N-Methyl-d-Aspartate (NMDA) Receptor Blockade Prevents Neuronal Death Induced by Zika Virus Infection

Abstract

Zika virus (ZIKV) infection is a global health emergency that causes significant neurodegeneration. Neurodegenerative processes may be exacerbated by N-methyl-d-aspartate receptor (NMDAR)-dependent neuronal excitoxicity. Here, we have exploited the hypothesis that ZIKV-induced neurodegeneration can be rescued by blocking NMDA overstimulation with memantine. Our results show that ZIKV actively replicates in primary neurons and that virus replication is directly associated with massive neuronal cell death. Interestingly, treatment with memantine or other NMDAR blockers, including dizocilpine (MK-801), agmatine sulfate, or ifenprodil, prevents neuronal death without interfering with the ability of ZIKV to replicate in these cells. Moreover, in vivo experiments demonstrate that therapeutic memantine treatment prevents the increase of intraocular pressure (IOP) induced by infection and massively reduces neurodegeneration and microgliosis in the brain of infected mice. Our results indicate that the blockade of NMDARs by memantine provides potent neuroprotective effects against ZIKV-induced neuronal damage, suggesting it could be a viable treatment for patients at risk for ZIKV infection-induced neurodegeneration.IMPORTANCE Zika virus (ZIKV) infection is a global health emergency associated with serious neurological complications, including microcephaly and Guillain-Barré syndrome. Infection of experimental animals with ZIKV causes significant neuronal damage and microgliosis. Treatment with drugs that block NMDARs prevented neuronal damage both in vitro and in vivo These results suggest that overactivation of NMDARs contributes significantly to the neuronal damage induced by ZIKV infection, and this is amenable to inhibition by drug treatment.

Keywords: NMDA receptor; Zika virus; intraocular pressure; memantine; microgliosis; mouse model; neuronal death.

FIG 1

Characterization of the disease parameters in WT and IFN-α/βR^−/− mice infected with a Brazilian strain of Zika virus. (A) WT and IFN-α/βR^−/− mice (n = 9 to 11) were inoculated i.v. with 4 × 10⁵ PFU of a Brazilian ZIKV strain (HS-2015-BA-01), and change in body weight was analyzed daily. Results are expressed as the percentage of initial weight loss. (B to I) Three or 6 days (peak of disease) after ZIKV inoculation, mice were culled and blood and tissues collected for the following analyses: (B) number of platelets, shown as platelets × 10³/ml of blood; (C) hematocrit index, expressed as percentage of volume occupied by red blood cells; (D) total and differential cell counts on blood, represented as the number of differential cell counts (leukocytes, mononuclear cells, and neutrophils) normalized by the percentage of total cell counts; (E) neutrophil influx to the brain; (F to I) concentrations of CXCL1, CCL5, IL-1β, and TNF-α in mouse brain, shown as picograms per 100 mg of brain tissue. All results are expressed as the mean ± standard error of the mean (SEM) and are representative of at least two independent experiments. *, P < 0.05 compared to control uninfected mice (MOCK). WT, SV129 mice; KO (knockout), IFN-α/βR^−/− mice. p.i., postinfection.

FIG 2

Viral load, intraocular pressure, and histopathological analysis of brain and eyes from ZIKV-infected mice. (A to F) WT (SV129) and IFN-α/βR^−/− mice (n = 8 to 11 mice per group) were inoculated i.v. with 4 × 10⁵ PFU of a Brazilian ZIKV strain (HS-2015-BA-01), and at day 3 or 6 of infection, tissues were harvested for plaque assay analysis of brain (A) and optic nerve (B). The results are shown as the log PFU per gram of brain or optic nerve, respectively. (C) Intraocular pressure (IOP) measurement after ZIKV infection. Results are expressed as millimeters of Hg increase in IOP. (D and E) Semiquantitative analysis (histopathological score) after H&E staining of retinal ganglionar cells (RGC) and brain sections of control uninfected (MOCK) and ZIKV-infected mice 6 days after infection. (F) Representative pictures from brain (left) and optic nerve (right) sections on day 6 of infection. Results are expressed as the median (A and B) or mean ± SEM (C to E) and are representative of at least two experiments. Asterisks indicate necrotic/apoptotic cells, and arrows indicate meningeal inflammation. Original magnification, ×200. Scale bar, 100 µm. Insert magnification, ×400. Inset scale bar, 50 µm.

FIG 3

ZIKV infection induces neurodegeneration and microgliosis in the cortex and hippocampus of IFN-α/βR^−/− mice. (A) WT and IFN-α/βR^−/− mice (n = 5 to 7 mice per group) were inoculated i.v. with 4 × 10⁵ PFU of a Brazilian ZIKV strain (HS-2015-BA-01). At day 6 of infection, mice were culled, and brain tissue was dissected and sliced (100-µm slices). Staining with fluoro-jade C (FJC) (neurodegeneration) and IBA-I (microgliosis) was performed in cortical hippocampal slices, followed by analysis. (A and B) The left panels show the number of neurons positive for fluoro-jade C (indicative of neurodegeneration). The right panels show representative pictures from cortical and hippocampal slices. (C and D) The left panels show the number of microglia positive for IBA-1 (microgliosis). The right panels show representative pictures from cortical and hippocampal slices. All results are expressed as mean ± SEM and are representative of at least two independent experiments. *, P < 0.05 compared to control uninfected mice (MOCK). Original magnification, ×200. Scale bar, 50 µm.

FIG 4

Kinetics of ZIKV infection on primary glial and neuronal cells and characterization of neuronal cell death after infection. (A) Culture of primary glial cells from a newborn C57BL/6 mouse. Viral loads were recovered from culture supernatant at different time points after infection with ZIKV (MOI of 1). (B) Primary culture of cortical-striatal neurons from C56BL/6 embryos (E15) on day 5 of differentiation in vitro (DIV5). The viral load was recovered from culture supernatant at different time points after infection with ZIKV (MOI of 1). (C to E) Neuronal death was assessed using the LIVE/DEAD assay in primary neurons on DIV5. (C) The panel represents the percentage of dead neurons after 12 to 96 h of infection. (D) Representative pictures from primary cultured neurons after 72 h of ZIKV infection labeled with calcein AM (green indicates live cells) and ethidium homodimer (red indicates dead cells). All results are expressed as the median (A and B) or mean ± SEM (C to E) and are representative of three to four experiments. The dashed line represents the median of remaining ZIKV titers recovered from culture supernatant at 0 h (just after the adsorption period). MOCK, uninfected. *, P < 0.05 compared to control uninfected neurons. Original magnification, ×200. Scale bar, 50 µm.

FIG 5

NMDAR blockade prevents ZIKV-induced neuronal cell death in vitro. Shown are primary cultured cortical-striatal neurons from C56BL/6 embryos (E15) on day 5 of differentiation in vitro. (A and B) NMDAR antagonist treatment was performed after infection (adsorption time) and every 24 h. Analyses were performed 72 h after ZIKV infection. Memantine, MK-801, and agmatine were given at different concentrations: 10 and 100 nM and 1, 10, and 30 μM for memantine, 1, 10, and 100 μM for MK-801, and 0.4, 2, 10, and 50 μM for agmatine. For ifenprodil dose-response, the concentrations were 0.0001, 0.001, and 0.01 μM. (A) Memantine’s effect on neuronal cell death was analyzed by LIVE/DEAD assay 72 h after ZIKV infection (MOI of 1). Results are represented as percentage of dead neurons. (B) Viral load was recovered from culture supernatant 72 h after ZIKV infection. Results are shown as PFU per milliliter of culture supernatant. (C) Cell death was analyzed by LIVE/DEAD assay 72 h after ZIKV infection following MK-801 treatment (MOI of 1). Results are represented as percentage of dead neurons. (D) Cell death was analyzed by LIVE/DEAD assay 72 h after ZIKV infection following agmatine sulfate treatment (MOI of 1). Results are represented as percentage of dead neurons. (E) Cell death was analyzed by LIVE/DEAD assay 72 h after ZIKV infection following ifenprodil treatment (MOI of 1). Results are represented as percentage of dead neurons. Results are expressed as mean ± SEM (A, C, and D) or the median (B) and are representative of four independent experiments. *, P < 0.05 compared to control uninfected neurons; #, P < 0.05 compared to ZIKV-infected neurons.

FIG 6

Memantine treatment prevents the increase in intraocular pressure (IOP) and brain damage induced by ZIKV. (A to F) IFN-α/βR^−/− mice (n = 5 to 11 mice per group) were inoculated i.v. with 4 × 10⁵ PFU of a Brazilian ZIKV strain (HS-2015-BA-01). Memantine (MEM) treatment (from days 3 to 6) of infection was performed orally b.i.d. (30 mg/kg). At day 6 of infection, optic nerve (B) and brain (A) were harvested for the plaque assay analysis. Results are shown as the log PFU per gram of optic nerve or brain. (C) Intraocular pressure (IOP) measurement after ZIKV infection. Results are expressed as millimeter Hg increase in IOP. (D and E) Semiquantitative analysis (histopathological score) after H&E staining of eye and brain sections of control uninfected (MOCK) and ZIKV-infected mice 6 days after infection. (F) Images are representative of each group on day 6 of infection. Results are expressed as the median (A and B) or mean ± SEM (C to E) and are representative of at least two independent experiments. *, P < 0.05 compared to control uninfected mice; #, P < 0.05 compared to ZIKV-infected mice. Asterisks indicate necrotic/apoptotic cells, and arrows indicate meningeal inflammation. Original magnification, ×200. Scale bar, 100 µm. Inset magnification, ×400. Inset scale bar, 50 µm.

FIG 7

Memantine treatment prevents neurodegeneration and microgliosis induced by ZIKV infection in the cortex and hippocampus of IFN-α/βR^−/− mice. (A) IFN-α/βR^−/− mice (n = 5 mice per group) were inoculated i.v. with 4 × 10⁵ PFU of a Brazilian ZIKV strain (HS-2015-BA-01). Memantine (MEM) treatment from days 3 to 6 of infection was performed orally b.i.d. (30 mg/kg per mouse). At day 6 of infection, mice were culled and brain tissue was dissected and sliced (100-µm slices). (A and B) Staining with fluoro-jade C (FJC) (neurodegeneration) and (C and D) IBA-1 (microgliosis) was performed in cortical and hippocampal slices, followed by analysis. (A and B) The left panels show the number of neurons positive for fluoro-jade C (indicative of neurodegeneration). The right panels show representative pictures from cortical and hippocampal slices. (C and D) The left panels show the number of microglia positive for IBA-1 (microglial activation). The right panels show representative pictures from cortical and hippocampal slices. All results are expressed as mean ± SEM and are representative of at least two independent experiments. MOCK, uninfected. *, P < 0.05 compared to the uninfected group; #, P < 0.05 compared to the ZIKV-infected group. Original magnification, ×200. Scale bar, 50 µm.