory sensitivity.
T. gondii infection severely impaired spatial memory
The link between T. gondii infection and AD has not been fully elucidated, and epidemiological studies are contradictory. One study found that patients with AD had higher seropositivity rates for anti-T. gondii IgG antibodies compared to healthy controls, indicating a possible link between T. gondii infection and AD . Two other studies reported no difference in the presence of anti-T. gondii IgG antibodies between AD patients and their healthy counterparts [55, 56]. Moreover, countries with high T. gondii seropositivity rates do not have higher rates of AD. For example, France had a T. gondii seroprevalence of 70% in the 1970s , but an AD prevalence of only 3.0% among French people ≥ 60 years of age in 2012 .
While T. gondii may not be the underlying cause of AD in the general population, it may initiate pathological events that over a life time can result in AD-like symptoms. We present direct evidence that T. gondii causes major signs of AD in mice that are not genetically manipulated towards AD (wild-type mice). We showed that infected wild-type mice showed major signs of AD including Aβ immunoreactivity, pTau expression, neuronal dysfunction, and behavior alterations. There is overwhelming evidence indicating that Aβ accumulation in the brain is the cause of neurodegeneration. In animal models, Aβ monomers and oligomers cause cognitive impairment, synaptic dysfunction, and neuronal loss . In cultured neurons and astrocytes, administration of Aβ-impaired glutamate uptake  which could hinder neurotransmission, memory formation, and learning. Treatment of mouse cortical neurons with Aβ oligomers (trimmers or tetramers) induced neuronal toxicity and death . We showed that Aβ immunoreactivity coincided with a decrease in NMDAR signal in the brains of infected mice as early as day 15 post infection. We conclude that T. gondii induced Aβ immunoreactivity which led to NMDAR loss. The loss of NMDAR disrupts the feedback inhibition signal by NMDA, GABA and VGLUTs and increases glutamate in the synaptic cleft, leading to neurotoxicity and neurodegeneration. Aβ enhances the endocytosis of NMDARs, thereby reducing their surface expression . Alternatively, T. gondii infection led to reduced NMDAR expression which then led to Aβ immunoreactivity. It is also possible that the inflammatory response caused by T. gondii infection caused Aβ immunoreactivity and eventual neuronal loss. Acute infection with T. gondii attracts neutrophils, microglia, and dendritic cells to the site of the infection which leads to the release of cytokines necessary to promote killing of the parasite and inhibit its replication . Inflammatory cytokines have also been implicated in the formation of Aβ plaques [63, 64] and neuronal cell death .
Our findings that T. gondii infection led to of Aβ immunoreactivity contradict two recent studies. The first one showed that T. gondii infection reduced Aβ plaque load in an AD transgenic mouse model (5xFAD) due to the recruitment of highly phagocytic monocytes . The second one found that the ME49 strain of T. gondii reduced plaque deposition, neurodegeneration, and improved cognition in Tg2576 mice . It is notable that both of these studies used AD transgenic mouse models. The observed reduction in plaque burden could be the result of T. gondii-driven clearance by hyperactivated astrocytes and microglial cells, as postulated by , or the result of T. gondii-driven acceleration of advanced signs of AD with resultant induction of cerebral amyloid angiopathy (CAA), which is the deposition of Aβ plaques in CNS vessels. However, neither study assessed the appearance of CAA or evaluated other advanced signs of AD. Alternatively, plaque formation could be a transient effect that Jung et al. missed by assessing plaque load and behavior in 9-month-old mice at 6 months post infection . In our studies, Aβ immunoreactivity and behavior were assessed within 60 to 90 days post infection in wild-type mice.
In addition to molecular signs, AD is characterized by alterations in social communication and social memory . We found clustering of T. gondii cysts and Aβ plaque formation in brain areas known to affect social behavior, namely the hippocampus and the prefrontal cortex [50, 51]. In line with these findings, we observed alterations in social novelty recognition in T. gondii-infected mice. This is consistent with other studies that have shown altered social behavior in mice genetically modified to overexpress Aβ . Since the animals had normal activity levels in the open field, hyper- or hypoactivity or motor defects are unlikely contributors to the altered recognition of social novelty.
T. gondii-infected mice showed a severe impairment in spatial learning and memory as assessed by Barnes maze. These mice showed normal activity levels, indicating motor defects are not responsible for the spatial memory defect. T. gondii has been consistently associated with behavioral changes in mice [11, 69, 70]. In humans, some studies show effects of T. gondii on cognitive decline. For example, Gajewski et al. found that adults with T. gondii-positive antibody status performed poorly in a verbal memory test and showed reduced working memory . However, the effects of T. gondii on cognition seem to depend largely on educational level, economic status, and ethnicity [10, 72].
We observed profound loss of VGLUT2 in olfactory sensory neurons indicating that there is also profound olfactory sensory neuronal loss which is one of the early signs of AD. The discrete upregulation of GAD67 in areas prevalent with T. gondii cysts, and increased VGLUT1 expression, which occurred near T. gondii cysts, may be a compensatory response to overcome neural inhibition and re-establish neural transmission. David et al. demonstrated that infection with T. gondii leads to a reduction in the primary astrocytic glutamate transporter GLT-1, increased extracellular concentrations of glutamate, and reduced dendritic spines . They also reported no induction of neuronal cell death by the parasite as well as reduced expression of VGLUT1, which contradicts our results. These authors used 20 T. gondii cysts to infect mice, while we used only 10. It is possible that the higher MOI used by David et al. induced excessive stimulation of glutamatergic signaling resulting in excitotoxicity. In fact, they observed synaptic changes in the prefrontal cortex including reduced spine density at 6 weeks post infection, which has been shown to contribute to cognitive decline . They also observed reduced brain wave activity as well as reduced expression of neuronal synaptic markers. We propose that Aβ induction and the high prevalence of cysts in the olfactory bulb contributed to loss of VGLUT2 on olfactory neurons, resulting in the upregulation of VGLUT1 and discrete increases in GAD67 to compensate for breakdown in neurotransmission at these sites. Together, these studies show that T. gondii infection caused loss of neurotransmission leading to severe neurodegeneration and major signs of AD in wild-type mice.
We also observed alterations in olfactory sensitivity which varied depending on sex. Mock-infected males spent more time inspecting all the tested scents compared to the female controls. This correlates with a previous study showing that male mice have a superior ability to discriminate between various odors compared to females . Olfactory sensitivity in infected males was reduced compared to mock-infected animals but was unaltered in female mice. T. gondii causes a mild attraction towards cat urine in female BALB/c mice , a finding that would indicate intact olfactory sensitivity. A second study showed that only infected females but not males exhibit an attraction for cat urine , which correlates with the unaltered olfactory sensitivity we observed in our infected female mice and the reduced olfactory sensitivity we see in male animals.
To the best of our knowledge, we are the first to show that T. gondii directly induces AD signs in non-genetically manipulated animals. This study is likely to inform many areas of science including, infectious disease, neuroscience, neuroimmunology, immunology, and neurodegeneration and behavior.
We thank Dr. JP Dubey for the kind gift of BAG1antibody.
This work was supported by NIH grant R01 NS063011 (to M.S. Bynoe).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
LT, DK, and SR conducted the experiments, performed the analysis, and wrote the manuscript. AY analyzed the data and imaging. TAC collaborated with us and facilitated the equipment and software to perform the behavior experiments. MSB designed the experiments and contributed to writing and editing the manuscript. All authors read and approved the final manuscript.
All animal work was done in accordance with PHS guidelines and was approved by Cornell’s Institutional Animal Care and Use Committee (Protocol # 2008–0092).
Consent for publication
The authors declare that they have no competing interests.
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