Distinct developmental and degenerative functions of SARM1 require NAD+ hydrolase activity.

SARM1 is the founding member of the TIR-domain family of NAD+ hydrolases and the central executioner of pathological axon degeneration. SARM1-dependent degeneration requires NAD+ hydrolysis. Prior to the discovery that SARM1 is an enzyme, SARM1 was studied as a TIR-domain adaptor protein with non-degenerative signaling roles in innate immunity and invertebrate neurodevelopment, including at the Drosophila neuromuscular junction (NMJ).

Structural basis of SARM1 activation, substrate recognition, and inhibition by small molecules.

The NADase SARM1 (sterile alpha and TIR motif containing 1) is a key executioner of axon degeneration and a therapeutic target for several neurodegenerative conditions. We show that a potent SARM1 inhibitor undergoes base exchange with the nicotinamide moiety of nicotinamide adenine dinucleotide (NAD) to produce the bona fide inhibitor 1AD. We report structures of SARM1 in complex with 1AD, NAD mimetics and the allosteric activator nicotinamide mononucleotide (NMN).

Constitutively active SARM1 variants that induce neuropathy are enriched in ALS patients.

Background: In response to injury, neurons activate a program of organized axon self-destruction initiated by the NAD hydrolase, SARM1. In healthy neurons SARM1 is autoinhibited, but single amino acid changes can abolish autoinhibition leading to constitutively active SARM1 enzymes that promote degeneration when expressed in cultured neurons.

Methods: To investigate whether naturally occurring human variants might disrupt SARM1 autoinhibition and potentially contribute to risk for neurodegenerative disease, we assayed the enzymatic activity of all 42 rare SARM1 alleles identified among 8507 amyotrophic lateral sclerosis (ALS) patients and 9671 controls.

Neurotoxins subvert the allosteric activation mechanism of SARM1 to induce neuronal loss.

SARM1 is an inducible TIR-domain NAD hydrolase that mediates pathological axon degeneration. SARM1 is activated by an increased ratio of NMN to NAD, which competes for binding to an allosteric activating site. When NMN binds, the TIR domain is released from autoinhibition, activating its NAD hydrolase activity.

Multiple domain interfaces mediate SARM1 autoinhibition.

Axon degeneration is an active program of self-destruction mediated by the protein SARM1. In healthy neurons, SARM1 is autoinhibited and, upon injury autoinhibition is relieved, activating the SARM1 enzyme to deplete NAD and induce axon degeneration. SARM1 forms a homomultimeric octamer with each monomer composed of an N-terminal autoinhibitory ARM domain, tandem SAM domains that mediate multimerization, and a C-terminal TIR domain encoding the NADase enzyme.

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.

Gene therapy targeting SARM1 blocks pathological axon degeneration in mice.

Axonal degeneration (AxD) following nerve injury, chemotherapy, and in several neurological disorders is an active process driven by SARM1, an injury-activated NADase. Axons of SARM1-null mice exhibit greatly delayed AxD after transection and in models of neurological disease, suggesting that inhibiting SARM1 is a promising strategy to reduce pathological AxD. Unfortunately, no drugs exist to target SARM1.

TIR Domain Proteins Are an Ancient Family of NAD-Consuming Enzymes.

The Toll/interleukin-1 receptor (TIR) domain is the signature signaling domain of Toll-like receptors (TLRs) and their adaptors, serving as a scaffold for the assembly of protein complexes for innate immune signaling [1, 2]. TIR domain proteins are also expressed in plants, where they mediate disease resistance [3, 4], and in bacteria, where they have been associated with virulence [5-9]. In pursuing our work on axon degeneration [10], we made the surprising discovery that the TIR domain of SARM1 (sterile alpha and TIR motif containing 1), a TLR adaptor protein, has enzymatic activity [11].

The SARM1 Toll/Interleukin-1 Receptor Domain Possesses Intrinsic NAD Cleavage Activity that Promotes Pathological Axonal Degeneration.

Axonal degeneration is an early and prominent feature of many neurological disorders. SARM1 is the central executioner of the axonal degeneration pathway that culminates in depletion of axonal NAD, yet the identity of the underlying NAD-depleting enzyme(s) is unknown. Here, in a series of experiments using purified proteins from mammalian cells, bacteria, and a cell-free protein translation system, we show that the SARM1-TIR domain itself has intrinsic NADase activity-cleaving NAD into ADP-ribose (ADPR), cyclic ADPR, and nicotinamide, with nicotinamide serving as a feedback inhibitor of the enzyme.

NMNAT1 inhibits axon degeneration via blockade of SARM1-mediated NAD depletion.

Overexpression of the NAD biosynthetic enzyme NMNAT1 leads to preservation of injured axons. While increased NAD or decreased NMN levels are thought to be critical to this process, the mechanism(s) of this axon protection remain obscure. Using steady-state and flux analysis of NAD metabolites in healthy and injured mouse dorsal root ganglion axons, we find that rather than altering NAD synthesis, NMNAT1 instead blocks the injury-induced, SARM1-dependent NAD consumption that is central to axon degeneration.

NFκB-inducing kinase inhibits NFκB activity specifically in neurons of the CNS.

The control of NFκB in CNS neurons appears to differ from that in other cell types. Studies have reported induction of NFκB in neuronal cultures and immunostaining in vivo, but others have consistently detected little or no transcriptional activation by NFκB in brain neurons. To test if neurons lack some component of the signal transduction system for NFκB activation, we transfected cortical neurons with several members of this signaling system along with a luciferase-based NFκB-reporter plasmid; RelA was cotransfected in some conditions.

Role of oxygen consumption in hypoxia protection by translation factor depletion.

The reduction of protein synthesis has been associated with resistance to hypoxic cell death. Which components of the translation machinery control hypoxic sensitivity and the precise mechanism has not been systematically investigated, although a reduction in oxygen consumption has been widely assumed to be the mechanism. Using genetic reagents in Caenorhabditis elegans, we examined the effect on organismal survival after hypoxia of knockdown of 10 factors functioning at the three principal steps in translation.

Protein misfolding induces hypoxic preconditioning via a subset of the unfolded protein response machinery.

Prolonged cellular hypoxia results in energy failure and ultimately cell death. However, less-severe hypoxia can induce a cytoprotective response termed hypoxic preconditioning (HP). The unfolded protein response pathway (UPR) has been known for some time to respond to hypoxia and regulate hypoxic sensitivity; however, the role of the UPR, if any, in HP essentially has been unexplored.

Unique aspects of transcriptional regulation in neurons--nuances in NFkappaB and Sp1-related factors.

The unique physiology and function of neurons create differences in their cellular physiology, including their regulation of gene expression. We began several years ago exploring the relationships between the NFkappaB transcription factor, neuronal survival, and glutamate receptor activation in telencephalic neurons. These studies led us to conclude that this population of cells is nearly incapable of activating the NFkappaB that is nonetheless expressed at reasonable levels.

Survival from hypoxia in C. elegans by inactivation of aminoacyl-tRNA synthetases.

Hypoxia is important in a wide range of biological processes, such as animal hibernation and cell survival, and is particularly relevant in many diseases. The sensitivity of cells and organisms to hypoxic injury varies widely, but the molecular basis for this variation is incompletely understood. Using forward genetic screens in Caenorhabditis elegans, we isolated a hypoxia-resistant reduction-of-function mutant of rrt-1 that encodes an arginyl-transfer RNA (tRNA) synthetase, an enzyme essential for protein translation.

Systematic identification of gene activities promoting hypoxic death.

The sensitivity of an organism to hypoxic injury varies widely across species and among cell types. However, a systematic description of the determinants of metazoan hypoxic sensitivity is lacking. Toward this end, we screened a whole-genome RNAi library for genes that promote hypoxic sensitivity in Caenorhabditis elegans.

Glutamate receptor activation evokes calpain-mediated degradation of Sp3 and Sp4, the prominent Sp-family transcription factors in neurons.

Sp-family transcription factors (Sp1, Sp3 and Sp4) contain a zinc-finger domain that binds to DNA sequences rich in G-C/T. As assayed by RT-PCR analysis of mRNA, western-blot analysis, immunofluorescence, and antibody-dependent "supershift" of DNA-binding assays, the prominent Sp-family factors in cerebral neurons were identified as Sp3 and Sp4. By contrast, glial cells were found to express Sp1 and Sp3.

Differential transcriptional control of the superoxide dismutase-2 kappaB element in neurons and astrocytes.

In addition to their conventional G-C/T target sequences, Sp1 family transcription factors (Sp-factors) can interact with a subset of the target sequences for NFkappaB. Due to the low level of bona fide NFkappaB activity in most resting cells, this interaction between Sp-factors and kappaB-sites could play important roles in cell function. Here we used mutagenesis of a canonical kappaB element from the immunoglobulin and HIV promoters to identify the GC-rich sequences at each end required for Sp-factor targeting.

NFkappaB in neurons? The uncertainty principle in neurobiology.

Nuclear factor kappaB (NFkappaB) is a dynamically modulated transcription factor with an extensive literature pertaining to widespread actions across species, cell types and developmental stages. Analysis of NFkappaB in a complex environment such as neural tissue suffers from a difficulty in simultaneously establishing both activity and location. Much of the available data indicate a profound recalcitrance of NFkappaB activation in neurons, as compared with most other cell types.

[Identification of peptides binding to Pisum sativum agglutinin from a phage-displayed random peptide library].

Objective: To obtain peptides binding specifically to Pisum sativum agglutinin (PSA) from a phage-displayed random peptide library.

Methods: (1) A phage-displayed random hexapeptide library was screened with PSA as target. (2) Dot blot was used to analyze the influence of the alpha-Met-D-mannoside on binding between PSA and phage-displayed peptides.

Molecular mechanisms of cytokine-induced neuroprotection: NFkappaB and neuroplasticity.

Since the first attempts to understand the mechanisms of learning, memory, development, and other instances of neuroplasticity, gene expression has been an attractive explanation for the persistence of such processes. It has been hypothesized that changes in the levels of expression of a gene, or a coordinated set of genes, would be necessary for dramatic structural changes like the growth of new neurites. And more subtle biochemical changes at existing synapses might also result from an alteration in the array of gene products being manufactured in the relevant cells.

Neuronal kappa B-binding factors consist of Sp1-related proteins. Functional implications for autoregulation of N-methyl-D-aspartate receptor-1 expression.

Neurons contain a protein factor capable of binding DNA elements normally bound by the transcription factor NF-kappaB. However, several lines of evidence suggest that this neuronal kappaB-binding factor (NKBF) is not bona fide NF-kappaB. We have identified NKBF from cultures of neocortical neurons as a complex containing proteins related to Sp1.