Permeant Fluorescent Probes Visualize the Activation of SARM1 and Uncover an Anti-neurodegenerative Drug Candidate

Li, Wan Hua; Huang, Ke; Cai, Yuyan; Wang, Qian Wen; Zhu, Wen Jie; Hou, Yun Nan; Wang, Sujing; Cao, Sheng; Zhao, Zhuo; Xie, Xu Jie; Du, Yang; Lee, Chi‐Sing; Lee, Hon Cheung; Zhang, Hongmin; Zhao, Yong

doi:10.1101/2021.02.24.432704

Cited by 10 publications

(33 citation statements)

References 35 publications

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“…The role of SARM1 as a driver of axon degeneration has been extensively studied in the context of traumatic brain injury, axotomy, and mitochondrial dysfunction, but mainly in B6 congenic Sarm1 -/- mice. The chemotherapeutic drug vincristine is widely reported to induce SARM1-dependent axon degeneration [14, 33, 34]. We exploited this to assess and confirm the loss of SARM1-dependent axonal degeneration in the Sarm1 em1.1Tftc , Sarm1 em1.2Tftc , and Sarm1 em1.3Tftc mice.…”

Section: Resultsmentioning

confidence: 99%

Next generation SARM1 knockout and epitope tagged CRISPR-Cas9-generated isogenic mice reveal that SARM1 does not participate in regulating nuclear transcription, despite confirmation of protein expression in macrophages

Doran¹,

Sugisawa²,

Carty³

et al. 2021

Preprint

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SARM1 is an ancient and highly conserved TIR-domain containing protein, with a diverse range of proposed roles in both innate immunity and neuronal death and degeneration. Murine SARM1 has been reported to regulate the transcription of specific chemokines in both neurons and macrophages, however the extent and mechanism by which SARM1 contributes to transcription regulation remains to be fully understood. Here, using RNA sequencing we identify differential gene expression in bone marrow-derived macrophages (BMDM) from C57BL/6 congenic 129 ES cell-derived Sarm1-/- mice compared to wild type (WT). However, we show that passenger genes which are derived from the 129 donor strain of mice flank the Sarm1 locus, confounding interpretation of results, since many of the identified differentially regulated genes come from the region containing passenger genes. To re-examine the transcriptional role of SARM1 in the absence of such passenger genes, we generated three different Sarm1-/- mice using CRISPR/Cas9 technology. Vincristine treatment of ex vivo cultured post-natal neurons from these mice confirmed SARM1's previously identified key function as an executor of axon degeneration. However, using these mice, we show that the absence of SARM1 has no impact on transcription of genes previously shown to be altered in macrophages or in the brainstem. To gain further insight into SARM1 function, we generated and characterized a mouse expressing epitope-tagged SARM1, as it has been difficult to date to confirm which cells and tissues express SARM1 protein. In these mice we see high SARM1 protein expression in the brain and brainstem, and lower but detectable levels in macrophages. Overall, the generation of these next generation SARM1 knockout and epitope-tagged mice has clarified that SARM1 is expressed in mouse macrophages but has no general role in transcriptional regulation in these cells, and has provided important new animal models to further explore SARM1 function.

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Section: Resultsmentioning

confidence: 99%

Next generation SARM1 knockout and epitope tagged CRISPR-Cas9-generated isogenic mice reveal that SARM1 does not participate in regulating nuclear transcription, despite confirmation of protein expression in macrophages

Doran¹,

Sugisawa²,

Carty³

et al. 2021

Preprint

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“…Animal and cell culture data strongly implicate SARM1-dependent axon loss in some peripheral neuropathies, glaucoma, Parkinson's disease and other conditions [1,3,59] and human genetic studies support its involvement in rare axonopathies involving NMNAT2 mutation [7,8] and in ALS [5,6]. This, together with the complete rescue of axons by SARM1 removal when the pathway is very specifically activated [10,20] has led to considerable focus on inhibiting and knocking down SARM1 as a therapeutic strategy [3,28,32,60]. Many studies are focusing on blocking the NADase activity specifically but our data now suggest the importance of targeting NADPase and base exchange too.…”

Section: Discussionmentioning

confidence: 99%

“…Many studies are focusing on blocking the NADase activity specifically but our data now suggest the importance of targeting NADPase and base exchange too. This is important because the most effective drugs will come from targeting the right enzyme activity, indeed small molecules are already known to influence different SARM1 activities in different ways [16,32].…”

Section: Discussionmentioning

confidence: 99%

“…This suggests that while CD38 is responsible for most NAD degradation under basal conditions, its primary product in vivo is not cADPR, while SARM1 is the main regulator of basal cADPR but other, possibly greater activities of SARM1 remain possible. Base exchange by SARM1 has been reported very recently in live cells [32] but, other than it being unaltered by NMN at pH 4.5 [16], little is known about this activity.…”

Section: Introductionmentioning

confidence: 99%

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Programmed axon death executor SARM1 is a multi-functional NAD(P)ase with prominent base exchange activity, all regulated by physiological levels of NMN, NAD, NADP and other metabolites

Angeletti

Amici

Gilley

et al. 2021

Preprint

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SARM1 is an NAD glycohydrolase and TLR adapter with an essential, prodegenerative role in programmed axon death (Wallerian degeneration). It has low basal NADase activity that becomes strongly activated by NAD precursor NMN. Very high levels of NAD oppose this activation, competing for the same allosteric site on SARM1′s regulatory ARM domain. Injury or diseases that deplete axons of NMNAT2, an essential enzyme converting NMN to NAD, cause SARM1 activation. The resulting NAD degradation by SARM1, combined with loss of NAD synthesis by NMNAT2, causes rapid depletion of axonal NAD. This NAD loss is widely assumed to mediate axon death and is consequently a key focus for therapeutic strategies for axonopathies. However, like other NAD(P) glycohydrolases, SARM1 has additional enzyme activities whose contributions, consequences and regulation need to be fully understood. Here, we compare the multiple actions and regulation of recombinant human SARM1 with those of two other NAD(P) glycohydrolases, human CD38 and Aplysia californica ADP ribosyl cyclase. We find that SARM1 has the highest transglycosidation (base exchange) activity of these enzymes at neutral pH and with some bases this dominates NAD(P) hydrolysis and cyclisation. Moreover, like its NADase and NADPase reactions, SARM1-mediated base exchange at neutral pH is activated by increases in the NMN:NAD ratio, which we show for the first time can act in the presence of physiological levels of both metabolites. We establish that SARM1 base exchange is the most likely physiological source of calcium mobilizing agent NaADP, and potentially of other NAD(P) analogues, which could contribute to axon and cell death. We also identify regulatory effects of free pyridine bases, of NADP and of nicotinic acid riboside (NaR) on SARM1 that represent further therapeutic opportunities. These data will help to pinpoint which of the multiple functions of SARM1 is responsible for axon degeneration and how it can be optimally targeted to block axon degeneration in disease.

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“…Antisense oligonucleotides that target SARM1 and lower SARM1 levels in axons by more than 50% were also shown to slow degeneration in vitro ( Gould et al, 2021 ). Furthermore, the first small molecule inhibitors of SARM1 have been discovered and are shown to slow axon degeneration in DRGs to the same level as Sarm1 −/− ( Hughes et al, 2021 ; Li et al, 2021 ). Interestingly, the Hughes et al paper indicated that the inhibitor could be added up to 3 h after injury with the same level of protection, and could even protect axons fated to degenerate through rotenone exposure and promote recovery from the latent stage of degeneration back towards healthier axons ( Hughes et al, 2021 ).…”

Section: Links To Diseases and Future Therapiesmentioning

confidence: 99%

A Novel NAD Signaling Mechanism in Axon Degeneration and its Relationship to Innate Immunity

Hopkins

Kobe

et al. 2021

Front. Mol. Biosci.

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Axon degeneration represents a pathological feature of many neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease where axons die before the neuronal soma, and axonopathies, such as Charcot-Marie-Tooth disease and hereditary spastic paraplegia. Over the last two decades, it has slowly emerged that a central signaling pathway forms the basis of this process in many circumstances. This is an axonal NAD-related signaling mechanism mainly regulated by the two key proteins with opposing roles: the NAD-synthesizing enzyme NMNAT2, and SARM1, a protein with NADase and related activities. The crosstalk between the axon survival factor NMNAT2 and pro-degenerative factor SARM1 has been extensively characterized and plays an essential role in maintaining the axon integrity. This pathway can be activated in necroptosis and in genetic, toxic or metabolic disorders, physical injury and neuroinflammation, all leading to axon pathology. SARM1 is also known to be involved in regulating innate immunity, potentially linking axon degeneration to the response to pathogens and intercellular signaling. Understanding this NAD-related signaling mechanism enhances our understanding of the process of axon degeneration and enables a path to the development of drugs for a wide range of neurodegenerative diseases.

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Permeant Fluorescent Probes Visualize the Activation of SARM1 and Uncover an Anti-neurodegenerative Drug Candidate

Cited by 10 publications

References 35 publications

Next generation SARM1 knockout and epitope tagged CRISPR-Cas9-generated isogenic mice reveal that SARM1 does not participate in regulating nuclear transcription, despite confirmation of protein expression in macrophages

Next generation SARM1 knockout and epitope tagged CRISPR-Cas9-generated isogenic mice reveal that SARM1 does not participate in regulating nuclear transcription, despite confirmation of protein expression in macrophages

Programmed axon death executor SARM1 is a multi-functional NAD(P)ase with prominent base exchange activity, all regulated by physiological levels of NMN, NAD, NADP and other metabolites

A Novel NAD Signaling Mechanism in Axon Degeneration and its Relationship to Innate Immunity

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