Sarm1 knockout protects against early but not late axonal degeneration in experimental allergic encephalomyelitis

Kenneth Viar; Daniel Njoku; Julie Secor McVoy; Unsong Oh

doi:10.1371/journal.pone.0235110

. 2020 Jun 25;15(6):e0235110. doi: 10.1371/journal.pone.0235110

Sarm1 knockout protects against early but not late axonal degeneration in experimental allergic encephalomyelitis

Kenneth Viar ¹, Daniel Njoku ¹, Julie Secor McVoy ¹, Unsong Oh ^1,^*

Editor: Thomas Forsthuber²

PMCID: PMC7316289 PMID: 32584865

Abstract

Programmed axonal degeneration, also known as Wallerian degeneration, occurs in immune-mediated central nervous system (CNS) inflammatory disorders such as multiple sclerosis and the animal model experimental allergic encephalomyelitis (EAE). Sterile alpha and TIR domain containing protein 1 (SARM1) functions to promote programmed axonal degeneration. To test the hypothesis that loss of SARM1 will reduce axonal degeneration in immune-mediated CNS inflammatory disorders, the course and pathology of EAE was compared in Sarm1 knockout mice and wild type littermates. The clinical course of EAE was similar in Sarm1 knockout and wild type. Analysis of EAE in mice expressing neuronal yellow fluorescent protein (YFP) showed significantly less axonal degeneration in Sarm1 knockout mice compared to wild type littermates at 14 days post-induction of EAE. At 21 days post-induction, however, difference in axonal degeneration was not significant. At 42 days post-induction, Sarm1 knockout mice were indistinguishable from wild type with respect to markers of axonal injury, and were similar with respect to axonal density in the lumbar cords. There was no significant change in peripheral immune activation or CNS inflammatory cell infiltration associated with EAE in Sarm1 knockout mice. In conclusion, Sarm1 deletion delayed axonal degeneration early in the course of CNS inflammation, but did not confer long-term protection from axonal degeneration in an animal model of immune-mediated CNS inflammation.

Introduction

Axonal degeneration is a major determinant of disability accumulation in chronic CNS inflammatory disorders such as multiple sclerosis [1,2]. Programmed axonal degeneration, also known as Wallerian degeneration, is known to occur in multiple sclerosis and in the animal model EAE [3,4]. However, whether or not targeting programmed axonal degeneration confers protection from long-term axonal loss in immune-mediated CNS inflammatory disorders such as multiple sclerosis and EAE remains unconfirmed.

Proteins involved in nicotinamide adenine dinucleotide (NAD) metabolism such as SARM1 and nicotinamide mononucleotide adenylyltransferase (NMNAT) were identified as key opposing factors that control programmed axonal degeneration [5,6]. SARM1 activation results in derepression of its NADase activity, which resides in its Toll/interleukin-1 (TIR) domain, leading to intra-axonal NAD depletion, local energetics failure and axonal degeneration [7]. SARM1-dependent neurodegeneration has been implicated in a number of different models of neuronal injury based on the finding that Sarm1 deletion was neuroprotective in models of traumatic, toxic and metabolic nervous system injury [8–10]. Thus prior research suggested that SARM1-dependent program of axonal degeneration might be a common mechanism contributing to neurodegeneration in a variety of contexts.

In addition to the function of neuronal SARM1 in programmed axonal degeneration, SARM1 expression in immune cells has the potential to influence immune response through its TIR domain, which can modulate Toll-like receptor signaling [11]. Prior reports suggested that the effect of SARM1 inactivation on the immune response depends on disease model or species studied. In human peripheral blood leukocytes, SARM1 functions to inhibit immune responses [11–13]. In animal models of CNS infections, however, Sarm1 deletion led to reduced CNS inflammation, suggesting that SARM1 functions to augment CNS anti-viral response [14,15]. The role of SARM1 in the immune response associated with immune-mediated “sterile” CNS inflammation such as EAE is unknown.

To test the hypothesis that Sarm1 deletion would reduce axonal degeneration in CNS inflammatory disorders, the course and pathology of EAE in Sarm1 knockout mice and wild type littermates were compared. Clinical course and markers of axonal degeneration were assessed. Immune activation and inflammatory infiltrates were also assessed to exclude the possibility that changes in the neuroimmunology of EAE in Sarm1 knockout mice might influence the outcome. We found that the incidence and clinical course of EAE were similar in Sarm1 knockout mice and wild type littermates. Sarm1 knockout mice showed substantially less axonal injury early in the course of EAE. However, the extent of axonal degeneration did not differ substantially later in the course of EAE.

Materials and methods

Reagents

All reagents were from Thermo Fisher Scientific unless otherwise specified.

Animals

Sarm1 knockout mice (B6.129X1-Sarm1^tm1Aidi/J) were obtained from Jackson Laboratory (Bar Harbor, ME) and maintained on a C57BL/6J background. Heterozygous mating produced homozygous Sarm1 knockout (i.e. Sarm1-/-), heterozygotes (i.e. Sarm1+/-) and wild type (i.e. Sarm1+/+) littermates. A colony of Sarm1 knockout mice and wild type littermates expressing neuronal YFP was generated by crossing Sarm1 knockout mice with Thy1-YFP-H transgenic mice (B6.Cg-Tg(Thy1-YFP)HJrs/J, Jackson Laboratory). Sarm1-/-YFP+ mice and Sarm1+/+YFP+ littermates were generated from Sarm1+/-YFP+ x Sarm1+/- mating pairs. Mice were genotyped using the following PCR primers. Sarm1 knockout forward and reverse primers were CTT GGG TGG AGA GGC TAT TC and AGG TGA GAT GAC AGG AGA TC, respectively. Wild type forward and reverse primers were GGG AGA GCC TTC CTC ATA CC and TAA GGA TGA ACA GGG CCA AG, respectively. YFP transgene expression was detected by the presence of neuronal YFP in ear punch samples under fluorescence microscopy. Animals were housed in littermate groups regardless of genotype, on a 12 h light-dark cycle and fed ad libitum. All animal procedures were performed in accordance with the Virginia Commonwealth University Animal Care and Use Program’s regulations under an approved protocol (protocol number: AD10000395).

Neuronal cultures, axotomy and oxidative stress

Cortex from E15.5 embryos of Sarm1 knockout and C57BL/6J mice were dissected then enzymatically dissociated in neurobasal medium containing trypsin (2.5 mg/ml, Sigma Aldrich) and DNase I (15 μg/ml, Sigma Aldrich) for 30 minutes at 37°C, washed in neurobasal medium, then triturated using fire-polished Pasteur pipettes. Cell suspensions were passed through a 70 μm strainer to remove debris. Cells were plated on poly-D-lysine (Sigma Aldrich) coated plates or glass-bottomed dish and cultured in neuronal culture media (neurobasal medium supplemented with B27, GlutaMax (0.5 mM) and penicillin/streptomycin/amphotericin B). Neuronal culture media were changed by one-half volume exchange every 3 days. Cytarabine (1 μM final, Sigma Aldrich) was added from day in vitro (DIV) 3 to 6 to inhibit glial proliferation. Axotomy was performed manually on DIV 12 to 14 neurons cultured in 35 mm glass-bottomed dish using the cutting edge of a 27-gauge sterile needle under visual guidance through a low magnification inverted light microscope. Neurons (DIV 10 to 12) were subjected to oxidative stress by 30-minute exposure to hydrogen peroxide (H₂O₂). First, neuronal culture media was replaced by 2 x volume exchange with plain neurobasal media. H₂O₂ in plain neurobasal media was added to wells by ½ media exchange to indicated final concentrations. After 30 minutes of exposure at 37°C, media was replaced by 2 x volume exchange to neuronal culture media. Cells were analyzed 24 hours later. Viability of cultured neurons was assessed by fluorescent dye exclusion (Ready Probes Cell Viability Imaging Kit Blue/Green) and expressed as percent of control (0 μM H₂O₂).

Neuronal immunocytochemistry

Tau, NeuN and ankyrin G were detected in cultured neurons by immunocytochemistry. Neurons were fixed for 10 minutes with 4% (w/v) paraformaldehyde, then washed 3 times in phosphate buffered saline (PBS). Cells were permeabilized in PBS containing 0.3% Triton-X100 and 10% normal goat serum, then incubated with primary antibodies against Tau (clone EP2456Y, Abcam, RRID: AB_1524475), NeuN antibody (clone A60, Millipore Sigma, RRID: AB_2298772) and ankyrin G (clone N106/36, Millipore Sigma, RRID: AB_2749806) for 1 hour at room temperature. After washing in PBS, cells were incubated with fluorochrome conjugated secondary antibodies for 1 hour at room temperature, then washed in PBS prior to microscopy. Tau immunocytochemistry was imaged on Zeiss LSM710 confocal laser scanning microscope on 40x/1.3 NA oil-immersion objective using a pin hole of 1 Airy disc unit and Nyquist sampling. For NeuN and ankyrin G immunocytochemistry, 3 non-overlapping fields of view were imaged from each well on a FLoid Cell Imaging Station using a 20x/0.45 NA objective.

EAE induction and clinical scoring

EAE was actively induced in 6- to 10-week old Sarm1 knockout mice and wild type littermates by subcutaneous injection of 200 μg of myelin oligodendrocyte glycoprotein peptide 35–55 (MOG_35-55, Anaspec) emulsified in complete Freund’s adjuvant (CFA) containing 500 μg M. Tuberculosis H37 RA (Difco) and intraperitoneal injection of 300 ng pertussis toxin (List Biological Laboratories), followed by a second intraperitoneal injection of 300 ng pertussis toxin 2 days later. Female and male mice were induced. Animals that died prior to onset of clinical EAE were included in the mortality analysis, but excluded from analysis of EAE clinical scores. Mice were scored daily as follows: 0 –no overt signs of disease; 1 –limp tail or loss of righting reflex but not both; 2 –limp tail and loss of righting reflex; 3 –partial hind limb paralysis; 4 –complete hind limb paralysis; 5 –moribund state or death. Clinical score rater was blinded to genotype. Primary clinical outcome was the mean total or cumulative clinical score defined as the sum of daily clinical scores from induction until 42 days post-induction [16].

Perfusion fixation and tissue processing

Following humane killing, animals underwent transcardiac perfusion with up to 50 ml of normal saline followed by perfusion with 100 ml of 4% (w/v) paraformaldehyde in PBS using a rate controlled pump. Following fixation, the entire CNS tissue was dissected out then cryoprotected in 30% sucrose in PBS for over 48 h. Spinal cords were cut into three 1 cm length sections measured from the cervicomedullary junction caudally under a dissecting microscope to approximate cervical, thoracic and lumbar spinal cord segments. Lumbar cord was further cut into two 0.5 cm segments—rostral half for transverse sections and caudal half for coronal sections. Tissue were then cryopreserved at -80°C in Optimal Cutting Temperature compound. Serial 20 μm sections through the entire lumbar cord in a ventral to dorsal direction (coronal sections) was obtained for each animal, and every 5th slide (~300 μm interval) was processed for YFP+ axon analysis or immunohistochemistry. Alternatively, transverse sections of lumbar cords at ~300 μm intervals were processed for SMI-31 immunohistochemistry.

Analysis of YFP+ axons

Coronal sections from the lumbar cords of EAE-induced Sarm1-/-YFP+ and Sarm1+/+YFP+ mice were rinsed 3 times in Tris buffered saline (TBS), them mounted with anti-fade mounting media (Vectashield, Vector Laboratories) and coverslips. Sections were imaged on a FLoid Cell Imaging Station using a 20x/0.45 NA objective. Six non-overlapping fields of view were acquired from each section. Slides and image files were coded to blind research personnel to genotype. Image analysis was performed using ImageJ by drawing 3 equidistant vertical lines over each image. Intact and fragmented axons that crossed each line was manually counted then averaged for each image. About 24 images were available and analyzed for each animal. Results were expressed as fragmented and total YFP+ axons per field of view per animal.

Immunohistochemistry

Animals were humanely killed at 42 days post-induction for immunohistochemical analysis of axonal degeneration. Tissue were processed as described above. Antigen-retrieval was performed for all samples by incubation of tissue sections in a citric acid buffer for 10 min at 45°C in a temperature-controlled microwave (BioWave Pro, Pelco), followed by 3 rinses in TBS. For APP immunohistochemistry, nonspecific antibody binding was blocked by 30 min incubation in a TBS blocking solution containing 4% cold water fish skin gelatin and 0.3% Triton-X 100. Sections were then incubated overnight at 4°C with anti-APP antibody (RRID: AB_2533275). After several washes in TBS, sections were incubated with appropriate secondary antibodies for 90 minutes at room temperature. After several washes, slides were mounted with anti-fade mounting media and coverslips for fluorescence microscopy. For SMI-31 and SMI-32 immunohistochemistry, sections were post-fixed in pre-chilled methanol at -20°C for 10 min, followed by 3 rinses in TBS prior to antigen-retrieval. Following methanol post-fixation and antigen retrieval, Mouse on Mouse kit (Vector Laboratories) was used according to manufacturer’s instructions to block non-specific binding. Sections were incubated overnight at 4°C with SMI-31 antibody (BioLegend, RRID: AB_2564642) or SMI-32 antibody (BioLegend, RRID: AB_2564641) for immunodetection of phosphorylated and non-phosphorylated neurofilament H, respectively. After several washes in TBS, biotinylated anti-mouse IgG followed by fluorescein conjugated Avidin D (Vector Laboratories) or Texas Red conjugated Avidin D (Vector Laboratories) were applied for SMI-31 and SMI-32 immunohistochemistry, respectively, according to manufacturer’s instructions. After several washes, slides were mounted with anti-fade mounting media and coverslips. DAPI nuclear stain was applied to sections prior to final washes for all immunohistochemistry samples.

Image acquisition and analysis

For all image acquisition and analysis, slides and image files were coded to blind research personnel to genotype. For APP immunohistochemistry, images were acquired using a Zeiss AxioImager Z2 microscope. Six non-overlapping field of view images from each lumbar cord section were taken with a 20x/0.8 NA objective. About 24 images were available and analyzed per animal. ImageJ was used to perform a region of interest (ROI) analysis comparing APP+ axon areas between the groups. APP+ signal thresholding at 0.1% was applied to each image. Three non-overlapping ROI (150 μm x 150 μm) were applied to each image and each ROI analyzed for APP+ area using the Analyze Particles (size 1 to infinity; circularity 0–1) function of ImageJ. Thus about 72 ROIs were analyzed per animal to derive the mean APP+ area per ROI per animal.

For SMI-32 immunohistochemistry, images were acquired on a FLoid Cell Imaging Station using a 20x/0.45 NA objective. Six non-overlapping fields of view were acquired from each section. About 24 images were available and analyzed per animal. Image analysis was performed using ImageJ. SMI-32 signal thresholding at 3% was applied to each image. Three non-overlapping ROI (200 μm x 200 μm) were superimposed on each image. Each ROI was analyzed for SMI-32+ area using the Analyze Particles (size 2 to infinity; circularity 0–1) function of ImageJ. Thus about 72 ROIs were analyzed per animal to derive the mean SMI-32+ area per ROI per animal.

For SMI-31 immunohistochemistry, images were acquired on a Zeiss AxioImager Z2 fluorescence microscope equipped with a motorized stage at 10x/0.45 NA objective. A single montage image of the entire transverse section of a lumbar cord was acquired by using the slide scanning function of Neurolucida 360 software (MBF Biosciences). Image analysis was performed using ImageJ. Images were converted to 8-bit images. Auto Threshold (Max Entropy) function of ImageJ was applied to each image. ROI consisting of an entire hemicord was outlined manually. SMI-31+ area was analyzed using the Analyze Particles (size 0 to infinity; circularity 0–1) function of ImageJ. Data from at least 4 hemicords per animal were averaged to obtain the mean SMI-31+ area per hemicord per animal.

Fluorescence-activated cell sorting (FACS) analysis

Following humane killing, animals underwent transcardiac perfusion with up to 50 ml of normal saline using a rate controlled pump. Thoracolumbar cords (from T2 to caudal end) were expelled out of the spinal column using hydraulic pressure manually applied through a 19-gauge needle and syringe filled with PBS. Cords were minced using a McIlwaine tissue chopper (Mickle Laboratory Eng. Co., UK), then enzymatically dissociated in RPMI media containing 2.5 mg/ml collagenase D (Roche Diagnostics) and DNase I (20 μg/ml, Sigma Aldrich) for 45 minutes at 37°C with constant rotation. Cells were passed through a 70 μm strainer, washed in RPMI, resuspended in 30% isotonic Percoll (GE Healthcare) in PBS, then centrifuged at 500 x g for 10 min. Supernatant was removed. Cell pellets were washed and resuspended in RPMI. Cells were aliquoted into tubes and washed in FACS buffer (0.1% sodium azide and 2% fetal calf serum in PBS). Cells were incubated with Fc block (anti-mouse CD16/CD32 antibody, BD Biosciences) for 5 minutes prior to addition of fluorochrome-conjugated antibodies against CD3 (clone 17A2, RRID: AB_395700), CD4 (clone GK1.5, RRID: AB_396633), CD8a (clone 53–6.7, RRID: AB_394570), CD11b (clone M1/70, RRID: AB_396679), CD11c (clone N418, RRID: AB_469590), CD19 (clone 1D3, RRID: AB_10853189), CD45 (clone 30-F11, RRID: AB_465667), Ly6C (clone HK1.4, RRID: AB_2616730) or Ly6G (clone 1A8-Ly6G, RRID: AB_2573307). Following a 30 min incubation in the dark at 4°C, cells were washed in FACS buffer. Count beads (CountBright, Thermo Fisher) were added to each sample to allow absolute count determination. FACS data was acquired on a flow cytometer (FACS Canto, BD Biosciences). Data were analyzed using FlowJo software (FlowJo, LLC). All analyses were performed on singlet cell-gated populations identified on FSC-H and FSC-A dot plots. CD45+ immune cell subsets were identified as follows: total leukocytes (total CD45+), microglia (CD3-Ly6G-CD11b+CD45int), macrophage (CD3-Ly6G-CD11b+CD45hi), pro-inflammatory monocyte/macrophage (Ly6G-CD11b+Ly6C^hi), myeloid dendritic cells (Ly6G-CD11b+CD11c+), T cells (CD3+, CD3+CD4+ and CD3+CD8+), B cells (CD3-CD19+) and neutrophils (CD45+Ly6G+).

Ex vivo antigen (MOG_35-55)-recall response

Following humane killing, spleens were removed from EAE-induced mice at 14 days post-induction. Single cell suspensions of splenocytes were prepared by pushing the spleen through a 70 μm strainer, then resuspending cells in ammonium-chloride-potassium buffer to lyse erythrocytes. Cells were washed and resuspended in RPMI supplemented with antibiotics and 10% fetal calf serum. Cells were plated in round-bottom 96-well plates at 1 x 10⁶ per well and stimulated for 72 hours with MOG_35-55 (20 μg/ml), phytohemagglutinin (2% v/v) or PBS at 37°C in 5% CO₂ incubator. Supernatant was collected and stored frozen and used later for detection of cytokines interleukin (IL)-4, IL-17 and interferon-γ (IFN-γ) using enzyme linked immunosorbent assay (ELISA) kits (R&D Systems) according to manufacturer’s instructions. Control (PBS-treated) samples showed no greater than 51 pg/ml of IFN-γ in any of the ELISA experiments. Splenocytes were fixed and permeabilized using a fixation/permeabilization kit (eBioscience) and used for intracellular antigen staining (Ki67 or FOXP3).

For intracellular staining of FOXP3 and Ki67, fixed and permeabilized splenocytes obtained from ex vivo antigen-recall response assay were incubated with normal goat serum (2% in FACS buffer) to block non-specific binding. Antibodies against the following antigens were added for 30 minutes in the dark: CD4 (clone RM4-5, RRID: AB_464896), CD3 (clone 17A2, RRID: AB_395700), CD25 (clone 7D4, RRID: AB_11149306), FOXP3 (clone FJK-16s, RRID: AB_465935) or Ki67 (clone B56, RRID: AB_10611874). Cells were then washed in permeabilization buffer (eBioscience) then resuspended in FACS buffer prior to FACS analysis.

Statistical analysis

Estimation statistics (www.estimationstats.com) were applied to EAE data determine the effect size (difference in means) with 95% confidence interval (CI) for two or multi-group comparisons. Mann-Whitney test or t-test with Holm-Sidak correction for multiple comparisons was used for null hypothesis testing with alpha less than 0.05 considered significant; Fisher’s exact test was used for two-group comparisons of categorical data; correlation was tested by linear regression and Pearson correlation testing (GraphPad Prism; San Diego, CA).

Results

Delayed axonal degeneration phenotype of Sarm1 knockout neurons in culture

Prior research indicated that cultured neurons from Sarm1 knockout mice show delayed axonal degeneration following axotomy or in response to oxidative stress [6,17]. To confirm the delayed axonal degeneration phenotype of Sarm1 knockout mice, cultured neurons from Sarm1 knockout and wild type mice were subjected to axotomy or exposed to hydrogen peroxide (H₂O₂). Tau immunocytochemistry 72 hours following axotomy of cultured neurons showed that whereas wild type axons lost Tau expression distal to axotomy, Tau expression was intact in distal axonal segments of Sarm1 knockout neurons, indicating delayed axonal degeneration (Fig 1A). To test the neuroprotective effects of Sarm1 deletion in the setting of oxidative stress, cortical neurons from Sarm1 knockout and wild type mice were subjected to a brief (30 minute) exposure to H₂O₂ in culture then assayed 24 hours later for viability by dye exclusion and for ankyrin G expression as a marker of axonal integrity. Ankyrin G immunocytochemistry showed short linear proximal segment labeling consistent with axon initial segments (AIS) in Sarm1 knockout and wild type neurons, with nearly 100% AIS expression on NeuN labeled cells in mock-treated (0 μM H₂O₂) neuronal cultures (Fig 1B). Following exposure to H₂O_2, neuronal viability was higher in Sarm1 knockout neurons compared to wild type neurons (Fig 1B). Whereas exposure to H₂O₂ resulted in loss of AIS in wild type neurons, Sarm1 knockout neurons showed significant sparing of AIS compared to wild type at 50 μM H₂O₂ (Fig 1B). These results confirmed the delayed axonal degeneration and neuroprotective phenotype of Sarm1 deletion, consistent with prior reports.

Fig 1 — (A) Cultured neurons from wild type (WT) and *Sarm1* KO mice were subjected to axotomy, then labeled for Tau 72 hours later. Confocal microscopy of Tau immunocytochemistry shows loss of Tau in distal axonal segments in WT, but not *Sarm1* KO, neurons at 72 h following axotomy. Representative images from 3 independent experiments. Scale bar, 50 μm. (B) Cultured neurons from WT and *Sarm1* KO mice were subjected to oxidative stress by 30 min exposure to 0, 12.5, 25 or 50 μM hydrogen peroxide (H₂O₂) then assayed 24 h later. Images show ankyrin G (red) and NeuN (green), detected by immunocytochemistry in cultured neurons from WT and *Sarm1* KO mice. Scale bar, 100 μm and inset scale bar 50 μm. Bar graph (left) shows mean neuronal viability +/- standard deviation at indicated H₂O₂ concentrations, assessed by dye exclusion and normalized to control (0 μM H₂O₂). * denotes p = 0.011, t-test with Holm-Sidak method for multiple comparisons. Bar graph (right) shows mean ankyrin G expression (% of NeuN) +/- standard deviation. ** denotes p < 0.001, t-test with Holm-Sidak method for multiple comparisons.

Clinical course of EAE in Sarm1 knockout mice compared to wild type littermates

To test the contribution of SARM1 to axonal degeneration in a model of chronic immune-mediated CNS inflammation, Sarm1 knockout and wild type littermates were actively induced to undergo EAE. Incidence of EAE was similar between wild type and Sarm1 knockout mice (100% vs. 96%; Fisher’s exact test p > 0.99). Mortality associated with EAE was also similar between wild type and Sarm1 knockout mice (17.4% vs. 18.5%; Fisher’s exact test p > 0.99). Onset and peak severity of EAE were similar in Sarm1 knockout mice and WT littermates (Fig 2A). Cumulative clinical scores did not differ substantially between Sarm1 knockout and wild type littermates that were observed up to 42 days post-induction: mean difference of 5.97 [95% CI -10.4, 25.8; p = 0.77, Mann-Whitney test] (Fig 2B).

Fig 2 — *Sarm1* KO mice and WT littermates were actively induced to undergo EAE and scored daily. (A) Mean daily clinical scores +/- 95% confidence interval. Data pooled from 8 independent experiments. N = 18 WT mice (11 females and 7 males) and N = 20 *Sarm1* KO mice (11 females and 9 males). (B) Mean cumulative clinical scores +/- 95% confidence interval for EAE-induced WT and *Sarm1* KO mice that were scored to 42 days post-induction. N = 11 WT mice (5 females and 6 males) and N = 10 *Sarm1* KO mice (5 females and 5 males). Mann-Whitney p-value.

Axonal degeneration is reduced early in the course of EAE in Sarm1 knockout mice

To assess the effect of Sarm1 deletion on axonal degeneration in EAE, Sarm1 knockout mice and wild type littermates expressing neuronal YFP (i.e. Sarm1-/-YFP+ and Sarm1+/+YFP+) were induced to undergo EAE. Control mice were injected with PBS in CFA and pertussis toxin. Neuronal YFP expression allowed identification of intact axons and fragmented axons that have undergone axonal transection and degeneration (Fig 3A). Analysis of YFP+ axons in the lumbar spinal cords of wild type EAE mice at 14 days post-induction showed evidence of axonal degeneration (Fig 3A and 3B). There was a reduction in total YFP+ axon counts in wild type, but not Sarm1 knockout, EAE mice compared to wild type control mice: mean difference of -2.03 counts per field of view [95% CI -3.03, -0.86] for wild type EAE vs. wild type control; mean difference of 0.59 counts per field of view [95% CI -0.58, 2.54] for Sarm1 knockout EAE vs. wild type control (Fig 3B). Fragmented YFP+ axon counts were significantly lower in Sarm1 knockout EAE mice compared to wild type EAE mice at 14 days post-induction: mean difference of -1.43 counts per field of view [95% CI -1.85, -0.734; p = 0.029, Mann-Whitney] (Fig 3A and 3B). Results were similar when fragmented YFP+ axon counts were analyzed as a percentage of total YFP+ axon counts. Percent fragmented YFP+ axons were significantly lower in Sarm1 knockout EAE compared to wild type EAE at 14 days post-induction: mean difference of -30.2% [95% CI -35.7, -24.4; p = 0.029, Mann-Whitney] (Fig 3B). These results indicated that Sarm1 knockout EAE mice showed less axonal degeneration at this early time point in the course of EAE. At 21 days post-induction, however, Sarm1 knockout EAE mice also showed substantial burden of axonal degeneration (Fig 3A and 3C). There was a substantial reduction in total YFP+ axon counts in Sarm1 knockout EAE mice compared to wild type control mice at 21 days post-induction: mean difference of -2.65 counts per field of view [95% CI -4.31, -0.934]. The difference in fragmented YFP+ axon counts between Sarm1 knockout EAE and wild type EAE mice was smaller at 21 days then at 14 days post-induction, and did not reach statistical significance on null hypothesis testing: mean difference of -0.96 counts per field of view [95% CI -1.59, -0.176; p = 0.079, Mann-Whitney]. There was no significant difference between Sarm1 knockout EAE mice and wild type EAE mice with respect to percent fragmented YFP+ axons at 21 days post-induction: mean difference of -9.72% [95% CI -21.7, 0.546; p = 0.22, Mann-Whitney] (Fig 3C). These results suggested that Sarm1 deletion conferred neuroprotection early in the course of EAE, but that protection against axonal degeneration in Sarm1 knockout mice was less robust later in the course of EAE.

Fig 3 — *Sarm1* KO mice and wild type (WT) littermates expressing neuronal yellow fluorescent protein (i.e. *Sarm1*-/-YFP+ and *Sarm1*+/+YFP+) were actively induced to undergo EAE (*Sarm1* KO EAE and WT EAE). Control mice were injected with phosphate buffered saline in complete Freund’s adjuvant and pertussis toxin (*Sarm1* KO control and WT control). (A) Representative images showing YFP+ axons (green) in the lumbar cords of control and EAE mice at 14 and 21 days post-induction (dpi). DAPI nuclear stain (blue). Scale bar, 100 μm. (B) YFP+ axon counts at 14 dpi. Scatter plots show total (left) and fragmented (middle) YFP+ axon counts per field of view (FOV) for each animal by experimental groups. Right scatter plot shows percent (of total) fragmented YFP+ axons for each animal by experimental groups. Mean +/- standard deviation are indicated by line and error bars. N = 4 WT EAE (2 females and 2 males) and N = 4 *Sarm1* KO EAE (2 females and 2 males). Mann-Whitney p-value. (C) YFP+ axon counts at 21 dpi. Scatter plots show total (left) and fragmented (middle) YFP+ axon counts per FOV for each animal by experimental groups. Right scatter plot shows percent (of total) fragmented YFP+ axons for each animal by experimental groups. Mean +/- standard deviation are indicated by line and error bars. N = 5 WT EAE (4 females and 1 male) and N = 5 *Sarm1* KO EAE (3 females and 2 males). Mann-Whitney p-value.

Sarm1 knockout does not confer long-term protection against axonal degeneration in EAE

Axonal integrity was assessed at 42 days post-induction by immunohistochemistry to determine the effect of Sarm1 deletion on long-term axonal degeneration in EAE. Accumulation of APP and non-phosphorylated neurofilament H (SMI-32) were used as markers of axonal injury [18,19]. Axonal density was assessed by SMI-31 immunohistochemistry to label phosphorylated neurofilament H [3]. Analysis of lumbar spine for APP immunohistochemistry showed no significant difference between Sarm1 knockout and wild type littermates at 42 days post-induction of EAE: mean difference of 2.8 μm² per ROI [95% CI -11.0, 20.2; p = 0.841, Mann-Whitney] (Fig 4A). Neither was there a significant difference between Sarm1 knockout mice and wild type littermates with respect to SMI-32 expressing axons at 42 days post-induction of EAE: mean difference of 66.0 μm² per ROI [95% CI -158, 378; p = 0.794, Mann-Whitney] (Fig 4B). Axonal density was assessed by SMI-31 immunohistochemistry (Fig 4C), and expressed as SMI-31+ area per hemicord. There was a significant inverse correlation between axonal density in the lumbar cords of EAE mice as measured by SMI-31 immunohistochemistry at 42 days post induction and their cumulative clinical scores: Pearson r = -0.606 [95% CI -0.836, -0.194; p = 0.0077] (Fig 4D), supporting the clinical relevance of this measure when assessed late in the course of EAE. Analysis of SMI-31 immunohistochemistry showed significant reduction in axonal density in the lumbar cords of both Sarm1 knockout and wild type littermates at 42 days post-induction of EAE compared to control mice immunized with PBS in CFA and pertussis toxin: mean difference between wild type EAE and wild type control was -5.96x10⁴ μm² per hemicord [95% CI -7.87x10⁴, -3.77x10⁴], and mean difference between Sarm1 knockout EAE and wild type control was -6.20x10⁴ μm² per hemicord [95% CI -8.09x10⁴, -4.14x10⁴] (Fig 4E). Two-group comparison between Sarm1 knockout EAE and wild type EAE mice showed no significant difference in axonal density at 42 days post-induction: mean difference of -2.39x10³ μm² per hemicord [95% CI -1.73x10⁴, 1.02x10⁴; p = 0.971, Mann-Whitney]. Together, these results indicated that Sarm1 deletion did not result in long-term protection from axonal degeneration in EAE-induced mice.

Fig 4 — Lumbar cords from EAE-induced mice at 42 days post-induction were assessed by immunohistochemistry for amyloid precursor protein (APP) and non-phosphorylated neurofilament H (SMI-32) as markers of axonal injury and for phosphorylated neurofilament H (SMI-31) to assess axonal density. (A) Images show representative APP (red) immunohistochemistry on lumbar cord sections from EAE-induced *Sarm1* knockout mice (*Sarm1* KO EAE) and wild type littermates (WT EAE) showing APP-labeled axons and spheroids. DAPI nuclear stain (blue). Scale bar, 100 μm. Inset scale bar 50 μm. The scatter plot shows the mean APP+ area per region of interest for each animal. Line and error bars indicate group means and standard deviations. N = 5 WT EAE mice (4 females, 1 male) and N = 5 *Sarm1* KO EAE mice (3 females, 2 males). Mann-Whitney p-value. (B) Images shows representative SMI-32 (red) immunohistochemistry on lumbar cords from *Sarm1* KO EAE and WT EAE showing SMI-32 labeled axons and spheroids. DAPI nuclear stain (blue). Scale bar, 100 μm. The scatter plot shows the mean SMI-32+ area per region of interest for each animal. Line and error bars indicate means and standard deviations for each group. N = 5 WT EAE mice (4 females, 1 male) N = 5 *Sarm1* KO EAE mice (3 females, 2 males). Mann-Whitney p-value. (C) Images shows representative SMI-31 (green) immunohistochemistry on lumbar cords from *Sarm1* KO EAE and WT EAE mice. Control mice (WT control and *Sarm1* KO control) were immunized with phosphate buffered saline in complete Freund’s adjuvant and pertussis toxin. Representative post-thresholding images with superimposed manual outline of hemicords are shown below to illustrate image analysis used to derive SMI-31+ area per hemicord. (D) Linear regression line with 95% confidence interval (dotted lines) showing the inverse relationship between axonal density (SMI-31+ area) and EAE cumulative clinical score. (E) Scatter plot of mean axonal density per hemicord for each animal as measured by SMI-31 immunohistochemistry. Line and error bars indicate means and standard deviations for each group. N = 10 WT EAE mice (5 females, 5 males) and N = 10 *Sarm1* KO EAE mice (5 females, 5 males). Mann-Whitney p-value.

Peripheral immune activation and CNS immune cell infiltration are not altered in EAE-induced Sarm1 knockout mice

Prior reports indicated that SARM1 inactivation could either promote or inhibit immune response depending on cell-type, disease model or species studied [11,15]. To exclude the possibility that SARM1 functions to influence the immune-mediated CNS inflammatory response of EAE and thereby alter its course, we compared the peripheral immune activation and CNS inflammatory cell infiltration associated with EAE in Sarm1 knockout and wild type littermates. Splenocytes were obtained at 14 days post-induction of EAE and tested for ex vivo recall response to MOG_35-55 peptide, assaying T helper cytokine production and proliferation. There was no significant difference between Sarm1 knockout and wild type EAE mice with respect to production of IL-4, IL-17 or IFN-γ (Table 1). In addition, there was no significant difference in the number of CD4+CD25+FOXP3+ regulatory T cells or in the number of proliferating T cells in response to ex vivo MOG_35-55 re-stimulation (Table 1). With respect to CNS inflammatory cell infiltration, phenotypic enumeration of cells from thoracolumbar spines of EAE-induced mice by FACS at 14 days post-induction showed no significant difference between Sarm1 knockout and wild type littermates with respect to the number of infiltrating immune cells (Table 1 and S1 Fig). These results suggest that SARM1 does not substantially influence peripheral immune activation or CNS inflammatory cell infiltration in EAE.

Table 1. Peripheral immune activation and CNS immune cell infiltration.

Immune activation	N (WT EAE, Sarm1 KO EAE)	*Effect size: Sarm1* KO EAE vs. WT EAE [95% confidence interval]**	Mann Whitney p
IL-4 (pg/ml)	7, 10	3.03 [-4.74, 14.7]	0.92
IL-17 (pg/ml)	7, 10	440 [–259, 1410]	0.96
IFN-γ (pg/ml)	7, 10	5.31x10³ [-2.25x10³, 1.22x10⁴]	0.30
CD25+Foxp3+ (%CD3+CD4+)	6, 7	-1.14 [-5.86, 3.96]	0.45
CD4+Ki67+ (%CD3+)	6, 7	-0.751 [-2.34, 1.17]	0.29
CNS inflammatory cells (absolute counts)	N (WT EAE, Sarm1 KO EAE)	*Effect size: Sarm1* KO EAE vs. WT EAE [95% confidence interval]**	Mann Whitney p
total leukocytes	6, 7	-1.96x10⁵ [-5.26x10⁵, 1.58x10⁵]	0.29
Microglia	6, 7	-2.42x10⁴ [-7.95x10⁴, 2.47x10⁴]	0.53
Macrophage	6, 7	-1.13x10⁵ [-3.24x10⁵, 9.51x10⁴]	0.37
CD11b+Ly6Chi	6, 7	-1.03x10⁵ [-2.39x10⁵, 6x10⁴]	0.37
CD11b+CD11c+	6, 7	-2.66x10⁴ [-9.88x10⁴, 3.99x10⁴]	0.73
T cells (CD3+)	6, 7	-3.07x10⁴ [-8.96x10⁴, 5.05x10⁴]	0.29
CD3+CD4+ T cells	6, 7	-8.15x10³ [-5.9x10⁴, 4.73x10⁴]	0.95
CD3+CD8+ T cells	6, 7	-2.09x10⁴ [-6.62x10⁴, 4.71x10²]	0.37
B cells	6, 7	2.31x10³ [-2.61x10³, 9.32x10³]	0.73
Neutrophils	6, 7	-1.45x10⁴ [-4.76x10⁴, 6.11x10³]	0.73

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Discussion

The contribution of SARM1-dependent program of axonal degeneration to the course and axonal pathology of immune-mediated CNS inflammatory disorders was previously unconfirmed. In this study, we compared the course, pathology and neuroimmunology of EAE in Sarm1 knockout mice and wild type littermates to test whether or not the loss of SARM1 confers neuroprotection in an animal model of immune-mediated CNS inflammatory disorder.

We confirmed the delayed axonal degeneration phenotype of Sarm1 knockout by demonstrating intact Tau protein expression in the distal stump of Sarm1 knockout cultured neurons at 72 h following axotomy. In addition, cortical neurons from Sarm1 knockout mice showed relative protection from cell death and axonal injury in the setting of oxidative stress, consistent with prior reports [6,17]. We assessed ankyrin G expression in the AIS as a marker of axonal integrity in cultured neurons. The loss of ankyrin G was previously shown to be a marker of axonal injury in EAE and in an in vitro model of oxidative stress-induced axonal degeneration [20,21]. Cultured neurons from Sarm1 knockout showed relative preservation of ankyrin G expression in the AIS compared to wild type in the setting of oxidative stress. These in vitro results suggest a neuroprotective potential for SARM1 deletion in the setting of CNS inflammation, based on prior knowledge that oxidative stress is a key contributor to acute axonal injury during immune-mediated CNS inflammation [22,23].

The clinical course of EAE, however, did not differ substantially between Sarm1 knockout and wild type littermates, suggesting that targeting SARM1 may not produce reliable clinical benefits in EAE or other immune-mediated CNS inflammatory disorders such as multiple sclerosis. There were no significant differences in the incidence, peak severity or mortality associated with EAE. At best, there was a modest difference in clinical scores at late time points. Effect size was small.

Sarm1 deletion did not lead to long-term axonal protection, despite relative preservation of axonal integrity early in the course of EAE. Axonal degeneration of EAE was assessed using 2 approaches. Intact and fragmented YFP+ axons were assessed in Sarm1 knockout and wild type littermates expressing neuronal YFP at 14 and 21 days post-induction of EAE [18]. Conventional immunohistochemistry was used to assess axonal integrity at 42 days post-induction. The results of YFP+ axon analysis suggested preservation of axonal integrity in Sarm1 knockout mice at an early, but not late, time point in EAE. Whereas Sarm1+/+YFP+ mice showed substantial loss and fragmentation of YFP+ axons at 14 days post-induction, Sarm1-/-YFP+ mice were similar to control animals with respect to total YFP+ axon counts and showed significantly less fragmented YFP+ axon counts compared to Sarm1+/+YFP+ mice. However, at 21 days post-induction, Sarm1-/-YFP+ mice also showed substantial burden of axonal degeneration. Total YFP+ axon counts were lower in Sarm1-/-YFP+ mice compared to controls at 21 days post induction, and the degree of YFP+ axon fragmentation was not significantly lower compared to Sarm1+/+YFP+ mice at this time point. At 42 days post-induction, Sarm1 knockout mice were indistinguishable from wild type littermates with respect to conventional immunohistochemical markers of axonal degeneration such as APP and SMI-32, and there was no significant difference between Sarm1 knockout and wild type littermates in axonal density as measured by SMI-31 immunohistochemistry. There was a significant inverse correlation between SMI-31+ axonal density at 42 days post induction and cumulative clinical scores, supporting the clinical relevance of axonal loss to neurological dysfunction late in the course of EAE. Together, these results indicated that Sarm1 deletion conferred early, but not long-term, axonal protection in EAE.

The key question addressed in this study was to what extent does programmed axonal degeneration contribute to neurodegeneration in immune-mediated CNS inflammatory disorders. Two proteins involved in NAD metabolism, SARM1 and NMNAT, are key opposing elements of a mechanism of programmed axonal degeneration [5–7]. The loss of NMNAT or the activation of SARM1 triggers intra-axonal depletion of NAD, leading to local energetic failure culminating in axonal degradation [24]. Prior research on the contribution of NMNAT to axonal degeneration in CNS inflammatory disorders produced conflicting results. Using the slow Wallerian degeneration (Wld^s) mice, which express a chimeric form of the axon survival factor NMNAT, one study reported a modest reduction in long-term axonal loss in the Wld^s mice undergoing EAE [25]. However, a second study showed no difference in long-term axonal loss in the EAE-induced Wld^s mice [4]. Mechanistically, SARM1 is downstream of NMNAT, and SARM1 inactivation can rescue the axon from degeneration even after the loss of NMNAT [26]. Prior research also showed that Sarm1 deletion conferred substantially longer neuroprotection over Wld^s in an animal model of axonopathy [27]. Therefore, it remained plausible that Sarm1 knockout mice might be more informative than the Wld^s mice with respect to delineating the full contribution of programmed axonal degeneration to the pathophysiology of EAE. The results of the current study, however, indicate that targeting SARM1 is no more likely than NMNAT to reduce long-term axonal degeneration associated with EAE. Considered together with results from prior research, the results presented herein suggest that SARM1/NMNAT-dependent mechanism of programmed axonal degeneration makes a modest contribution, if at all, to the overall burden of long-term axonal degeneration in this model of immune-mediated CNS inflammatory disorder.

Targeting SARM1 is neuroprotective in some models of neurological injury, but not others. Sarm1 knockout mice showed reduced clinical severity and reduced axonal loss in animal models of traumatic brain injury [10,28]. Sarm1 deletion was beneficial in the TDP-43^Q331K model of amyotrophic lateral sclerosis-frontotemporal dementia [29], but had no significant impact on axonal loss or clinical course of mutant SOD1 model of amyotrophic lateral sclerosis [30]. Therefore, the contribution of SARM1 is likely to be disease-specific. A number of inter-dependent pathogenic mechanisms have been proposed to explain inflammation-induced axonal degeneration in multiple sclerosis and EAE. These include oxidative stress [22], mitochondrial dysfunction [31], intra-axonal ionic dyshomeostasis [32] and excitotoxicity [33]. Prior research suggests that a distinction should be made between acute and chronic axonal injury in EAE and multiple sclerosis [34]. One possible explanation for the finding that Sarm1 deletion results in early but not late axonal protection is that the primary mechanistic drivers of axonal degeneration may differ between early and late axonal degeneration in EAE and multiple sclerosis. Whereas oxidative stress is thought to play a prominent role in acute axonal degeneration, which can occur independently of demyelination [23], intra-axonal ionic dyshomeostasis may be more critical to axonal degeneration along chronically demyelinated axons [35]. Sarm1 deletion may be more neuroprotective in the setting of oxidative stress associated with early, acute inflammation in EAE, but not as protective against axonal degeneration that occurs as a result of chronic demyelination. Alternatively, the results of the study may indicate that Sarm1 deletion simply delays the perhaps inevitable axonal degeneration that follows axonal transection, similar to that seen following axotomy [6].

The immune response and CNS inflammation in Sarm1 knockout mice were also examined in this study. Prior research showed that SARM1 function in immune cells can affect CNS inflammation [14,15]. Significant alteration in the immune-mediated CNS inflammatory response of EAE in Sarm1 knockout mice could have confounded the interpretation of the results, potentially masking a neuroprotective effect. The results of this study indicate that Sarm1 deletion does not lead to a significant alteration in peripheral immune activation or CNS inflammatory cell infiltration in the context of EAE. MOG-specific immune activation and CNS inflammatory infiltration were similar in Sarm1 knockout and wild type littermates. The lack of a significant change immune activation was further supported by the clinical data that showed similar onset and incidence of EAE between Sarm1 knockout mice and wild type littermate. These results are also consistent with a recent report that indicates that background effects in part explain previously reported effects of Sarm1 deletion on immune response, suggesting a more limited role for SARM1 in immunity [36].

This study has several limitations. Axonal YFP epifluorescence in the Thy1-YFP-H transgenic mice is susceptible to fluorescence quenching associated with blood-brain barrier breakdown and local edema [37]. Although YFP fluorescence quenching could lead to spuriously low total YFP+ axon counts, it is unlikely to result in the increased fragmented YFP+ axon counts that were observed in wild type and Sarm1 knockout EAE mice at 14 and 21 days post-induction, respectively. Immune responses were studied at day 14 post-induction, but not at other time points. The possibility that Sarm1 deletion might affect CNS inflammation earlier or later in the course of EAE has not been excluded. A broad, but not exhaustive, survey of CNS infiltrating immune cell subsets was performed in this study. The effect of Sarm1 deletion on demyelination was not examined in this study. SARM1 function is not known to be involved in myelin formation or oligodendrocyte survival, and thus Sarm1 deletion is unlikely to directly affect myelination. However, we have not excluded the possibility that early axonal protection may indirectly affect remyelination in EAE. Further work is needed to ascertain the impact of Sarm1 deletion on demyelination/remyelination in the setting of CNS inflammation. Another limitation of the study is that EAE recapitulates many but not all aspects of axonal degeneration observed in multiple sclerosis [38]. EAE alone may not adequately capture the impact of targeting SARM1 on neurodegeneration in multiple sclerosis.

In conclusion, Sarm1 deletion conferred protection from axonal degeneration early in the course of EAE, but did not confer long-term protection against axonal degeneration in this model of immune-mediated CNS inflammation.

Supporting information

S1 Fig. Phenotypic enumeration of CNS inflammatory cells by FACS analysis.

Representative plots of FACS analysis to enumerate CNS inflammatory cells from thoracolumbar cords of EAE mice are shown. A) Gating for singlets B) Gating for Count beads and live cells. C) Total CD45+ (leukocyte) gating. D) CD45+CD3+ (T cell) gating. E) CD45+Ly6G+ (neutrophil) gating. F) Ly6G-CD11b+CD11c+ (CD11b+ dendritic cell) gating. G) Ly6G-CD11b+Ly6C^hi (Ly6C high monocyte/macrophage) gating. H) Ly6G-CD11b+CD45^int (microglia) and Ly6G-CD11b+CD45^hi (macrophage) gating. I) CD3+CD4+ (CD4 T cell) gating. J) CD3+CD8+ (CD8 T cell) gating. K) CD19+ (B-cell) gating.

(TIF)

Click here for additional data file.^{(294.6KB, tif)}

Acknowledgments

Microscopy and flow cytometry were performed at the VCU Department of Anatomy & Neurobiology Microscopy Facility and at the VCU Massey Cancer Center Flow Cytometry Shared Resource.

Data Availability

Data are held in Open Science Framework repository: osf.io/5mzr6

Funding Statement

This study was supported by Virginia Commonwealth University Excellence Fund (internal funding to U.O.). There was no additional external funding received for this study.

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PLoS One. doi: 10.1371/journal.pone.0235110.r001

Decision Letter 0

Thomas Forsthuber

6 May 2020

PONE-D-20-10362

Sarm1 knockout delays but does not protect against axonal degeneration in experimental allergic encephalomyelitis

PLOS ONE

Dear Dr. Oh,

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We look forward to receiving your revised manuscript.

Kind regards,

Thomas Forsthuber

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: No

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is a clear and well designed study. The findings were communicated well and will be useful to the research community. Overall I had only relatively minor concerns:

1. The phenotypic data currently available in the supporting information is difficult to follow. A more extensive figure legend would be useful to be certain that the reader understands the data presented, including what populations are shown in each section and what gates are applicable to individual graphs.

2. The authors conclude that " SARM1 activation is not the critical trigger for irreversible axonal injury in CNS inflammatory disorders" and suggest that SARM1 involvement in axonal loss is insult dependent. However the authors do not note that this intermediate outcome, protection from early loss with slightly later loss of axon integrity is not typical of previous reports regarding SARM1 activity. The finding that SARM1 appears to block axonal damage at the peak of disease could, and should be discussed in greater detail. This will probably also have to modify the authors statement that "There was a significant inverse correlation between SMI-31+ axonal

density and cumulative clinical scores, supporting the clinical relevance of axonal loss to neurological

dysfunction in EAE."

3. The authors note that the lack of examination of demyelination is a limitation. It is worth discussing how the results reported may impact myelination/demyelination.

Reviewer #2: The authors test the EAE model of win wild type and SARM1 KO mice. SARM1 is a key driver of pathological axon degeneration, and so the authors assess whether SARM1 blocks axon loss in the EAE model of neuroinflammation. They find that SARM1 KO has a significant delay in axon loss, but that axon loss is not prevented indefinitely. They do not see any difference in measures of immune activation of disease score between wild type and SARM1 KO mice. They conclude that SARM1 KO delays but does not protect against axon degeneration in EAE and that SARM1 "is not the critical trigger for irreversible axonal injury in CNS inflammatory disorders." While this study is novel and performed is technically of high quality, some of the conclusions are not well supported by the data.

1) The authors show that axons are well protected for about 2 weeks, and then degenerate in the SARM1 Ko during EAE. This is almost the identical time course to the protection afforded by SARM1 following axotomy in vivo. Axons survive for about 2 weeks and then degenerate in a SARM1-independent manner, likely due to the absence of cell-body derived factors. In EAE, it is very likely that the immune attack causes severing of axons and so it quite similar to axotomy. Do the authors see evidence for axon severing in their wild type mice? This would also explain the inability of SARM1 KO to change the disease course. Severed axons obviously can't contribute to function. The authors should acknowledge that all their data can be explained by the occurrence of axon severing in the EAE model.

2) Based on their EAE results, the authors making sweeping conclusions about the role of SARM1 in CNS neuroinflammatory disease. This is dramatically overstating what can be concluded from this model. While it is clear that the EAE model is a reasonable model of the inflammatory component of MS, it is not known if EAE models the neurodegenerative component of MS. Indeed, based on the time course it seems quite unlikely. Hence, it is inappropriate to conclude that SARM1 is not involved in CNS neuroinflammatory disorders. These statements should be removed, and the limitations of the EAE model for studying the neurodegenerative component of MS should be clearly acknowledged.

3) The title states that SARM1 KO does not "protect against axonal degeneration" in EAE. However the data clearly show that it does "protect," even if it doesn't permanently block. Again, this is identical to the result with axotomy--nothing can permanently block degeneration of a severed axon. The title should be changed to reflect that SARM1 Ko does protect against axon degeneration in EAE, albeit not indefinitely.

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6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Jun 25;15(6):e0235110. doi: 10.1371/journal.pone.0235110.r002

Author response to Decision Letter 0

12 May 2020

Response to PLOS ONE reviewer comments

PONE-D-20-10362

Dear PLOS ONE Reviewers

Thank you for your constructive comments. The manuscript has been revised in response to your comments.

Reviewer #1

A more extensive figure legend has been provided for Supporting information S1. Fig 1.

The statement “SARM1 activation is not the critical trigger for irreversible axonal injury in CNS inflammatory disorders” has been removed in the revised manuscript. An alternative explanation for the finding that Sarm1 deletion protects early but not late axonal degeneration has been proposed in the discussion, based on mechanistic distinction between acute and chronic axonal degeneration in EAE and multiple sclerosis. The manuscript was also revised to qualify the statement regarding the inverse correlation between SMI-31+ axonal density and cumulative clinical scores to specify that this correlation applies to a late time point in the course of EAE.

3. The authors note that the lack of examination of demyelination is a limitation. It is worth

discussing how the results reported may impact myelination/demyelination.

The manuscript has been revised to discuss possible impact on myelination/demyelination: “SARM1 function is not known to be involved in myelin formation or oligodendrocyte survival, and thus Sarm1 deletion is unlikely to directly affect myelination. However, we have not excluded the possibility that early axonal protection may indirectly affect remyelination in EAE. Further work is needed to ascertain the impact of Sarm1 deletion on demyelination/ remyelination in the setting of CNS inflammation.”

Reviewer #2

1) The authors show that axons are well protected for about 2 weeks, and then degenerate in the SARM1 Ko during EAE. This is almost the identical time course to the protection afforded by SARM1 following axotomy in vivo. Axons survive for about 2 weeks and then degenerate in a SARM1- independent manner, likely due to the absence of cell-body derived factors. In EAE, it is very likely that the immune attack causes severing of axons and so it quite similar to axotomy. Do the authors see evidence for axon severing in their wild type mice? This would also explain the inability of SARM1 KO to change the disease course. Severed axons obviously can't contribute to function. The authors should acknowledge that all their data can be explained by the occurrence of axon severing in the EAE model.

The manuscript has been revised to acknowledge the consequence of axonal transection: “Alternatively, the results of the study may indicate that Sarm1 deletion simply delays the perhaps inevitable axonal degeneration that follows axonal transection, similar to that seen following axotomy”

The manuscript has been revised to remove the statement “SARM1 activation is not the critical trigger for irreversible axonal injury in CNS inflammatory disorders”. An alternative explanation has been proposed in the discussion based on mechanistic distinction between acute and chronic axonal degeneration in EAE and multiple sclerosis. Furthermore, the manuscript has been revised to acknowledge the limitations of EAE in modeling neurodegeneration of multiple sclerosis.

In response to reviewer comments, the manuscript title has been revised to “Sarm1 knockout protects against early but not late axonal degeneration in experimental allergic encephalomyelitis”. In addition the conclusion has also been revised to state early but not late protection of axonal degeneration in Sarm1 KO in EAE.

Attachment

Submitted filename: Response to PLOS ONE Reviewers.docx

Click here for additional data file.^{(14KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0235110.r003

Decision Letter 1

Thomas Forsthuber

2 Jun 2020

PONE-D-20-10362R1

Sarm1 knockout protects against early but not late axonal degeneration in experimental allergic encephalomyelitis

PLOS ONE

Dear Dr. Oh,

Please make sure to address the remaining minor concerns.

Please submit your revised manuscript by Jul 17 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Thomas Forsthuber

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: The authors made most of the requested changes but missed a couple of spots. In addition there are a few missing references.

1) I had asked the authors not claim that there findings were valid for all CNS inflammatory disorders, but just for this one model. They made the changes most places, but missed line 451 and line 471. Please change to "model of" here as well.

2) The authors discuss the potential role of SARM1 in the immune system, but do not mention an important new paper that suggests most of the referenced studies are an artifact of the genetic background in the line used by the authors. The authors should acknowledge this issue and reference the paper (Uccellini 2020).

3) The authors mention one study that did not fine a role for SARM1 in an ALS model, but leave out another study that did find a role (White 2019). In fact this paper was similar to the author's work, showing some axonal protection but not behavioral protection. This second study should be acknowledged.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Jun 25;15(6):e0235110. doi: 10.1371/journal.pone.0235110.r004

Author response to Decision Letter 1

5 Jun 2020

PONE-D-20-10362R1

Response to Reviewers

Reviewer #2: The authors made most of the requested changes but missed a couple of spots. In addition there are a few missing references. 1) I had asked the authors not claim that there findings were valid for all CNS inflammatory disorders, but just for this one model. They made the changes most places, but missed line 451 and line 471. Please change to "model of" here as well. 2) The authors discuss the potential role of SARM1 in the immune system, but do not mention an important new paper that suggests most of the referenced studies are an artifact of the genetic background in the line used by the authors. The authors should acknowledge this issue and reference the paper (Uccellini 2020). 3) The authors mention one study that did not fine a role for SARM1 in an ALS model, but leave out another study that did find a role (White 2019). In fact this paper was similar to the author's work, showing some axonal protection but not behavioral protection. This second study should be acknowledged.

The manuscript has been revised in response to reviewer comments.

1) We agree with the reviewer and line 471 has been changed in the revised manuscript to:

471 “... degeneration in this model of immune-mediated CNS inflammatory disorder.”

However, we are confounded by the reviewer’s request for change in line 451 which reads as follows in the resubmitted manuscript:

451 “Sarm1 deletion conferred early, but not long-term, axonal protection in EAE.”

There is no claim to validity for CNS inflammatory disorders in general in line 451, therefore we are at a loss as to how that should to be changed.

2) The paper by Uccellini et al reporting that background effects in part explain some of the previously reported effect of SARM1 on immune response has been acknowledged and referenced in the revised Discussion.

3) The paper by White et al was also acknowledged in the revised Discussion.

Attachment

Submitted filename: Response to reviewers 060220.docx

Click here for additional data file.^{(13.4KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0235110.r005

Decision Letter 2

Thomas Forsthuber

10 Jun 2020

Sarm1 knockout protects against early but not late axonal degeneration in experimental allergic encephalomyelitis

PONE-D-20-10362R2

Dear Dr. Oh,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Thomas Forsthuber

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #2: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

PLoS One. doi: 10.1371/journal.pone.0235110.r006

Acceptance letter

Thomas Forsthuber

15 Jun 2020

PONE-D-20-10362R2

Sarm1 knockout protects against early but not late axonal degeneration in experimental allergic encephalomyelitis

Dear Dr. Oh:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Thomas Forsthuber

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Phenotypic enumeration of CNS inflammatory cells by FACS analysis.

(TIF)

Click here for additional data file.^{(294.6KB, tif)}

Attachment

Submitted filename: Response to PLOS ONE Reviewers.docx

Click here for additional data file.^{(14KB, docx)}

Attachment

Submitted filename: Response to reviewers 060220.docx

Click here for additional data file.^{(13.4KB, docx)}

Data Availability Statement

Data are held in Open Science Framework repository: osf.io/5mzr6

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PERMALINK

Sarm1 knockout protects against early but not late axonal degeneration in experimental allergic encephalomyelitis

Kenneth Viar

Daniel Njoku

Julie Secor McVoy

Unsong Oh

Roles

Abstract

Introduction

Materials and methods

Reagents

Animals

Neuronal cultures, axotomy and oxidative stress

Neuronal immunocytochemistry

EAE induction and clinical scoring

Perfusion fixation and tissue processing

Analysis of YFP+ axons

Immunohistochemistry

Image acquisition and analysis

Fluorescence-activated cell sorting (FACS) analysis

Ex vivo antigen (MOG35-55)-recall response

Statistical analysis

Results

Delayed axonal degeneration phenotype of Sarm1 knockout neurons in culture

Fig 1. Delayed axonal degeneration phenotype of Sarm1 knockout (KO) neurons in culture.

Clinical course of EAE in Sarm1 knockout mice compared to wild type littermates

Fig 2. Clinical course of EAE in Sarm1 knockout (KO) mice compared to wild type (WT) littermates.

Axonal degeneration is reduced early in the course of EAE in Sarm1 knockout mice

Fig 3. Axonal degeneration is reduced early in the course of EAE in Sarm1 knockout (KO) mice.

Sarm1 knockout does not confer long-term protection against axonal degeneration in EAE

Fig 4. Sarm1 knockout does not confer long-term protection against axonal degeneration in EAE.

Peripheral immune activation and CNS immune cell infiltration are not altered in EAE-induced Sarm1 knockout mice

Table 1. Peripheral immune activation and CNS immune cell infiltration.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Thomas Forsthuber

Roles

Author response to Decision Letter 0

Decision Letter 1

Thomas Forsthuber

Roles

Author response to Decision Letter 1

Decision Letter 2

Thomas Forsthuber

Roles

Acceptance letter

Thomas Forsthuber

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Ex vivo antigen (MOG_35-55)-recall response