The SARM1 TIR domain produces glycocyclic ADPR molecules as minor products.

Sterile alpha and TIR motif-containing 1 (SARM1) is a protein involved in programmed death of injured axons. Following axon injury or a drug-induced insult, the TIR domain of SARM1 degrades the essential molecule nicotinamide adenine dinucleotide (NAD+), leading to a form of axonal death called Wallerian degeneration. Degradation of NAD+ by SARM1 is essential for the Wallerian degeneration process, but accumulating evidence suggest that other activities of SARM1, beyond the mere degradation of NAD+, may be necessary for programmed axonal death. In this study we show that the TIR domains of both human and fruit fly SARM1 produce 1''-2' and 1''-3' glycocyclic ADP-ribose (gcADPR) molecules as minor products. As previously reported, we observed that SARM1 TIR domains mostly convert NAD+ to ADPR (for human SARM1) or cADPR (in the case of SARM1 from Drosophila melanogaster). However, we now show that human and Drosophila SARM1 additionally convert ~0.1-0.5% of NAD+ into gcADPR molecules. We find that SARM1 TIR domains produce gcADPR molecules both when purified in vitro and when expressed in bacterial cells. Given that gcADPR is a second messenger involved in programmed cell death in bacteria and likely in plants, we propose that gcADPR may play a role in SARM1-induced programmed axonal death in animals.

Loss of Sarm1 reduces retinal ganglion cell loss in chronic glaucoma.

Glaucoma is one of the leading causes of irreversible blindness worldwide and vision loss in the disease results from the deterioration of retinal ganglion cells (RGC) and their axons. Metabolic dysfunction of RGC plays a significant role in the onset and progression of the disease in both human patients and rodent models, highlighting the need to better define the mechanisms regulating cellular energy metabolism in glaucoma. This study sought to determine if Sarm1, a gene involved in axonal degeneration and NAD+ metabolism, contributes to glaucomatous RGC loss in a mouse model with chronic elevated intraocular pressure (IOP). Our data demonstrate that after 16 weeks of elevated IOP, Sarm1 knockout (KO) mice retain significantly more RGC than control animals. Sarm1 KO mice also performed significantly better when compared to control mice during optomotor testing, indicating that visual function is preserved in this group. Our findings also indicate that Sarm1 KO mice display mild ocular developmental abnormalities, including reduced optic nerve axon diameter and lower visual acuity than controls. Finally, we present data to indicate that SARM1 expression in the optic nerve is most prominently associated with oligodendrocytes. Taken together, these data suggest that attenuating Sarm1 activity through gene therapy, pharmacologic inhibition, or NAD+ supplementation, may be a novel therapeutic approach for patients with glaucoma.

Impacts of H2O2, SARM1 inhibition, and high NAm concentrations on Huntington's disease laser-induced degeneration.

Axonal degeneration is a key component of neurodegenerative diseases such as Huntington's disease (HD), Alzheimer's disease, and amyotrophic lateral sclerosis. Nicotinamide, an NAD+ precursor, has long since been implicated in axonal protection and reduction of degeneration. However, studies on nicotinamide (NAm) supplementation in humans indicate that NAm has no protective effect. Sterile alpha and toll/interleukin receptor motif-containing protein 1 (SARM1) regulates several cell responses to axonal damage and has been implicated in promoting neuronal degeneration. SARM1 inhibition seems to result in protection from neuronal degeneration while hydrogen peroxide has been implicated in oxidative stress and axonal degeneration. The effects of laser-induced axonal damage in wild-type and HD dorsal root ganglion cells treated with NAm, hydrogen peroxide (H2O2), and SARM1 inhibitor DSRM-3716 were investigated and the cell body width, axon width, axonal strength, and axon shrinkage post laser-induced injury were measured.

ARMC5 controls the degradation of most Pol II subunits, and ARMC5 mutation increases neural tube defect risks in mice and humans.

BACKGROUND: Neural tube defects (NTDs) are caused by genetic and environmental factors. ARMC5 is part of a novel ubiquitin ligase specific for POLR2A, the largest subunit of RNA polymerase II (Pol II). RESULTS: We find that ARMC5 knockout mice have increased incidence of NTDs, such as spina bifida and exencephaly. Surprisingly, the absence of ARMC5 causes the accumulation of not only POLR2A but also most of the other 11 Pol II subunits, indicating that the degradation of the whole Pol II complex is compromised. The enlarged Pol II pool does not lead to generalized Pol II stalling or a generalized decrease in mRNA transcription. In neural progenitor cells, ARMC5 knockout only dysregulates 106 genes, some of which are known to be involved in neural tube development. FOLH1, critical in folate uptake and hence neural tube development, is downregulated in the knockout intestine. We also identify nine deleterious mutations in the ARMC5 gene in 511 patients with myelomeningocele, a severe form of spina bifida. These mutations impair the interaction between ARMC5 and Pol II and reduce Pol II ubiquitination. CONCLUSIONS: Mutations in ARMC5 increase the risk of NTDs in mice and humans. ARMC5 is part of an E3 controlling the degradation of all 12 subunits of Pol II under physiological conditions. The Pol II pool size might have effects on NTD pathogenesis, and some of the effects might be via the downregulation of FOLH1. Additional mechanistic work is needed to establish the causal effect of the findings on NTD pathogenesis.

SARM1 regulates NAD+-linked metabolism and select immune genes in macrophages.

Sterile alpha and HEAT/armadillo motif-containing protein (SARM1) was recently described as a NAD+-consuming enzyme and has previously been shown to regulate immune responses in macrophages. Neuronal SARM1 is known to contribute to axon degeneration due to its NADase activity. However, how SARM1 affects macrophage metabolism has not been explored. Here, we show that macrophages from Sarm1-/- mice display elevated NAD+ concentrations and lower cyclic ADP-ribose, a known product of SARM1-dependent NAD+ catabolism. Further, SARM1-deficient macrophages showed an increase in the reserve capacity of oxidative phosphorylation and glycolysis compared to WT cells. Stimulation of macrophages to a proinflammatory state by lipopolysaccharide (LPS) revealed that SARM1 restricts the ability of macrophages to upregulate glycolysis and limits the expression of the proinflammatory gene interleukin (Il) 1b, but boosts expression of anti-inflammatory Il10. In contrast, we show macrophages lacking SARM1 induced to an anti-inflammatory state by IL-4 stimulation display increased oxidative phosphorylation and glycolysis, and reduced expression of the anti-inflammatory gene, Fizz1. Overall, these data show that SARM1 fine-tunes immune gene transcription in macrophages via consumption of NAD+ and altered macrophage metabolism.

SARM1 is responsible for calpain-dependent dendrite degeneration in mouse hippocampal neurons.

Sterile alpha and toll/interleukin receptor motif-containing 1 (SARM1) is a critical regulator of axon degeneration that acts through hydrolysis of NAD+ following injury. Recent work has defined the mechanisms underlying SARM1's catalytic activity and advanced our understanding of SARM1 function in axons, yet the role of SARM1 signaling in other compartments of neurons is still not well understood. Here, we show in cultured hippocampal neurons that endogenous SARM1 is present in axons, dendrites, and cell bodies and that direct activation of SARM1 by the neurotoxin Vacor causes not just axon degeneration, but degeneration of all neuronal compartments. In contrast to the axon degeneration pathway defined in dorsal root ganglia, SARM1-dependent hippocampal axon degeneration in vitro is not sensitive to inhibition of calpain proteases. Dendrite degeneration downstream of SARM1 in hippocampal neurons is dependent on calpain 2, a calpain protease isotype enriched in dendrites in this cell type. In summary, these data indicate SARM1 plays a critical role in neurodegeneration outside of axons and elucidates divergent pathways leading to degeneration in hippocampal axons and dendrites.

Sarm1 knockout prevents type 1 diabetic bone disease in females independent of neuropathy.

Patients with diabetes have a high risk of developing skeletal diseases accompanied by diabetic peripheral neuropathy (DPN). In this study, we isolated the role of DPN in skeletal disease with global and conditional knockout models of sterile-α and TIR-motif-containing protein-1 (Sarm1). SARM1, an NADase highly expressed in the nervous system, regulates axon degeneration upon a range of insults, including DPN. Global knockout of Sarm1 prevented DPN, but not skeletal disease, in male mice with type 1 diabetes (T1D). Female wild-type mice also developed diabetic bone disease but without DPN. Unexpectedly, global Sarm1 knockout completely protected female mice from T1D-associated bone suppression and skeletal fragility despite comparable muscle atrophy and hyperglycemia. Global Sarm1 knockout rescued bone health through sustained osteoblast function with abrogation of local oxidative stress responses. This was independent of the neural actions of SARM1, as beneficial effects on bone were lost with neural conditional Sarm1 knockout. This study demonstrates that the onset of skeletal disease occurs rapidly in both male and female mice with T1D completely independently of DPN. In addition, this reveals that clinical SARM1 inhibitors, currently being developed for treatment of neuropathy, may also have benefits for diabetic bone through actions outside of the nervous system.

Loss of SARM1 ameliorates secondary thalamic neurodegeneration after cerebral infarction.

Ischemic stroke causes secondary neurodegeneration in the thalamus ipsilateral to the infarction site and impedes neurological recovery. Axonal degeneration of thalamocortical fibers and autophagy overactivation are involved in thalamic neurodegeneration after ischemic stroke. However, the molecular mechanisms underlying thalamic neurodegeneration remain unclear. Sterile /Armadillo/Toll-Interleukin receptor homology domain protein (SARM1) can induce Wallerian degeneration. Herein, we aimed to investigate the role of SARM1 in thalamic neurodegeneration and autophagy activation after photothrombotic infarction. Neurological deficits measured using modified neurological severity scores and adhesive-removal test were ameliorated in Sarm1-/- mice after photothrombotic infarction. Compared with wild-type mice, Sarm1-/- mice exhibited unaltered infarct volume; however, there were markedly reduced neuronal death and gliosis in the ipsilateral thalamus. In parallel, autophagy activation was attenuated in the thalamus of Sarm1-/- mice after cerebral infarction. Thalamic Sarm1 re-expression in Sarm1-/- mice increased thalamic neurodegeneration and promoted autophagy activation. Auotophagic inhibitor 3-methyladenine partially alleviated thalamic damage induced by SARM1. Moreover, autophagic initiation through rapamycin treatment aggravated post-stroke neuronal death and gliosis in Sarm1-/- mice. Taken together, SARM1 contributes to secondary thalamic neurodegeneration after cerebral infarction, at least partly through autophagy inhibition. SARM1 deficiency is a potential therapeutic strategy for secondary thalamic neurodegeneration and functional deficits after stroke.

SARM1 Promotes Neurodegeneration and Memory Impairment in Mouse Models of Alzheimer's Disease.

Neuroinflammation plays a crucial role in the pathogenesis and progression of Alzheimer's disease (AD). The Sterile Alpha and Toll Interleukin Receptor Motif-containing protein 1 (SARM1) has been shown to promote axonal degeneration and is involved in neuroinflammation. However, the role of SARM1 in AD remains unclear. In this study, we found that SARM1 was reduced in hippocampal neurons of AD model mice. Interestingly, conditional knockout (CKO) of SARM1 in the central nervous system (CNS, SARM1Nestin-CKO mice) delayed the cognitive decline in APP/PS1 AD model mice. Furthermore, SARM1 deletion reduced the Aß deposition and inflammatory infiltration in the hippocampus and inhibited neurodegeneration in APP/PS1 AD model mice. Further investigation into the underlying mechanisms revealed that the signaling of tumor necrosis factor-α (TNF-α) was downregulated in the hippocampus tissues of APP/PS1;SARM1Nestin-CKO mice, thereby alleviating the cognitive decline, Aß deposition and inflammatory infiltration. These findings identify unrecognized functions of SARM1 in promoting AD and reveal the SARM1-TNF-α pathway in AD model mice.

Targeting SARM1 improves autophagic stress-induced axonal neuropathy.

ABBREVIATIONS: AAV: adeno-associated virus; ATF3: activating transcription factor 3; ATG7: autophagy related 7; AVIL: advillin; cADPR: cyclic ADP ribose; CALC: calcitonin/calcitonin-related polypeptide; CMT: Charcot-Marie-Tooth disease; cKO: conditional knockout; DEG: differentially expressed gene; DRG: dorsal root ganglion; FE-SEM: field emission scanning electron microscopy; IF: immunofluorescence; NCV: nerve conduction velocity; PVALB: parvalbumin; RAG: regeneration-associated gene; ROS: reactive oxygen species; SARM1: sterile alpha and HEAT/Armadillo motif containing 1; SYN1: synapsin I.

A phase transition reduces the threshold for nicotinamide mononucleotide-based activation of SARM1, an NAD(P) hydrolase, to physiologically relevant levels.

Axonal degeneration is a hallmark feature of neurodegenerative diseases. Activation of the NAD(P)ase sterile alpha and toll-interleukin receptor motif containing protein 1 (SARM1) is critical for this process. In resting neurons, SARM1 activity is inhibited, but upon damage, SARM1 is activated and catalyzes one of three NAD(P)+ dependent reactions: (1) NAD(P)+ hydrolysis to form ADP-ribose (ADPR[P]) and nicotinamide; (2) the formation of cyclic-ADPR (cADPR[P]); or (3) a base exchange reaction with nicotinic acid (NA) and NADP+ to form NA adenine dinucleotide phosphate. Production of these metabolites triggers axonal death. Two activation mechanisms have been proposed: (1) an increase in the nicotinamide mononucleotide (NMN) concentration, which leads to the allosteric activation of SARM1, and (2) a phase transition, which stabilizes the active conformation of the enzyme. However, neither of these mechanisms have been shown to occur at the same time. Using in vitro assay systems, we show that the liquid-to-solid phase transition lowers the NMN concentration required to activate the catalytic activity of SARM1 by up to 140-fold. These results unify the proposed activation mechanisms and show for the first time that a phase transition reduces the threshold for NMN-based SARM1 activation to physiologically relevant levels. These results further our understanding of SARM1 activation and will be important for the future development of therapeutics targeting SARM1.

Phosphorylated SARM1 is involved in the pathological process of rotenone-induced neurodegeneration.

Sterile alpha and Toll/interleukin receptor motif-containing protein 1 (SARM1) is a NAD+ hydrolase that plays a key role in axonal degeneration and neuronal cell death. We reported that c-Jun N-terminal kinase (JNK) activates SARM1 through phosphorylation at Ser-548. The importance of SARM1 phosphorylation in the pathological process of Parkinson's disease (PD) has not been determined. We thus conducted the present study by using rotenone (an inducer of PD-like pathology) and neurons derived from induced pluripotent stem cells (iPSCs) from healthy donors and a patient with familial PD PARK2 (FPD2). The results showed that compared to the healthy neurons, FPD2 neurons were more vulnerable to rotenone-induced stress and had higher levels of SARM1 phosphorylation. Similar cellular events were obtained when we used PARK2-knockdown neurons derived from healthy donor iPSCs. These events in both types of PD-model neurons were suppressed in neurons treated with JNK inhibitors, Ca2+-signal inhibitors, or by a SARM1-knockdown procedure. The degenerative events were enhanced in neurons overexpressing wild-type SARM1 and conversely suppressed in neurons overexpressing the SARM1-S548A mutant. We also detected elevated SARM1 phosphorylation in the midbrain of PD-model mice. The results indicate that phosphorylated SARM1 plays an important role in the pathological process of rotenone-induced neurodegeneration.

[Macronodular adrenal hyperplasia causing Cushing's syndrome due to ARMC5 gene mutation.] / Cushing-szindrómát okozó macronodularis mellékvese-hyperplasia ARMC5-génmutáció következtében.

Our 69-year-old female patient was investigated for a 20 kg weight gain over 2 years. The patient's medical history included hypertension, hyperuricemia, bilateral cataract surgery and musculosceletal complaints. Diabetes mellitus was not found. Physical examination revealed abdominal obesity, proximal myopathy and atrophic, vulnerable skin. The "overnight", low-dose and long, low-dose dexamethasone suppression tests indicated autonomous cortisol overproduction (plasma cortisol level: 172.6 and 153.2 nmol/L, cut-off: 50 nmol/L). The suppressed ACTH (<1.11 pmol/L, normal value: 1.12-10.75 pmol/L) suggested ACTH-independent hypercortisolism. Abdominal CT described macronodular enlargement of both adrenals. The size of the largest nodule was 23 × 20 mm in the right, and 24 × 30 mm on the left side (with -33 ± 37 HU density values on native scans). The 131I-cholesterol adrenal scintigraphy and SPECT/CT showed almost equally intensive radiopharmacon uptake on both sides. Based on the clinical results, bilateral macronodular adrenal hyperplasia associated with ACTH-independent hypercortisolism was diagnosed. Genomic DNA was obtained from the peripheral blood leukocytes. Targeted sequencing of 25 genes potentially involved in adrenal tumorigenesis revealed a new disease-causing armadillo repeat-containing 5 (ARMC5) gene mutation (c.1724del28 bp, g.31,476,067-31,476,094). Because of the autosomal dominant inheritance of this genetic alteration, the patient's two children underwent genetic screening for the ARMC5 mutation. The same mutation was found in the younger child of our patient. To the best of our knowledge, this is the first published Hungarian case of ARMC5 mutation with bilateral macronodular adrenal hyperplasia and ACTH-independent Cushing's syndrome. The genetic alteration is present in two generations of the family of the index patient. Orv Hetil. 2023; 164(32): 1271-1277.

Mitophagy suppresses motor neuron necroptotic mitochondrial damage and alleviates necroptosis that converges to SARM1 in acrylamide-induced dying-back neuropathy.

Acrylamide (ACR), a common industrial ingredient that is also found in many foodstuffs, induces dying-back neuropathy in humans and animals. However, the mechanisms remain poorly understood. Sterile alpha and toll/interleukin 1 receptor motif-containing protein 1 (SARM1) is the central determinant of axonal degeneration and has crosstalk with different cell death programs to determine neuronal survival. Herein, we illustrated the role of SARM1 in ACR-induced dying-back neuropathy. We further demonstrated the upstream programmed cell death mechanism of this SARM1-dependent process. Spinal cord motor neurons that were induced to overexpress SARM1 underwent necroptosis rather than apoptosis in ACR neuropathy. Mechanically, non-canonical necroptotic pathways mediated mitochondrial permeability transition pore (mPTP) opening, reactive oxygen species (ROS) production, and mitochondrial fission. What's more, the final executioner of necroptosis, phosphorylation-activated mixed lineage kinase domain-like protein (MLKL), aggregated in mitochondrial fractions. Rapamycin intervention removed the impaired mitochondria, inhibited necroptosis for axon maintenance and neuronal survival, and alleviated ACR neuropathy. Our work clarified the functional links among mitophagy, necroptosis, and SARM1-dependent axonal destruction during ACR intoxication, providing novel therapeutic targets for dying-back neuropathies.

SARM1 promotes the neuroinflammation and demyelination through IGFBP2/NF-κB pathway in experimental autoimmune encephalomyelitis mice.

AIM: Multiple sclerosis (MS) is an autoimmune disease, and its typical characteristics are neuroinflammation and the demyelination of neurons in the central nervous system (CNS). Sterile alpha and TIR motif containing 1 (SARM1) is an essential factor mediating axonal degeneration and SARM1 deletion reduces the neuroinflammation in spinal cord injury. This study aimed to explore the roles of SARM1 and its underlying mechanisms in MS. METHODS: Experimental autoimmune encephalomyelitis (EAE, a model of MS) model was established. Immunostaining, western blot, electron microscope, and HE staining were used to examine the pathological manifestations such as inflammation, demyelination, and neuronal death in SARM1f/f EAE mice and SARM1Nestin -CKO EAE mice. In addition, RNA-seq, real-time PCR and double-immunostaining were used to examine the underlying mechanism of SARM1 in EAE mice. RESULTS: SARM1 was upregulated in neurons of the spinal cords of EAE mice. SARM1 knockout in CNS ameliorated EAE with less neuroinflammation, demyelination, and dead neurons. Mechanically, SARM1 knockout resulted in the reduction of insulin-like growth factor (IGF)-binding protein 2 (IGFBP2) in neurons of EAE mice, which might inhibit the neuroinflammation through inhibiting NF-κB signaling. Finally, activation of NF-κB partially aggravated the neuroinflammation and demyelination deficits of SARM1Nestin -CKO EAE mice. CONCLUSIONS: These results identified the unknown role of SARM1 in the promotion of neuroinflammation and demyelination and revealed a novel drug target pathway of SARM1/IGFBP2/NF-κB for MS.

Morphological Harbingers of ARMC5-Pathogenic Variant-Related Bilateral Macronodular Adrenocortical Disease.

Bilateral macronodular adrenocortical disease (BMAD) is a neoplastic disease associated with a high frequency of germline disease. Armadillo repeat containing 5 (ARMC5) pathogenic variants (PVs) have not been widely studied to determine the morphological and immunohistochemical characteristics of BMAD. We carried out a detailed morphologic review of 22 surgical specimens excised from patients with BMAD and compared them with PV of ARMC5 (PV + , n = 14) and those without (PV - , n = 8), and further comparing them with a control group of adrenals excised from patients with renal cancer (n = 11). No patients presented with a genetic syndrome related to BMAD. Overt Cushing's syndrome was present in 12/22 patients, 10 PV + and 2 PV - (p = 0.074). We also evaluated the expression of Ki-67, BCL-2, BAX, p53, CYP11B1, and ARMC5 protein. The pseudo-glandular and trabecular architectural patterns were strongly associated with the PV + group (both p < 0.001), as well as capsular extrusion (p < 0.001). There was no predictive value in the distinction of ARMC5 variants in Hsiao subtyping. ARMC5 diffuse cytoplasmic staining was observed in all 11 control samples. The ARMC5 expression was significantly lower in BMAD than in the control group (p < 0.001). In all the specimens, expression of BCL-2 was identified only in the medulla, and expression of BAX was observed in adrenocortical cells. CYP11B1 diffuse immunoexpression was identified in all the specimens of BMAD and in the fasciculata zone in the control group. The mitotic count and Ki-67 proliferation index was very low in all three groups (controls, PV + , and PV - BMAD). None of the specimens stained positive for the p53 protein. Although our series is limited, the presence of pseudo-glandular and/or trabecular patterns and capsular extrusion indicated the presence of pathogenic variants of ARMC5 in BMAD. The gland enlargement does not seem to be related to the increase of mitotic count or a higher proliferation index (Ki-67).

Primary bilateral macronodular adrenocortical hyperplasia (PBMAH) patient with ARMC5 mutations.

BACKGROUND: Primary bilateral macronodular adrenocortical hyperplasia (PBMAH) is a highly heterogeneous disease with divergent manifestations ranging from asymptomatic subclinical Cushing syndrome (CS) to overt Cushing syndrome with severe complications. ARMC5 mutations occur in 20 to 55% PBMAH patients usually with more severe phenotypes. Different ARMC5 mutations might be associated with diverse phenotypes of PBMAH. CASE PRESENTATION: A 39-year-old man was admitted to our hospital with progressive weight gain and severe hypertension. He presented typical CS and its classical metabolic and bone complications like hypertension and osteoporosis. The laboratory results showed high levels of cortisol and low levels of ACTH. Low- and high-dosed dexamethasone suppression tests were negative. Contrast-enhanced computed tomography (CT) revealed multiple bilateral irregular macronodular adrenal masses. Adrenal venous sampling (AVS) confirmed that the right adrenal gland with larger nodules secreted more hormone that the left side did. Right adrenalectomy and subsequent contralateral subtotal resection were conducted. His blood pressure and CS symptoms as well as comorbidities including backache and muscle weakness improved. Whole exome sequencing identified one ARMC5 germline mutation (c.1855C > T, p. R619*), five ARMC5 somatic mutations (four novel mutations) in his right and left adrenal nodules. CONCLUSIONS: This PBMAH patient was identified with one ARMC5 germline mutation and five different somatic ARMC5 mutations (four novel mutations) in the different nodules of the bilateral adrenal masses. AVS combined with CT imagine could be helpful to determine the dominant side for adrenalectomy. Genetic testing is important for the diagnosis and management of the patient with PBMAH.

SARM1 deletion delays cerebellar but not spinal cord degeneration in an enhanced mouse model of SPG7 deficiency.

Hereditary spastic paraplegia is a neurological condition characterized by predominant axonal degeneration in long spinal tracts, leading to weakness and spasticity in the lower limbs. The nicotinamide adenine dinucleotide (NAD+)-consuming enzyme SARM1 has emerged as a key executioner of axonal degeneration upon nerve transection and in some neuropathies. An increase in the nicotinamide mononucleotide/NAD+ ratio activates SARM1, causing catastrophic NAD+ depletion and axonal degeneration. However, the role of SARM1 in the pathogenesis of hereditary spastic paraplegia has not been investigated. Here, we report an enhanced mouse model for hereditary spastic paraplegia caused by mutations in SPG7. The eSpg7 knockout mouse carries a deletion in both Spg7 and Afg3l1, a redundant homologue expressed in mice but not in humans. The eSpg7 knockout mice recapitulate the phenotypic features of human patients, showing progressive symptoms of spastic-ataxia and degeneration of axons in the spinal cord as well as the cerebellum. We show that the lack of SPG7 rewires the mitochondrial proteome in both tissues, leading to an early onset decrease in mito-ribosomal subunits and a remodelling of mitochondrial solute carriers and transporters. To interrogate mechanisms leading to axonal degeneration in this mouse model, we explored the involvement of SARM1. Deletion of SARM1 delays the appearance of ataxic signs, rescues mitochondrial swelling and axonal degeneration of cerebellar granule cells and dampens neuroinflammation in the cerebellum. The loss of SARM1 also prevents endoplasmic reticulum abnormalities in long spinal cord axons, but does not halt the degeneration of these axons. Our data thus reveal a neuron-specific interplay between SARM1 and mitochondrial dysfunction caused by lack of SPG7 in hereditary spastic paraplegia.

TIR-1/SARM1 inhibits axon regeneration and promotes axon degeneration.

Growth and destruction are central components of the neuronal injury response. Injured axons that are capable of repair, including axons in the mammalian peripheral nervous system and in many invertebrate animals, often regenerate and degenerate on either side of the injury. Here we show that TIR-1/dSarm/SARM1, a key regulator of axon degeneration, also inhibits regeneration of injured motor axons. The increased regeneration in tir-1 mutants is not a secondary consequence of its effects on degeneration, nor is it determined by the NADase activity of TIR-1. Rather, we found that TIR-1 functions cell-autonomously to regulate each of the seemingly opposite processes through distinct interactions with two MAP kinase pathways. On one side of the injury, TIR-1 inhibits axon regeneration by activating the NSY-1/ASK1 MAPK signaling cascade, while on the other side of the injury, TIR-1 simultaneously promotes axon degeneration by interacting with the DLK-1 mitogen-activated protein kinase (MAPK) signaling cascade. In parallel, we found that the ability to cell-intrinsically inhibit axon regeneration is conserved in human SARM1. Our finding that TIR-1/SARM1 regulates axon regeneration provides critical insight into how axons coordinate a multidimensional response to injury, consequently informing approaches to manipulate the response toward repair.

Calaxin stabilizes the docking of outer arm dyneins onto ciliary doublet microtubule in vertebrates.

Outer arm dynein (OAD) is the main force generator of ciliary beating. Although OAD loss is the most frequent cause of human primary ciliary dyskinesia, the docking mechanism of OAD onto the ciliary doublet microtubule (DMT) remains elusive in vertebrates. Here, we analyzed the functions of Calaxin/Efcab1 and Armc4, the two of five components of vertebrate OAD-DC (docking complex), using zebrafish spermatozoa and cryo-electron tomography. Mutation of armc4 caused complete loss of OAD, whereas mutation of calaxin caused only partial loss of OAD. Detailed structural analysis revealed that calaxin-/- OADs are tethered to DMT through DC components other than Calaxin, and that recombinant Calaxin can autonomously rescue the deficient DC structure and the OAD instability. Our data demonstrate the discrete roles of Calaxin and Armc4 in the OAD-DMT interaction, suggesting the stabilizing process of OAD docking onto DMT in vertebrates.