TNFR1 signaling converging on FGF14 controls neuronal hyperactivity and sickness behavior in experimental cerebral malaria.

BACKGROUND: Excess tumor necrosis factor (TNF) is implicated in the pathogenesis of hyperinflammatory experimental cerebral malaria (eCM), including gliosis, increased levels of fibrin(ogen) in the brain, behavioral changes, and mortality. However, the role of TNF in eCM within the brain parenchyma, particularly directly on neurons, remains underdefined. Here, we investigate electrophysiological consequences of eCM on neuronal excitability and cell signaling mechanisms that contribute to observed phenotypes. METHODS: The split-luciferase complementation assay (LCA) was used to investigate cell signaling mechanisms downstream of tumor necrosis factor receptor 1 (TNFR1) that could contribute to changes in neuronal excitability in eCM. Whole-cell patch-clamp electrophysiology was performed in brain slices from eCM mice to elucidate consequences of infection on CA1 pyramidal neuron excitability and cell signaling mechanisms that contribute to observed phenotypes. Involvement of identified signaling molecules in mediating behavioral changes and sickness behavior observed in eCM were investigated in vivo using genetic silencing. RESULTS: Exploring signaling mechanisms that underlie TNF-induced effects on neuronal excitability, we found that the complex assembly of fibroblast growth factor 14 (FGF14) and the voltage-gated Na+ (Nav) channel 1.6 (Nav1.6) is increased upon tumor necrosis factor receptor 1 (TNFR1) stimulation via Janus Kinase 2 (JAK2). On account of the dependency of hyperinflammatory experimental cerebral malaria (eCM) on TNF, we performed patch-clamp studies in slices from eCM mice and showed that Plasmodium chabaudi infection augments Nav1.6 channel conductance of CA1 pyramidal neurons through the TNFR1-JAK2-FGF14-Nav1.6 signaling network, which leads to hyperexcitability. Hyperexcitability of CA1 pyramidal neurons caused by infection was mitigated via an anti-TNF antibody and genetic silencing of FGF14 in CA1. Furthermore, knockdown of FGF14 in CA1 reduced sickness behavior caused by infection. CONCLUSIONS: FGF14 may represent a therapeutic target for mitigating consequences of TNF-mediated neuroinflammation.

Light-Sheet Microscopy of the Optic Nerve Reveals Axonal Degeneration and Microglial Activation in NMDA-Induced Retinal Injury.

Purpose: Optic nerve degeneration is a feature of neurodegenerative eye diseases and causes irreversible vision loss. Therefore, understanding the degenerating patterns of the optic nerve is critical to find the potential therapeutic target for optic neuropathy. However, the traditional method of optic nerve degeneration has the limitations of losing spatiotemporal tissue information. Light sheet fluorescence microscopy (LSFM) is a fluorescence microscopy technique that allows capturing 3D images rapidly with a high spatial optical resolution. In this study, we evaluated the availability of LSFM on the optic nerve with NMDA injected Thy1-CFP mice. Methods: NMDA injected to both eyes of Thy1-CFP mice. After 7 days from the injection, the retina and optic nerve were collected and immunostained with anti-Iba1 antibody. NMDA excitotoxicity induced RGC, and its axon loss and microglial activation in the retina were observed using confocal microscopy. The immunostained optic nerve was completed the optical clearing process with TDE and mounted for LSFM imaging. Results: We found that retinal flatmounts confirmed significant loss of CFP-expressing RGC and axon degradation and loss in Thy1-CFP mice at 7 days after NMDA injection. Together with these data verifying that NMDA induces RGC and its axon loss, we confirmed that NMDA excitotoxicity induced microglia activation and leukostasis, such as increased microglia number, transform its morphology to ameboid or round, and increase in attached leukocytes in vessels. Using LSFM, we observed that CFP expressing nerve fiber was well organized and arranged parallel in vehicle treated optic nerve, whileas NMDA injected optic nerve showed axon swelling and fragmentation and loss of axon density from the anterior to the posterior regions. Furthermore, LSFM enabled the observation of microglia phenotype transformation in the entire optic nerve. Unlike microglia in vehicle injected optic nerve, microglia in NMDA injected optic nerve displayed larger soma and short process with high Iba1 expression through the entire optic nerve from the anterior to posterior. Conclusions: In summary, we examined the applicability of the modified optic clearing protocol for the optic nerve and verified it enabled to acquiring of the 3D images of the optic nerve successfully revealing the complex spatial relationships between the axons, microglia and vasculature throughout the entire organ with single acquisitions. With these optimized techniques, we successfully obtained the high-resolution 3D images of NMDA-induced optic neuropathy, including the clues for optic nerve degeneration such as axon swelling, axonal fragmentation, and microglia activation. Overall, we believe that our current study could help understand the pathology of the optic nerve in neurodegenerative diseases, and it will be the basis for translational research.

Correction: Role of microglia in the dissemination of Zika virus from mother to fetal brain.

[This corrects the article DOI: 10.1371/journal.pntd.0008413.].

Role of microglia in the dissemination of Zika virus from mother to fetal brain.

Global Zika virus (ZIKV) outbreaks and their link to microcephaly have raised major public health concerns. However, the mechanism of maternal-fetal transmission remains largely unknown. In this study, we determined the role of yolk sac (YS) microglial progenitors in a mouse model of ZIKV vertical transmission. We found that embryonic (E) days 6.5-E8.5 were a critical window for ZIKV infection that resulted in fetal demise and microcephaly, and YS microglial progenitors were susceptible to ZIKV infection. Ablation of YS microglial progenitors significantly reduced the viral load in both the YS and the embryonic brain. Taken together, these results support the hypothesis that YS microglial progenitors serve as "Trojan horses," contributing to ZIKV fetal brain dissemination and congenital brain defects.