In vivo functions of p75NTR: challenges and opportunities for an emerging therapeutic target.

The p75 neurotrophin receptor (p75NTR) functions at the molecular nexus of cell death, survival, and differentiation. In addition to its contribution to neurodegenerative diseases and nervous system injuries, recent studies have revealed unanticipated roles of p75NTR in liver repair, fibrinolysis, lung fibrosis, muscle regeneration, and metabolism. Linking these various p75NTR functions more precisely to specific mechanisms marks p75NTR as an emerging candidate for therapeutic intervention in a wide range of disorders. Indeed, small molecule inhibitors of p75NTR binding to neurotrophins have shown efficacy in models of Alzheimer's disease (AD) and neurodegeneration. Here, we outline recent advances in understanding p75NTR pleiotropic functions in vivo, and propose an integrated view of p75NTR and its challenges and opportunities as a pharmacological target.

Pharmacological inhibition of P2RX7 ameliorates liver injury by reducing inflammation and fibrosis.

Extracellular adenosine triphosphate (eATP) released by damaged cells, and its purinergic receptors, comprise a crucial signaling network after injury. Purinergic receptor P2X7 (P2RX7), a major driver of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and IL-1ß processing, has been shown to play a role in liver injury in murine diet- and chemically-induced liver injury models. It is unclear, however, whether P2RX7 plays a role in non-alcoholic steatohepatitis (NASH) and which cell type is the main target of P2RX7 pharmacological inhibition. Here, we report that P2RX7 is expressed by infiltrating monocytes and resident Kupffer cells in livers from NASH-affected individuals. Using primary isolated human cells, we demonstrate that P2RX7 expression in CD14+ monocytes and Kupffer cells primarily mediates IL-1ß release. In addition, we show that pharmacological inhibition of P2RX7 in monocytes and Kupffer cells, blocks IL-1ß release, reducing hepatocyte caspase 3/7 activity, IL-1ß-mediated CCL2 and CCL5 chemokine gene expression and secretion, and hepatic stellate cell (HSC) procollagen secretion. Consequently, in a chemically-induced nonhuman primate model of liver fibrosis, treatment with a P2RX7 inhibitor improved histological characteristics of NASH, protecting from liver inflammation and fibrosis. Taken together, these findings underscore the critical role of P2RX7 in the pathogenesis of NASH and implicate P2RX7 as a promising therapeutic target for the management of this disease.

A high-fat diet promotes depression-like behavior in mice by suppressing hypothalamic PKA signaling.

Obesity is associated with an increased risk of depression. The aim of the present study was to investigate whether obesity is a causative factor for the development of depression and what is the molecular pathway(s) that link these two disorders. Using lipidomic and transcriptomic methods, we identified a mechanism that links exposure to a high-fat diet (HFD) in mice with alterations in hypothalamic function that lead to depression. Consumption of an HFD selectively induced accumulation of palmitic acid in the hypothalamus, suppressed the 3', 5'-cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, and increased the concentration of free fatty acid receptor 1 (FFAR1). Deficiency of phosphodiesterase 4A (PDE4A), an enzyme that degrades cAMP and modulates stimulatory regulative G protein (Gs)-coupled G protein-coupled receptor signaling, protected animals either from genetic- or dietary-induced depression phenotype. These findings suggest that dietary intake of saturated fats disrupts hypothalamic functions by suppressing cAMP/PKA signaling through activation of PDE4A. FFAR1 inhibition and/or an increase of cAMP signaling in the hypothalamus could offer potential therapeutic targets to counteract the effects of dietary or genetically induced obesity on depression.

Fibrinogen Activates BMP Signaling in Oligodendrocyte Progenitor Cells and Inhibits Remyelination after Vascular Damage.

Blood-brain barrier (BBB) disruption alters the composition of the brain microenvironment by allowing blood proteins into the CNS. However, whether blood-derived molecules serve as extrinsic inhibitors of remyelination is unknown. Here we show that the coagulation factor fibrinogen activates the bone morphogenetic protein (BMP) signaling pathway in oligodendrocyte progenitor cells (OPCs) and suppresses remyelination. Fibrinogen induces phosphorylation of Smad 1/5/8 and inhibits OPC differentiation into myelinating oligodendrocytes (OLs) while promoting an astrocytic fate in vitro. Fibrinogen effects are rescued by BMP type I receptor inhibition using dorsomorphin homolog 1 (DMH1) or CRISPR/Cas9 activin A receptor type I (ACVR1) knockout in OPCs. Fibrinogen and the BMP target Id2 are increased in demyelinated multiple sclerosis (MS) lesions. Therapeutic depletion of fibrinogen decreases BMP signaling and enhances remyelination in vivo. Targeting fibrinogen may be an upstream therapeutic strategy to promote the regenerative potential of CNS progenitors in diseases with remyelination failure.

Microglial complement receptor 3 regulates brain Aß levels through secreted proteolytic activity.

Recent genetic evidence supports a link between microglia and the complement system in Alzheimer's disease (AD). In this study, we uncovered a novel role for the microglial complement receptor 3 (CR3) in the regulation of soluble ß-amyloid (Aß) clearance independent of phagocytosis. Unexpectedly, ablation of CR3 in human amyloid precursor protein-transgenic mice results in decreased, rather than increased, Aß accumulation. In line with these findings, cultured microglia lacking CR3 are more efficient than wild-type cells at degrading extracellular Aß by secreting enzymatic factors, including tissue plasminogen activator. Furthermore, a small molecule modulator of CR3 reduces soluble Aß levels and Aß half-life in brain interstitial fluid (ISF), as measured by in vivo microdialysis. These results suggest that CR3 limits Aß clearance from the ISF, illustrating a novel role for CR3 and microglia in brain Aß metabolism and defining a potential new therapeutic target in AD.

p75 Neurotrophin Receptor Regulates Energy Balance in Obesity.

Obesity and metabolic syndrome reflect the dysregulation of molecular pathways that control energy homeostasis. Here, we show that the p75 neurotrophin receptor (p75(NTR)) controls energy expenditure in obese mice on a high-fat diet (HFD). Despite no changes in food intake, p75(NTR)-null mice were protected from HFD-induced obesity and remained lean as a result of increased energy expenditure without developing insulin resistance or liver steatosis. p75(NTR) directly interacts with the catalytic subunit of protein kinase A (PKA) and regulates cAMP signaling in adipocytes, leading to decreased lipolysis and thermogenesis. Adipocyte-specific depletion of p75(NTR) or transplantation of p75(NTR)-null white adipose tissue (WAT) into wild-type mice fed a HFD protected against weight gain and insulin resistance. Our results reveal that signaling from p75(NTR) to cAMP/PKA regulates energy balance and suggest that non-CNS neurotrophin receptor signaling could be a target for treating obesity and the metabolic syndrome.

Nuclear pore complex remodeling by p75(NTR) cleavage controls TGF-ß signaling and astrocyte functions.

Astrocytes modulate neuronal activity and inhibit regeneration. We show that cleaved p75 neurotrophin receptor (p75(NTR)) is a component of the nuclear pore complex (NPC) required for glial scar formation and reduced gamma oscillations in mice via regulation of transforming growth factor (TGF)-ß signaling. Cleaved p75(NTR) interacts with nucleoporins to promote Smad2 nucleocytoplasmic shuttling. Thus, NPC remodeling by regulated intramembrane cleavage of p75(NTR) controls astrocyte-neuronal communication in response to profibrotic factors.

p75 neurotrophin receptor is a clock gene that regulates oscillatory components of circadian and metabolic networks.

The p75 neurotrophin receptor (p75(NTR)) is a member of the tumor necrosis factor receptor superfamily with a widespread pattern of expression in tissues such as the brain, liver, lung, and muscle. The mechanisms that regulate p75(NTR) transcription in the nervous system and its expression in other tissues remain largely unknown. Here we show that p75(NTR) is an oscillating gene regulated by the helix-loop-helix transcription factors CLOCK and BMAL1. The p75(NTR) promoter contains evolutionarily conserved noncanonical E-box enhancers. Deletion mutagenesis of the p75(NTR)-luciferase reporter identified the -1039 conserved E-box necessary for the regulation of p75(NTR) by CLOCK and BMAL1. Accordingly, gel-shift assays confirmed the binding of CLOCK and BMAL1 to the p75(NTR-)1039 E-box. Studies in mice revealed that p75(NTR) transcription oscillates during dark and light cycles not only in the suprachiasmatic nucleus (SCN), but also in peripheral tissues including the liver. Oscillation of p75(NTR) is disrupted in Clock-deficient and mutant mice, is E-box dependent, and is in phase with clock genes, such as Per1 and Per2. Intriguingly, p75(NTR) is required for circadian clock oscillation, since loss of p75(NTR) alters the circadian oscillation of clock genes in the SCN, liver, and fibroblasts. Consistent with this, Per2::Luc/p75(NTR-/-) liver explants showed reduced circadian oscillation amplitude compared with those of Per2::Luc/p75(NTR+/+). Moreover, deletion of p75(NTR) also alters the circadian oscillation of glucose and lipid homeostasis genes. Overall, our findings reveal that the transcriptional activation of p75(NTR) is under circadian regulation in the nervous system and peripheral tissues, and plays an important role in the maintenance of clock and metabolic gene oscillation.

Whole-organism screening for gluconeogenesis identifies activators of fasting metabolism.

Improving the control of energy homeostasis can lower cardiovascular risk in metabolically compromised individuals. To identify new regulators of whole-body energy control, we conducted a high-throughput screen in transgenic reporter zebrafish for small molecules that modulate the expression of the fasting-inducible gluconeogenic gene pck1. We show that this in vivo strategy identified several drugs that affect gluconeogenesis in humans as well as metabolically uncharacterized compounds. Most notably, we find that the translocator protein ligands PK 11195 and Ro5-4864 are glucose-lowering agents despite a strong inductive effect on pck1 expression. We show that these drugs are activators of a fasting-like energy state and, notably, that they protect high-fat diet-induced obese mice from hepatosteatosis and glucose intolerance, two pathological manifestations of metabolic dysregulation. Thus, using a whole-organism screening strategy, this study has identified new small-molecule activators of fasting metabolism.

p75 neurotrophin receptor regulates glucose homeostasis and insulin sensitivity.

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. p75(NTR) 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, p75(NTR) 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.

p75 (NTR) and hypoxia: a breath of fresh air in neurotrophin receptor signaling.

Comment on: Le Moan N, et al. Mol Cell 2011; 44:476-90.

Fibrinogen drives dystrophic muscle fibrosis via a TGFbeta/alternative macrophage activation pathway.

In the fatal degenerative Duchenne muscular dystrophy (DMD), skeletal muscle is progressively replaced by fibrotic tissue. Here, we show that fibrinogen accumulates in dystrophic muscles of DMD patients and mdx mice. Genetic loss or pharmacological depletion of fibrinogen in these mice reduced fibrosis and dystrophy progression. Our results demonstrate that fibrinogen-Mac-1 receptor binding, through induction of IL-1beta, drives the synthesis of transforming growth factor-beta (TGFbeta) by mdx macrophages, which in turn induces collagen production in mdx fibroblasts. Fibrinogen-produced TGFbeta further amplifies collagen accumulation through activation of profibrotic alternatively activated macrophages. Fibrinogen, by engaging its alphavbeta3 receptor on fibroblasts, also directly promotes collagen synthesis. These data unveil a profibrotic role of fibrinogen deposition in muscle dystrophy.

Interleukin-6 is an essential regulator of satellite cell-mediated skeletal muscle hypertrophy.

Skeletal muscles adapt to increasing workload by augmenting their fiber size, through mechanisms that are poorly understood. This study identifies the cytokine interleukin-6 (IL-6) as an essential regulator of satellite cell (muscle stem cell)-mediated hypertrophic muscle growth. IL-6 is locally and transiently produced by growing myofibers and associated satellite cells, and genetic loss of IL-6 blunted muscle hypertrophy in vivo. IL-6 deficiency abrogated satellite cell proliferation and myonuclear accretion in the preexisting myofiber by impairing STAT3 activation and expression of its target gene cyclin D1. The growth defect was indeed muscle cell intrinsic, since IL-6 loss also affected satellite cell behavior in vitro, in a STAT3-dependent manner. Myotube-produced IL-6 further stimulated cell proliferation in a paracrine fashion. These findings unveil a role for IL-6 in hypertrophic muscle growth and provide mechanistic evidence for the contribution of satellite cells to this process.

Genetic analysis of p38 MAP kinases in myogenesis: fundamental role of p38alpha in abrogating myoblast proliferation.

The p38 mitogen-activated protein kinase (MAPK) pathway plays a critical role in skeletal muscle differentiation. However, the relative contribution of the four p38 MAPKs (p38alpha, p38beta, p38gamma and p38delta) to this process is unknown. Here we show that myoblasts lacking p38alpha, but not those lacking p38beta or p38delta, are unable to differentiate and form multinucleated myotubes, whereas p38gamma-deficient myoblasts exhibit an attenuated fusion capacity. The defective myogenesis in the absence of p38alpha is caused by delayed cell-cycle exit and continuous proliferation in differentiation-promoting conditions. Indeed, activation of JNK/cJun was enhanced in p38alpha-deficient myoblasts leading to increased cyclin D1 transcription, whereas inhibition of JNK activity rescued the proliferation phenotype. Thus, p38alpha controls myogenesis by antagonizing the activation of the JNK proliferation-promoting pathway, before its direct effect on muscle differentiation-specific gene transcription. More importantly, in agreement with the defective myogenesis of cultured p38alpha(Delta/Delta) myoblasts, neonatal muscle deficient in p38alpha shows cellular hyperproliferation and delayed maturation. This study provides novel evidence of a fundamental role of p38alpha in muscle formation in vitro and in vivo.

The plasminogen activation system in skeletal muscle regeneration: antagonistic roles of urokinase-type plasminogen activator (uPA) and its inhibitor (PAI-1).

The plasminogen activation (PA) system is an extensively used mechanism for the generation of proteolytic activity in the extracellular matrix, where it contributes to tissue remodeling in a wide range of physiopathological processes. Despite the limited information available at present on plasminogen activators, their inhibitors and cognate receptors in skeletal muscle, increasing evidence is accumulating on their important roles in the homeostasis of muscle fibers and their surrounding extracellular matrix. The development of mice deficient for the individual components of the PA system has provided an incisive approach to test the proposed muscle functions in vivo. Skeletal muscle regeneration induced by injury has been analyzed in urokinase-type plasminogen activator (uPA)-, tissue-type plasminogen activator (tPA)-, plasminogen (Plg)- and plasminogen activator inhibitor-1 (PAI-1)-deficient mice and has demonstrated profound effects of these molecules on the fibrotic state and the inflammatory response, which contribute to muscle repair. In particular, the opposite roles of uPA and its inhibitor PAI-1 in this process are highlighted. Delineating the mechanisms by which the different plasminogen activation system components regulate tissue repair will be of potential therapeutic value for severe muscle disorders.

p38 MAPK-induced nuclear factor-kappaB activity is required for skeletal muscle differentiation: role of interleukin-6.

p38 MAPK and nuclear factor-kappaB (NF-kappaB) signaling pathways have been implicated in the control of skeletal myogenesis. However, although p38 is recognized as a potent activator of myoblast differentiation, the role of NF-kappaB remains controversial. Here, we show that p38 is activated only in differentiating myocytes, whereas NF-kappaB activity is present both in proliferation and differentiation stages. NF-kappaB activation was found to be dependent on p38 activity during differentiation, being NF-kappaB an effector of p38, thus providing a novel mechanism for the promyogenic effect of p38. Activation of p38 in C2C12 cells induced the activity of NF-kappaB, in a dual way: first, by reducing IkappaBalpha levels and inducing NF-kappaB-DNA binding activity and, second, by potentiating the transactivating activity of p65-NF-kappaB. Finally, we show that interleukin (IL)-6 expression is induced in C2C12 differentiating myoblasts, in a p38- and NF-kappaB-dependent manner. Interference of IL-6 mRNA reduced, whereas its overexpression increased, the extent of myogenic differentiation; moreover, addition of IL-6 was able to rescue significantly the negative effect of NF-kappaB inhibition on this process. This study provides the first evidence of a crosstalk between p38 MAPK and NF-kappaB signaling pathways during myogenesis, with IL-6 being one of the effectors of this promyogenic mechanism.