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. We have shown previously that a sublethal hypoxic exposure of the nematode Caenorhabditis elegans induces a protein chaperone component of the UPR (L. L. Anderson, X. Mao, B. A. Scott, and C. M. Crowder, Science 323:630-633, 2009). Here, we show that HP induces the UPR and that the pharmacological induction of misfolded proteins is itself sufficient to stimulate a delayed protective response to hypoxic injury that requires the UPR pathway proteins IRE-1, XBP-1, and ATF-6. HP also required IRE-1 but not XBP-1 or ATF-6; instead, GCN-2, which is known to suppress translation and induce an adaptive transcriptional response under conditions of UPR activation or amino acid deprivation, was required for HP. The phosphorylation of the translation factor eIF2α, an established mechanism of GCN-2-mediated translational suppression, was not necessary for HP. These data suggest a model where hypoxia-induced misfolded proteins trigger the activation of IRE-1, which along with GCN-2 controls an adaptive response that is essential to HP

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 (NA

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. We, therefore, developed SARM1 dominant-negatives that potently block AxD in cellular models of axotomy and neuropathy. To assess efficacy in vivo, we used adeno-associated virus-mediated expression of the most potent SARM1 dominant-negative and nerve transection as a model of severe AxD. While axons of vehicle-treated mice degenerate rapidly, axons of mice expressing SARM1 dominant-negative can remain intact for \u3e10 d after transection, similar to the protection observed in SARM1-null mice. We thus developed a novel in vivo gene therapeutic to block pathological axon degeneration by inhibiting SARM1, an approach that may be applied clinically to treat manifold neurodegenerative diseases characterized by axon loss

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 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. We then intrathecally injected mice with virus expressing SARM1 constructs to test the capacity of an ALS-associated constitutively active SARM1 variant to promote neurodegeneration in vivo. RESULTS: Twelve out of 42 SARM1 missense variants or small in-frame deletions assayed exhibit constitutive NADase activity, including more than half of those that are unique to the ALS patients or that occur in multiple patients. There is a \u3e 5-fold enrichment of constitutively active variants among patients compared to controls. Expression of constitutively active ALS-associated SARM1 alleles in cultured dorsal root ganglion (DRG) neurons is pro-degenerative and cytotoxic. Intrathecal injection of an AAV expressing the common SARM1 reference allele is innocuous to mice, but a construct harboring SARM1 CONCLUSIONS: These results implicate rare hypermorphic SARM1 alleles as candidate genetic risk factors for ALS and other neurodegenerative conditions

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). Here we explore whether the NADase activity of SARM1 also contributes to developmental signaling. We developed transgenic Drosophila lines that express SARM1 variants with normal, deficient, and enhanced NADase activity and tested their function in NMJ development. We find that NMJ overgrowth scales with the amount of NADase activity, suggesting an instructive role for NAD+ hydrolysis in this developmental signaling pathway. While degenerative and developmental SARM1 signaling share a requirement for NAD+ hydrolysis, we demonstrate that these signals use distinct upstream and downstream mechanisms. These results identify SARM1-dependent NAD+ hydrolysis as a heretofore unappreciated component of developmental signaling. SARM1 now joins sirtuins and Parps as enzymes that regulate signal transduction pathways via mechanisms that involve NAD+ cleavage, greatly expanding the potential scope of SARM1 TIR NADase functions

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 SARM

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

SARM1 is an inducible TIR-domain NA

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 NFκB 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 NFκB that is nonetheless expressed at reasonable levels. A subset of the κB cis elements are instead bound by members of the Sp1 family in neurons. Also surprising was our discovery that Sp1 itself, typically described as ubiquitous, is severely restricted in expression within forebrain neurons; Sp4 seems to be substituted during neuronal differentiation. These findings and their implications for neuronal differentiation – as well as potential dedifferentiation during degenerative processes – are discussed here

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. RNAi knockdown of 198 genes conferred an invariant hypoxia-resistant phenotype (Hyp-r). Eighty-six per cent of these hyp genes had strong homologs in other organisms, 73 with human reciprocal orthologs. The hyp genes were distributed among multiple functional categories. Transcription factors, chromatin modifying enzymes, and intracellular signaling proteins were highly represented. RNAi knockdown of about half of the genes produced no apparent deleterious phenotypes. The hyp genes had significant overlap with previously identified life span extending genes. Testing of the RNAi's in a mutant background defective in somatic RNAi machinery showed that most genes function in somatic cells to control hypoxic sensitivity. DNA microarray analysis identified a subset of the hyp genes that may be hypoxia regulated. siRNA knockdown of human orthologs of the hyp genes conferred hypoxia resistance to transformed human cells for 40% of the genes tested, indicating extensive evolutionary conservation of the hypoxic regulatory activities. The results of the screen provide the first systematic picture of the genetic determinants of hypoxic sensitivity. The number and diversity of genes indicates a surprisingly nonredundant genetic network promoting hypoxic sensitivity

High-Resolution Observations of Upwelling and Front in Daya Bay, South China Sea

Field observations of coastal regions are important for studying physical and biological features. Observations of high-resolution coastal phenomena were obtained by using a tow-yo instrument and a turbulence profiler at Daya Bay in the South China Sea in October 2015. Details of coastal phenomena, including warm water from a nuclear plant discharge, as well as an upwelling, and front, were obtained. The upwelling, with a width of 2 km, resulted in saltier and more turbid water near the bottom, with low chlorophyll-a and dissolved oxygen contents being transported upward to the surface layer and changing the local water environment. The front, with the lateral salinity variations as large as 0.7 psu across 1 km, was active at the water intersection of the South China Sea and Daya Bay. Such events commonly form during weak stratification periods in autumn. Continuous measurements from VMP-250 profiler over circa 22 h revealed active fronts and an averaged dissipation rate of 8 × 10−8 W/kg and diffusivity of 5.8 × 10−5 m2/s (i.e., one order of magnitude larger than in the open ocean) in the thermocline. The front was accompanied by strong mixing, indicating that it had formed at the intersection of different water masses and played an important role in energy dissipation in Daya Bay, further affecting the distribution of ecological elements