Sarm1 haploinsufficiency or low expression levels after antisense oligonucleotides delay programmed axon degeneration.

Activation of the pro-degenerative protein SARM1 after diverse physical and disease-relevant injuries causes programmed axon degeneration. Original studies indicate that substantially decreased SARM1 levels are required for neuroprotection. However, we demonstrate, in Sarm1 haploinsufficient mice, that lowering SARM1 levels by 50% delays programmed axon degeneration in vivo after sciatic nerve transection and partially prevents neurite outgrowth defects in mice lacking the pro-survival factor NMNAT2. In vitro, the rate of degeneration in response to traumatic, neurotoxic, and genetic triggers of SARM1 activation is also slowed. Finally, we demonstrate that Sarm1 antisense oligonucleotides decrease SARM1 levels by more than 50% in vitro, which delays or prevents programmed axon degeneration. Combining Sarm1 haploinsufficiency with antisense oligonucleotides further decreases SARM1 levels and prolongs protection after neurotoxic injury. These data demonstrate that axon protection occurs in a Sarm1 gene dose-responsive manner and that SARM1-lowering agents have therapeutic potential, making Sarm1-targeting antisense oligonucleotides a promising therapeutic strategy.

Neutrophils Facilitate Prolonged Inflammasome Response in the DAMP-Rich Inflammatory Milieu.

Aberrant inflammasome activation contributes to various chronic inflammatory diseases; however, pyroptosis of inflammasome-active cells promptly terminates local inflammasome response. Molecular mechanisms underlying prolonged inflammasome signaling thus require further elucidation. Here, we report that neutrophil-specific resistance to pyroptosis and NLRP3 desensitization can facilitate sustained inflammasome response and interleukin-1ß secretion. Unlike macrophages, inflammasome-activated neutrophils did not undergo pyroptosis, indicated by using in vitro cell-based assay and in vivo mouse model. Intriguingly, danger-associated molecular patterns (DAMP)-rich milieu in the inflammatory region significantly abrogated NLRP3-activating potential of macrophages, but not of neutrophils. This macrophage-specific NLRP3 desensitization was associated with DAMP-induced mitochondrial depolarization that was not observed in neutrophils due to a lack of SARM1 expression. Indeed, valinomycin-induced compulsory mitochondrial depolarization in neutrophils restored inflammasome-dependent cell death and ATP-induced NLRP3 desensitization in neutrophils. Alongside prolonged inflammasome-activating potential, neutrophils predominantly secreted interleukin-1ß rather than other proinflammatory cytokines upon NLRP3 stimulation. Furthermore, inflammasome-activated neutrophils did not trigger efferocytosis-mediated M2 macrophage polarization essential for the initiation of inflammation resolution. Taken together, our results indicate that neutrophils can prolong inflammasome response via mitochondria-dependent resistance to NLRP3 desensitization and function as major interleukin-1ß-secreting cells in DAMP-rich inflammatory region.

Small Molecule SARM1 Inhibitors Recapitulate the SARM1-/- Phenotype and Allow Recovery of a Metastable Pool of Axons Fated to Degenerate.

Axonal degeneration is responsible for disease progression and accumulation of disability in many neurodegenerative conditions. The axonal degenerative process can generate a metastable pool of damaged axons that remain structurally and functionally viable but fated to degenerate in the absence of external intervention. SARM1, an NADase that depletes axonal energy stores upon activation, is the central driver of an evolutionarily conserved program of axonal degeneration. We identify a potent and selective small molecule isoquinoline inhibitor of SARM1 NADase that recapitulates the SARM1-/- phenotype and protects axons from degeneration induced by axotomy or mitochondrial dysfunction. SARM1 inhibition post-mitochondrial injury with rotenone allows recovery and rescues axons that already entered the metastable state. We conclude that SARM1 inhibition with small molecules has the potential to treat axonopathies of the central and peripheral nervous systems by preventing axonal degeneration and by allowing functional recovery of a metastable pool of damaged, but viable, axons.

Regulation of degenerative spheroids after injury.

Neuronal injury leads to rapid, programmed disintegration of axons distal to the site of lesion. Much like other forms of axon degeneration (e.g. developmental pruning, toxic insult from neurodegenerative disorder), Wallerian degeneration associated with injury is preceded by spheroid formation along axons. The mechanisms by which injury leads to formation of spheroids and whether these spheroids have a functional role in degeneration remain elusive. Here, using neonatal mouse primary sympathetic neurons, we investigate the roles of players previously implicated in the progression of Wallerian degeneration in injury-induced spheroid formation. We find that intra-axonal calcium flux is accompanied by actin-Rho dependent growth of calcium rich axonal spheroids that eventually rupture, releasing material to the extracellular space prior to catastrophic axon degeneration. Importantly, after injury, Sarm1-/- and DR6-/-, but not Wlds (excess NAD+) neurons, are capable of forming spheroids that eventually rupture, releasing their contents to the extracellular space to promote degeneration. Supplementation of exogenous NAD+ or expressing WLDs suppresses Rho-dependent spheroid formation and degeneration in response to injury. Moreover, injured or trophically deprived Sarm1-/- and DR6-/-, but not Wlds neurons, are resistant to degeneration induced by conditioned media collected from wild-type axons after spheroid rupture. Taken together, these findings place Rho-actin and NAD+ upstream of spheroid formation and may suggest that other mediators of degeneration, such as DR6 and SARM1, mediate post-spheroid rupture events that lead to catastrophic axon disassembly.

Role of Armadillo repeat 2 and kinesin-II motor subunit Klp64D for wingless signaling in Drosophila.

Armadillo (Arm) is crucial for transducing Wingless (Wg) signaling. Previously, we have shown that Klp64D, a motor subunit of Drosophila kinesin-II, interacts with Arm for Wg signaling. Molecular basis for this interaction has remained unknown. Here we identify a critical Arm repeat (AR) required for binding Klp64D and Wg signaling. Arm/[Formula: see text]-catenin family proteins contain a conserved domain of 12 Arm repeats (ARs). Five of these ARs can interact with Klp64D, but only the second AR (AR2) binds to the cargo/tail domain of Klp64D. Overexpression of AR2 in wing imaginal disc is sufficient to cause notched wing margin. This phenotype by AR2 is enhanced or suppressed by reducing or increasing Klp64D expression, respectively. AR2 overexpression inhibits Wg signaling activity in TopFlash assay, consistent with its dominant-negative effects on Klp64D-dependent Wg signaling. Overexpression of the Klp64D cargo domain also results in dominant-negative wing notching. Genetic rescue data indicate that both AR2 and Klp64D cargo regions are required for the function of Arm and Klp64D, respectively. AR2 overexpression leads to an accumulation of Arm with GM130 Golgi marker in Klp64D knockdown. This study suggests that Wg signaling for wing development is regulated by specific interaction between AR2 and the cargo domain of Klp64D.

The SARM1 axon degeneration pathway: control of the NAD+ metabolome regulates axon survival in health and disease.

Axons are essential for nervous system function and axonal pathology is a common hallmark of many neurodegenerative diseases. Over a century and a half after the original description of Wallerian axon degeneration, advances over the past five years have heralded the emergence of a comprehensive, mechanistic model of an endogenous axon degenerative process that can be activated by both injury and disease. Axonal integrity is maintained by the opposing actions of the survival factors NMNAT2 and STMN2 and pro-degenerative molecules DLK and SARM1. The balance between axon survival and self-destruction is intimately tied to axonal NAD+ metabolism. These mechanistic insights may enable axon-protective therapies for a variety of human neurodegenerative diseases including peripheral neuropathy, traumatic brain injury and potentially ALS and Parkinson's.

SARM1 deficiency promotes rod and cone photoreceptor cell survival in a model of retinal degeneration.

Retinal degeneration is the leading cause of incurable blindness worldwide and is characterised by progressive loss of light-sensing photoreceptors in the neural retina. SARM1 is known for its role in axonal degeneration, but a role for SARM1 in photoreceptor cell degeneration has not been reported. SARM1 is known to mediate neuronal cell degeneration through depletion of essential metabolite NAD and induction of energy crisis. Here, we demonstrate that SARM1 is expressed in photoreceptors, and using retinal tissue explant, we confirm that activation of SARM1 causes destruction of NAD pools in the photoreceptor layer. Through generation of rho -/- sarm1 -/- double knockout mice, we demonstrate that genetic deletion of SARM1 promotes both rod and cone photoreceptor cell survival in the rhodopsin knockout (rho -/- ) mouse model of photoreceptor degeneration. Finally, we demonstrate that SARM1 deficiency preserves cone visual function in the surviving photoreceptors when assayed by electroretinography. Overall, our data indicate that endogenous SARM1 has the capacity to consume NAD in photoreceptor cells and identifies a previously unappreciated role for SARM1-dependent cell death in photoreceptor cell degeneration.

Novel role of SARM1 mediated axonal degeneration in the pathogenesis of rabies.

Neurotropic viral infections continue to pose a serious threat to human and animal wellbeing. Host responses combatting the invading virus in these infections often cause irreversible damage to the nervous system, resulting in poor prognosis. Rabies is the most lethal neurotropic virus, which specifically infects neurons and spreads through the host nervous system by retrograde axonal transport. The key pathogenic mechanisms associated with rabies infection and axonal transmission in neurons remains unclear. Here we studied the pathogenesis of different field isolates of lyssavirus including rabies using ex-vivo model systems generated with mouse primary neurons derived from the peripheral and central nervous systems. In this study, we show that neurons activate selective and compartmentalized degeneration of their axons and dendrites in response to infection with different field strains of lyssavirus. We further show that this axonal degeneration is mediated by the loss of NAD and calpain-mediated digestion of key structural proteins such as MAP2 and neurofilament. We then analysed the role of SARM1 gene in rabies infection, which has been shown to mediate axonal self-destruction during injury. We show that SARM1 is required for the accelerated execution of rabies induced axonal degeneration and the deletion of SARM1 gene significantly delayed axonal degeneration in rabies infected neurons. Using a microfluidic-based ex-vivo neuronal model, we show that SARM1-mediated axonal degeneration impedes the spread of rabies virus among interconnected neurons. However, this neuronal defense mechanism also results in the pathological loss of axons and dendrites. This study therefore identifies a potential host-directed mechanism behind neurological dysfunction in rabies infection. This study also implicates a novel role of SARM1 mediated axonal degeneration in neurotropic viral infection.

Loss of Sarm1 does not suppress motor neuron degeneration in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

Axon degeneration occurs in all neurodegenerative diseases, but the molecular pathways regulating axon destruction during neurodegeneration are poorly understood. Sterile Alpha and TIR Motif Containing 1 (Sarm1) is an essential component of the prodegenerative pathway driving axon degeneration after axotomy and represents an appealing target for therapeutic intervention in neurological conditions involving axon loss. Amyotrophic lateral sclerosis (ALS) is characterized by rapid, progressive motor neuron degeneration and muscle atrophy, causing paralysis and death. Patient tissue and animal models of ALS show destruction of upper and lower motor neuron cell bodies and loss of their associated axons. Here, we investigate whether loss of Sarm1 can mitigate motor neuron degeneration in the SOD1G93A mouse model of ALS. We found no change in survival, behavioral, electrophysiogical or histopathological outcomes in SOD1G93A mice null for Sarm1. Blocking Sarm1-mediated axon destruction alone is therefore not sufficient to suppress SOD1G93A-induced neurodegeneration. Our data suggest the molecular pathways driving axon loss in ALS may be Sarm1-independent or involve genetic pathways that act in a redundant fashion with Sarm1.

Role of SARM1 and DR6 in retinal ganglion cell axonal and somal degeneration following axonal injury.

Optic neuropathies such as glaucoma are characterized by the degeneration of retinal ganglion cells (RGCs) and the irreversible loss of vision. In these diseases, focal axon injury triggers a propagating axon degeneration and, eventually, cell death. Previous work by us and others identified dual leucine zipper kinase (DLK) and JUN N-terminal kinase (JNK) as key mediators of somal cell death signaling in RGCs following axonal injury. Moreover, others have shown that activation of the DLK/JNK pathway contributes to distal axonal degeneration in some neuronal subtypes and that this activation is dependent on the adaptor protein, sterile alpha and TIR motif containing 1 (SARM1). Given that SARM1 acts upstream of DLK/JNK signaling in axon degeneration, we tested whether SARM1 plays a similar role in RGC somal apoptosis in response to optic nerve injury. Using the mouse optic nerve crush (ONC) model, our results show that SARM1 is critical for RGC axonal degeneration and that axons rescued by SARM1 deficiency are electrophysiologically active. Genetic deletion of SARM1 did not, however, prevent DLK/JNK pathway activation in RGC somas nor did it prevent or delay RGC cell death. These results highlight the importance of SARM1 in RGC axon degeneration and suggest that somal activation of the DLK/JNK pathway is activated by an as-yet-unidentified SARM1-independent signal.

Structural and functional dissection of Toxoplasma gondii armadillo repeats only protein.

Rhoptries are club-shaped, regulated secretory organelles that cluster at the apical pole of apicomplexan parasites. Their discharge is essential for invasion and the establishment of an intracellular lifestyle. Little is known about rhoptry biogenesis and recycling during parasite division. In Toxoplasma gondii, positioning of rhoptries involves the armadillo repeats only protein (ARO) and myosin F (MyoF). Here, we show that two ARO partners, ARO-interacting protein (AIP) and adenylate cyclase ß (ACß) localize to a rhoptry subcompartment. In absence of AIP, ACß disappears from the rhoptries. By assessing the contribution of each ARO armadillo (ARM) repeat, we provide evidence that ARO is multifunctional, participating not only in positioning but also in clustering of rhoptries. Structural analyses show that ARO resembles the myosin-binding domain of the Caenorhabditis elegans myosin chaperone UNC-45. A conserved patch of aromatic and acidic residues denotes the putative MyoF-binding site, and the overall arrangement of the ARM repeats explains the dramatic consequences of deleting each of them. Finally, Plasmodium falciparum ARO functionally complements ARO depletion and interacts with the same partners, highlighting the conservation of rhoptry biogenesis in Apicomplexa.

Kinesin-II recruits Armadillo and Dishevelled for Wingless signaling in Drosophila.

Wingless (Wg)/Wnt signaling is fundamental in metazoan development. Armadillo (Arm)/ß-catenin and Dishevelled (Dsh) are key components of Wnt signal transduction. Recent studies suggest that intracellular trafficking of Wnt signaling components is important, but underlying mechanisms are not well known. Here, we show that Klp64D, the Drosophila homolog of Kif3A kinesin II subunit, is required for Wg signaling by regulating Arm during wing development. Mutations in klp64D or RNAi cause wing notching and loss of Wg target gene expression. The wing notching phenotype by Klp64D knockdown is suppressed by activated Arm but not by Dsh, suggesting that Klp64D is required for Arm function. Furthermore, klp64D and arm mutants show synergistic genetic interaction. Consistent with this genetic interaction, Klp64D directly binds to the Arm repeat domain of Arm and can recruit Dsh in the presence of Arm. Overexpression of Klp64D mutated in the motor domain causes dominant wing notching, indicating the importance of the motor activity. Klp64D shows subcellular localization to intracellular vesicles overlapping with Arm and Dsh. In klp64D mutants, Arm is abnormally accumulated in vesicular structures including Golgi, suggesting that intracellular trafficking of Arm is affected. Human KIF3A can also bind ß-catenin and rescue klp64D RNAi phenotypes. Taken together, we propose that Klp64D is essential for Wg signaling by trafficking of Arm via the formation of a conserved complex with Arm.

Sarm1, a neuronal inflammatory regulator, controls social interaction, associative memory and cognitive flexibility in mice.

Impaired neurodevelopment leads to several psychiatric disorders, including autism, schizophrenia and attention deficiency hyperactivity disorder. Our prior study showed that sterile alpha and TIR motif-containing 1 protein (Sarm1) regulates neuronal morphogenesis through at least two pathways. Sarm1 controls neuronal morphogenesis, including dendritic arborization, axonal outgrowth and establishment of neuronal polarity, through the MKK-JNK pathway. Neuronally expressed Sarm1 also regulates the expression of inflammatory cytokines in the brain, which have also been shown to impact brain development and function. Because the reduction of Sarm1 expression negatively influences neuronal development, here we investigated whether Sarm1 controls mouse behaviors. We analyzed two independent Sarm1 transgenic mouse lines using a series of behavioral assays, and found that the reduction of Sarm1 protein levels had a limited effect on locomotion and anxiety. However, Sarm1 knockdown mice exhibited impairments in cued and contextual fear conditioning as well as cognitive flexibility. Moreover, the three-chambered social test, reciprocal social interaction and social transmission of food preference further illustrated deficiencies in Sarm1 knockdown mice in social interaction. These findings suggest that Sarm1, a molecule that regulates innate immunity and neuronal morphogenesis, regulates social behaviors and cognition. We conclude that Sarm1 is involved in immune response, neural development and psychiatric disorders.

Gudu, an Armadillo repeat-containing protein, is required for spermatogenesis in Drosophila.

The Drosophila annotated gene CG5155 encodes a protein that contains 10 Armadillo-repeats and has an unknown function. To fill this gap, we performed loss-of-function studies using RNAi. By analysis of four independent Drosophila RNAi lines targeting two non-overlapping regions of the CG5155 transcript, we demonstrate that this gene is required for male fertility. Therefore, we have named this gene Gudu. The transcript of Gudu is highly enriched in adult testes. Knockdown of Gudu by a ubiquitous driver leads to defects in the formation of the individualization complex that is required for spermatid maturation, thereby impairing spermatogenesis. Furthermore, testis-specific knockdown of Gudu by crossing the RNAi lines with the bam-Gal4 driver is sufficient to cause the infertility and defective spermatogenesis. Since Gudu is highly homologous to vertebrate ARMC4, also an Armadillo-repeat-containing protein enriched in testes, our results suggest that Gudu and ARMC4 are a subfamily of Armadillo-repeat containing proteins that may have an evolutionarily conserved function in spermatogenesis.

The Armadillo repeat gene ZAK IXIK promotes Arabidopsis early embryo and endosperm development through a distinctive gametophytic maternal effect.

The proper balance of parental genomic contributions to the fertilized embryo and endosperm is essential for their normal growth and development. The characterization of many gametophytic maternal effect (GME) mutants affecting seed development indicates that there are certain classes of genes with a predominant maternal contribution. We present a detailed analysis of the GME mutant zak ixik (zix), which displays delayed and arrested growth at the earliest stages of embryo and endosperm development. ZIX encodes an Armadillo repeat (Arm) protein highly conserved across eukaryotes. Expression studies revealed that ZIX manifests a GME through preferential maternal expression in the early embryo and endosperm. This parent-of-origin-dependent expression is regulated by neither the histone and DNA methylation nor the DNA demethylation pathways known to regulate some other GME mutants. The ZIX protein is localized in the cytoplasm and nucleus of cells in reproductive tissues and actively dividing root zones. The maternal ZIX allele is required for the maternal expression of miniseed3. Collectively, our results reveal a reproductive function of plant Arm proteins in promoting early seed growth, which is achieved through a distinct GME of ZIX that involves mechanisms for maternal allele-specific expression that are independent of the well-established pathways.

dSarm/Sarm1 is required for activation of an injury-induced axon death pathway.

Axonal and synaptic degeneration is a hallmark of peripheral neuropathy, brain injury, and neurodegenerative disease. Axonal degeneration has been proposed to be mediated by an active autodestruction program, akin to apoptotic cell death; however, loss-of-function mutations capable of potently blocking axon self-destruction have not been described. Here, we show that loss of the Drosophila Toll receptor adaptor dSarm (sterile α/Armadillo/Toll-Interleukin receptor homology domain protein) cell-autonomously suppresses Wallerian degeneration for weeks after axotomy. Severed mouse Sarm1 null axons exhibit remarkable long-term survival both in vivo and in vitro, indicating that Sarm1 prodegenerative signaling is conserved in mammals. Our results provide direct evidence that axons actively promote their own destruction after injury and identify dSarm/Sarm1 as a member of an ancient axon death signaling pathway.

Adherens junction assembly and function in the Drosophila embryo.

Adherens junctions are essential for the development and physiology of epithelial tissues. The Drosophila embryo is an excellent model for understanding adherens junction assembly, maintenance, and regulation during tissue development. Here, I review our current state of knowledge in this model system. The review begins by outlining the structure of the cadherin-catenin complex in Drosophila including core (DE-cadherin, Armadillo, α-catenin, and p120-catenin) and peripheral proteins. Then, it summarizes adherens junction assembly at cellularization and maturation at gastrulation. Finally, the regulation of adherens junctions during tissue morphogenesis is discussed. This discussion compares major morphogenetic events in the embryo (invagination of the ventral furrow, convergent extension of the germband, flattening of the amnioserosa, maintenance of tissue borders, epithelial branching, lumen formation, cell delamination, cell division, apoptosis, and dorsal closure) and common mechanisms involved (myosin activity, endocytosis, and mesenchymal-to-epithelial transitions).

Plakophilin-2: a cell-cell adhesion plaque molecule of selective and fundamental importance in cardiac functions and tumor cell growth.

Within the characteristic ensemble of desmosomal plaque proteins, the armadillo protein plakophilin-2 (Pkp2) is known as a particularly important regulatory component in the cytoplasmic plaques of various other cell-cell junctions, such as the composite junctions (areae compositae) of the myocardiac intercalated disks and in the variously-sized and -shaped complex junctions of permanent cell culture lines derived therefrom. In addition, Pkp2 has been detected in certain protein complexes in the nucleoplasm of diverse kinds of cells. Using a novel set of highly sensitive and specific antibodies, both kinds of Pkp2, the junctional plaque-bound and the nuclear ones, can also be localized to the cytoplasmic plaques of diverse non-desmosomal cell-cell junction structures. These are not only the puncta adhaerentia and the fasciae adhaerentes connecting various types of highly proliferative non-epithelial cells growing in culture but also some very proliferative states of cardiac interstitial cells and cardiac myxomata, including tumors growing in situ as well as fetal stages of heart development and cultures of valvular interstitial cells. Possible functions and assembly mechanisms of such Pkp2-positive cell-cell junctions as well as medical consequences are discussed.

Cell-cell connectivity: desmosomes and disease.

Cell-cell connectivity is an absolute requirement for the correct functioning of cells, tissues and entire organisms. At the level of the individual cell, direct cell-cell adherence and communication is mediated by the intercellular junction complexes: desmosomes, adherens, tight and gap junctions. A broad spectrum of inherited, infectious and auto-immune diseases can affect the proper function of intercellular junctions and result in either diseases affecting specific individual tissues or widespread syndromic conditions. A particularly diverse group of diseases result from direct or indirect disruption of desmosomes--a consequence of their importance in tissue integrity, their extensive distribution, complex structure, and the wide variety of functions their components accomplish. As a consequence, disruption of desmosomal assembly, structure or integrity disrupts not only their intercellular adhesive function but also their functions in cell communication and regulation, leading to such diverse pathologies as cardiomyopathy, epidermal and mucosal blistering, palmoplantar keratoderma, woolly hair, keratosis, epidermolysis bullosa, ectodermal dysplasia and alopecia. Here, as well as describing the importance of the other intercellular junctions, we focus primarily on the desmosome, its structure and its role in disease. We will examine the various pathologies that result from impairment of desmosome function and thereby demonstrate the importance of desmosomes to tissues and to the organism as a whole.