Chronic infection of T. gondii has been reported to cause cognitive behavioral abnormalities and is considered a potential cause of many mental illnesses such as AD. A large number of epidemiological statistics show that the seroprevalence of Toxoplasma antibodies in AD patients is significantly higher than that in the control group[33, 34]. This suggests a possible link between T. gondii infection and AD. However, there are some data showing the opposite trend. This may be due to the different experimental models and toxoplasma strains used in different experiments. At present, although it is known that there is a relationship between T. gondii infection and cognitive behavioral abnormality, the research on the relationship between T. gondii Chinese 1 genotype (ToxoDB # 9) and cognitive behavioral abnormality is not enough.
In our studies, we used TgCtwh6, one of representative strains of ToxoDB # 9 to infect mice constructing model of cognitive behavioral abnormalities. Our results indicated that the mice suffered from TgCtwh6 infection showed abnormal appearance and posture compared with the control mice. Furthermore, the OFT displayed that the infected mice appeared obvious corner preference which is related to increased anxiety. It has been reported that the anxiety-like behavior is also common in AD patients[34, 36]; meanwhile, the data from the MWMT revealed that TgCtwh6 infection damaged the mice memory retention and spatial learning. However, the infected mice have normal mobility, suggesting that T. gondii infection hardly affects motor function in mice [37, 38]. After dissecting the brain of mice, cysts were found in the infected group, suggesting that the mice were infected successfully with TgCtwh6 which had reached the mice brain tissue. These results verified that TgCtwh6 infection can result in cognitive behavioral impairment in C57BL/6 mice. So in the following experiments, we tried to investigate the mechanism with which TgCtwh6 infection triggers cognitive behavioral abnormalities.
Our histopathological research in vivo showed that the hippocampus neurons reduced in number, disordered in the arrangement and deeply stained in the nucleus in the infected mice, which suggested that the changes in morphology and decrease in number of hippocampal neurons might be related to cognitive behavioral abnormalities. It has been reported that Toxoplasma infection can cause neuron loss in mice . As we all know neuron loss, Aβ deposition and neuroinflammation are the vital histopathological features of AD disease; moreover, theneuron apoptosis is the significant cause of neuron loss, so we further confirmed whether TgCtwh6 infection can lead to neuronal apoptosis and Aβ deposition in vivo and in vitro.
Our study in vivo showed that Bax, Caspase3 expression increased and Bcl-XL expression decreased in infected mice hippocampus tissue, indicating TgCtwh6 infection could cause hippocampal nervous cells (probably including neurons) apoptosis. In addition, the expressions of BACE1, APP and Aβ in mice hippocampus tissue were significantly higher in the infected group than that in the control group, which suggested that Aβ deposition and neuronal apoptosis were implicated in cognitive behavioral abnormalities in mice infected with TgCtwh6. A large number of data have validated that BACE1 is the main (but not the only) secretion enzyme in vivo, and Aβ is derived from the sequential cleavage of APP by BACE1. Aβ has a strong neurotoxic effect after abnormal processing and accumulation in the cellular matrix, and it plays an important role in the progression of AD. Studies have shown that the addition of Aβ to animal and cell models can impede neurotransmission and cause cognitive impairment [43, 44].
In order to verify the mechanism with which TgCtwh6 induce neuron apoptosis and Aβ deposition in neuron, we carried out the separated or co-culture experiments in transwell device with HT22 and BV2 in vitro. The results manifested that HT22 apoptosis was initiated and Aβ expression was increased in HT22 when the HT22 was infected with TgCtwh6; moreover, BV2 was activated to M1 with increased expression of IL-6, TNF-α and iNOS when the BV2 was infected with TgCtwh6. Interestingly, the activated BV2 can induce HT22 apoptosis and APP production. Further study confirmed that Aβ expression was increased via NF-κB pathway in HT22 infected with TgCtwh6. In our study, when HT22 was infected with TgCtwh6, the p-NF-κBp65 and NF-κBp65 expression were up-regualated in cytoplasm, and then p-NF-κBp65 was transferred into nucleus, promoting BACE1, APP and Aβ expression. Now, more and more studies have affirmed that NF-κB signaling is closely associated with Aβ generation in neurological diseases[45, 46]. NF-κBp65 interacts with NF-κB binding elements to regulate BACE1 at the level of transcription and the BACE1 promoter contains specific NF-κB binding elements. The expression of BACE1 and the production of Aβ are induced via the NF-κB pathway.
Additionally, many studies have shown that apoptosis is induced via the NF-κB signaling, too. Mathilde et al. reported apoptosis was elicited through NF-κB pathway in cystic fibrosis cells. Shao et al. also showed miR-146a-5p promoted IL-1β-induced chondrocyte apoptosis via the TRAF6-mediated NF-kB pathway. On the contrary, Robert E et al. have demonstrated NF-κB activation after T. gondii RH strain infection is involved in the increase of anti-apoptotic gene expression, which plays a pivotal role in the T. gondii-mediated blockade of apoptosis. The difference between the two results may be due to the different T. gondii genetype and cell strain used in the experiments. Because T. gondii type Ⅱ strain dense granule protein 15 (GRA15Ⅱ), one of the genotype-associated effectors of T. gondii Ⅱ strain, could activate the NF-kB signaling, we speculate that GRA15Ⅱ derived from TgCtwh6 might induce HT22 apoptosis and Aβ production through NF-kB signaling. We will explore this hypothesis in our future work. In addition, our previous results show that the GRA15Ⅱ protein derived from T. gondii (Pru stain) can effectively promote the polarization of macrophages to M1. Some other studies demonstrated that GRA15Ⅱ can activate the NF-κB pathway of macrophages, thereby inducing the expression of pro-inflammatory M1-type related genes and transferring macrophages to M1. Besides, ROP16Ⅰ/Ⅲ of T. gondii type Ⅰ/Ⅲ can directly phosphorylate the STAT6 pathway of macrophages and polarize macrophages to M2[25, 26, 53]. So, further research is required to clarify whether GRA15Ⅱ derived from TgCtwh6 could induce microglia to polarize into M1 via NF-κB signaling.
Neuroinflammation has been considered as a possible pathological mechanism for cognitive behavioral disorders. Microglia, one of inherent immnune cells, is key mediator of the neuroinflammatory response in brain. Inflammatory response of microglia is an important factor in cognitive abnormalities. In our study, we found that microglia can be activated into M1 by TgCtwh6 infection, and can secret some pro-inflammatory factors which probably induce HT22 apoposis and APP production in HT22. Especially, we found that TgCtwh6 infection activated microglia perhaps via Notch signaling. Some researchers reported that microglia can be activated by infection (eg., parasites), trauma, and other factors, producing a variety of immune effector molecules that not only mediate chronic inflammation and apoptosis, but also lead to degenerative diseases of the nervous system . The pro-inflammatory cytokines, such as IFN-γ, IL-1β, and TNF-α can attenuate microglia's phagocytic activity, and transform microglia into M1 types. Moreover, some studies have identified that BV2 was activated into M1 through Notch pathway . Notch signaling is involved in regulating microglia activation after hypoxia partly through the cross talk between TLR4/MyD88/TRAF6/NF-κB pathways in brain damage. Cao et al. evidenced when LPS stimulated BV2 cells, both Notch and NF-κB/p65 proteins expression increased significantly, and the expression of Hes-1, TNF-α and IL-1β increased successively. Moreover, they considered Notch signaling can trans-activate NF-NF-κB/p65 by amplifying NF-κB/p65-dependent pro-inflammatory functions in activated BV2 cells. What effector molecule derived from TgCtwh6 can activate Notch signaling, and how the Notch signaling lead to NF-κB activation which promote microglia polarization, Aβ generation and neuron apoptosis are very important and interesting topics we will focus on in the future.
Aβ is neurotoxic, which can mediate neuronal apoptosis [26, 59]. Studies have shown that the presence of endogenous Aβ stimulation in the environment can continuously activate the M1 pro-inflammatory response and eventually lead to irreversible neuron loss. Wu et al. further demonstrated that the pro-inflammatory factors from microglia stimulated by Aβ cause extensive death of apoptotic neurons . So, we supposed that HT22 apoptosis is partly induced by excess Aβ secreted from HT22. Furthermore, Aβ may be used as immune micro-endogenous stimuli in the tissue micro-environment to constantly activate microglia to maintain the M1 pro-inflammatory response.