Edited by: Pabitra Sahoo, University of South Carolina, United States
Reviewed by: Yongcheol Cho, Daegu Gyeongbuk Institute of Science and Technology (DGIST), South Korea; Yasushi Kitaoka, St. Marianna University School of Medicine, Japan
This article was submitted to Molecular Signalling and Pathways, a section of the journal Frontiers in Molecular Neuroscience
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Zika virus (ZIKV) is a neurotropic flavivirus recently linked to congenital ZIKV syndrome in children and encephalitis and Guillain-Barré syndrome in adults. Neurotropic viruses often use axons to traffic to neuronal or glial cell somas where they either remain latent or replicate and proceed to infect new cells. Consequently, it has been suggested that axon degeneration could represent an evolutionarily conserved mechanism to limit viral spread. Whilst it is not known if ZIKV transits in axons, we previously reported that ZIKV infection of glial cells in a murine spinal cord-derived cell culture model of the CNS is associated with a profound loss of neuronal cell processes. This, despite that postmitotic neurons are relatively refractory to infection and death. Here, we tested the hypothesis that ZIKV-associated degeneration of neuronal processes is dependent on activation of Sterile alpha and armadillo motif-containing protein 1 (SARM1), an NADase that acts as a central executioner in a conserved axon degeneration pathway. To test this, we infected wild type and
The emergence of mosquito-borne Zika virus (ZIKV; a positive-strand RNA virus of the
SARM1 (Sterile alpha and armadillo motif-containing protein 1) has a well-established role in Wallerian degeneration and is regarded as a central executioner in this conserved axon degeneration pathway involving NAD-related metabolism. The SARM1 TIR domain has NAD+ consuming glycohydrolase (NADase) activity, considered critical for its prodegenerative capacity (Essuman et al.,
Notably, SARM1 also has a role in innate immunity since it contains a highly conserved Toll/Il-1 receptor (TIR) domain-containing protein, classified as a member of the toll-like receptor (TLR) adaptor family (Carty and Bowie,
The SARM1-dependent loss of axons and dendrites with absent or delayed neuronal cell death following infection by bunyavirus (Mukherjee et al.,
Type I interferon receptor deficient (
Myelinating cell cultures were established using E13 mouse spinal cord as described in detail previously (Thomson et al.,
Genomic DNA was extracted from ear (adult) or tail (embryo) biopsies using a protocol modified from Truett et al. (
The Brazilian strain of ZIKV, ZIKV/H. sapiens/Brazil/PE243/2015 (GenBank accession number KX197192; abbreviated ZIKV PE243; referred to subsequently as ZIKV) has been described previously (Donald et al.,
For immunocytochemistry, cultures were fixed at 24 h post infection (hpi) or 6 days post infection (dpi) in 8% paraformaldehyde for 1 h at room temperature and subsequently stored in PBS at 4°C before staining. For NAD+ assay, cells were collected at 24 hpi.
Samples were prepared for NAD+ quantification using the NAD/NADH-Glo™ assay (G9071, Promega) according to the manufacturer's instructions. Briefly, at 24 hpi, a cell scraper was used to remove cell monolayers from glass coverslips (2 × 35 mm dish per sample, 3 coverslips per dish) and these were transferred to a microcentrifuge tube. The cells were pelleted by centrifugation on a benchtop centrifuge at 13,000 RPM for 30 s. Supernatant was discarded and cells rinsed once in 1 ml PBS containing cOmplete™ EDTA-free protease inhibitor (11873580001, Sigma-Aldrich) before repeat centrifugation. The cells were lysed in 100 μl of bicarbonate-based lysis buffer (100 mM Na2CO3, 20 mM NaHCO3, 10 mM nicotinamide, 0.05% Triton-X 100, 1% dodecyltrimethylammonium bromide) by vortexing for ~1 min. Cell debris and insoluble material was then pelleted by centrifugation at 13,000 RPM for 10 min before the supernatant was transferred to a fresh 1.5 ml reaction tube. Pierce™ BCA Protein Assay Kit (23225, Thermo Fisher) was used according to the manufacturer's instructions to determine protein concentrations. Cell lysates were prepared to 0.5 mg/ml in lysis solution. 12.5 μg lysate was then incubated in 12.5 μl 0.4 M HCl at 60°C for 15 min, allowed to cool to room temperature, and neutralized with 12.5 μl 0.5 M Tris base. The samples were then snap-frozen and transported between institutions on dry ice. For determination of NAD+ levels, 10 μl of each neutralized reaction was mixed with 10 μl of the NAD-Glo detection reagent (prepared following manufacturer's instructions) on ice, in wells of a 384-well white polystyrene microplate (Corning). The plate and contents were incubated at room temperature for 50 min before luminescence was read using a GloMax® Explorer plate reader (Promega). Concentrations of NAD+ are expressed as nmol/mg of protein.
Post-fixation, cells were permeabilized in ethanol (−20°C; 10 min) and incubated in primary antibodies in blocking buffer (10% goat serum in PBS containing 2x NaCl [274 mM final] and 1% BSA) overnight at 4°C. Mouse anti-ZIKV (Aalto Bio, clone 0302156; 1 in 500) was used in combination with rabbit anti-NeuN (Sigma-Aldrich, ABN78; 1 in 500). Mouse IgG1 SMI31 anti-phosphorylated heavy and medium chain neurofilament (BioLegend, 801601; 1 in 1,500) and mouse IgG2a anti-β-Tubulin III (Sigma-Aldrich, T8578; 1 in 200) were applied simultaneously. After washing, secondary antibodies (goat anti-mouse IgG1 Alexa 488/568/647 and goat anti-rabbit IgG or goat anti-mouse IgG2a Alexa 568/647; 1 in 1,000; Invitrogen) were applied for 1 h at room temperature. Coverslips were mounted on glass slides in Mowiol® 4-88 (Sigma-Aldrich) mounting medium with DAPI (1 μg/ml; D1306, Invitrogen).
For quantification of cell density and neurofilament staining, fluorescence microscopy and image capture were performed using a Zeiss Axio Imager M2 fluorescent microscope with standard epifluorescence optics and Zen Blue software (Carl Zeiss AG, Germany). To avoid bias, fields of view (FoV) were selected in the blue (DAPI) channel and images were captured (10 images from across each coverslip) at x20 magnification in the red, far red, green and blue channels. Representative images for illustration were obtained using the same microscope and software or with a Zeiss LSM 880 Confocal Microscope using a Zeiss Plan-Apochromat 63 × /1.4 oil immersion objective and Zen Black software.
A rectangular area of interest (AoI) of 111,488 μm2 was placed on each image and immunostained cells or DAPI +ve nuclei, respectively, within and touching west and north borders were quantified. Only immunopositive cells with a DAPI +ve nucleus qualified. The average cell density per AOI was converted to cells/mm2 using the formula, cell density per AOI/area of AOI μm2 × 1,000,000. Pyknotic nuclei were distinguished from healthy nuclei on the basis of size and homogeneity and intensity of DAPI staining; pyknotic nuclei being condensed and intensely labeled (Cummings and Schnellmann,
Digital images of representative fields of view obtained from cell cultures immunostained with antibody SMI31 that recognizes phosphorylated heavy (H) and medium (M) chain neurofilament, were used to quantify the densities of neuronal processes. A Fiji (Schindelin et al.,
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Analyses were performed using GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA). Significance is indicated as <0.05 (*), <0.001 (***) and <0.0001 (****). A one-way ANOVA was used to compare cell densities or neurofilament stained area across three
Embryonic murine spinal cord-derived myelinating cell cultures contain all major neural cell types including choline acetyltransferase positive motor neurons that project to the body's periphery
MOI ZIKV used in experiments quantifying neurofilament and β-tubulin III staining.
MOI 0.3 | 1 | 3 | 3 |
MOI 0.6 | 2 | 2 | 2 |
3 | 5 | 5 |
SARM1 depletion delays degeneration of neuronal processes.
To confirm that the observed decrease in degeneration of neuronal processes in the absence of SARM1 does not simply reflect reduced infection in
SARM1 does not enhance susceptibility to ZIKV infection.
SARM1 is an NADase that depletes axons of NAD+ following a variety of insults. Furthermore, the NS3 domain of ZIKV itself has been shown to deplete NAD+ by a PARP1-dependent mechanism in HeLa cells (Xu et al.,
ZIKV infection depletes NAD+ levels independent of SARM1 at 24 hpi.
As some neurotropic viruses use microtubule-dependent axon transport to reach the neuronal soma, the changes observed in microtubule staining at 24 hpi led us to ask whether SARM1-dependent mechanisms might limit infection of neuronal somas. To address this, we examined infected NeuN +ve somas across the three
SARM1-dependent degeneration of neuronal processes limits infection and death of neurons.
Our data provide novel evidence of a SARM1-dependent mechanism of degeneration of neuronal processes following ZIKV infection. Further, this involves neither an increase in ZIKV infected cells nor increased susceptibility of neuronal somas to cell death. On the contrary, at least at the time point tested, the proportions of infected and/or dying neurons were significantly enhanced in the absence of SARM1. Furthermore, we provide evidence in primary CNS cell cultures for the physiological relevance of a previous report of ZIKV-induced NAD+ depletion in HeLa cells (Xu et al.,
One possible explanation for these findings is that degeneration of neuronal processes in this context could result from one of the additional enzyme activities of SARM1, which include NADP hydrolysis and cyclisation, and base exchange of nicotinamide of NAD(P) with other endogenous pyridine bases such as nicotinic acid (Zhao et al.,
One limitation of our study is that the NAD+ assay is based on cell culture lysates and therefore it cannot discriminate cell or cell compartment-specific effects. Regarding this, it is worth noting that ZIKV protein NS3 itself potently activates a nuclear poly (ADP-ribose) polymerase, PARP1, in HeLa and glioma cells that results in NAD+ depletion and cell death at 48 h post-treatment in the former (Xu et al.,
Degeneration of neuronal processes following ZIKV infection occurs independently of overt infection or death of neuronal somas in
It is noteworthy that widespread changes in neuronal-specific β-tubulin III immunostaining were observed as early as 24 h post-ZIKV infection. This raises the intriguing possibility that, more generally, activation of evolutionarily conserved SARM1 hampers the retrograde transportation of some viruses from the site of entry at the body's periphery, even prior to axon destruction. In support of this suggestion is the fact that SARM1 participates in microtubule posttranslational modification (Chen et al.,
The initial trigger for SARM1-dependent degeneration of neuronal processes following ZIKV infection could act through gain or loss of function, for example, through cytotoxicity, failure of trophic support from neighboring glia, or a combination of both. If a soluble cytotoxic factor is involved, neuronal processes seem particularly susceptible since all cell types are, in principle, equally exposed in cell culture where they are bathed in media. Nitric oxide is one potential cytotoxic molecule that causes Wallerian degeneration changes in electrically active axons at low micromolar concentrations (Smith et al.,
CNS complications of ZIKV infection in adults are rare, but include encephalitis, meningitis, myelitis, meningoencephalitis, transverse myelitis and neuropsychiatric symptoms (Carteaux et al.,
The original contributions presented in the study are included in the article/
The animal study was reviewed and approved by Ethics Review Committee of the University of Glasgow.
JE and MC: study design and supervision. CC, CA, LK, and VS: experimentation. CD: viral propagation. PM: genotyping. CC, LK, and JE: data analysis. JE, MC, CA, and CC: manuscript draft. JE, CC, and LK: figure composition. All authors: manuscript editing and approval. HW, JE, AK, MC, SB, and CL: funding. All authors contributed to the article and approved the submitted version.
This project was partially funded through the European Union's Horizon 2020 research and innovation programme under ZikaPLAN Grant Agreement No. 734584 (HW, JE, SB, and CL); the UK Medical Research Council (MC_UU_12014/8 and MR/N017552/1) (AK); CSO and University of Glasgow SPRINT PhD programme studentship to CLC (JE); an MRC DTP Award and Gates Scholarship (CA); the John and Lucille van Geest Foundation (MC).
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
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