We have obtained converging evidence for an interference of chronic T. gondii infection with the protein composition of synapses by combining proteomics and candidate-based protein expression studies in synaptosomes. Immunoblot and immunofluorescence data point to a marked interference of T. gondii-related neuroinflammation with synaptic protein composition. The recovery levels of glutamatergic synaptic protein abundances upon sulfadiazine treatment of infected mice, which were similar to basal levels, support the view that neuroinflammation affects synaptic protein composition and thereby, most likely, function. These effects were more pronounced for glutamatergic transmission in both the proteomic (Fig. 1, Additional file 3, Additional file 6) and candidate-based investigations (Figs. 3 and 4), but the proteomic data also indicate a reduction in GABAergic synapse components in synaptosomal preparations (Additional file 7). Our investigation was largely based on purified synaptosomal fractions, a well-established approach which has been “exhaustively characterized in functional terms” [60, 61]. Importantly with respect to our results, previous investigations of synaptosomal protein composition in the developing mouse brain have revealed that, besides pre- and postsynaptic proteins, synaptosomes also contain multiple inflammation-related proteins [62]. Among those proteins were interferon-inducible immunity-related GTPase Irgm1, interleukin enhancer-binding factor 2 (ILF2) as well as glial proteins like GFAP and proteoglycans of the synapse surrounding extracellular matrix proteoglycans. The presence of proteins indicating immune response has been also highlighted in a recent publication on proteomic analyses of the human frontal lobe tissue from T. gondii-infected humans co-infected with HIV [63]. The quality of our proteomic data on changes in the glutamatergic and GABAergic systems as well as calcium homeostasis and extracellular matrix components is strongly supported by the same study. There is an overlap of 98 proteins (approx. one third) found in our screening with the data reported there. Strikingly, these common proteins are almost exclusively synaptic proteins comprising of glutamate and GABA receptors, neurotransmitter and/or ion transporters, calcium channels, extracellular matrix components, and neuronal cell adhesion molecules. A recent proteomic study used the same synaptosomal approach in a mouse model for neurodegeneration [64]. In a meta-analysis, a set of more than 130 common biological processes between both datasets became obvious and might be indicative for certain similarity to our experimental system, underlining a multipartite concept of the synapse [65].

Alterations of the synaptosomal proteome

Evidently, a large number of synaptosomal proteins was found upregulated in the proteomic investigations, and meta-analyses revealed that this was mostly observed for proteins involved in MHC class I-dependent antigen presentation, protein processing in the ER, and, most prominently, poly(A)-RNA binding. The latter observation is compatible with a recent transcriptome study demonstrating upregulation of proteins related to nuclear RNA processing in T. gondii-infected primary neural cultures [48]. Polyadenylation is a critical step in mRNA synthesis and serves to stabilize mRNAs after transcription. The most straightforward explanation for the increased abundance of proteins involved in poly(A)-RNA binding may be an overall increase in protein synthesis or turnover, which in turn may be related to increased expression of proteins related to inflammatory and immunological processes, such as MHC class I-dependent antigen recognition, which also showed a significant upregulation (Fig. 1). Poly(A)-RNA processing has been increasingly implicated in synaptic plasticity and neurodegenerative disorders [66, 67], and disruption of normal synaptic poly(A)-RNA processing leads to impaired synaptic protein synthesis and cognitive dysfunction [68]. Future research should thus assess the functional consequences of up regulation of poly(A) RNA-binding proteins in T. gondii-induced neuroinflammation. Interestingly, our proteomic data confirm to a large degree results from a previous study using full genome microarrays to analyze RNA levels in chronically T. gondii-infected mouse brains [69].

Although fewer proteins were downregulated rather than upregulated, a remarkable observation is the clustering of downregulated proteins in excitatory and inhibitory synaptic transmission, plasticity, and learning. This observation expands previously reported transcriptome alterations in pathways related to epilepsy and neurodegeneration and also malignancy [48]. In that study, pathways related to synaptic transmission and plasticity were not reported among the most strongly affected functional systems. One reason for this may be that synaptic changes at the mRNA level may be more transient and thereby less readily detectable than the more long-lasting changes in protein composition. Additionally, differential alterations of protein turnover rates caused, e.g., by changes in protein stability or degradation may contribute to variations in protein composition, while not being detectable at the mRNA level. Furthermore, Ngô and colleagues investigated primary neuronal cultures, while the present study was conducted using synaptosomes prepared from in vivo infected mice and may therefore be more likely to capture the effects of T. gondii infection on the molecular organization of synapses in otherwise normally developing brains, i.e., under more physiological—or pathophysiological—conditions.

The results of meta-analyses using IPA™ are summarized in Additional file 9. In addition to confirming functional aspects extracted from the proteomic datasets already revealed by DAVID and GeneCodis analyses, additional strong links to neurological diseases and psychiatric disorders emerge. This is also indicative of a functional relevance of our data as they are in agreement with earlier observations in which T. gondii infection status correlates with higher incidences of neurological and psychiatric disorders [8, 17, 70]. Concerning molecular and cellular functions, we found our proteomics data strongly related to changes in cell-to-cell signaling and interaction, cell morphology, cellular development, and also cellular assembly and organization. At the physiological level, the dataset identifies major aspects of nervous system development and function as changed by persistent T. gondii infection (Additional file 9). Further IPA™ results point to increases in the antigen presentation pathway (Additional file 10) and the lipid antigen presentation by the CD1 pathway (Additional file 11). Our proteomics data also revealed a significant reduction of synaptic proteins related to glutamatergic transmission (Additional file 6) and to GABAergic signaling (Additional file 7).

Interference of T. gondii with glutamatergic neurotransmission

Downregulation of synaptosomal proteins in T. gondii-infected mice was most pronounced for proteins involved in calcium signaling, glutamatergic and GABAergic synapse function, and, generally, in neural plasticity and learning and memory. Regarding glutamatergic transmission, major results of our proteomic investigations were confirmed by candidate-based experiments. In the synaptosomes, we observed downregulation of EAAT2, Shank3, and the AMPA-type glutamate receptor subunit GluA2. In the hippocampal and neocortical tissue homogenates, we found reduced abundances of the EAAT2, Shank3, the AMPA receptor subunit GluA1, and the NMDA receptor subunit GluN1, respectively. Infection-related downregulation of EAAT2 has been observed previously in the forebrain homogenates of mice with chronic toxoplasmosis [45]. Beyond confirming those results, we have shown that this downregulation was particularly pronounced in synaptosomes. Furthermore, using immunofluorescence staining, we have also demonstrated infection-related alterations of EAAT2 distribution throughout the brain. The downregulation of major glutamatergic synapse components (GluA2 and Shank3) observed here calls into question whether chronic T. gondii infection merely results in excess glutamatergic signaling due to reduced uptake as suggested [45, 71]. Instead, our results point to a more global impairment of glutamatergic synapse function related to T. gondii-induced neuroinflammation. One explanation for this is the simplification of neuronal architecture and a significant reduction of spine density [8] and spine numbers in the prefrontal cortex [45]. Another possibility could be a negative feedback effect, that is, excess synaptic glutamate might result in adaptive downregulation of proteins involved in neurotransmitter release as well as glutamate receptors and their interacting proteins. In line with this interpretation, both presynaptic scaffolding proteins like Piccolo, Bassoon, or RIM and postsynaptic adapter molecules like PSD-95/Dlg4, SAPAPs/DLGPs, or Shank proteins were found among the top 100 downregulated proteins in our proteomic investigations. Furthermore, changes detected in synaptic proteomes related to GABAergic transmission might also contribute to chronic T. gondii infection-associated alterations in behavior or correlations to neuropsychiatric disorders as a consequence of subtle alterations in the balance of excitatory and inhibitory neurotransmission.

With respect to the reduced expression and changed distribution of EAAT2 in infected mice, it remains yet to be determined, to what extent that reduction could be attributed to glial versus neuronal EAAT2. While the molecule is most strongly expressed in glial cells, it also shows a certain level of neuronal expression [72]. Neuronal EAAT2 is actually mainly found at axon terminals, particularly as part of synaptosomal preparations [73] and may therefore have contributed substantially to the reduced EAAT2 expression observed in the synaptosomes of infected mice. On the other hand, in the present study, EAAT2 was also reduced in the brain homogenates. Considering that, at least under standard conditions, 80% of EAAT2 is expressed in astrocytes [73], it is thus implausible to assume that the reduced EAAT2 abundances in infected animals purely reflected a reduction of neuronal EAAT2. In this context, it should also be noted that at least a fraction of the EAAT2 protein found to be reduced in synaptosomes may actually be of glial origin, as astrocytes can form protrusions into the synaptic cleft, thereby allowing rapid EAAT2-dependent synaptic glutamate clearance [74]. These astrocytic “endfeet” are known to copurify partly with synaptosomes, suggesting that also in the synaptosomal preparations, both neuronal and glial EAAT2 abundances were most likely reduced upon T. gondii infection. It should be noted that downregulation of EAAT2 was paralleled by upregulation of the glial fibrillary acidic protein (GFAP), which is exclusively expressed in astrocytes (Fig. 6). This observation is noteworthy as it indicates that, at least with respect to astrocytes, the observed alterations in synaptosomal protein composition could not be attributed to a mere change in predominant cell types.

While immunoblot analyses essentially confirmed this overall pattern of synaptic protein downregulation in T. gondii-infected mice, this could not be confirmed for all candidate proteins in the brain homogenates, which in turn might be indicative for a specific synaptic regulation of protein abundance. Particularly, GluN2B downregulation was not significant in the hippocampus and only moderate in the cortex when compared to other synaptic proteins. One explanation for this may be the relatively small number of animals investigated and the resulting lack of statistical power to detect subtle differences. Alternatively, it may be speculated that the GluN2B subunit is primarily a component of extrasynaptic NMDA receptors, whereas GluN1 is a prominent component of synaptic NMDA receptors [75]. A robust downregulation of synaptic NMDA receptors, accompanied by a less pronounced downregulation of extrasynaptic NMDA receptors, might shift the balance between synaptic and extrasynaptic NMDA receptor-dependent glutamate signaling—a molecular mechanism that has been implicated in learning impairment or ultimately excitotoxicity [76].

Although we could confirm and expand previous observations regarding dysregulation of glutamatergic neurotransmission in chronic toxoplasmosis [8, 45, 71], results of the GABAergic signaling are less clear. In analogy to the changes observed in the glutamatergic system, reductions of GABAergic synapse components in infected mice were observed in our proteomic analyses. However, in immunoblot analysis, synaptic expression levels of GABA A -α1 receptor subunits were not significantly different between infected and control animals. Considering the superior sensitivity of MS-based techniques over the traditional immunodetection, there is little reason to question the changes observed in the GABAergic system. In a previous study by Brooks et al. [47], no changes in global GAD67 expression levels had been found, but loss of the typical synaptic localization of GAD67 was observed. We therefore suggest that GABAergic alterations in T. gondii-infected mice may be rather subtle, but its impact should not be underestimated, particularly in conjunction with the more pronounced alterations in glutamatergic neurotransmission. Hence, changes in the balance between excitation and inhibition in the chronic infection status are very likely and might contribute to pathophysiological changes implicated in neuropsychiatric disorders [71].

Inflammation-related modulation of synaptic and neural protein expression patterns

In contrast to the downregulation of proteins related to synaptic transmission in synaptosomes of T. gondii-infected mice, inflammation-related proteins were strongly upregulated after infection (Fig. 1). MS results revealed an infection-related increase of the synaptosomal levels of proteins like interferon-inducible GTPase 1 (IIGP1), immunity-related GTPase (IGTP), interferon-induced protein with tetratricopeptide repeats 3 (IFIT3), and guanylate-binding protein 2 (GBP2), which are all signature molecules for the infection with the intracellular pathogen T. gondii. Moreover, the signaling molecule signal transducer and activator of transcription 1-alpha/beta (STAT1) was elevated during chronic infection, indicating an active host defense mechanism. The astrocyte marker glial fibrillary acidic protein (GFAP) was also strongly upregulated, most likely indicating inflammation-related astrocyte activation. This finding was corroborated by our immunoblot analyses demonstrating GFAP upregulation in tissue homogenates. The rather uniform increase of GFAP levels is indicative of a general response to the infection. GFAP is exclusively expressed in astrocytes, and its upregulation has been demonstrated during brain development, regeneration, or reactive gliosis [57]. Increased GFAP expression has also been suggested as an indicator of neuroinflammatory responses [56] and that might also apply to our current observations (Fig. 6) [57]. Thus, locally restricted parasite cysts in a small number of infected neurons may trigger a systemic inflammatory response.

Pro-inflammatory cytokines are critically involved in the physiology and pathophysiology of neuroinflammatory responses. Our qRT-PCR results are compatible with the previously reported pivotal role of IFN-γ and TNF in the neuroinflammatory response to T. gondii. IFN-γ, which is the key cytokine to control T. gondii infection, is produced at high levels initially by microglia and neutrophil granulocytes and by lymphocytes at later stages of the infection [6, 77]. Accumulating evidence suggests a role for IFN-γ in synaptic plasticity and neurodegeneration [38]. Upregulation of IFN-γ levels upon infection, as observed in our study, might effect MHC-1 expression by neurons and subsequent synaptic pruning by innate immune cells [78, 79]. Elimination of the synapses by mononuclear immune cells follows similar patterns and has been reported in diseases like multiple sclerosis [80] and Alzheimer’s [81]. More evidence suggests interference of IFN-γ with glutamatergic signaling, with AMPA receptors being a key player in IFN-γ-triggered excitotoxicity and neurodegeneration. Complex formation of active IFN-γR and GluA1 can result in calcium influx and production of nitric oxide, followed by dendritic bead formation [82]. Especially, the effect of chronic IFN-γ was shown to modulate AMPA receptor expression in the hippocampal neurons, which would be a possible explanation for our results, as sulfadiazine treatment was associated with reduced IFN-γ levels and synaptic protein expression in infected mice, similar to basal levels. In addition to IFN-γ, the specific inflammatory milieu with TNF, IL-6, and IL-1β might be responsible for the observed neuronal changes. Previous reports, reviewed by Klein et al. [33], indicate the involvement of IFN-γ and IL-1β in adult hippocampal neurogenesis as well as in learning and memory. Alterations include inhibitory (IL-1β, IFN-γ) or promoting (TNF) effects on long-term potentiation (LTP) as well as promoting (IFN-γ, TNFR2) or inhibiting (TNFR1) effects on neurogenesis in adults. Sulfadiazine treatment resulted in reduced tachyzoite numbers which was followed by downregulation of the cytokines IFN-γ, TNF, and IL-6, and IL-1β levels were sligthly affected. Thus, it is likely that the changes in the inflammatory milieu could influence neuronal alterations and synaptic protein expression.

Sulfadiazine treatment results in decreased SAG1 levels

The antiparasitic drug sulfadiazine is a para-aminobenzoic acid inhibitor, interfering with the folic acid synthesis pathway of the fast-replicating tachyzoites. Our results indicate that the sulfadiazine treatment, starting on day 10 post-infection, diminished tachyzoite (SAG1) levels and reduced parasite dissemination to the CNS. Cyst numbers were also affected, as cyst development starts as early as the tachyzoites enter the CNS, infecting neurons, and continues alongside the infection. Thus, reduced tachyzoite levels after treatment resulted in partially diminished cyst development as reported previously [69]. Cyst levels reached high numbers on day 20 and remained comparable on day 35 as previously indicated in studies with C57BL/6 mice [83, 84]. Importantly, cyst numbers on day 35 were not significantly reduced by the sulfadiazine treatment, only a trend could be observed, indicating the comparable principal presence of intraneuronal cysts in both experimental groups.

Synaptic protein levels after sulfadiazine treatment of infected mice

Protein levels of EAAT2, Shank3, and GluA2 were at least partly restored by treatment of infected animals with the sulfonamide antibiotic sulfadiazine, and a similar trend could also be observed for GABA A receptor α1 subunits. GFAP levels, on the other hand, were lower in sulfadiazine-treated compared to untreated animals, compatible with a reduction of the infection-related neuroinflammatory response. Since sulfadiazine reduces tachyzoites and infection-related neuroinflammation, but not cysts, we suggest that the T. gondii-induced changes in synaptic protein composition are most likely an indirect effect mediated by inflammatory agents. A previous study associated the larger volume of inflammatory infiltrates, observed by histopathology with more pronounced abnormal behavior in mice chronically infected with Toxoplasma gondii [69]. This study described for the first time in details the neuroinflammatory changes in outbred mice upon infection. IFN-γ and TNF are both critically involved in the control of T. gondii infection [85] and were downregulated by sulfadiazine treatment in our present study. They have previously been shown to impair synaptic plasticity mechanisms like LTP [32, 86]. Therefore, our results suggest that neuroinflammation-related impairment in neural plasticity may partially result from subtle modulations of synaptic ultrastructure. It is a candidate pathophysiological mechanism that may also be relevant for neuroinflammation of another origin than T. gondii infection.

Clinical implications

With the advance of genome-wide association studies (GWAS), several components of the glutamatergic synapse have been identified as risk factors for the major psychiatric disorders like schizophrenia, bipolar disorder, and major depression. These include neurocan [52, 87], Homer-1 [53], and Piccolo [88], all of which were downregulated in synaptosomes of T. gondii-infected animals. Similarly, the genes of the Shank proteins, which also showed reduced abundance in infected animals, have been linked to autism spectrum disorders [89, 90]. Moreover, proteins related to MHC class I-dependent immune responses were upregulated in infected animals, and GWAS for schizophrenia have actually detected a cluster of risk loci in genes related to the MHC class I complex [91]. Similar associations have been found for bipolar disorder [92]. This dual overlap of genetic findings in the major psychoses with alterations of synaptic protein composition in latent toxoplasmosis—which is considered a risk factor for the very same disorders—convergingly raises the possibility that inflammation-induced changes in synaptic ultrastructure and ultimately function may constitute an overarching pathomechanism, by which diverse genetic and environmental factors may affect disease risk.