Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Apr 13:15:860410.
doi: 10.3389/fnmol.2022.860410. eCollection 2022.

SARM1 Depletion Slows Axon Degeneration in a CNS Model of Neurotropic Viral Infection

Colin L Crawford  1 , Christina Antoniou  2 , Lina Komarek  1   3 , Verena Schultz  1 , Claire L Donald  4 , Paul Montague  1 , Susan C Barnett  1 , Christopher Linington  1 , Hugh J Willison  1 , Alain Kohl  4 , Michael P Coleman  2 , Julia M Edgar  1   3
Affiliations

SARM1 Depletion Slows Axon Degeneration in a CNS Model of Neurotropic Viral Infection

Colin L Crawford et al. Front Mol Neurosci. .

Abstract

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 Sarm1 homozygous or heterozygous null cell cultures with ZIKV and examined NAD+ levels as well as the survival of neurons and their processes. Unexpectedly, ZIKV infection led to a rapid SARM1-independent reduction in NAD+. Nonetheless, the subsequent profound loss of neuronal cell processes was SARM1-dependent and was preceded by early changes in the appearance of β-tubulin III staining. Together, these data identify a role for SARM1 in the pathogenesis of ZIKV infection, which may reflect SARM1's conserved prodegenerative function, independent of its NADase activity.

Keywords: Zika virus; glia; neurofilament; nicotinamide adenine dinucleotide; tubulin (microtubules).

PubMed Disclaimer

Conflict of interest statement

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.

Figures

Figure 1
Figure 1
SARM1 depletion delays degeneration of neuronal processes. (A) Mock infected Sarm1+/+ cultures appeared healthy at 6 dpi, with numerous pale DAPI +ve nuclei (yellow arrowhead in inset), relatively few pyknotic nuclei (white arrowhead) and dense neurofilament (NF) and β-tubulin III (β-TubIII) positive cell processes. (B) ZIKV infected cultures appeared altered at the same time point, with several pyknotic nuclei (white arrowheads in insets) and fragmented neurofilament and β-tubulin III-stained neuronal processes. This was most obvious in Sarm1+/+ and Sarm1+/− cultures whereas neurofilament appeared relatively preserved in Sarm1−/− cultures. Bar: 50 μm. (C) Quantification of neurofilament positive pixels as a proportion of all pixels demonstrated a significant preservation of neuronal processes in Sarm1−/− cultures compared to Sarm1+/+ or +/−. Bars represent mean ± S.E.M. ***p < 0.001.
Figure 2
Figure 2
SARM1 does not enhance susceptibility to ZIKV infection. (A) Fluorescence micrographs of ZIKV infected cells at 6 dpi in Sarm1 +/+, +/− and −/− cultures. Across all three genotypes, many cells are ZIKV +ve (yellow arrowheads). Few of these are NeuN +ve, indicating that most infected cells are glia. Bar: 50 μm. (B) Graph of percentage DAPI +ve nuclei that are ZIKV +ve, demonstrates that SARM1 status does not result in significant difference in proportions of infected cells, although there is a trend toward an increased proportion of infected cells in the absence of SARM1. (C) Graphs of DAPI +ve cell densities demonstrates there is no significant difference in cell densities across the three Sarm1 genotypes. However, there appears to be a tendency for increased cell death (pyknotic nuclei) in the absence of SARM1. Bars represent mean ± S.E.M.
Figure 3
Figure 3
ZIKV infection depletes NAD+ levels independent of SARM1 at 24 hpi. (A) Mock infected Sarm1+/+ cultures appear healthy at 24 hpi. DAPI +ve cell nuclei are abundant and neurofilament (NF) and β-tubulin III-stained neuronal processes appear dense and smooth. Bar: 50 μm. (B) At 24 hpi with ZIKV, DAPI +ve cell nuclei appear similar in terms of density and appearance across all three Sarm1 genotypes, although there is already evidence of subtle injury to neuronal cell processes in Sarm1+/+ (arrows indicate irregularly stained neurofilament +ve structures) and Sarm1+/− cultures, most obvious with β-tubulin III staining. In contrast, neuronal processes in Sarm1−/− cultures appear dense and smooth, as in the mock-infected controls (see also Supplementary Figure 1). (C) Neurofilament positive pixels as a percentage of all pixels per AOI is similar across all three genotypes suggesting that neuronal processes remain viable at this timepoint. (D) Compared to levels in matched mock-infected controls (black circles), NAD+ levels are significantly reduced in ZIKV-infected cultures (gray squares) at 24 hpi. (E) The relative reduction in NAD+ in ZIKV infected cultures in comparison to their mock-infected controls is similar across all three genotypes. Bars represent mean ± S.E.M. ****p < 0.0001.
Figure 4
Figure 4
SARM1-dependent degeneration of neuronal processes limits infection and death of neurons. (A) Fluorescence micrographs of ZIKV infected Sarm1+/+, Sarm1+/− and Sarm1−/− cultures at 6 dpi. In wild type cultures (Sarm1+/+), most NeuN +ve neurons are not ZIKV +ve (yellow arrowheads) whereas in Sarm1−/− cultures, many NeuN +ve neurons are ZIKV +ve (orange arrowheads). In Sarm1+/− cultures, both ZIKV +ve (orange arrowheads) and -ve (yellow arrowheads) NeuN +ve cells are evident. Bar: 50 μm. (B) Graph of proportions of NeuN +ve infected cells, demonstrates a significantly greater proportion of NeuN +ve neurons are ZIKV +ve in Sarm1−/− cultures compared to Sarm1+/+ control. (C) This does not reflect a significant difference in NeuN +ve cell densities in Sarm1−/− cultures compared to Sarm1+/+ controls. (D) There is a significant increase in the proportion of pyknotic NeuN +ve nuclei in Sarm1−/− cultures compared to Sarm1+/+ controls. Bars represent mean ± S.E.M. *p < 0.05 (defined in M&M).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Angeletti C., Amici A., Gilley J., Loreto A., Trapanotto A. G., Antoniou C., et al. . (2021). SARM1 is a multi-functional NAD(P)ase with prominent base exchange activity, all regulated by multiple physiologically-relevant NAD metabolites. iScience. 25:103812. 10.1016/j.isci.2022.103812 - DOI - PMC - PubMed
    1. Baud D., Musso D., Vouga M., Alves M. P., Vulliemoz N. (2017). Zika virus: a new threat to human reproduction. Am. J. Reprod. Immunol. 77. 10.1111/aji.12614 - DOI - PubMed
    1. Baxter P. S., Hardingham G. E. (2016). Adaptive regulation of the brain's antioxidant defences by neurons and astrocytes. Free Radic. Biol. Med. 100, 147–152. 10.1016/j.freeradbiomed.2016.06.027 - DOI - PMC - PubMed
    1. Bijland S., Thomson G., Euston M., Michail K., Thümmler K., Mücklisch S., et al. . (2019). An in vitro model for studying CNS white matter: functional properties and experimental approaches. [version 1; peer review: 2 approved]. F1000Research 8, 117. 10.12688/f1000research.16802.1 - DOI - PMC - PubMed
    1. Brito Ferreira M. L., Militão de Albuquerque M., de F. P., de Brito C. A. A., de Oliveira França R. F., Porto Moreira Á. J., de Morais Machado, et al. . (2020). Neurological disease in adults with Zika and chikungunya virus infection in Northeast Brazil: a prospective observational study. Lancet Neurol. 19, 826–839. 10.1016/S1474-4422(20)30232-5 - DOI - PMC - PubMed