Activation of the Innate Signaling Molecule MAVS
by Bunyavirus Infection Upregulates
Piyali Mukherjee,1Tyson A. Woods,1Roger A. Moore,1and Karin E. Peterson1,*
1Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 S. 4thStreet, Hamilton,
MT 59840, USA
La Crosse virus (LACV), a zoonotic Bunyavirus, is
a major cause of pediatric viral encephalitis in the
United States. A hallmark of neurological diseases
caused by LACV and other encephalitic viruses is
the induction of neuronal cell death. Innate immune
damage, but no mechanism has been elucidated.
By using in vitro studies in primary neurons and
in vivo studies in mice, we have shown that LACV
infection induced the RNA helicase, RIG-I, and mito-
chondrial antiviral signaling protein (MAVS) signaling
pathway, resulting in upregulation of the sterile alpha
and TIR-containing motif 1 (SARM1), an adaptor
molecule that we found to be directly involved in
neuronal damage. SARM1-mediated cell death was
associated with induced oxidative stress response
and mitochondrial damage. These studies provide
an innate-immune signaling mechanism for virus-
induced neuronal death and reveal potential targets
for development of therapeutics to treat encephalitic
The innate immune response protects against virus infections
by production of type I interferons that mediate antiviral host
responses and cytokines that recruit inflammatory cells. This
innate immune response can also induce cellular damage
(Chattopadhyay et al., 2010; Fink et al., 2008; Lei et al., 2009;
McAllister and Samuel, 2009). Studies have demonstrated
direct innate immune signaling-mediated cell death with activa-
tion of pattern recognition receptors such as Toll-like receptors
(TLRs) or RNA helicase receptors (RLRs) leading to cellular
damage and, sometimes, apoptosis (Cameron et al., 2007;
Lathia et al., 2008; Ma et al., 2006, 2007; Tang et al., 2008). In
the brain, innate immune-induced apoptosis following virus
infection may be a contributing factor to neuronal damage
and neuronal dropout. Identification of specific targets that
initiate apoptosis during virus infection of neurons will offer
important insight into the mechanisms of neurodegeneration
and will provide targets for the development of antiviral
One protein that may have a role in innate immune signaling-
mediated cell death is sterile alpha and Toll/interleukin-1 (IL-1)
receptor (TIR) motif-containing 1 protein (SARM1, MyD88-5).
This protein is a member of the TIR-containing adaptor family
and, in immune cells, acts as a negative regulator of TLR-
mediated NF-kB activation (Carty et al., 2006; Peng et al.,
2010) and contributes to T cell apoptosis (Panneerselvam
et al., 2013). In neurons, SARM1 interacts with syndecan 2
and regulates neuronal morphogenesis (Chen et al., 2011).
Studies using GFP-tagged SARM1 show that under condi-
tions of metabolic stress in neurons, SARM1 translocates
to the mitochondria, interacts with c-Jun N-terminal kinase 3
(JNK3), and mediates neuronal apoptosis (Kim et al., 2007).
SARM1 has also been identified as mediating axonal death,
although the mechanism is unknown (Osterloh et al., 2012).
The role of SARM1 in the innate immune response and
neuronal or axon death has prompted questions about the
function for SARM1 in inducing neuronal damage during virus
infections in the central nervous system (CNS) and whether
this damage would be mediated through innate immune
To investigate the role of SARM1 in virus-induced neuronal
death, we utilized La Crosse virus (LACV), an enveloped triseg-
mented negative-sense RNA virus belonging to the family
Bunyaviridae. LACV is a major cause of pediatric viral enceph-
alitis in the USA and is an emerging pathogen due to increased
vector hosts and range (Gerhardt et al., 2001; Haddow and
Odoi, 2009; McJunkin et al., 2001). Neurons are the pre-
dominant cell type infected with the virus in the CNS and
LACV-mediated encephalitis is associated with degenerative
neuronal changes characteristic of apoptotic cells, including
nuclear vacuolization and cell shrinkage (Bennett et al., 2008;
Kalfayan, 1983; Pekosz et al., 1996). In this study, we have
demonstrated a clear role for SARM1 in mediating LACV-
induced neuronal apoptosis. We have also determined the
mechanisms for SARM1 induction and SARM1-induced cell
death during LACV infection. We show that both protective
type I interferon (IFN) and damaging SARM1-induced re-
sponses are generated following virus stimulation of the RNA
helicase, retinoic acid-inducible gene 1 protein (RIG-I), and
subsequent activation of mitochondrial antiviral signaling
protein (MAVS) signaling pathway in neurons.
Immunity 38, 705–716, April 18, 2013 ª2013 Elsevier Inc. 705
SARM1 Is Induced in LACV-Infected Neurons
SARM1 is highly conserved from chordates to humans (Mink
et al., 2001), suggesting a conserved function. Given that
SARM1 influences neuronal death following oxygen and glucose
deprivation (OGD) or axonal injury (Osterloh et al., 2012; Yuan
et al., 2010; Kim et al., 2007), we investigated whether SARM1
also influenced virus-mediated neuronal damage. Primary
cortical neurons were infected with LACV at different multiplici-
ties of infection (MOI) and followed for gene expression and
cell death. LACV RNA was detectable in infected neuronal
cultures as early as 6 hr postinfection (hpi) and increased loga-
rithmically until reaching a plateau between 18 to 36 hpi,
depending on MOI (Figure 1A). LACV-induced neuronal death
started at 24 hpi in cultures infected with the highest MOI of
virus with widespread cell death by 72 hpi for all MOIs tested
(Figure 1B). Dying neurons were TUNEL positive (Figure 1C)
and associated with increased caspase-3 activity, typical of
apoptotic neurons (Figure 1D). Analysis of SARM1 expres-
sion during LACV infection demonstrated increased Sarm1
messenger RNA (mRNA) as early as 18 hpi at the highest MOI
and by 24 to 30 hpi for the lower MOIs (Figure 1E). A correspond-
ing increase was observed with SARM1 protein in whole-cell
lysates of LACV-infected neurons compared to mock-infected
controls (Figure 1F). Thus, LACV infection resulted in the upregu-
lation of Sarm1 mRNA and protein expression.
SARM1 Contributes to LACV-Mediated Neuronal Death
Because SARM1 was induced during LACV infection and upre-
gulated prior to apoptosis, we examined whether SARM1 had
a role in LACV-mediated cell death. Transfection of neurons
with small interfering RNA (siRNA) targeted to Sarm1 prior to
infection significantly reduced LACV-mediated neuronal death
(Figure 2A). Similar results were observed with neurons from
Sarm1?/?mice as determined by both MTT assay and TUNEL
staining (Figures 2B–2D), with a significant inhibition or delay in
neuronal death compared to C57BL/6 wild-type (WT) neurons.
SARM1 deficiency did not inhibit virus replication with cells
from Sarm1?/?cultures expressing high amounts of virus (red
fluorescence) (Figure 2E), but not undergoing the same amount
of cell death (green fluorescence) as observed in infected cells
Figure 1. LACV Infection of Primary Cortical Neurons Induces Apoptotic Death and Increased Production of SARM1
(A) Cortical neurons were infected with LACV at MOIs of 1, 0.1, and 0.01. Virus RNA was measured at 4, 8, 12, 18, 24, 36, and 48 hpi by real-time PCR and
calculated as percent of Gapdh expression.
(B) Cell viability of neurons following LACV infection was measured by MTT assay.
(C) Neurons infected with LACV (MOI 0.01) were stained with TUNEL reagent. Cells were counted from five to six images for each group and a percentage of
TUNEL-positive cells per total number of nuclei per image determined.
(D) At 36 hpi, neurons were analyzed for caspase-3 activity.
(E) RNA from cells in (A) was analyzed for Sarm1 mRNA by real-time PCR and calculated as percentage of Gapdh mRNA expression. (A–E) Data are the mean ±
SEM for three or more samples per group per time point and are representative of duplicate experiments.
(F) Immunoblot analysis of SARM1 and b-actin in whole cell lysates of mock and LACV infected neurons from WT and Sarm1?/?mice at 36 hpi.
Neuronal Death Mediated by MAVS Induction of SARM1
706 Immunity 38, 705–716, April 18, 2013 ª2013 Elsevier Inc.
from WT cultures (Figures 2C–2E). Analysis of viral RNA expres-
sion in neuronal cultures, controlled for the number of cells by
Gapdh mRNA expression, showed similar amounts of virus
RNA (Figure 2F). Thus, SARM1 mediates neuronal death during
LACV infection, by a mechanism unrelated to suppression of
virus replication in neurons.
SARM1 Deficiency Suppresses Viral Pathogenesis
of LACV Infection In Vivo
To examine whether SARM1 deficiency would alter LACV path-
ogenesis in vivo, we infected C57BL/6 WT and Sarm1?/?mice
with 103plaque forming units (PFU) of LACV intraperitoneally,
a dose that induces 100% incidence of neurological disease in
3-week-old WT mice (Figure 3A). Sarm1?/?mice developed
neurological disease at a significantly lower incidence than WT
mice,indicating adetrimental roleforSARM1duringLACVinfec-
tion. In comparison, deficiency in MyD88, a family member of
SARM1, did not significantly affect neurological disease devel-
opment. Analysis of viral RNA from brain tissue of wild-type
and Sarm1?/?mice at 5 dpi, just prior to the onset of disease
in wild-type mice, revealed similar amounts of viral RNA in
Sarm1?/?mice compared to wild-type controls (Figure 3B). In
in neuronal cell bodies (see Figure S1A available online).
However, in focal areas in the cortex of LACV infected mice,
SARM1 was observed localized to the axons of neurons (Fig-
ure S1B). Analysis of 5 dpi preclinical WT mice demonstrated
numerous TUNEL-positive cells (green) in areas of virus infection
(red) (Figure 3C; Figure S1G). In contrast, Sarm1?/?mice at 5 dpi
had significantly fewer TUNEL-positive cells, despite large areas
of virus infection (Figures 3D and 3E; Figure S1H). Thus, SARM1
deficiency inhibits LACV-induced damage and death in vivo,
through a mechanism independent of virus replication, similar
to the results from neuronal cultures in vitro.
SARM1 Localizes to the Mitochondria and Is Associated
with Mitochondrial Damage
The N-terminal domain of SARM1 contains a mitochondrial
localization signal sequence (Panneerselvam et al., 2012).
Analysis of LACV-infected neurons demonstrated increased
controls, which was not observed in the cytosol fraction (Fig-
ure 4A). Similar results were observed in the mitochondrial
fraction of brain tissue from LACV-infected mice (Figure S1C).
The c-Jun N-terminal kinase 3 (JNK3), which associates with
SARM1 following metabolic stress (Kim et al., 2007), was also
increased in mitochondrial fractions following LACV infection
as was phosphorylated JNK (Figure 4A).
Despite mitochondrial localization, the mechanism by which
SARM1 induces neuronal apoptosis is unknown. Analysis of
neurons from WT mice showed numerous neurons containing
swollen and/or degenerative mitochondria in cell bodies and
axons in LACV-infected cultures (Figure 4C), but not in mock-
infected controls (Figure 4B). In contrast, LACV-infection of
neurons did not induce mitochondrial damage
(Figures 4D and 4E). Mitochondrial damage can be induced via
reactive oxygen species (ROS) formation, which has been impli-
cated in some cases of neuronal damage (Pan et al., 2009).
Staining with Mitosox red, an indicator of mitochondrial super-
oxide production, indicated that LACV-infected neurons had
increased superoxide production following LACV infection
(Figures 4F and 4G). However, superoxide production was not
detected in LACV-infected neurons from Sarm1?/?mice indi-
cating that SARM1 was necessary for LACV-induced generation
of ROS (Figure 4H). Furthermore, analysis of genes that are often
induced in response to oxidative stress demonstrated increased
mRNA expression following LACV infection in WT neurons,
but not in Sarm1?/?neurons (Figure S2A). Differences in
mRNA expression of oxidative stress response genes were
Figure 2. SARM1 Deficiency Limits LACV-
Mediated Neuronal Death but Not Virus
Infection or Replication in Neurons
(A) Neurons were treated with Sarm1 siRNA or
nontargeting (NT) siRNA starting at 3 days
postculture. Twenty-four hours later, cells were
infected with LACV and cell viability wasmeasured
in neurons treated with NT or Sarm1 siRNA is
shown in inset on right.
(B) Neurons from WT and Sarm1?/?mice were
infected with LACV, and cell viability was mea-
sured at 24, 30, and 36 hr by MTT assay.
(C–E) Cells were stained for DNA fragmentation by
TUNEL analysis at 36 hpi and counted as percent
positive as described in Figure 1. (D and E)
Immunofluorescence analysis of neurons from (D)
WT and (E) Sarm1?/?mice infected with LACV
at 36 hpi with TUNEL-stained nuclei (green) and
LACV (red). The relative lack of cells in (D)
compared to (E) is due to cell death. Scale bar
represents 25 mm.
(F) Viral RNA expression in neurons at 36 hpi as
determined by real-time PCR. All data are shown
as mean ± SEM of 3–6 samples per group per time
point. Data are representative of 2 or 3 replicate
experiments per panel. See also Figure S3.
Neuronal Death Mediated by MAVS Induction of SARM1
Immunity 38, 705–716, April 18, 2013 ª2013 Elsevier Inc. 707
also observed in vivo to a lesser extent (data not shown). These
data indicate that SARM1 is necessary for oxidative stress
response to virus infection and contributes to LACV-induced
SARM1-Mediated Damage Is Independent of Interleukin
1 or Type I IFN Pathways
Mechanisms of innate immune signaling-mediated cell death
have been described including caspase 1-dependent IL-1 medi-
ated pyroptosis, IFN-mediated apoptosis, and IFN regulatory
topadhyay et al., 2010; Fink et al., 2008). We therefore investi-
gated whether SARM1-mediated neuronal death was influenced
not related to pyroptosis because caspase-1 inhibitors did
not suppress neuronal death in vitro, SARM1 deficiency did not
affect Il1a or Il1b mRNA expression, and LACV infection did
not induce pro-IL-1b cleavage (Figures S3A–S3C, data not
Figure 3. SARM1 Deficiency Protects Mice from LACV-Induced Neuronal Damage
(A) Mice at 20 to 21 days of age were infected with 103PFU of LACV by intraperitoneal infection and followed for the development of clinical disease. Survival
curve for each strain was determined using Kaplan-Meier analysis of 32 WT, 16 Irf3?/?and Irf7?/?, 38 Myd88?/?, 19 Mavs?/?, and 29 Sarm1?/?mice. P value
for Sarm1?/?versus WT was < 0.01.
(B) Viral RNA and Ifna4 mRNA was quantified from brains of WT and Sarm1?/?mice at 5 dpi. No difference was observed in viral RNA between WT and Sarm1?/?
mice. A decrease in Ifna4 mRNA was observed in Mavs?/?mice. Data are shown as mean ± SEM of 5–8 samples per group.
(C–E) Immunohistochemical analysis of TUNEL (green) and LACV (red) in the cortex of (C) WT and (D) Sarm1?/?mice at 5 dpi. Scale bar represents 100 mm. (E)
Number ofTUNEL-positivecells per fieldofview (2003)fromfocalareaofvirusinfection inbrain tissuefromWT andSarm1?/?mice.Dataareshownasindividual
points with mean +/? SEM plotted. See also Figure S1.
Neuronal Death Mediated by MAVS Induction of SARM1
708 Immunity 38, 705–716, April 18, 2013 ª2013 Elsevier Inc.
shown). Stimulation of neurons with type I IFNs did not signifi-
cantly alter Sarm1 mRNA expression either in the presence or
absence of LACV infection (Figure S3D). Additionally, deficiency
in Irf3 and Irf7 or IFN-a receptor (Ifnar1) did not influence LACV-
induced cell death or SARM1 expression, suggesting that
SARM1-induced neuronal death was not dependent on IRF3 or
type I IFN responses (Figure S3E; data not shown). SARM1 defi-
ciency did not suppress type I IFN responses of neurons either
in vitro or in vivo (Figure S2B; data not shown), indicating that
SARM1 does not mediate antiviral type I IFN responses.
Figure 4. LACV Infection Induces Mitochondrial Localization of SARM1 and Mitochondrial Damage
at 36 hpi. CoxIV and b-actin were used as loading controls. SARM1 whole-cell lysate is shown in Figure 1F. Data are representative of duplicate or triplicate
experiments. Graphs below are the mean amounts of SARM1 protein in the mitochondrial and cytosol fractions from mock and LACV-infected neurons. Data are
the mean ± SEM of densitometry readings from three experiments.
(B–E)Morphologicalchanges inthemitochondriaassociatedwithLACVinfection.TEMimageswereacquiredat36hpifrom(Band D)mock- and(CandE)LACV-
infected neurons. Data are representative of 5–6 fields for each group. Scale bars represent 100 mm (F–J). Images of (F) mock- and (G–J) LACV-infected neurons
from (F–G) WT or (H) Sarm1?/?, (I) Mavs?/?, or (J) Mavs?/?cells treated with imiquimod at 36 hpi stained with 5 uM of Mitosox red. First panel for each image is
single channel for Mitosox red and the second panel is Mitosox red plus DAPI. Images are representative of cells in culture. Scale bar represents 10 mm. See also
Neuronal Death Mediated by MAVS Induction of SARM1
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Protein Interactions with SARM1 at the Mitochondria
following LACV Infection
In studies using transfected SARM1 in different cells, SARM1
was shown to interact with Syndecan 2 and JNK3 (Chen et al.,
2011; Kim et al., 2007). Additionally, SARM1 was predicted to
interact with the NOD-like receptor protein, NLRX1 based on
computational studies (Li et al., 2011). Surprisingly, immuno-
precipitation (IP) of endogenous SARM1 from the mitochondrial
fraction of uninfected or infected cortical neurons did not readily
precipitate detectable amounts of these proteins (Figure 5A,
Figure 5. SARM1 Associates with MAVS at
(A) IP with anti-SARM1 or IgG control (IgG) from
the mitochondrial fractions of mock and LACV-
infected WT neurons at 36 hpi, subjected to
immunoblotting by using anti-MAVS, anti-SARM1,
anti-NLRX1, and anti-JNK3 (left panel).
(B) SARM1 IP readily pulled down SARM1 and
MAVS (arrows) in WT, but not Sarm1?/?mice.
Arrow indicates correct band size. Lower band for
MAVS immunoblot is IgG, which is not shown in
(A), (C), or (G).
(C) IP with anti-ATP synthase or IgG control from
mitochondrial fractions of brain tissue from mock
or LACV-infected mice at 5 dpi. IPs were run on
a SDS-PAGE, transferred to a blot, and probed for
SARM1 or MAVS.
(D) Immunoblot analysis of mitochondria fraction
or whole-cell lysate from mock or LACV-infected
WT neurons at 36 hpi probed for ATP synthase,
MAVS, NLXR1, Cox IV, and/or b-actin.
(E and F) MAVS was IP from the (E) mitochondria
or (F) whole-cell lyaste of neurons at 36 hpi. The
blots were probed with anti-SARM1 to confirm
coIP of SARM1.
(G) HEK293T cells were transfected with GFP-
tagged full-length SARM1 and then infected with
LACV. Mitochondria were isolated at 36 hpi for
IP analysis using anti-SARM1 antibody. The
membrane was probed with anti-GFP or anti-
MAVS. (H) Mitochondrial fraction of neurons from
WT and Mavs?/?mice at 36 hpi, probed with anti-
SARM1. Densitometry quantitation of bands is
displayed below in arbitrary units. All data are
representative of at least two replicate experi-
ments. See also Figure S4.
data not shown). Instead, IP studies
with SARM1 analyzed either by tandem
mass-spectroscopy (MS/MS) or immu-
noblot indicated two readily detectable
protein interactions with SARM1; ATP
synthase and MAVS (Figure 5A; Fig-
ure S5). Specificity of the IP with anti-
SARM1 in precipitating SARM1 was
confirmed using MS analysis (Figure S4)
as well as the lack of precipitation of
SARM1 in cells from Sarm1?/?mice (Fig-
ure 5B). Coimmunoprecipitation (coIP) of
these proteins was also detectable by IP
of mitochondrial fractions from LACV-
infected brain tissue (Figure 5C). Immu-
noblot analysis on mitochondrial fractions from LACV-infected
neurons showed increased amounts of MAVS and ATP synthase
at the mitochondria during LACV infection (Figure 5D).
MAVS Localizes with SARM1 during LACV Infection
MAVS is located on mitochondria, mitochondrial associated
membranes (MAMs), and peroxisomes, and forms a signaling
complex following activation of RIG-I and other RLRs (Horner
et al., 2011; Seth et al., 2005). LACV infection induces the activa-
tion of RIG-I (Verbruggen et al., 2011) and RIG-I protein
Neuronal Death Mediated by MAVS Induction of SARM1
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expression was elevated in brain tissue from LACV-infected
mice compared to WT controls (Figure S3F). To confirm MAVS
interaction with SARM1, mitochondrial fractions from mock
and LACV-infected neurons were immunoprecipitated using
anti-MAVS, which resulted in the precipitation of SARM1 from
mitochondria (Figure 5E), although it was undetectable in an IP
of the whole cell lysate (Figure 5F). CoIP of MAVS with SARM1
was confirmed in the mitochondrial fraction of HEK cells trans-
fected with green-fluorescent protein (GFP)-tagged SARM1
Confocal microscopy analysis also demonstrated colocaliza-
tion of SARM1 and MAVS in LACV-infected neurons compared
to controls (Figure S6). Pearson’s correlation coefficient anal-
ysis, which measures degree of colocalization, was higher in
LACV-infected neurons (range of 0.067 to 0.624) than in mock-
infected cultures (range of ?0.312 to ?0.078) indicating an
increase in colocalization of SARM1 with MAVS following infec-
tion. This was particularly true in axons, where SARM1 and
MAVS aggregated in specific regions in LACV-infected neurons
(Figures S5B and S5D), but not in mock-infected neurons
drial marker TOMM20 in infected neurons (Figures S5C and
S5E), correlating with the IP of SARM1 with MAVS in mitochon-
drial fractions (Figure 5E). Thus, both IP and colocalization
studies demonstrate interactions of SARM1 with MAVS are
increased by LACV-infection.
MAVS Deficiency Inhibits SARM1 Upregulation and
SARM1-Induced Neuronal Apoptosis
The interaction of SARM1 with MAVS suggested that MAVS may
also influence LACV-induced neuronal death. To examine the
role of MAVS in SARM-mediated neuronal death, we utilized
neurons from Mavs?/?mice. LACV-infection of Mavs?/?neurons
did not result in increased SARM1 at the mitochondria as
observed in neurons from WT mice (Figure 5H) and did not
produce detectable ROS (Figure 4I). Additionally, MAVS-
deficient neurons had a delay in the onset of LACV-induced
neuronal death (Figures 6A–6C). In contrast, deficiency in
MyD88, which is an adaptor molecule for TLR signaling and
has been shown to interact with amphioxus SARM1 (Yuan
et al., 2010), had no such effect (Figure 6D). This indicated that
MAVS, but not MyD88, was required for SARM1-mediated
neuronal death during LACV infection. Furthermore, siRNA
targeted against RIG-I, which induces MAVS activation, also
inhibited LACV-induced cell death (Figure S3H). As expected,
deficiency in MAVS inhibited the Ifnb1 mRNA response to virus
infection (Figure 6E). However, Sarm1 mRNA expression was
also inhibited in Mavs?/?neurons (Figure 6F) indicating that acti-
vation through MAVS was required for LACV-induced upregula-
mitochondria, MAVS also appears to be important for the induc-
tion of SARM1 during LACV infection.
The role for MAVS in SARM1-mediated neuronal death sug-
gested that MAVS may contribute to neuronal pathogenesis
in vivo. However, the MAVS pathway may also have an impor-
tant role in the induction of type I IFN responses produced in
response to LACV infection, which are protective in vivo as
shown by Ifnar1?/?mice (Blakqori et al., 2007; Hefti et al.,
1999) and Irf3?/?, Irf7?/?mice (Figure 3A). Therefore, in vivo,
MAVS may have both protective as well as pathogenic roles
during LACV infection. Mavs?/?mice infected with 103PFU
of virus had similar incidence and kinetics of neurological
disease development comparable to WT mice (Figure 3A),
despite reduced type I IFN responses (Figure 3B; data not
shown). Thus, MAVS deficiency does not provide the same
protection in vivo as SARM1 deficiency, despite the similar
effect observed in reducing LACV-induced neuronal death,
Figure 6. MAVS Is Required for SARM1
(A) Cell death in neurons from WT and Mavs?/?
mice infected with LACV. MTT assay was per-
formed as described in Figure 2. P value for
two-way ANOVA for strain difference was < 0.01.
(B) Neurons from WT (left panel) or Mavs?/?mice
stained with TUNEL (green) and LACV (red).
(C) Quantification of the number of TUNEL-
positive cells in WT and Mavs?/?cultures as
described in Figure 1.
(D) Neurons from WT and either Mavs?/?or
Myd88?/?mice were infected with LACV and
death was compared to WT control cultures
generated the same day for each deficient strain
and each experiment.
(E) Ifnb1 and (F) Sarm1 mRNA in the mock and
LACV-infected neurons from WT and Mavs?/?
mice were determined quantitatively by real time
PCR at 24, 30, and 36 hpi. P value for two-way
ANOVA for strain difference between LACV-
infected groups was < 0.001 All data are the
mean ± SEM of 3 to 6 samples per point and are
representative of 2 to 4 replicate experiments. See
also Figure S5.
Neuronal Death Mediated by MAVS Induction of SARM1
Immunity 38, 705–716, April 18, 2013 ª2013 Elsevier Inc. 711
MAVS Influences SARM1 Localization to the
Mitochondria during Virus Infection
The above study indicated that MAVS could influence SARM1-
first in the upregulation of SARM1 (Figure 6F) and second at the
mitochondria where MAVS directly interacts with SARM1 (Fig-
ure 5). Because SARM1 also contributes to neuronal death in
instances where MAVS would not be activated, such as OGD,
we examined whether MAVS was required for OGD induced
neuronal death. Mavs?/?neurons underwent OGD at a similar
rate as WT controls (Figure S6), whereas Sarm1?/?neurons
did not, indicating that MAVS was not required for SARM1-
mediated neuronal death induced by OGD.
To determine whetherwecould circumvent the MAVS require-
ment for SARM1 upregulation during LACV infection, we stimu-
lated Mavs?/?neurons with the TLR7 agonist, imiquimod. Stim-
ulation with imiquimod did increase SARM1 mRNA and protein
expression in Mavs?/?neurons (Figure 7A; data not shown)
and induced a low amount of cell death (Figure 7C; data not
shown). However, neurons from Mavs?/?mice had significantly
lower amounts of cell death compared to wild-type controls
even in the presence of imiquimod-induced SARM1 expression
(Figure 7C). Furthermore, even though SARM1 was induced by
imiquimod stimulation in Mavs?/?neurons, SARM1 did not
localize to the mitochondria in the absence of MAVS (Figure 7B).
two time points during LACV infection, first in the induction of
Sarm1 mRNA expression and then in translocation of SARM1
protein to the mitochondria resulting in oxidative damage and
RLR-induced MAVS activation has an important role in the
generation of type I IFN response and inhibition of virus repli-
cation (Scott, 2010). The current study provides evidence that
the RLR pathway is also involved in virus-mediated neuronal
of our studies, MAVS activation during LACV-infection of
neurons results in increased expression of SARM1. SARM1
then localizes to the mitochondria where it interacts with MAVS
and induces oxidative stress, mitochondrial damage, and
ultimately neuronal death. These studies provide mechanistic
details for both SARM1 induction in neurons as well as innate
immune signaling-mediated neuronal death.
Our current results indicate that SARM1-mediated cell
damage, not virus replication per se, is a key mediator of
bunyavirus-induced neuronal death. Cell death was significantly
lower in Sarm1?/?neurons compared to WT neurons despite
similar amounts of viral RNA and widespread infection of
cultures. Similar results were observed in vivo, where virus
RNA expression was similar between WT and Sarm1?/?mice,
despite the difference in clinical outcome. The ability to inhibit
or delay the onset of neuronal damage in the presence of an
insult is important not only for therapeutic potential in virus-
mediated disease but could also be important for other neurode-
generative diseases where neuronal cell death is a hallmark of
This study also demonstrates a direct role for the innate
immune response in mediating neuronal damage during virus
infection. The requirement for MAVS in the induction of SARM1
expression and the interaction of MAVS with SARM1 at the mito-
chondria demonstrates that the MAVS activation in the neuron
can be harmful. Studies with TLR ligands also demonstrated
innate immune signaling-mediated neuronal damage (Butchi
et al., 2010; Cameron et al., 2007; Lathia et al., 2008; Ma et al.,
2006, 2007; Tang et al., 2008), although the mechanism behind
TLR-induced cell death is still unknown. The induction of
neuronal cell death by stimulation of pattern recognition recep-
tors (PRRs) may offset the benefits of the type I IFN response
and pathogenesis may explain why MAVS deficiency did not
significantly alter the incidence of LACV-induced neurological
disease in vivo. Possibly, the balance between the SARM1
Figure 7. MAVS Is Necessary for SARM1 Localization to the Mito-
chondria during LACV Infection
Neurons from Mavs?/?mice were treated with 5 uM imiquimod at the time of
infection, and mitochondria and whole cell lysate were generated at 36 hpi.
(A and B)Immunoblot of SARM1 inthe(A) whole celllysate or (B)mitochondrial
fraction of cells. Blots from wild-type and MAVS deficient neurons were pro-
cessed under identical conditions.
(C) Cell viability at 36 hpi from above experiment. Data are shown as the
mean ± SEM of 3–6 samples per group per time point. A low amount of
imiquimod-induced cell death was observed in most, but not all, experiments.
See also Figure S6.
Neuronal Death Mediated by MAVS Induction of SARM1
712 Immunity 38, 705–716, April 18, 2013 ª2013 Elsevier Inc.
pathway and the type I IFN response may determine whether the
innate immune response is pathogenic or protective in neu-
rovirulent viral diseases.
In contrast to our results with LACV infection, SARM1 did not
alter viral pathogenesis during West Nile Virus (WNV) infection in
mice (Szretter et al., 2009). Although both LACV and WNV are
RNA viruses that activate the RLR pathway (Verbruggen et al.,
2011; Fredericksen and Gale, 2006), they belong to separate
virus families and replicate via different mechanisms. Both
viruses are also known to express virus proteins that subvert
the host immune response (Verbruggen et al., 2011; Freder-
icksen and Gale, 2006). The different role of SARM1 in the path-
ogenesis of these viruses may be partially due to different viral
proteins affecting neuronal cell death. It will be important to
determine whether other encephalitic RNA viruses induce
SARM1 expression in neurons or perhaps have mechanisms to
limit the SARM1 induction of cell death.
SARM1 has been associated with cell death in other systems,
including OGD-induced neuronal death, axonal injury, and
most recently T cell death (Kim et al., 2007; Osterloh et al.,
2012; Panneerselvam et al., 2013). SARM1-mediated damage
appears mechanistically to be similar among different cell types,
with SARM1 leading to ROS generation and mitochondrial
damage as demonstrated in our study with neurons and as
recently shown for T cells (Panneerselvam et al., 2013). How-
ever, the mechanism of SARM1 upregulation appears to be
through distinct pathways, with innate immune responses
leadingto SARM1 expressionin neurons, whereas SARM1 upre-
gulation in T cells was associated with activation-induced cell
death and neglect-induced cell death pathways (Panneerselvam
et al., 2013).
MAVS also contributes to apoptosis of other cell types (Lei
et al., 2009; McAllister and Samuel, 2009; Yu et al., 2010).
MAVS-mediated cell death in these systems is independent of
the type I IFN response, similar to our current study where defi-
ciency in IFNAR1 or IRF3 and IRF7 did not affect cell death.
However, MAVS induces NF-kB activation (Sun et al., 2006),
which is also observed following LACV infection (data not
shown), and this pathway may be responsible for the increase
in SARM1. It is possible that increased SARM1 expression is
a common response to MAVS activation and that other factors,
such as the expression of antiapoptotic molecules, determine
whether MAVS activation leads to cell death. Overexpression
of SARM1 through MAVS stimulation may have therapeutic
potential in diseases where targeted cell death is the goal as in
the case of tumors.
Other proteins may also influence MAVS and SARM1 activa-
tion during LACV infection. MAVS forms a signaling complex at
the mitochondria or MAMs where it interacts with the ubiquitin
protein ligase TRAF6 to induce signaling (Yoshida et al., 2008).
NLRX1 can also interact with MAVS at the complex to negatively
regulate MAVS (Allen et al., 2011). Although NLRX1 was pre-
dicted to interact with SARM1 based on computational studies
(Li et al., 2011), we did not observe an interaction between
SARM1 and NLRX1 in neurons. Furthermore, SARM1 deficiency
did not affect type I IFN or other cytokine production suggesting
that SARM1 does not influence the MAVS signaling pathway.
ATP synthase interacted with SARM1 both in vitro and in vivo.
Disrupted ATP synthase expression and/or function has been
correlated with the generation of ROS in multiple studies,
including ROS generation in neurons (Natera-Naranjo et al.,
2012; Comelli et al., 1998). Although our current studies have
not identified suppression of ATP synthase activity by SARM1,
SARM1 interactions with ATP synthase may affect ATP synthase
function and influence the generation of ROS.
In the current study, SARM1 interacted with MAVS at the
mitochondria. Deficiency in MAVS resulted in a decrease in
SARM1 localization to the mitochondria, even when SARM1
was independently induced by imiquimod stimulation. However,
SARM1 also contributes to neuronal death in instances where
MAVS may not be activated, such as OGD or axonal damage
(Kim et al., 2007; Osterloh et al., 2012). Indeed, MAVS was not
necessary for OGD-induced cell death, suggesting that other
mechanisms can induce mitochondrial localization of SARM1.
SARM1 contains a mitochondrial signaling sequence (Panneer-
selvam et al., 2012) and overexpression of SARM1 constructs
in COS-cells or HEK cells is sufficient to induce mitochondrial
SARM1 localization (Kim et al., 2007; Panneerselvam et al.,
2013). However, SARM1 also interacts with the cytoplasmic
domain of syndecan 2 in neurons (Chen et al., 2011). Perhaps
in neurons, SARM1 needs a trigger for mitochondrial localization
by interacting with a complex containing MAVS or other mito-
chondrial proteins. Although the amount of SARM1 induced by
imiquimod stimulation was similar to that induced by LACV
infection alone, it may be insufficient to induce mitochondrial
localization in the absence of critical cofactor. Further investiga-
tion into the mechanisms by which SARM1 localizes to the
mitochondria in other disease models will be essential in under-
standing how SARM1 contributes to cell death.
Our studies demonstrate a mechanism of virus-induced
neuronal cell death that involves activation of the innate immune
response and SARM1 induction. Because SARM1 is normally
expressed in neurons and has a clear role in dendritic arboriza-
tion (Chen et al., 2011), the overexpression of SARM1 and
resulting oxidative damage may simply be an unintended conse-
quence of stimulation of neurons through pathways that are
primarily required for neuronal development. Developing thera-
peutics that target SARM1, but not MAVS, may be a viable
strategy for inhibiting neuronal damage during virus infection
while still allowing antiviral responses induced by RIG-I and
Infection of Mice with LACV
Sarm1?/?and Irf3?/?, Irf7?/?mice were kindly provided by Michael Diamond,
Washington University (Szretter et al., 2009; Daffis et al., 2009). These mice as
well as Myd88?/?mice (Adachi et al., 1998) and Ifnar1?/?mice (Auerbuch
et al., 2004) were maintained on a C57BL/6 background. Mavs?/?mice were
purchased from Jackson Laboratories. These strains, as well as Inbred Rocky
Mountain White (IRW) mice, were maintained at Rocky Mountain Laboratories
(RML). WT refers to the C57BL/6 strain unless otherwise noted. All of the
animal procedures were conducted in accordance with the RML Animal
Care and Use Committee guidelines under protocol RML2011-65. LACV
human 1978 stock was a kind gift from Richard Bennett (NIAID, NIH) and
has been previously described (Bennett et al., 2007). At 3 weeks of age,
mice were inoculated intraperitoneally with 103PFU of LACV. Mice were
observed daily for signs of encephalitis. Mice at 5 dpi or at the time of clinical
signs were euthanized and brains were removed for histology and RNA
Neuronal Death Mediated by MAVS Induction of SARM1
Immunity 38, 705–716, April 18, 2013 ª2013 Elsevier Inc. 713
Primary Cultures of Cortical Neurons and LACV Infection
Primary culturesof cortical neurons werepreparedfrom 14to16day gestation
mouse embryos from indicated mouse strains or from IRW mice. Mouse
cortices were digested in CMF-HBSS containing 0.125% Trypsin followed
by dissociation by repeated pipetting. Cells were plated in amine-coated
plates (BDBiosciences)at83105cells/ml.Following attachment, themedium
was replaced with neurobasal medium containing 2% B-27 and 0.5 mM
glutamine. After overnight culture, primary neurons were infected with LACV
at a MOI of 0.01 unless otherwise indicated. Mock-infected cultures were
treated with equivalent amounts of supernatants from uninfected Vero cells.
Samples were collected for RNA analysis or MTT assay at 36 hpi unless
otherwise indicated. For caspase inhibition studies, cells were treated with
200 mM of Z-VAD-FMK or 10 mM of Z-WEHD-FMK (R&D Systems) at the
time of LACV infection. For imiquimod studies, 5 mM of imiquimod was added
to cultures at the time of LACV infection. For OGD studies, neurons were
cultured in OGD buffer as described (Kim et al., 2007) and placed in an
incubator containing 95% N2and 5% CO2 for 1–3 hr.
Mouse brains were fixed in 10% neutral-buffered formalin, embedded in
paraffin, and cut in 4 mm sections. Immunohistochemical analysis was
completed using an antigen-retrieval protocol (Du et al., 2010). SARM was
detected using an anti-SARM1 (Proscience) and detected using Alexa Fluor
555 conjugated goat anti-rabbit. SARM1 specificity was confirmed by staining
tissue sections from Sarm1?/?mice. Cell body staining of SARM1 was
confirmed as specific (Figures S1D–S1F, yellow arrow), however, nonspecific
staining of nuclei was observed in both WT and Sarm1?/?mice as shown in
spinal cord sections (Figures S1D–S1F, white arrow). Neurons were detected
by using mouse anti-MAP2 (Millipore) with an Alexa Fluor 488 conjugated
anti-mouse. LACV infection was detected using anti-G2 monoclonal (QED
Biosciences, 18572) and Alexa Fluor 555 goat anti-mouse. TUNEL was
detected by using the in situ cell death detection kit (Roche Applied Science).
Slides were mounted with ProLong Gold antifade reagent (Invitrogen) with or
without DAPI. Slides incubated without primary antibodies or with isotype
controls were used to confirm specificity.
RNA was isolated from primary cortical neurons using the RNA isolation kit
(Zymo Research). cDNA was prepared from RNA samples as described
(Du et al., 2010). Primers were designed using primer3 website (Rozen and
Skaletsky, 2000) with a Tm of 60?C. SYBR green dye with ROX (Bio-Rad)
was used for measurement of real-time PCR amplification. Data for each
sample was calculated as the percent difference in CT value (DCT =
CTGapdh - CTgene of interest). The data was plotted as mean percent Gapdh
values for each gene of interest for each sample. Superarray analysis was
conducted as described (Du et al., 2010). Data were calculated as fold differ-
ences between mock and LACV infected neurons and brain homogenates for
both WT and Sarm1?/?mice.
MTT Assay to Determine Cell Viability
Neurons were cultured in 96 well plates and inoculated with mock superna-
tants or LACV. At specific time points, triplicate or quadruplicate wells per
treatment were incubated with MTT reagent (Invitrogen) at a concentration
of 0.5 mg/ml for 3 hr. The MTT solution was aspirated and the cells were
lysed in DMSO. The formazan concentration in each well was measured
by absorbance at 540 nm using a cell plate reader (Synergy 4, BioTek).
Data were compared with mock-infected cultures to determine percent
cell death. Cultures from deficient mice were directly compared to WT
control cultures generated the same day and treated with the same virus
Measurement of DNA Fragmentation by Using TUNEL Assay
Apoptotic cell death following LACV infection was analyzed in the primary
cortical neurons by TUNEL reaction using the in situ cell death detection kit
(Roche). Cells were further stained with a mouse antibody against the G2
envelope protein of LACV (QED Bioscience, Inc.) and anti-mouse Alexa Fluor
Caspase-3 activity was determined using a caspase-3 colorimetric assay kit
(GenScript). Cells were lysed, centrifuged at 10,000 rpm, and the supernatant
was collected. We incubated 300 mg of protein with the caspase-3 substrate.
The samples were incubated for 6 hr and the extinction values obtained using
Synergy 4, Biotek spectrophotometer at 405 nm.
Transfection of Primary Neurons with SARM1, Mda5, or Rig-I siRNA
Primary cortical neurons were cultured for 3 days and transfected with 50 nM
of Sarm1 (Dharmacon, Thermo Scientific), Mda5, or Rig-I siRNA (Santa Cruz
Biotechnology) using 0.5 ml of HiPerFect (QIAGEN) for 24 hr. For confirmation
of Sarm1 inhibition, siRNA for Sarm1 from Santa Cruz Biotechnology was also
used and yieldedsimilar results.All siRNAs were apoolof three target-specific
siRNAs. Cells also were transfected with a nontargeting siRNA (Dharmacon,
Thermo Scientific). Neurons were then infected with LACV at a MOI of 0.01
and cultured for 36 hpi. Transfection efficiency was ?35% as confirmed with
cGFP siRNA transfection.
Transfection of HEK Cells with GFP-Tagged SARM1
HEK293T cells were seeded at a density of 4 3 106cells/ml. Cells were trans-
fected with mouse GFP-SARM1 (OriGene) and LTX reagent (Invitrogen).
Following transfection, cells were infected with LACV (MOI 0.01). At 36 hpi,
cells and immunoprecipitated using SARM1 antibody (Proscience).
Immunoprecipitation and Immunoblot
Mitochondria and cytosolic fractions from mock and LACV-infected primary
neurons were isolated by using a mitochondria and cytosol fractionation kit
(Millipore). The IP matrix from ImmunoCruz IP/WB Optima F system (Santa
Cruz) was used for all IP. For each reaction, the IP-matrix was incubated over-
night with anti-SARM1 (Proscience). Intact mitochondrial fractions were lysed
in 0.1% NP-40, 50 mM Tris, pH 7.4, and 25 mM NaCl, precleared with non-
specific IgG and added to IP-matrix beads and incubated overnight prior to
processing for immunoblot analysis. Immunoblotting was done using anti-
rabbit SARM1 (Genetex), anti-MAVS (Cell Signaling), anti-NLRX1 (Millipore),
and anti-mouse JNK3 (Novus Biologicals). Cox IV (Abcam) was used as a
mitochondrial loading control. Protein was detected using a Typhoon scanner
and analyzed with ImageQuant TL software (GE Healthcare). Specificity of
the SARM1 antibody was confirmed by comparison to cells from Sarm1?/?
mice (Figure 1F).
Tandem Mass Spectrometry Analysis
Samples isolated by IP were separated by SDS-PAGE and stained with
Coomassie blue. Those bands selected for analysis by HPLC-based
nanospray LC-MS/MS were processed and analyzed as described previ-
ously (Moore et al., 2010). Peak lists were searched using MASCOT Daemon
(Perkins et al., 1999). The proteins identified by MASCOT were visualized
using the proteomics software ProteoIQ (NuSep, Inc Athens, GA, USA).
Proteins were considered present in the sample only if they were identified
by at least two peptides having distinct sequences and Mascot ion scores
Transmission Electron Microscopy
Primary cortical neurons were cultured on Aclar coverslips precoated with
poly-D-Lysine (0.1 mg/ml) (Sigma). Cells were infected with LACV at a MOI
of 0.01 and following 36 hpi, cells were fixed with 2.5% glutaraldehyde.
Samples were prepared for TEM following standard protocols and analyzed
using a H7500 microscope (Hitachi high Technologies, Tokyo, Japan). Images
were acquired in single-blind experiments.
For confocal microscopy, primary cortical neurons were grown on poly-D-
lysine coated chamber slides and infected with LACV at a MOI of 0.01. For
colocalization studies, cells were fixed and permeabilized at 36 hpi and
costained with anti-SARM1 (a gift from Aihao Ding, Cornell University) and
anti-MAVS (Cell Signaling). The slides were analyzed using a Zeiss 510 Meta
Neuronal Death Mediated by MAVS Induction of SARM1
714 Immunity 38, 705–716, April 18, 2013 ª2013 Elsevier Inc.
Detection of Mitochondrial Superoxide Generation
To detect relative superoxide generation, MitoSox Red (Invitrogen) was used.
Primary cortical neurons were cultured in 8-well chamber slides. At 36 hpi with
LACV, cells were incubated with 5 uM of MitoSox Red for 10 min. The relative
intensity of mitochondrial superoxide generation was analyzed using a Zeiss
510 Meta confocal microscope. Images were acquired in single-blinded
All statistical analysis was completed using Graphpad Prism. *p < 0.05,
**p < 0.01, and ***p < 0.001. For a comparison of two values, a two-tailed
Mann-Whitney analysis was performed. For comparison of more than two
values with one variable, a one-way ANOVA was used with a Bonferroni’s
post-test. For comparison of two or more values with more than one variable,
a two-way ANOVA was used with a Bonferroni’s post-test.
Supplemental Information includes six figures and can be found with this
article online at http://dx.doi.org/10.1016/j.immuni.2013.02.013.
The authors thank Sonja Best and Vinod Nair for confocal microscopy
assistance and David Dorward for transmission electron microscopy assis-
tance. The authors also thank Austin Athman for graphics assistance and
Sonja Best, SuePriola, Byron Caughey, and Kim Hasenkrug for critical reading
of the manuscript. The project was supported by the Division of Intramural
Research, National Institutes of Health, National Institute of Allergy and
Received: September 6, 2012
Accepted: February 25, 2013
Published: March 14, 2013
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