Neural progenitor cell pyroptosis contributes to Zika virus-induced brain atrophy and represents a therapeutic target

Abstract

Mounting evidence has associated Zika virus (ZIKV) infection with congenital malformations, including microcephaly, which raises global alarm. Nonetheless, mechanisms by which ZIKV disrupts neurogenesis and causes microcephaly are far from being understood. In this study, we discovered direct effects of ZIKV on neural progenitor cell development by inducing caspase-1- and gasdermin D (GSDMD)-mediated pyroptotic cell death, linking ZIKV infection with the development of microcephaly. Importantly, caspase-1 depletion or its inhibitor VX-765 treatment reduced ZIKV-induced inflammatory responses and pyroptosis, and substantially attenuated neuropathology and brain atrophy in vivo. Collectively, our data identify caspase-1- and GSDMD-mediated pyroptosis in neural progenitor cells as a previously unrecognized mechanism for ZIKV-related pathological effects during neural development, and also provide treatment options for ZIKV-associated diseases.

Keywords: Zika virus; caspase-1; microcephaly; neurogenesis; pyroptosis.

Fig. 1.

ZIKV infection leads to severe neuropathological alterations and brain atrophy in mice. Three-day-old mouse pups of immunocompetent mice were subcutaneously injected with 5 × 10⁵ PFUs of ZIKV, or were mock-infected. (A) Body weight curves of mock- and ZIKV-infected mice (n = 12). (B) Representative images of brains obtained from mock- and ZIKV-infected mice (21 dpi). (Scale bar, 1 cm.) (C) Quantification of dorsal brain area is shown in the histogram (n = 10). (D) ZIKV RNA in mice specimens was quantified by qRT-PCR (21 dpi) (n = 5). Dotted line depicts the limit of detection for the assays. (E) Representative micrographs of histopathological analysis with H&E staining of brain sections (21 dpi). Black arrows: necrotic loci; red arrowheads: perivascular cuffing. (Scale bar, 100 μm.) (F) Representative micrographs of the hippocampus, cortex, and thalamus of ZIKV- and mock-infected mice brain (21 dpi). (Scale bar, 50 μm.) All data are presented as mean ± SD, Student’s t test, ***P < 0.001.

Fig. 2.

ZIKV infection induces NPCs pyroptosis in vivo. (A) Representative micrographs of the brain tissue costained for SOX2, cleaved caspase-1 (p10), and ZIKV NS3 protein (21 dpi). The nuclei were stained with DAPI. (Scale bar, 25 μm.) (B) Representative micrographs of the brain tissue costained for SOX2 and PI (21 dpi). The nuclei were stained with DAPI. (Scale bar, 25 μm.) (C and D) Relative expression levels of inflammasome-associated genes were determined using qRT-PCR in mice brain specimens (21 dpi) (n = 5). (E) Examination of the proteolytic cleavage of caspase-1 and GSDMD in mice brain specimens with ZIKV- or mock- infection, using immunoblotting analysis (21 dpi). GSDMD-FL, full-length GSDMD; GSDMD-N, the N-terminal cleavage product of GSDMD. All data are presented as mean ± SD, Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.

ZIKV infects cultured hNPCs and induces pyroptosis. (A) Phase-contrast cell images of hNPCs with ZIKV or mock infection (white arrows, pyroptotic cells). (Scale bar, 50 μm.) (B) Representative micrographs of hNPCs double-stained for ZIKV (E protein) and SOX2 at 36 hpi (MOI = 1). The nuclei were stained with DAPI. (Scale bar, 400 μm.) (C) Representative transmission electron micrographs of mock- and ZIKV-infected hNPCs. Red arrows: membrane pores, Yellow arrowheads: mitochondria with collapsed cristae; red arrowheads: immature viral particles. (Scale bars, 2 μm.) (D) Caspase-1 activity was determined in cell lysates from mock- and ZIKV-infected hNPCs. (E) Representative micrographs of PI and Hoechst 33342 staining of hNPCs with ZIKV or mock infection. Red: PI dye; blue: Hoechst 33342 dye. (Scale bar, 100 μm.) (F) Quantification of the number of PI⁺ cells relative to mock infection is shown in the histogram. (G) LDH release was measured in supernatant derived from mock- and ZIKV-infected hNPCs transfected with small-interfering RNAs (siRNAs) targeting caspase 1. (H) Proteolytic cleavage of caspase-1 and GSDMD in hNPCs with ZIKV or mock infection was examined by immunoblotting analysis. Nigericin induced abundant caspase-1 and GSDMD activation in uninfected cells served as a positive control. GSDMD-FL, full-length GSDMD; GSDMD-N, the N-terminal cleavage product of GSDMD. All data are presented as mean ± SD, Student’s t test, *P < 0.05, **P < 0.01.

Fig. 4.

ZIKV infects human neurospheres and induces pyroptosis. (A and B) Representative micrographs of human neurospheres infected with ZIKV (MOI = 0.5; 96 hpi) or mock infection. White arrows: pyroptotic cells. (Scale bar, 200 μm in A and 100 μm in B.) (C and D) Quantification of the diameter (C) and number (D) of neurospheres is shown in the histogram. (E) Representative micrographs of neurospheres double-stained for active caspses-1 with a FLICA probe conjugated with FAM (green), and PI (red) at 96 hpi (MOI = 0.5). Nuclei were visualized by staining with Hoechst 33342 (blue). (Scale bar, 100 μm.) (F) Representative micrographs of neurospheres double-stained for ZIKV (E protein) and SOX2 at 96 hpi (MOI = 0.5). The nuclei were stained with DAPI. (Scale bar, 100 μm.) (G) LDH release was measured in supernatant taken from cultured mock- and ZIKV-infected neurospheres. All data are presented as mean ± SD, Student’s t test, **P < 0.01, ***P < 0.001.

Fig. 5.

Caspase-1 KO restores neuropathological alterations and brain atrophy induced by ZIKV in mice. Three-day-old mouse pups of WT or Casp1^−/− were subcutaneously infected with 5 × 10⁵ PFUs of ZIKV or mock-infected. (A) Body weight curves of WT or Casp1^−/− mice with ZIKV and mock infection (n = 10). (B) Representative images of brains taken from WT or Casp1^−/− mice infected with ZIKV or mock infection (21 dpi). (Scale bar, 1 cm.) (C) Dorsal brain area was analyzed and shown in the histogram (21 dpi) (n = 10). (D) Representative micrographs of the hippocampus of WT or Casp1^−/− mice (21 dpi). (Scale bars, 100 μm [Upper], 50 μm [Lower].) Black arrowheads: necrotic loci; red arrowheads: perivascular cuffing. (E and F) IL-1β and IL-18 were measured with ELISA in brain samples of WT and Casp1^−/− mice with ZIKV and mock infection (21 dpi). (G) Proteolytic cleavage of caspase-1 and GSDMD in brain specimens of WT and Casp1^−/− mice with ZIKV- and mock-infection was determined by immunoblotting analysis (21 dpi). GSDMD-FL, full-length GSDMD; GSDMD-N, the N-terminal cleavage product of GSDMD. All data are presented as mean ± SD, Student’s t test, **P < 0.01, ***P < 0.001. ns, not significant.

Fig. 6.

VX-765 alleviates neuroinflammation and brain atrophy in ZIKV-infected mice. Three-day-old mouse pups were subcutaneously infected with 1 × 10⁶ PFUs of ZIKV- or mock-infected, and subsequently treated with VX-765 (50 mg/kg, i.p.), or Z-DEVD-FMK (50 mg/kg, i.p.), or with vehicle (PBS/DMSO) every 2 d. (A) Body weight curves of ZIKV- and mock-infected mice treated with VX-765, Z-DEVD-FMK, or control vehicle (n = 16). (B) Survival curves of ZIKV- and mock-infected mice treated with VX-765, Z-DEVD-FMK, or control vehicle (n = 16). (C) Representative images of brains from mock- and ZIKV-infected mice treated with VX-765 or vehicle (30 dpi). (Scale bar, 1 cm.) (D) Dorsal brain area of mice was measured and shown in the histogram (n = 10). (E) Representative micrographs of histopathological analysis with H&E staining of the hippocampus of ZIKV- and mock-infected mice treated with VX-765, Z-DEVD-FMK, or vehicle (30 dpi). (Scale bars, 100 μm [Upper], 50 μm [Lower].) Black arrowheads: necrotic loci; red arrowheads: perivascular cuffing. (F) Representative micrographs of immunohistochemistry of brain sections stained for Iba-1 and GFAP (30 dpi). (Scale bars, 50 μm.) (G and H) IL-1β and IL-18 were measured with ELISA in brain specimens of ZIKV- and mock-infected mice treated with VX-765, Z-DEVD-FMK or vehicle (30 dpi). (I) Proteolytic cleavage of caspase-1 and GSDMD in brain specimens of indicated mice was determined by immunoblotting analysis (30 dpi). GSDMD-FL, full-length GSDMD; GSDMD-N, the N-terminal cleavage product of GSDMD. All data are presented as mean ± SD, Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001.