Concentrations of acetyl–coenzyme A and nicotinamide adenine dinucleotide (NAD+) affect histone acetylation and thereby couple cellular metabolic status and transcriptional regulation. We report that the ketone body d-β-hydroxybutyrate (βOHB) is an endogenous and specific inhibitor of class I histone deacetylases (HDACs). Administration of exogenous βOHB, or fasting or calorie restriction, two conditions associated with increased βOHB abundance, all increased global histone acetylation in mouse tissues. Inhibition of HDAC by βOHB was correlated with global changes in transcription, including that of the genes encoding oxidative stress resistance factors FOXO3A and MT2. Treatment of cells with βOHB increased histone acetylation at the Foxo3a and Mt2 promoters, and both genes were activated by selective depletion of HDAC1 and HDAC2. Consistent with increased FOXO3A and MT2 activity, treatment of mice with βOHB conferred substantial protection against oxidative stress.

Blood-brain barrier disruption, microglial activation and neurodegeneration are hallmarks of multiple sclerosis. However, the initial triggers that activate innate immune responses and their role in axonal damage remain unknown. Here we show that the blood protein fibrinogen induces rapid microglial responses toward the vasculature and is required for axonal damage in neuroinflammation. Using in vivo two-photon microscopy, we demonstrate that microglia form perivascular clusters before myelin loss or paralysis onset and that, of the plasma proteins, fibrinogen specifically induces rapid and sustained microglial responses in vivo. Fibrinogen leakage correlates with areas of axonal damage and induces reactive oxygen species release in microglia. Blocking fibrin formation with anticoagulant treatment or genetically eliminating the fibrinogen binding motif recognized by the microglial integrin receptor CD11b/CD18 inhibits perivascular microglial clustering and axonal damage. Thus, early and progressive perivascular microglial clustering triggered by fibrinogen leakage upon blood-brain barrier disruption contributes to axonal damage in neuroinflammatory disease.

By its ability to engage in a variety of redox reactions and coordinating metals, cysteine serves as a key residue in mediating enzymatic catalysis, protein oxidative folding and trafficking, and redox signaling. The thiol redox system, which consists of the glutathione and thioredoxin pathways, uses the cysteine residue to catalyze thiol-disulfide exchange reactions, thereby controlling the redox state of cytoplasmic cysteine residues and regulating the biological functions it subserves. Here, we consider the thiol redox systems of Escherichia coli and Saccharomyces cerevisiae, emphasizing the role of genetic approaches in the understanding of the cellular functions of these systems. We show that although prokaryotic and eukaryotic systems have a similar architecture, they profoundly differ in their overall cellular functions.

Multiple sclerosis (MS) is an autoimmune disease in which myelin is progressively degraded. Because degraded myelin may both initiate and accelerate disease progression, clearing degraded myelin from extracellular spaces may be critical. In this study, we prepared myelin vesicles (MV) from rat brains as a model of degraded myelin. Murine embryonic fibroblasts (MEFs) rapidly internalized MVs, which accumulated in lysosomes only when these cells expressed low-density lipoprotein receptor-related protein (LRP1). Receptor-associated protein (RAP), which binds LRP1 and inhibits interaction with other ligands, blocked MV uptake by LRP1-expressing MEFs. As a complementary approach, we prepared primary cultures of rat astrocytes, microglia and oligodendrocytes. All three cell types expressed LRP1 and mediated MV uptake, which was inhibited by RAP. LRP1 gene-silencing in oligodendrocytes also blocked MV uptake. Myelin basic protein (MBP), which was expressed as a recombinant protein, bound directly to LRP1. MBP-specific antibody inhibited MV uptake by oligodendrocytes. In experimental autoimmune encephalomyelitis in mice, LRP1 protein expression was substantially increased in the cerebellum and spinal cord. LRP1 colocalized with multiple CNS cell types. These studies establish LRP1 as a major receptor for phagocytosis of degraded myelin, which may function alone or in concert with co-receptors previously implicated in myelin phagocytosis.

Protein thiol oxidation subserves important biological functions and constitutes a sequel of reactive oxygen species toxicity. We developed two distinct thiol-labeling approaches to identify oxidized cytoplasmic protein thiols in Saccharomyces cerevisiae. In one approach, we used N-(6-(biotinamido)hexyl)-3 -(2 -pyridyldithio)-propionamide to purify oxidized protein thiols, and in the other, we used N-[ 14 C]ethylmaleimide to quantify this oxidation. Both approaches showed a large number of the same proteins with oxidized thiols (ϳ200), 64 of which were identified by mass spectrometry. We show that, irrespective of its mechanism, protein thiol oxidation is dependent upon molecular O 2 . We also show that H 2 O 2 does not cause de novo protein thiol oxidation, but rather increases the oxidation state of a select group of proteins. Furthermore, our study reveals contrasted differences in the oxidized proteome of cells upon inactivation of the thioredoxin or GSH pathway suggestive of very distinct thiol redox control functions, assigning an exclusive role for thioredoxin in H 2 O 2 metabolism and the presumed thiol redox buffer function for GSH. Taken together, these results suggest the high selectivity of cytoplasmic protein thiol oxidation.The amino acid cysteine subserves important biological functions due to the unique redox properties of the sulfur atom of its thiol side chain (1). By engaging in a wide variety of redox reactions and coordinating metals, cysteine serves as a key residue in enzyme catalysis, protein oxidative folding (2-4) and trafficking (5), and redox signaling and regulation (6 -8). However, these unique redox properties also make the cysteine residue vulnerable to reaction with a wide spectrum of non-physiological electrophiles, especially the reactive oxygen and nitrogen species, potentially leading to unwanted redox modifications and protein loss of function (9).The cysteine residue exists in vivo in the fully reduced free thiol form (-SH or -S Ϫ ) and in different oxidation forms: the thiyl radical (-S ⅐ ); the disulfide bond (Cys-S-S-Cys); the sulfenic (-SOH), sulfinic (SO 2 H), and sulfonic (-SO 3 H) acid forms; and the S-nitrosylated form (-S-NO) (1, 7, 10). The cysteine thiyl radical and cysteine sulfenic acid are very unstable because of their highly reactive nature and thus cannot be easily identified biochemically. In contrast, the cysteine sulfinic and sulfonic acids are irreversible forms of protein oxidation, although the cysteine sulfinic acid that forms in the peroxide-reducing enzyme peroxiredoxin is enzymatically retroreduced by sulfiredoxin (11, 12). Disulfide bonds are relatively stable, reversing to the reduced state by thioldisulfide exchange with kinetics depending on the protein context (10). Their occurrence is thought to be restricted to specific subcellular compartments. In the endoplasmic reticulum, disulfide bond formation drives the correct folding of secreted proteins and is catalyzed by a FAD-containing sulfhydryl oxidase (Ero1) and protein-disulfide isomerase ...

SUMMARY Homeostatic control of oxygen availability allows cells to survive oxygen deprivation. Although the transcription factor hypoxia-inducible factor 1α (HIF-1α) is the main regulator of the hypoxic response, the upstream mechanisms required for its stabilization remain elusive. Here, we show that p75 neurotrophin receptor (p75NTR) undergoes hypoxia-induced γ-secretase-dependent cleavage to provide a positive feed-forward mechanism required for oxygen-dependent HIF-1α stabilization. The intracellular domain of p75NTR directly interacts with the evolutionary conserved zinc finger domains of the E3 RING ubiquitin ligase seven in absentia homologue 2 (Siah2), which regulates HIF-1α degradation. p75NTR stabilizes Siah2 by decreasing its auto-ubiquitination. Genetic loss of p75NTR dramatically decreases Siah2 abundance, HIF-1α stabilization and induction of HIF-1α target genes in hypoxia.p75NTR−/− mice show reduced HIF-1α stabilization, vascular endothelial growth factor (VEGF) expression and neoangiogenesis after retinal hypoxia. Thus, hypoxia-induced intramembrane proteolysis of p75NTR constitutes an apical oxygen-dependent mechanism to control the magnitude of the hypoxic response.

Insulin resistance is a key factor in the etiology of type 2 diabetes. Insulin-stimulated glucose uptake is mediated by the glucose transporter 4 (GLUT4), which is expressed mainly in skeletal muscle and adipose tissue. Insulin-stimulated translocation of GLUT4 from its intracellular compartment to the plasma membrane is regulated by small guanosine triphosphate hydrolases (GTPases) and is essential for the maintenance of normal glucose homeostasis. Here we show that the p75 neurotrophin receptor (p75 NTR ) is a regulator of glucose uptake and insulin resistance. p75NTR knockout mice show increased insulin sensitivity on normal chow diet, independent of changes in body weight. Euglycemic-hyperinsulinemic clamp studies demonstrate that deletion of the p75 NTR gene increases the insulin-stimulated glucose disposal rate and suppression of hepatic glucose production. Genetic depletion or shRNA knockdown of p75 NTR in adipocytes or myoblasts increases insulin-stimulated glucose uptake and GLUT4 translocation. Conversely, overexpression of p75 NTR in adipocytes decreases insulin-stimulated glucose transport. In adipocytes, p75NTR forms a complex with the Rab5 family GTPases Rab5 and Rab31 that regulate GLUT4 trafficking. Rab5 and Rab31 directly interact with p75 NTR primarily via helix 4 of the p75 NTR death domain. Adipocytes from p75 NTR knockout mice show increased Rab5 and decreased Rab31 activities, and dominant negative Rab5 rescues the increase in glucose uptake seen in p75 NTR knockout adipocytes. Our results identify p75 NTR as a unique player in glucose metabolism and suggest that signaling from p75 NTR to Rab5 family GTPases may represent a unique therapeutic target for insulin resistance and diabetes.3T3L1 | brain-derived neurotrophic factor | Rho | peptide array | obesity

Astrocytes play critical roles in neuronal activity and inhibition of regeneration. Here we show that the cleaved p75 neurotrophin receptor (p75NTR) is a component of the nuclear pore complex (NPC) required for glial scar formation and reduced gamma oscillations in mice via regulation of TGF-β signaling. The cleaved p75NTR interacts with nucleoporins to promote Smad2 nucleocytoplasmic shuttling. Thus, NPC remodeling by regulated intramembrane cleavage of p75NTR controls astrocyte-neuronal communication in response to profibrotic factors.