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| ZIKV infection depletes NAD + levels independent of SARM1 at 24 hpi. (A) Mock infected Sarm1 +/+ cultures appear healthy at 24 hpi. DAPI +ve cell nuclei are abundant and neurofilament (NF) and β-tubulin III-stained neuronal processes appear dense and smooth. Bar: 50 µm. (B) At 24 hpi with ZIKV, DAPI +ve cell nuclei appear similar in terms of density and appearance across all three Sarm1 genotypes, although there is already evidence of subtle injury to neuronal cell processes in Sarm1 +/+ (arrows indicate irregularly stained neurofilament +ve structures) and Sarm1 +/− cultures, most obvious with β-tubulin III staining. In contrast, neuronal processes in Sarm1 −/− cultures appear dense and smooth, as in the mock-infected controls (see also Supplementary Figure 1). (C) Neurofilament positive pixels as a percentage of all pixels per AOI is similar across all three genotypes suggesting that neuronal processes remain viable at this timepoint. (D) Compared to levels in matched mock-infected controls (black circles), NAD + levels are significantly reduced in ZIKV-infected cultures (gray squares) at 24 hpi. (E) The relative reduction in NAD + in ZIKV infected cultures in comparison to their mock-infected controls is similar across all three genotypes. Bars represent mean ± S.E.M. ****p < 0.0001.
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Zika virus (ZIKV) is a neurotropic flavivirus recently linked to congenital ZIKV syndrome in children and encephalitis and Guillain-Barré syndrome in adults. Neurotropic viruses often use axons to traffic to neuronal or glial cell somas where they either remain latent or replicate and proceed to infect new cells. Consequently, it has been suggested...
Contexts in source publication
Context 1
... protection of neuronal processes does not result from there being reduced numbers of productively infected cells in the absence of SARM1. Figure 1), suggesting the absence of overt ZIKV infection-related cell death at this time point, as quantified previously (Cumberworth et al., ). Nonetheless, despite that staining for phosphorylated H-and M-neurofilament (antibody clone SMI31) demonstrated similar positive pixel densities across all the three genotypes ( Figure 3C), subtle changes in morphology of some neuronal processes in ZIKV-infected Sarm1 +/+ cultures could be seen at this time point (arrows; Figure 3B). This was confirmed by β-tubulin III staining which provided evidence for fragmentation of the microtubule network in ZIKV-infected Sarm1 +/+ and Sarm1 +/− , but not Sarm1 −/− cultures or mock-infected controls (Figures 3A,B and Supplementary Figure 1). ...
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... protection of neuronal processes does not result from there being reduced numbers of productively infected cells in the absence of SARM1. Figure 1), suggesting the absence of overt ZIKV infection-related cell death at this time point, as quantified previously (Cumberworth et al., ). Nonetheless, despite that staining for phosphorylated H-and M-neurofilament (antibody clone SMI31) demonstrated similar positive pixel densities across all the three genotypes ( Figure 3C), subtle changes in morphology of some neuronal processes in ZIKV-infected Sarm1 +/+ cultures could be seen at this time point (arrows; Figure 3B). This was confirmed by β-tubulin III staining which provided evidence for fragmentation of the microtubule network in ZIKV-infected Sarm1 +/+ and Sarm1 +/− , but not Sarm1 −/− cultures or mock-infected controls (Figures 3A,B and Supplementary Figure 1). ...
Context 3
... was confirmed by β-tubulin III staining which provided evidence for fragmentation of the microtubule network in ZIKV-infected Sarm1 +/+ and Sarm1 +/− , but not Sarm1 −/− cultures or mock-infected controls (Figures 3A,B and Supplementary Figure 1). Quantification of NAD + levels at 24 hpi demonstrated a similar significant decrease in NAD + levels in ZIKV-infected cultures of each of the three Sarm1 genotypes, in comparison to their matched mockinfected controls ( Figure 3D). The degree of NAD + reduction was most consistent in Sarm1 −/− cultures and more variable in wild type and heterozygous controls, most particularly when NAD + levels were below 2 nmol/mg −1 protein in mockinfected control cultures. ...
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... degree of NAD + reduction was most consistent in Sarm1 −/− cultures and more variable in wild type and heterozygous controls, most particularly when NAD + levels were below 2 nmol/mg −1 protein in mockinfected control cultures. On average, NAD + levels were reduced to ∼60% of mock-infected control levels, across all three genotypes ( Figure 3E). Together, these data demonstrate that whilst degeneration of neuronal processes is expediated by SARM1, NAD + depletion at 24 hpi is independent of SARM1. ...
Citations
... However, these animals are kept in highly controlled, pathogen-free environments, which could mask potential detrimental effects of SARM1 deficiency. For instance, several studies suggest that SARM1 may have important roles in preventing viral damage or spread [52][53][54] . Other research also suggests a physiological role of SARM1 in regulating synaptic plasticity 55 and preventing local inflammation 56 . ...
SARM1 is a key regulator of a conserved program of axon degeneration increasingly linked to human neurodegenerative diseases. Pathological SARM1 activation causes rapid NAD consumption, disrupting cellular homeostasis and leading to axon degeneration. In this study, we develop antisense oligonucleotides targeting human SARM1, demonstrating robust neuroprotection against morphological, metabolic, and mitochondrial impairment in human iPSC-derived dopamine neurons induced by the lethal neurotoxin vacor, a potent SARM1 activator. Furthermore, our findings reveal that axon fragmentation can be prevented, and mitochondrial dysfunction reversed using the NAD precursor nicotinamide, a form of vitamin B 3 , even after SARM1 activation has occurred, when neurons are already unhealthy. This research identifies ASOs as a promising therapeutic strategy to block SARM1, and provides an extensive characterisation and further mechanistic insights that demonstrate the reversibility of SARM1 toxicity in human neurons. It also identifies the SARM1 activator vacor as a specific and reversible neuroablative agent in human neurons.
... Both show ocular toxicity as well as damage to other types of neuron [18,93,94] (Fig. 2). Final, SARM1-dependent axon loss is caused by several viruses, including rabies and zika, with related phenomena also reported for West Nile virus [95][96][97][98]. As the eye is an important route of infection for a number of viruses and many cause optic neuropathies, including (but not limited to) herpes viruses, West Nile virus, Epstein-Barr virus [99], it will be important to determine whether viral activation of SARM1 plays a role in any retinal disorders. ...
Programmed axon death is a druggable pathway of axon degeneration that has garnered considerable interest from pharmaceutical companies as a promising therapeutic target for various neurodegenerative disorders. In this review, we highlight mechanisms through which this pathway is activated in the retina and optic nerve, and discuss its potential significance for developing therapies for eye disorders and beyond. At the core of programmed axon death are two enzymes, NMNAT2 and SARM1, with pivotal roles in NAD metabolism. Extensive preclinical data in disease models consistently demonstrate remarkable, and in some instances, complete and enduring neuroprotection when this mechanism is targeted. Findings from animal studies are now being substantiated by genetic human data, propelling the field rapidly toward clinical translation. As we approach the clinical phase, the selection of suitable disorders for initial clinical trials targeting programmed axon death becomes crucial for their success. We delve into the multifaceted roles of programmed axon death and NAD metabolism in retinal and optic nerve disorders. We discuss the role of SARM1 beyond axon degeneration, including its potential involvement in neuronal soma death and photoreceptor degeneration. We also discuss genetic human data and environmental triggers of programmed axon death. Lastly, we touch upon potential therapeutic approaches targeting NMNATs and SARM1, as well as the nicotinamide trials for glaucoma. The extensive literature linking programmed axon death to eye disorders, along with the eye’s suitability for drug delivery and visual assessments, makes retinal and optic nerve disorders strong contenders for early clinical trials targeting programmed axon death.
... Loss of IENFs is largely dependent on Wallerian degeneration and SARM1 activity (38-41). While direct links between inflammation and SARM1 activity in the peripheral nerve are emerging (42,43), SARM1 is known to be heavily involved in responses evoked by pathogen-and damage-associated molecular patterns (DAMPs and PAMPs) (44,45). Combined with the breadth of infectious and autoimmune diseases exhibiting IENF loss described here, this suggests interplay between pattern recognition receptors and the Wallerian degeneration pathway may contribute to axon retraction. ...
Background
Intraepidermal nerve fiber density (IENFD) has become an important biomarker for neuropathy diagnosis and research. The consequences of reduced IENFD can include sensory dysfunction, pain, and a significant decrease in quality of life. We examined the extent to which IENFD is being used as a tool in human and mouse models and compared the degree of fiber loss between diseases to gain a broader understanding of the existing data collected using this common technique.
Methods
We conducted a scoping review of publications that used IENFD as a biomarker in human and non-human research. PubMed was used to identify 1,004 initial articles that were then screened to select articles that met the criteria for inclusion. Criteria were chosen to standardize publications so they could be compared rigorously and included having a control group, measuring IENFD in a distal limb, and using protein gene product 9.5 (PGP9.5).
Results
We analyzed 397 articles and collected information related to publication year, the condition studied, and the percent IENFD loss. The analysis revealed that the use of IENFD as a tool has been increasing in both human and non-human research. We found that IENFD loss is prevalent in many diseases, and metabolic or diabetes-related diseases were the most studied conditions in humans and rodents. Our analysis identified 73 human diseases in which IENFD was affected, with 71 reporting IENFD loss and an overall average IENFD change of −47%. We identified 28 mouse and 21 rat conditions, with average IENFD changes of −31.6% and −34.7%, respectively. Additionally, we present data describing sub-analyses of IENFD loss according to disease characteristics in diabetes and chemotherapy treatments in humans and rodents.
Interpretation
Reduced IENFD occurs in a surprising number of human disease conditions. Abnormal IENFD contributes to important complications, including poor cutaneous vascularization, sensory dysfunction, and pain. Our analysis informs future rodent studies so they may better mirror human diseases impacted by reduced IENFD, highlights the breadth of diseases impacted by IENFD loss, and urges exploration of common mechanisms that lead to substantial IENFD loss as a complication in disease.
... It has been postulated that one of the purposes of SARM1, a toll like adapter protein and remnant of the innate immune system, and the wider axon degeneration machinery is to bring about compartmentalized neurodegeneration to prevent spread of viral pathogens throughout the nervous system (Tsunoda, 2008). In mouse CNS myelinating cocultures, loss of Sarm1 protects neuronal somas against infection and cell death (Crawford et al., 2022). Zika virus preferentially infects oligodendroglia and astroglia in neuron/glia cocultures and causes glia cell death (Cumberworth et al., 2017). ...
Since SARM1 mutations have been identified in human neurological disease,
SARM1 inhibition has become an attractive therapeutic strategy to preserve
axons in a variety of disorders of the peripheral (PNS) and central nervous
system (CNS). While SARM1 has been extensively studied in neurons, it
remains unknown whether SARM1 is present and functional in myelinating
glia? This is an important question to address. Firstly, to identify whether
SARM1 dysfunction in other cell types in the nervous system may contribute to
neuropathology in SARM1 dependent diseases? Secondly, to ascertain whether
therapies altering SARM1 function may have unintended deleterious impacts
on PNS or CNS myelination? Surprisingly, we find that oligodendrocytes
express sarm1 mRNA in the zebrafish spinal cord and that SARM1 protein is
readily detectable in rodent oligodendrocytes in vitro and in vivo. Furthermore,
activation of endogenous SARM1 in cultured oligodendrocytes induces rapid
cell death. In contrast, in peripheral glia, SARM1 protein is not detectable
in Schwann cells and satellite glia in vivo and sarm1/Sarm1 mRNA is
detected at very low levels in Schwann cells, in vivo, in zebrafish and
mouse. Application of specific SARM1 activators to cultured mouse Schwann
cells does not induce cell death and nicotinamide adenine dinucleotide
(NAD) levels remain unaltered suggesting Schwann cells likely contain no
functionally relevant levels of SARM1. Finally, we address the question of
whether SARM1 is required for myelination or myelin maintenance. In the zebrafish and mouse PNS and CNS, we show that SARM1 is not required for
initiation of myelination and myelin sheath maintenance is unaffected in the
adult mouse nervous system. Thus, strategies to inhibit SARM1 function to treat
neurological disease are unlikely to perturb myelination in humans.
... To impede the viral spread, neurons are thought to have evolved a mechanism of rapid axon degeneration to clear the damaged and unhealthy neurons. Consistent with this notion, SARM1 has been shown to regulate axon degeneration during lyssavirus (Sundaramoorthy and others 2020) and Zika virus infections (Crawford and others 2022), although the downstream pathways may differ from injury-induced degeneration. Axon degeneration induced by lyssavirus is regulated by NAD + loss and calpain activation (Sundaramoorthy and others 2020), while Crawford and others (2022) showed that NAD + loss observed following Zika virus infection is independent of SARM1 NADase activity at 24 h postinfection. ...
... Consistent with this notion, SARM1 has been shown to regulate axon degeneration during lyssavirus (Sundaramoorthy and others 2020) and Zika virus infections (Crawford and others 2022), although the downstream pathways may differ from injury-induced degeneration. Axon degeneration induced by lyssavirus is regulated by NAD + loss and calpain activation (Sundaramoorthy and others 2020), while Crawford and others (2022) showed that NAD + loss observed following Zika virus infection is independent of SARM1 NADase activity at 24 h postinfection. Other functions of SARM1, such as hydrolyzing NADP + and performing base exchange reactions, as well as the immunity-related activities, may play a role in the early stages of infection. ...
Axons are an essential component of the nervous system, and axon degeneration is an early feature of many neurodegenerative disorders. The NAD+ metabolome plays an essential role in regulating axonal integrity. Axonal levels of NAD+ and its precursor NMN are controlled in large part by the NAD+ synthesizing survival factor NMNAT2 and the pro-neurodegenerative NADase SARM1, whose activation triggers axon destruction. SARM1 has emerged as a promising axon-specific target for therapeutic intervention, and its function, regulation, structure, and role in neurodegenerative diseases have been extensively characterized in recent years. In this review, we first introduce the key molecular players involved in the SARM1-dependent axon degeneration program. Next, we summarize recent major advances in our understanding of how SARM1 is kept inactive in healthy neurons and how it becomes activated in injured or diseased neurons, which has involved important insights from structural biology. Finally, we discuss the role of SARM1 in neurodegenerative disorders and environmental neurotoxicity and its potential as a therapeutic target.
... Loss of IENFs is largely dependent on Wallerian degeneration and SARM1 activity [37][38][39][40]. While direct links between inflammation and SARM1 activity in the peripheral nerve are emerging [41,42], SARM1 is known to be heavily involved in responses evoked by pathogen-and damage-associated molecular patterns (DAMPs and PAMPs) [43,44]. Combined with the breadth of infectious and autoimmune diseases exhibiting IENF loss described here, this suggests interplay between pattern recognition receptors and the Wallerian degeneration pathway may contribute to axon retraction. ...
Background: Intraepidermal nerve fiber density (IENFD) has become an important biomarker for neuropathy diagnosis and research. The consequences of reduced IENFD can include sensory dysfunction, pain, and a significant decrease in quality of life. We examined the extent to which IENFD is being used as a tool in human and mouse models and compared the degree of fiber loss between diseases to gain a broader understanding of the existing data collected using this common technique.
Methods: We conducted a scoping review of publications that used IENFD as a biomarker in human and non-human research. PubMed was used to identify 1,004 initial articles that were then screened to select articles that met the criteria for inclusion. Criteria were chosen to standardize publications so they could be compared rigorously and included having a control group, measuring IENFD in a distal limb, and using protein gene product 9.5 (PGP9.5).
Results: We analyzed 397 articles and collected information related to publication year, the condition studied, and the percent IENFD loss. The analysis revealed that the use of IENFD as a tool has been increasing in both human and non-human research. We found that IENFD loss is prevalent in many diseases, and metabolic or diabetes-related diseases were the most studied conditions in humans and rodents. Our analysis identified 74 human diseases in which IENFD was affected, with 71 reporting IENFD loss and an overall average IENFD change of -47%. We identified 28 mouse and 21 rat conditions, with average IENFD changes of -31.6 % and -34.7% respectively. Additionally, we present data describing sub-analyses of IENFD loss according to disease characteristics in diabetes and chemotherapy treatments in humans and rodents.
Interpretation: Reduced IENFD occurs in a surprising number of human disease conditions. Abnormal IENFD contributes to important complications, including poor cutaneous vascularization, sensory dysfunction, and pain. Our analysis informs future rodent studies so they may better mirror human diseases impacted by reduced IENFD, highlights the breadth of diseases impacted by IENFD loss, and urges exploration of common mechanisms that lead to substantial IENFD loss as a complication in disease.
... It has been postulated that one of the purposes of SARM1, a toll like adapter protein and remnant of the innate immune system, and the wider axon degeneration machinery is to bring about compartmentalized neurodegeneration to prevent spread of viral pathogens throughout the nervous system (Tsunoda, 2008). In mouse CNS myelinating cocultures, loss of Sarm1 protects neuronal somas against infection and cell death (Crawford et al., 2022). ...
SARM1 is a central regulator of programmed axon death and is required to initiate axon self-destruction after traumatic and toxic insults to the nervous system. Abnormal activation of this axon degeneration pathway is increasingly recognized as a contributor to human neurological disease and SARM1 knockdown or inhibition has become an attractive therapeutic strategy to preserve axon loss in a variety of disorders of the peripheral and central nervous system. Despite this, it remains unknown whether Sarm1 /SARM1 is present in myelinating glia and whether it plays a role in myelination in the PNS or CNS. It is important to answer these questions to understand whether future therapies inhibiting SARM1 function may have unintended deleterious impacts on myelination. Here we show that Sarm1 mRNA is present in oligodendrocytes in zebrafish but only detectable at low levels in Schwann cells in both zebrafish and mice. We find SARM1 protein is readily detectable in murine oligodendrocytes in vitro and in vivo and activation of endogenous SARM1 in oligodendrocytes induces cell death. In contrast, SARM1 protein is not detectable in Schwann cells and satellite glia in the adult murine nervous system. Cultured Schwann cells contain negligible functional SARM1 and are insensitive to specific SARM1 activators. Using zebrafish and mouse Sarm1 mutants, we show that SARM1 is not required for initiation of myelination nor myelin sheath maintenance by oligodendrocytes and Schwann cells. Thus, strategies to inhibit SARM1 function in the nervous system to treat neurological disease are unlikely to perturb myelination in humans.
Main Points
SARM1 protein is detectable in oligodendrocytes but not in Schwann cells
Oligodendrocytes but not Schwann cells die in response to endogenous SARM1 activation
CNS nor PNS myelination, in zebrafish and mice, is hindered by loss of sarm1/Sarm1
... Although this prodegenerative role and its regulation in axons was discovered using the experimental platform of axon injury, SARM1 can kill the neuronal soma directly too, for example when it is becomes constitutively active through gain-of-function (GoF) mutation [5,6], or when it is activated by a toxin [7,8]. SARM1 also responds to some viruses in ways that are less well understood [9,10,11]. What is the evidence so far supporting a role for SARM1 in ALS, and what more do we need to know to confirm this and to understand how widespread its involvement is? And which genetic, environmental and other factors could lead to SARM1 activation in ALS? ...
... Finally, the recent finding that zika virus causes SARM1dependent neuronal death [9], along with earlier indications of similar effects with both rabies and West Nile virus [10,11], albeit so far by unknown mechanisms, raise the important question of whether endemic viruses could make an as-yet unrecognised contribution to neurodegenerative disorders such as ALS by acting on SARM1. At present, this can be only speculative, but since an environmental component in sporadic ALS of around 40% needs to be accounted for [74], and since some viruses including rabies and zika have indeed been associated with ALS risk and motor neuron death [75,76,77], it will be important to consider. ...
This review addresses the longstanding debate over whether amyotrophic lateral sclerosis (ALS) is a ‘dying back’ or ‘dying forward’ disorder in the light of new gene identifications and the increased understanding of mechanisms of action for previously identified ALS genes. While the topological pattern of pathology in animal models, and more anecdotally in patients is indeed ‘dying back’, this review discusses how this fits with the fact that many of the major initiating events are thought to occur within the soma. It also discusses how widely varying ALS risk factors, including some impacting axons directly, may combine to drive a common pathway involving TAR DNA binding protein 43 (TDP-43) and neuromuscular junction (NMJ) denervation. The emerging association between sterile alpha and TIR motif-containing 1 (SARM1), a protein so far mostly associated with axon degeneration, and sporadic ALS is another major theme. The strengths and limitations of the current evidence supporting an association are considered, along with ways in which SARM1 could become activated in ALS. The final section addresses SARM1-based therapies along with the prospects for targeting other axonal steps in ALS pathogenesis.
Sterile alpha and Toll/interleukin-1 receptor motif containing 1 (SARM1), a nicotinamide adenine dinucleotide (NAD)-utilizing enzyme, mediates axon degeneration (AxD) in various neurodegenerative diseases. It is activated by nicotinamide mononucleotide (NMN) to produce a calcium messenger, cyclic ADP-ribose (cADPR). This activity is blocked by elevated NAD level. Here, we verified this metabolic regulation in somatic HEK-293T cells by overexpressing NMN-adenyltransferase to elevate cellular NAD, which resulted not only in inhibition of their own SARM1 from producing cADPR but, surprisingly, also in the 5–10 neighboring wildtype cells in mixed cultures via connexin (Cx)-43. Direct visualization of gap junction intercellular communication (GJIC) was achieved by incubating cells with a permeant probe, PC11, which is converted by SARM1 into PAD11, a fluorescent NAD analog capable of traversing GJs. Extending the findings to dorsal root ganglion neurons, we further showed that CZ-48, a permeant NMN analog, or axotomy, activated SARM1 and the produced PAD11 was transferred to contacting axons via GJIC. The gap junction involved was identified as Cx36 instead. This neuronal GJIC was demonstrated to be functional, enabling healthy neurons to protect adjacent axotomized axons from degeneration. Inhibition of GJIC in mice by AAV-PHP.eB-mediated knockdown of Cx36 in brain induced neuroinflammation, which in turn activated SARM1 and resulted in axon degeneration as well as behavioral deficits. Our results demonstrate a novel intercellular regulation mechanism of SARM1 and reveal a protective role of healthy tissue against AxD induced by injury or neuroinflammation.
Classifications: BIOLOGICAL SCIENCES -- Neuroscience; Biochemistry; Cell Biology.
Severe acute respiratory coronavirus 2 (SARS‐CoV‐2) causes neurological disease in the peripheral and central nervous system (PNS and CNS, respectively) of some patients. It is not clear whether SARS‐CoV‐2 infection or the subsequent immune response are the key factors that cause neurological disease. Here, we addressed this question by infecting human induced pluripotent stem cell‐derived CNS and PNS neurons with SARS‐CoV‐2. SARS‐CoV‐2 infected a low number of CNS neurons and did not elicit a robust innate immune response. On the contrary, SARS‐CoV‐2 infected a higher number of PNS neurons. This resulted in expression of interferon (IFN) λ1, several IFN‐stimulated genes and proinflammatory cytokines. The PNS neurons also displayed alterations characteristic of neuronal damage, as increased levels of sterile alpha and Toll/interleukin receptor motif‐containing protein 1, amyloid precursor protein and α‐synuclein, and lower levels of cytoskeletal proteins. Interestingly, blockade of the Janus kinase and signal transducer and activator of transcription pathway by Ruxolitinib did not increase SARS‐CoV‐2 infection, but reduced neuronal damage, suggesting that an exacerbated neuronal innate immune response contributes to pathogenesis in the PNS. Our results provide a basis to study coronavirus disease 2019 (COVID‐19) related neuronal pathology and to test future preventive or therapeutic strategies.
