This study aimed to assess the risk of damage from the anti-ZIKV antibody complex in neurosphere cultures obtained from the SVZ of neonatal rats. To simulate a re-exposure to ZIKV, neurospheres were pre-exposed to IgG serum reactive to ZIKV and then incubated with the virus for 24 hours.
Recent studies suggest that ZIKV has the potential to alter the morphology of neurospheres and brain organoids, leading to changes in phenotype and a size reduction, indicating increased precursor cell death. However, the methodology employed in this study for the morphological evaluation of neurospheres may not have been sufficiently sensitive to demonstrate the impact of ZIKV on neurosphere morphology.
The protocol used for obtaining neurospheres displayed morphological variability, both in terms of total area and sphere phenotype, showing irregularity even before exposure to IgG + serum or ZIKV. However, IgG + serum for ZIKV appeared to exhibit a protective potential on the cells, resulting in a significant decrease in neurosphere irregularity when exposed to lower virus titration (MOI 0.001).
The expression of the NOTCH protein is crucial for various fundamental roles within an organism. Insufficiency or absence of NOTCH expression can contribute to a range of diseases, including neuronal disorders. Genes such as NOTCH-1, along with NOTCH-3, play pivotal roles in neuronal cell differentiation and the formation of structures like the dentate gyrus [10]. Furthermore, maintaining regular expression of the NOTCH-1 gene is essential for promoting nerve cell growth, as this protein plays a significant role in the development of the central nervous system [11].
In our findings, we observed that ZIKV infection can increase NOTCH-1 expression as early as 24 hours post-infection (p.i.), and this elevated expression persists until the last time point analyzed (72 hours p.i.). This effect could be attributed to the intrinsic nature of these cells, which rely on tightly regulated developmental pathways for their growth. ZIKV infection appears to interfere with the maintenance and proliferation of stem cells, often altering genes associated with development, particularly NOTCH [11]. Abnormal expression of NOTCH-1 has been linked to various pathologies, including bicuspid aortic valve, astrocytic hypertrophy, and Alzheimer's disease [12, 13, 14]. In our study, we observed a significant alteration in NOTCH-1 expression, with levels being elevated compared to those in the control group. It is conceivable that under the influence of ZIKV, these cells initiated a premature differentiation process, leading to increased NOTCH-1 expression levels. This phenomenon could potentially contribute to the depletion of progenitor cells [15].
Our findings align with recent literature indicating that ZIKV has a preference for infecting neural progenitor cells (NPCs), resulting in increased NOTCH-1 expression and disruption of the viability and growth of these cells [16]. Moreover, recent studies have demonstrated that ZIKV promotes the degradation of the Numb protein, which serves as an inhibitor of Notch signaling [17]. Therefore, this mechanism may contribute to the observed increase in NOTCH-1 expression induced by ZIKV infection.
Our results indicate a decrease in HES-1 expression levels at time 0h for groups that underwent any intervention, whether exposed to IgG + serum, ZIKV, or both (ZIKV group, IgG+/ZIKV group, or IgG+), highlighting the high heterogeneity of this gene expression. HES-1 serves as a fundamental transcriptional repressor in cell proliferation and differentiation during embryogenesis [18]. Studies emphasize the significance of HES-1 expression in young cells, as reduced expression levels of this gene during gestation or shortly after birth can lead to severe neurological defects in rodents, potentially resulting in death [19]. Additionally, other studies have demonstrated that low levels of HES-1 expression in mice caused hypoplasia of certain organs due to the depletion of precursor cells [20], underscoring the importance of maintaining regular levels of HES-1 in precursor cells
However, at 72 hours post-exposure to ZIKV, HES-1 expression levels appeared to be normalized in the IgG + and IgG+/ZIKV groups compared to the control group. Only the ZIKV group showed a persistent decrease in HES-1 expression at higher virus titers (MOI 0.1 and 0.01). Thus, ZIKV appears to directly influence decreasing the expression levels of this gene, while conversely, IgG + serum for ZIKV could modulate expression levels, returning to normal levels over time post-exposure to ZIKV. Considering that HES-1 participates in the proper control of embryonic neurogenesis and neural tube formation, IgG + reactive to ZIKV could play a neuroprotective role by inhibiting the downregulation of this gene upon re-exposure to ZIKV.
Subsequently, when we evaluated the expression levels of the HEY-1 gene, which is also implicated in embryonic development outcomes and neurogenesis, it does not seem to be affected by exposure to ZIKV at any of the viral titrations. Surprisingly, only the neurosphere group exposed to IgG + for ZIKV showed a persistent decrease in HEY-1 expression levels. Acutely, a decrease in gene expression levels was also observed in the group that was previously exposed to IgG + serum and subsequently incubated with ZIKV only at time 0 h, with levels normalized at 72 h after virus exposure.
The anti-inflammatory activity in neurospheres previously immunized with IgG + serum for ZIKV and subsequently exposed to the virus, based on levels of GCSF and IL-10. GCSF, with receptors widely distributed in the central nervous system, exhibits a multimodal neuroprotective effect, including anti-apoptotic activity in neurons [21, 22, 23], stimulation of endogenous neurogenesis [24], regeneration and repair of vascularization, and stimulation of angiogenesis [25], along with an anti-inflammatory effect [26]. Similarly, IL-10 plays crucial roles in maintaining tissue homeostasis by restricting excessive inflammatory responses, positively regulating innate immunity, and promoting tissue repair mechanisms [27, 28]. However, the changes in GCSF or IL-10 levels observed in our study were not significant for any of the experimental groups.