Human Neural Stem Cell Systems to Explore Pathogen-Related Neurodevelopmental and Neurodegenerative Disorders.

Building and functioning of the human brain requires the precise orchestration and execution of myriad molecular and cellular processes, across a multitude of cell types and over an extended period of time. Dysregulation of these processes affects structure and function of the brain and can lead to neurodevelopmental, neurological, or psychiatric disorders. Multiple environmental stimuli affect neural stem cells (NSCs) at several levels, thus impairing the normal human neurodevelopmental program. In this review article, we will delineate the main mechanisms of infection adopted by several neurotropic pathogens, and the selective NSC vulnerability. In particular, TORCH agents, i.e., Toxoplasma gondii, others (including Zika virus and Coxsackie virus), Rubella virus, Cytomegalovirus, and Herpes simplex virus, will be considered for their devastating effects on NSC self-renewal with the consequent neural progenitor depletion, the cellular substrate of microcephaly. Moreover, new evidence suggests that some of these agents may also affect the NSC progeny, producing long-term effects in the neuronal lineage. This is evident in the paradigmatic example of the neurodegeneration occurring in Alzheimer's disease.

Trypanosoma evansi impacts on embryonic neural progenitor cell functions.

Trypanosoma evansi appears to have a significant tropism for brain tissue in its chronic and acute phases. The most common symptoms of this brain infection are motor incoordination, meningoencephalitis, demyelination, and anemia. There have only been few studies of the effects of T. evansi infection on neuronal differentiation and brain plasticity. Here, we investigated the impact of the congenital T. evansi infection on brain development in mice. We collected telencephalon-derived neural progenitor cells (NPCs) from T. evansi uninfected and infected mice, and cultivated them into neurospheres. We found that T. evansi significantly decreased the number of cells during development of neurospheres. Analysis of neurosphere differentiation revealed that T. evansi infection significantly increased neural migration. We also observed that T. evansi promoted expression of glial fibrillary acidic protein (GFAP) in infected cells. These data suggest that congenital T. evansi infection may affect embryonic brain development.

Resveratrol as a Therapy to Restore Neurogliogenesis of Neural Progenitor Cells Infected by Toxoplasma gondii.

The intracellular protozoan Toxoplasma gondii may cause congenital toxoplasmosis and serious brain damage in fetus. However, the underlying mechanism of neuropathogenesis in brain toxoplasmosis remains unclear. For this study, neural progenitor cells (NPCs) were obtained from embryo telencephalons (embryonic day 13) and induced to proliferation in the presence of growth factors (GFs). For gathering insights into the biological effects of resveratrol (RSV) on neurogenesis, this study aimed to investigate effects of RSV concentrations (0.1 to 100 µM) on proliferation, migration and differentiation of NPCs infected by T. gondii. T. gondii infection increased the presence of cells in Sub G1 phase, reducing the global frequency of undifferentiated cells in S and G2/M phases of cell cycle and reduced cell viability/mithochondrial activity of infected NPCs. Moreover T. gondii stimulated neural migration and gliogenesis during neutral differentation. However, the treatment with RSV stimulated cell proliferation, restored cellular viability of infected NPCs and exerted an inhibitory effect on gliogenesis of infected NPCs favorecing neuronal maturation during toxoplasmosis infection. Thus, we have successfully to demonstrated that RSV is promising as therapeutic for congenital toxoplasmosis.

A mechanistic study of Toxoplasma gondii ROP18 inhibiting differentiation of C17.2 neural stem cells.

BACKGROUND: Congenital infection of Toxoplasma gondii is an important factor causing birth defects. The neural stem cells (NSCs) are found to be one of the target cells for the parasite during development of the brain. As a key virulence factor of the parasite that hijacks host cellular functions, ROP18 has been demonstrated to mediate the inhibition of host innate and adaptive immune responses through specific binding different host immunity related molecules. However, its pathogenic actions in NSCs remain elusive. RESULTS: In the present study, ROP18 recombinant adenovirus (Ad-ROP18) was constructed and used to infect C17.2 NSCs. After 3d- or 5d-culture in differentiation medium, the differentiation of C17.2 NSCs and the activity of the Wnt/ß-catenin signaling pathway were detected. The results showed that the protein level of ßIII-tubulin, a marker of neurons, in the Ad-ROP18-transfected C17.2 NSCs was significantly decreased, indicating that the differentiation of C17.2 NSCs was inhibited by the ROP18. The ß-catenin level in the Ad-ROP18-transfected C17.2 NSCs was found to be lower than that in the Ad group. Also, neurogenin1 (Ngn1) and neurogenin2 (Ngn2) were downregulated significantly (P < 0.05) in the Ad-ROP18-transfected C17.2 NSCs compared to the Ad group. Accordingly, the TOP flash/FOP flash dual-luciferase report system showed that the transfection of Ad-ROP18 decreased the Wnt/ß-catenin pathway activity in the C17.2 NSCs. CONCLUSIONS: The inhibition effect of the ROP18 from T. gondii (TgROP18) on the neuronal differentiation of C17.2 NSCs was at least partly mediated through inhibiting the activity of the Wnt/ß-catenin signaling pathway, eventually resulting in the downregulation of Ngn1 and Ngn2. The findings help to better understand potential mechanisms of brain pathology induced by TgROP18.

Toxoplasma gondii inhibits differentiation of C17.2 neural stem cells through Wnt/ß-catenin signaling pathway.

Toxoplasma gondii is a major cause of congenital brain disease. T. gondii infection in the developing fetus frequently results in major neural developmental damage; however, the effects of the parasite infection on the neural stem cells, the key players in fetal brain development, still remain elusive. This study is aiming to explore the role of T. gondii infection on differentiation of neural stem cells (NSCs) and elucidate the underlying molecular mechanisms that regulate the inhibited differentiation of NSCs induced by the infection. Using a differentiation medium, i.e. , DMEM: F12 (1:1 mixture) supplemented with 2% N2, C17.2 neural stem cells (NSCs) were able to differentiate to neurons and astrocytes, respectively evidenced by immunofluorescence staining of differentiation markers including ßIII-tubulin and glial fibrillary acidic protein (GFAP). After 5-day culture in the differentiation medium, the excreted-secreted antigens of T. gondii (Tg-ESAs) significantly down-regulated the protein levels of ßIII-tubulin and GFAP in C17.2 NSCs in a dose-dependent manner. The protein level of ß-catenin in the nucleus of C17.2 cells treated with both wnt3a (a key activator for Wnt/ß-catenin signaling pathway) and Tg-ESAs was significantly lower than that in the cells treated with only wnt3a, but significantly higher than that in the cells treated with only Tg-ESAs. In conclusion, the ESAs of T. gondii RH blocked the differentiation of C17.2 NCSs and downregulated the expression of ß-catenin, an essential component of Wnt/ß-catenin signaling pathway. The findings suggest a new mechanism underlying the neuropathogenesis induced by T. gondii infection, i.e. inhibition of the differentiation of NSCs via blockade of Wnt/ß-catenin signaling pathway, such as downregulation of ß-catenin expression by the parasite ESAs.

Toxoplasma gondii prevalent in China induce weaker apoptosis of neural stem cells C17.2 via endoplasmic reticulum stress (ERS) signaling pathways.

BACKGROUND: Toxoplasma gondii, an obligate intracellular pathogen, has a strong affinity for the nervous system. TgCtwh3, a representative Chinese 1 Toxoplasma strain prevalent in China, has the polymorphic features of the effectors ROP16I/III with type I and GRA15II with type II Toxoplasma strains. The interaction of this atypical strain with host cells remains extremely elusive. METHODS: Using a transwell system, neural stem cells C17.2 were co-cultured with the tachyzoites of TgCtwh3 or standard type I RH strain. The apoptosis levels of C17.2 cells and the expression levels of related proteins in the endoplasmic reticulum stress (ERS)-mediated pathway were detected by flow cytometry and Western blotting. RESULTS: The apoptosis level of C17.2 cells co-cultured with TgCtwh3 had a significant increase compared to the negative control group; however, the apoptosis level in the TgCtwh3 group was significantly lower than that in the RH co-culture group. Western blotting analyses reveal that, after the C17.2 cells were co-cultured with TgCtwh3 and RH tachyzoites, the expression levels of caspase-12, CHOP and p-JNK in the cells increased significantly when compared to the control groups. After the pretreatment of Z-ATAD-FMK, an inhibitor of caspase-12, the apoptosis level of the C17.2 cells co-cultured with TgCtwh3 or RH tachyzoites had an apparent decline, and correspondingly, the expression levels of those related proteins were notably decreased. CONCLUSIONS: Our findings suggest that TgCtwh3 may induce the apoptosis of the C17.2 cells by up-regulation of caspase-12, CHOP, and p-JNK, which are associated with ERS signaling pathways. This work contributes to better understanding the possible mechanism of brain pathology induced by T. gondii Chinese 1 isolates prevalent in China, and also reveals the potential value of ERS inhibitors to treat such related diseases in the future.

[Effect of Toxoplasma gondii infection on the embryonic neural stem cells in rats].

OBJECTIVE: To investigate the effect of Toxoplasma gondii infection on the proliferation, differentiation and migration of the embryonic neural stem cells (NSCs) in early pregnancy of rat. METHODS: Twelve pregnant Sprague-Dawley rats were randomly divided into control and infection groups. Rats in the infection group were each inoculated intraperitoneally with 1 x 10(5) T. gondii RH strain tachyzoites at day 1 (E1 day). Same amount of physiological saline was intraperitoneally injected for rats in control group. At E5 day, blood samples were taken from caudal vein and Giemsa staining of blood cells was performed to find T. gondii. At E9, E10 and E11 day, two rats in each group per time point were sacrificed and reverse transcription PCR (RT-PCR) was performed to detect B1 gene expression of T. gondii in amniotic fluid to confirm T. gondii infection. NSCs were cultured in vitro. The proliferation level was detected by methyl thiazolyl tetrazolium (MTT) assay. After differentiation culture of NSCs, the immunofluorescence assay was conducted to detect the expression of nestin, microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) to calculate the ratio of NSCs which differentiated to neurons and astrocytes. The embryonic nerve tissues at E9, E10 and E11 day in each group were taken to make frozen sections. The immunofluorescence assay was carried out to detect the expression of neuronal cell adhesion molecule (NCAM) in the nerve tissues at different developmental stages. RESULTS: Both the results of blood smears and RT-PCR confirmed that the pregnant rats and embryos were all infected by T. gondii in infection group. The morphology of the cultured NSCs under microscope was consistent with the characteristics of the normal NSCs. In addition, the NSC biomarker nestin protein was stained positive. The MTT assay showed that the proliferation level was lower in infection group than that of the control, and statistical differences were found between the two groups at day 3 and 4 after passages (P < 0.05). The immunofluorescence staining of MAP2 and GFAP showed that the percentage of neuron differentiation was 15.15% (55/363) in control group and 8.73% (31/355) in infection group, respectively, with a statistical difference (P < 0.05), and the percentage of astrocyte differentiation was 53.35% (199/374) and 67.48% 249/369), respectively (P > 0.05). In both groups, NCAM protein was found expressed at E9, E10 and E11 day in embryo nerve tissues. The fluorescence became stronger with time. The expression level in control group was significantly higher than that in infection group (P < 0.01). CONCLUSION: T. gondii infection at early gestation may inhibit the proliferation, differentiation and migration of neural stem cells in rats.