Catastrophic NAD+ Depletion in Activated T Lymphocytes through Nampt Inhibition Reduces Demyelination and Disability in EAE

Nicotinamide phosphoribosyltransferase (Nampt) inhibitors such as FK866 are potent inhibitors of NAD⁺ synthesis that show promise for the treatment of different forms of cancer. Based on Nampt upregulation in activated T lymphocytes and on preliminary reports of lymphopenia in FK866 treated patients, we have investigated FK866 for its capacity to interfere with T lymphocyte function and survival. Intracellular pyridine nucleotides, ATP, mitochondrial function, viability, proliferation, activation markers and cytokine secretion were assessed in resting and in activated human T lymphocytes. In addition, we used experimental autoimmune encephalomyelitis (EAE) as a model of T-cell mediated autoimmune disease to assess FK866 efficacy in vivo. We show that activated, but not resting, T lymphocytes undergo massive NAD⁺ depletion upon FK866-mediated Nampt inhibition. As a consequence, impaired proliferation, reduced IFN-γ and TNF-α production, and finally autophagic cell demise result. We demonstrate that upregulation of the NAD⁺-degrading enzyme poly-(ADP-ribose)-polymerase (PARP) by activated T cells enhances their susceptibility to NAD⁺ depletion. In addition, we relate defective IFN-γ and TNF-α production in response to FK866 to impaired Sirt6 activity. Finally, we show that FK866 strikingly reduces the neurological damage and the clinical manifestations of EAE. In conclusion, Nampt inhibitors (and possibly Sirt6 inhibitors) could be used to modulate T cell-mediated immune responses and thereby be beneficial in immune-mediated disorders.     

Crystal Structure of the PIM2 Kinase in Complex with an Organoruthenium Inhibitor

Background

The serine/threonine kinase PIM2 is highly expressed in human leukemia and lymphomas and has been shown to positively regulate survival and proliferation of tumor cells. Its diverse ATP site makes PIM2 a promising target for the development of anticancer agents. To date our knowledge of catalytic domain structures of the PIM kinase family is limited to PIM1 which has been extensively studied and which shares about 50% sequence identity with PIM2.

Principal Findings

Here we determined the crystal structure of PIM2 in complex with an organoruthenium complex (inhibition in sub-nanomolar level). Due to its extraordinary shape complementarity this stable organometallic compound is a highly potent inhibitor of PIM kinases.

Significance

The structure of PIM2 revealed several differences to PIM1 which may be explored further to generate isoform selective inhibitors. It has also demonstrated how an organometallic inhibitor can be adapted to the binding site of protein kinases to generate highly potent inhibitors.

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Functional characterization of a synthetic abscisic acid analog with anti-inflammatory activity on human granulocytes and monocytes

The phytohormone abscisic acid (ABA), in addition to regulating several important physiological functions in plants, is also produced and released by human granulocytes and monocytes where it stimulates cell activities involved in the innate immune response.Here we describe the properties of an ABA synthetic analog that competes with the hormone for binding to human granulocyte membranes and to purified recombinant LANCL2 (the human ABA receptor) and inhibits several ABA-triggered inflammatory functions of granulocytes and monocytes in vitro: chemotaxis, phagocytosis, reactive oxygen species production and release of prostaglandin E₂ (PGE₂) by human granulocytes, release of PGE₂ and of monocyte chemoattractant protein-1 by human monocytes. This observation provides a proof of principle that ABA antagonists may represent a new class of anti-inflammatory agents.     

The Diadenosine Homodinucleotide P18 Improves In Vitro Myelination in Experimental Charcot‐Marie‐Tooth Type 1A

Charcot‐Marie‐Tooth 1A (CMT1A) is a demyelinating hereditary neuropathy whose pathogenetic mechanisms are still poorly defined and an etiologic treatment is not yet available. An abnormally high intracellular Ca²⁺ concentration ([Ca²⁺]_i) occurs in Schwann cells from CMT1A rats (CMT1A SC) and is caused by overexpression of the purinoceptor P2X7. Normalization of the Ca²⁺ levels through down‐regulation of P2X7 appears to restore the normal phenotype of CMT1A SC in vitro. We recently demonstrated that the diadenosine 5′,5′′′‐P1, P2‐diphosphate (Ap2A) isomer P18 behaves as an antagonist of the P2X7 purinergic receptor, effectively blocking channel opening induced by ATP. In addition, P18 behaves as a P2Y11 agonist, inducing cAMP overproduction in P2Y11‐overexpressing cells. Here we investigated the in vitro effects of P18 on CMT1A SC. We observed that basal levels of intracellular cAMP ([cAMP]_i), a known regulator of SC differentiation and myelination, are significantly lower in CMT1A SC than in wild‐type (wt) cells. P18 increased [cAMP]_i in both CMT1A and wt SC, and this effects was blunted by NF157, a specific P2Y11 antagonist. Prolonged treatment of organotypic dorsal root ganglia (DRG) cultures with P18 significantly increased expression of myelin protein zero, a marker of myelin production, in both CMT1A and wt cultures. Interestingly, P18 decreased the content of non‐phosphorylated neurofilaments, a marker of axonal damage, only in CMT1A DRG cultures. These results suggest that P2X7 antagonists, in combination with [cAMP]_i‐increasing agents, could represent a therapeutic strategy aimed at correcting the molecular derangements causing the CMT1A phenotype. J. Cell. Biochem. 115: 161–167, 2014. © 2013 Wiley Periodicals, Inc.     

Extremophile Microalgae: the potential for biotechnological application

Microalgae are photosynthetic microorganisms that use sunlight as an energy source, and convert water, carbon dioxide, and inorganic salts into algal biomass. The isolation and selection of microalgae, which allow one to obtain large amounts of biomass and valuable compounds, is a prerequisite for their successful industrial production. This work provides an overview of extremophile algae, where their ability to grow under harsh conditions and the corresponding accumulation of metabolites are addressed. Emphasis is placed on the high-value products of some prominent algae. Moreover, the most recent applications of these microorganisms and their potential exploitation in the context of astrobiology are taken into account.     

Beta-Amyloid Monomer and Insulin/IGF-1 Signaling in Alzheimer's Disease

Alzheimer's disease is the most common form of dementia among older people and is still untreatable. While ??-amyloid protein is recognized as the disease determinant with a pivotal role in inducing neuronal loss and dementia, an impaired brain insulin signaling seems to account in part for the cognitive deficit associated with the disease. The origin of this defective signaling is uncertain. Accumulating toxic species of ??-amyloid, the so-called oligomers, has been proposed to be responsible for downregulation of neuronal insulin receptors. We have found that the nontoxic form of ??-amyloid, the monomer, is able to activate insulin/insulin-like growth factor-1 (IGF-1) receptor signaling and thus behaves as a neuroprotectant agent. Our suggestion is that depletion of ??-amyloid monomers, occurring in the preclinical phase of Alzheimer's disease, might be the cause of early insulin/IGF-1 signaling disturbances that anticipate cognitive decline.     

Extracellular NAD+ is an agonist of the human P2Y11 purinergic receptor in human granulocytes

Micromolar concentrations of extracellular beta-NAD+ (NAD(e)+) activate human granulocytes (superoxide and NO generation and chemotaxis) by triggering: (i) overproduction of cAMP, (ii) activation of protein kinase A, (iii) stimulation of ADP-ribosyl cyclase and overproduction of cyclic ADP-ribose (cADPR), a universal Ca2+ mobilizer, and (iv) influx of extracellular Ca2+. Here we demonstrate that exposure of granulocytes to millimolar rather than to micromolar NAD(e)+ generates both inositol 1,4,5-trisphosphate (IP3) and cAMP, with a two-step elevation of intracellular calcium levels ([Ca2+]i): a rapid, IP3-mediated Ca2+ release, followed by a sustained influx of extracellular Ca2+ mediated by cADPR. Suramin, an inhibitor of P2Y receptors, abrogated NAD(e)+-induced intracellular increases of IP3, cAMP, cADPR, and [Ca2+]i, suggesting a role for a P2Y receptor coupled to both phospholipase C and adenylyl cyclase. The P2Y(11) receptor is the only known member of the P2Y receptor subfamily coupled to both phospholipase C and adenylyl cyclase. Therefore, we performed experiments on hP2Y(11)-transfected 1321N1 astrocytoma cells: micromolar NAD(e)+ promoted a two-step elevation of the [Ca2+]i due to the enhanced intracellular production of IP3, cAMP, and cADPR in 1321N1-hP2Y(11) but not in untransfected 1321N1 cells. In human granulocytes NF157, a selective and potent inhibitor of P2Y(11), and the down-regulation of P2Y(11) expression by short interference RNA prevented NAD(e)+-induced intracellular increases of [Ca2+]i and chemotaxis. These results demonstrate that beta-NAD(e)+ is an agonist of the P2Y(11) purinoceptor and that P2Y(11) is the endogenous receptor in granulocytes mediating the sustained [Ca2+]i increase responsible for their functional activation.     

NAADP+ synthesis from cADPRP and nicotinic acid by ADP-ribosyl cyclases

ADP-ribosyl cyclases (ADPRCs) are present from lower Metazoa to mammals and synthesize the Ca2+-active (di)nucleotides cyclic ADP-ribose (cADPR), NAADP+, and ADP-ribose (ADPR), involved in the regulation of important cellular functions. NAADP+ can be synthesized by ADPRCs from NADP+ through a base-exchange reaction, which substitutes nicotinamide for nicotinic acid (NA). Here we demonstrate that ADPRCs from both lower and higher Metazoa (including human CD38) can also synthesize NAADP+ starting from 2'-phospho-cyclic ADP-ribose (cADPRP) and NA. Comparison, on the two substrates cADPRP and NADP+, of the relative rates of the reactions introducing NA and hydrolyzing/cyclizing the substrate, respectively, indicates that with all ADPRCs tested cADPRP is preferentially transformed into NAADP+, while NADP+ is preferentially cyclized or hydrolyzed to cADPRP/2'-phospho-ADP-ribose. cADPRP was detectable in retinoic acid-differentiated, CD38+ HL-60 cells, but not in undifferentiated, CD38- cells. These results suggest that cADPRP may be a NAADP+ precursor in ADPRC+ cells.