On the Role of Protein Disulfide Isomerase in the Retrograde Cell Transport of Secreted Phospholipases A2

Following the finding that ammodytoxin (Atx), a neurotoxic secreted phospholipase A₂ (sPLA₂) in snake venom, binds specifically to protein disulfide isomerase (PDI) in vitro we show that these proteins also interact in living rat PC12 cells that are able to internalize this group IIA (GIIA) sPLA₂. Atx and PDI co-localize in both differentiated and non-differentiated PC12 cells, as shown by fluorescence microscopy. Based on a model of the complex between Atx and yeast PDI (yPDI), a three-dimensional model of the complex between Atx and human PDI (hPDI) was constructed. The Atx binding site on hPDI is situated between domains b and b’. Atx interacts hPDI with an extensive area on its interfacial binding surface. The mammalian GIB, GIIA, GV and GX sPLA₂s have the same fold as Atx. The first three sPLA₂s have been detected intracellularly but not the last one. The models of their complexes with hPDI were constructed by replacement of Atx with the respective mammalian sPLA₂ in the Atx—hPDI complex and molecular docking of the structures. According to the generated models, mammalian GIB, GIIA and GV sPLA₂s form complexes with hPDI very similar to that with Atx. The contact area between GX sPLA₂ and hPDI is however different from that of the other sPLA₂s. Heterologous competition of Atx binding to hPDI with GV and GX sPLA₂s confirmed the model-based expectation that GV sPLA₂ was a more effective inhibitor than GX sPLA₂, thus validating our model. The results suggest a role of hPDI in the (patho)physiology of some snake venom and mammalian sPLA₂s by assisting the retrograde transport of these molecules from the cell surface. The sPLA₂–hPDI model constitutes a valuable tool to facilitate further insights into this process and into the (patho)physiology of sPLA₂s in relation to their action intracellularly.     

Groups IV, V, and X phospholipases A2s in human neutrophils: role in eicosanoid production and gram-negative bacterial phospholipid hydrolysis.

The bacterial tripeptide formyl-Met-Leu-Phe (fMLP) induces the secretion of enzyme(s) with phospholipase A(2) (PLA(2)) activity from human neutrophils. We show that circulating human neutrophils express groups V and X sPLA(2) (GV and GX sPLA(2)) mRNA and contain GV and GX sPLA(2) proteins, whereas GIB, GIIA, GIID, GIIE, GIIF, GIII, and GXII sPLA(2)s are undetectable. GV sPLA(2) is a component of both azurophilic and specific granules, whereas GX sPLA(2) is confined to azurophilic granules. Exposure to fMLP or opsonized zymosan results in the release of GV but not GX sPLA(2) and most, if not all, of the PLA(2) activity in the extracellular fluid of fMLP-stimulated neutrophils is due to GV sPLA(2). GV sPLA(2) does not contribute to fMLP-stimulated leukotriene B(4) production but may support the anti-bacterial properties of the neutrophil, because 10-100 ng per ml concentrations of this enzyme lead to Gram-negative bacterial membrane phospholipid hydrolysis in the presence of human serum. By use of a recently described and specific inhibitor of cytosolic PLA(2)-alpha (group IV PLA(2)alpha), we show that this enzyme produces virtually all of the arachidonic acid used for the biosynthesis of leukotriene B(4) in fMLP- and opsonized zymosan-stimulated neutrophils, the major eicosanoid produced by these pro-inflammatory cells.     

Time-resolved fluoroimmunoassays of the complete set of secreted phospholipases A2 in human serum

Time-resolved fluoroimmunoassays (TR-FIA) were developed for all human secreted phospholipases A(2) (PLA(2)), viz. group (G) IB, GIIA, GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA(2) and the GXIIB PLA(2)-like protein. Antibodies were raised in rabbits against recombinant human PLA(2) proteins and used in sandwich-type TR-FIAs as both catching and detecting antibodies, the latter after labeling with Europium. The antibodies were non-cross-reactive. The analytical sensitivities were 1 microg/L for the TR-FIA for GIB PLA(2), 1 microg/L (GIIA), 35 microg/L (GIID), 3 microg/L (GIIE), 4 microg/L (GIIF), 14 microg/L (GIII), 11 microg/L (GV), 2 microg/L (GX), 92 microg/L (GXIIA) and 242 microg/L (GXIIB). All secreted PLA(2)s were assayed by these TR-FIAs in serum samples from 34 patients (23 men and 11 women, mean age 53.2 years) treated in an intensive care unit for septic infections, and in control samples from 28 volunteer blood donors (14 men and 14 women, mean age 57.0 years). Five serum samples (3 in the sepsis group and 2 in the blood donor group) gave high TR-FIA signals that were reduced to background (blank) levels by the addition of non-immune rabbit IgG to the sera. This reactivity was assumed to be due to the presence of heterophilic antibodies in these subjects. In all other subjects, including septic patients and healthy blood donors, the TR-FIA signals for GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA(2) and the GXIIB PLA(2)-like protein were at background (blank) levels. Four patients in the sepsis group had pancreatic involvement and elevated concentration of GIB PLA(2) in serum (median 19.0 microg/L, range 13.1-33.7 microg/L, n = 4) as compared to the healthy blood donors (median 1.8 microg/L, range 0.8-3.4 microg/L, n = 28, P < 0.0001). The concentration of GIIA PLA(2) in the sera of septic patients (median 315.7 microg/L, range 15.9-979.6 microg/L, n = 34) was highly elevated as compared to that of the blood donors (median 1.8 microg/L, range 0.8-5.8 microg/L, n = 28, P < 0.0001). Our current results confirmed elevated concentrations of GIB and GIIA PLA(2) in the sera of patients suffering from acute pancreatitis or septic infections, respectively, as compared to healthy subjects. However, in the same serum samples, the concentrations of the other secreted PLA(2)s, viz. GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA(2) and the GXIIB PLA(2)-like protein were below the respective analytical sensitivities of the TR-FIAs. It is concluded that generalized bacterial infections do not lead to elevated serum levels of GIIE, GIIF, GIII, GV and GX PLA(2)s above the detection limits of the current TR-FIAs.     

Design of new potent and selective secretory phospholipase A2 inhibitors. Part 5: synthesis and biological activity of 1-alkyl-4-[4,5-dihydro-1,2,4-[4H]-oxadiazol-5-one-3-ylmethylbenz-4'-yl(oyl)] piperazines

Among the different PLA(2)s identified to date, the group IIA secretory PLA(2) (sPLA(2) GIIA) is implied in diverse pathological conditions. In this work we describe the synthesis, inhibitory activities, and structure-activity relationships (SAR) of a new class of substituted piperazine derivatives. The in vitro fluorimetric assay using two groups of enzymes, GIB and GIIA, revealed several compounds as highly potent inhibitors (IC(50)=0.1 microM). The in vivo activity assessed by ip or per os administration in a carrageenan-induced edema test in rats showed that two compounds proved to be as potent as indomethacin (10 mg/kg).     

Effect of NGF on the Subcellular Localization of Group IIA Secretory Phospholipase A2 (GIIA) in PC12 Cells: Role in Neuritogenesis

Phospholipases A(2) (PLA(2)s) are involved in neuritogenesis but the identity of the isoforms(s) contributing to this process is still not defined. Several reports have focused on secretory PLA(2)s (sPLA(2)) as the administration of exogenous sPLA(2)s to PC12 neuronal cells stimulates neurite outgrowth. The present study demonstrates that the endogenous group IIA sPLA(2) (GIIA), constitutively expressed in mammalian neural cells, changes its subcellular localization when PC12 cells are induced to differentiate by NGF treatment. Indeed, confocal analysis showed a time-dependent accumulation of GIIA in growth cones and neurite tips. Under identical conditions the subcellular distribution of another isoform (GV) was unaffected by NGF. Contrary to GX, another sPLA(2) isoform expressed by PC12 cells, the contribution of GIIA to neuritogenesis does not require its release in the extracellular medium.     

The interrelations between malfunctioning DNA damage response (DDR) and the functionality of the neuro-glio-vascular unit

A hallmark of neurodegenerative diseases is impairment of certain aspects of “brain functionality”. Brain functionality is defined as the total input and output of the brain's neural circuits and networks. A given brain degenerative disorder does not deregulate total brain functionality but rather the activity of specific circuits in a given network, affecting their organization and topology, their cell numbers, their cellular functionality, and the interactions between neural circuits. Similarly, our concept of neurodegenerative diseases, which for many years revolved around neural survival or death, has now been extended to emphasize the role of glia. In particular, the role of glial cells in neuro-vascular communication is now known to be central to the effect of insults to the nervous system. In addition, a malfunctioning vascular system likely plays a role in the etiology of certain neurodegenerative diseases. Thus, the symptoms of neurodegenerative or more correctly brain degenerative disease are, to a very large extent, a result of impairment in glial cells that lead to pathological neuro-vascular interactions that, in turn, generate a rather “hostile” environment in which the neurons fail to function. These events lead to systematic neural cell death on a scale that appears to be proportional to the severity of the neurological deficit.     

NGF Induces the Expression of Group IIA Secretory Phospholipase A2 in PC12 Cells: The Newly Synthesized Enzyme Is Addressed to Growing Neurites

We proposed that group IIA secretory phospholipase A₂ (GIIA) participates in neuritogenesis based on our observations that the enzyme migrates to growth cones and neurite tips when PC12 cells are induced to differentiate by nerve growth factor (NGF) (Ferrini et al., Neurochem Res 35:2168–2174, 2010). The involvement of other secretory PLA₂ isoforms in neuronal development has been suggested by others but through different mechanisms. In the present study, we compared the subcellular distribution of GIIA and group X sPLA₂ (GX) after stimulation of PC12 cells with NGF. We found that GIIA, but not GX, localized at the neuritic tips after treatment with NGF, as demonstrated by immunofluorescence analysis. We also found that NGF stimulated the expression and the activity of GIIA. In addition, NGF induced the expressed myc-tagged GIIA protein to migrate to neurite tips in its active form. We propose that GIIA expression, activity, and subcellular localization is regulated by NGF and that the enzyme may participate in neuritogenesis through intracellular mechanisms, most likely by facilitating the remodelling of glycerophospholipid molecular species by deacylation–reacylation reactions necessary for the incorporation of polyunsaturated fatty acids.     

Epoxyeicosanoid signaling in CNS function and disease

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites of cytochrome P450 epoxygenase enzymes recognized as key players in vascular function and disease, primarily attributed to their potent vasodilator, anti-inflammatory and pro-angiogenic effects. Although EETs’ actions in the central nervous system (CNS) appear to parallel those in peripheral tissue, accumulating evidence suggests that epoxyeicosanoid signaling plays different roles in neural tissue compared to peripheral tissue; roles that reflect distinct CNS functions, cellular makeup and intercellular relationships. This is exhibited at many levels including the expression of EETs-synthetic and -metabolic enzymes in central neurons and glial cells, EETs’ role in neuro-glio-vascular coupling during cortical functional activation, the capacity for interaction between epoxyeicosanoid and neuroactive endocannabinoid signaling pathways, and the regulation of neurohormone and neuropeptide release by endogenous EETs. The ability of several CNS cell types to produce and respond to EETs suggests that epoxyeicosanoid signaling is a key integrator of cell–cell communication in the CNS, coordinating cellular responses across different cell types. Under pathophysiological conditions, such as cerebral ischemia, EETs protect neurons, astroglia and vascular endothelium, thus preserving the integrity of cellular networks unique to and essential for proper CNS function. Recognition of EETs’ intimate involvement in CNS function in addition to their multi-cellular protective profile has inspired the development of therapeutic strategies against CNS diseases such as cerebral ischemia, tumors, and neural pain and inflammation that are based on targeting the cellular actions of EETs or their biosynthetic and metabolizing enzymes. Based upon the emerging importance of epoxyeicosanoids in cellular function and disease unique to neural systems, we propose that the actions of “neuroactive EETs” are best considered separately, and not in aggregate with all other peripheral EETs functions.     

Unstructured loop is essential for the activation of mGluR2

Metabotropic Glutamate Receptors (mGluRs) are Class C G-protein coupled receptors (GPCRs) that are expressed throughout the central nervous system and are involved in several neurological and psychiatric disorders. Although, many studies focused on Glutamate induced activation of mGluR2, however, the role of unstructured loop (or “BC loop”) in activation of metabotropic Glutamate receptors is currently unknown. Here, using Förster Resonance Energy Transfer (FRET) based assay in live cells we show that unstructured loop is required for Glutamate induced conformation and hence the activation of the receptor.     

Inhibitors of secreted phospholipase A2 suppress the release of PGE2 in renal mesangial cells

The upregulation of PGE₂ by mesangial cells has been observed under chronic inflammation condition. In the present work, renal mesangial cells were stimulated to trigger a huge increase of PGE₂ synthesis and were treated in the absence or presence of known PLA₂ inhibitors. A variety of synthetic inhibitors, mainly developed in our labs, which are known to selectively inhibit each of GIVA cPLA₂, GVIA iPLA₂, and GIIA/GV sPLA₂, were used as tools in this study. Synthetic sPLA₂ inhibitors, such as GK115 (an amide derivative based on the non-natural amino acid (R)-γ-norleucine) as well as GK126 and GK241 (2-oxoamides based on the natural (S)-α-amino acid leucine and valine, respectively) presented an interesting effect on the suppression of PGE₂ formation.     

Fibrosis of two: Epithelial cell-fibroblast interactions in pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by the progressive and ultimately fatal accumulation of fibroblasts and extracellular matrix in the lung that distorts its architecture and compromises its function. IPF is now thought to result from wound-healing processes that, although initiated to protect the host from injurious environmental stimuli, lead to pathological fibrosis due to these processes becoming aberrant or over-exuberant. Although the environmental stimuli that trigger IPF remain to be identified, recent evidence suggests that they initially injure the alveolar epithelium. Repetitive cycles of epithelial injury and resultant alveolar epithelial cell death provoke the migration, proliferation, activation and myofibroblast differentiation of fibroblasts, causing the accumulation of these cells and the extracellular matrix that they synthesize. In turn, these activated fibroblasts induce further alveolar epithelial cell injury and death, thereby creating a vicious cycle of pro-fibrotic epithelial cell-fibroblast interactions. Though other cell types certainly make important contributions, we focus here on the “pas de deux” (steps of two), or perhaps more appropriate to IPF pathogenesis, the “folie à deux” (madness of two) of epithelial cells and fibroblasts that drives the progression of pulmonary fibrosis. We describe the signaling molecules that mediate the interactions of these cell types in their “fibrosis of two”, including transforming growth factor-β, connective tissue growth factor, sonic hedgehog, prostaglandin E₂, angiotensin II and reactive oxygen species. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.     

Suppression of Brain Mast Cells Degranulation Inhibits Microglial Activation and Central Nervous System Inflammation

Brain inflammation has a critical role in the pathophysiology of brain diseases. Microglia, the resident immune cells in the brain, play an important role in brain inflammation, while brain mast cells are the “first responder” in the injury rather than microglia. Functional aspects of mast cell-microglia interactions remain poorly understood. Our results demonstrated that site-directed injection of the “mast cell degranulator” compound 48/80 (C48/80) in the hypothalamus induced mast cell degranulation, microglial activation, and inflammatory factor production, which initiated the acute brain inflammatory response. “Mast cell stabilizer” disodium cromoglycate (cromolyn) inhibited this effect, including decrease of inflammatory cytokines, reduced microglial activation, inhibition of MAPK and AKT pathways, and repression of protein expression of histamine receptor 1 (H₁R), histamine receptor 4 (H₄R), protease-activated receptor 2 (PAR2), and toll-like receptor 4 (TLR4) in microglia. We also demonstrated that C48/80 had no effect on microglial activation in mast cell-deficient Kit^W-sh/W-sh mice. These results implicate that activated brain mast cells trigger microglial activation and stabilization of mast cell inhibits microglial activation-induced central nervous system (CNS) inflammation. Interactions between mast cells and microglia could constitute a new and unique therapeutic target for CNS immune inflammation-related diseases.     

Neural stem cells—trends and advances

For many years, accepted dogma held that brain is a static organ with no possibility of regeneration of cells in injured or diseased human brain. However, recent preclinical reports have shown regenerative potential of neural stem cells using various injury models. This has resulted in renewed hope for those suffering from spinal cord injury and neural damage. As the potential of stem cell therapy gained impact, these claims, in particular, led to widespread enthusiasm that acute and chronic injury of the nervous system would soon be a problem of the past. The devastation caused by injury or diseases of the brain and spinal cord led to wide premature acceptance that “neural stem cells (NSCs)” derived from embryonic, fetal or adult sources would soon be effective in reversing neural and spinal trauma. However, neural therapy with stem cells has not been realized to its fullest extent. Although, discrete population of regenerative stem cells seems to be present in specific areas of human brain, the function of these cells is unclear. However, similar cells in animals seem to play important role in postnatal growth as well as recovery of neural tissue from injury, anoxia, or disease. J. Cell. Biochem. 114: 764–772, 2013. © 2012 Wiley Periodicals, Inc.     

A comparative study of electronic and neural networks involved in pattern recognition

The comparative study of electronic and neural networks involved in pattern recognition starts with the analogies of structure and function which exist between the electronic “basic integrative unit” and the neuron. Both elements represent the basic components in each system of networks and may be considered as functionally equivalent.According to the kind of response given to a standard input signal, four types of integrative units, either electronic or neural, may be distinguished: the fixed, the accommodative, the signal prolongating and the adaptive type.The integrative units perform many different functions. Those involved in pattern recognition, however, can all be grouped into three categories according to one of the following functions they perform: contrast detection, pattern detection and pattern discrimination. A “contrast detecting unit” gives responses in two senses, positive or negative, according to the position of the stimulus over its receptive field. A “pattern detecting unit” gives responses in one sense only, with a maximum for a pattern having the spatial distribution corresponding to the positive acting receptors of its receptive field. For performing the function of discrimination, which leads to reliable identification of any pattern, a network arrangement called a “maximum amplitude filter” is necessary. Examples of such units and arrangements existing in the nervous system are provided.It is concluded that a “logical analysis of neural networks” based on engineering principles is possible and that this could provide a new tool to the neurophysiologist in the study of the nervous system.     

The stem cells of the neural crest

In the vertebrate embryo, the neurectodermal neural crest cells (NCC) have remarkably broad potencies, giving rise, after a migratory phase, to neurons and glial cells in the peripheral nervous system, and to skin melanocytes, being all designated here as “neural” derivatives. NC-derived cells also include non-neural, “mesenchymal” cell types like chondrocytes and bone cells, myofibroblasts and adipocytes, which largely contribute to the head structures in amniotes. Similar to the blood cell system, the NC is therefore a valuable model to investigate the mechanisms of cell lineage diversification in vertebrates. Whether NCC are endowed with multiple differentiation potentials or if, conversely, they are a mosaic of different committed cells is an important ongoing issue to understand the ontogeny of NC derivatives in normal development and pathological conditions. Here we focus on recent findings that established the presence in the early migratory NC of the avian embryo, of a multipotent progenitor endowed with both mesenchymal and neural differentiation capacities. This “mesenchymal-neural” clonogenic cell lies upstream of all the other NC progenitors known so far and shows increased frequency when single cell cultures are treated with the Sonic Hedgehog signaling molecule. These findings are discussed in the context of the broad potentials of NC stem cells recently evidenced in certain adult mammalian tissues.     

Hypoxia or in situ normoxia: The stem cell paradigm

Although O₂ concentrations are considerably lowered in vivo, depending on the tissue and cell population in question (some cells need almost anoxic environment for their maintenance) the cell and tissue cultures are usually performed at atmospheric O₂ concentration (20–21%). As an instructive example, the relationship between stem cells and micro‐environmental/culture oxygenation has been recapitulated. The basic principle of stem cell biology, “the generation‐age hypothesis,” and hypoxic metabolic properties of stem cells are considered in the context of the oxygen‐dependent evolution of life and its transposition to ontogenesis and development. A hypothesis relating the self‐renewal with the anaerobic and hypoxic metabolic properties of stem cells and the actual O₂ availability is elaborated (“oxygen stem cell paradigm”). Many examples demonstrated that the cellular response is substantially different at atmospheric O₂ concentration when compared to lower O₂ concentrations which better approximate the physiologic situation. These lower O₂ concentrations, traditionally called “hypoxia” represent, in fact, an in situ normoxia, and should be used in experimentation to get an insight of the real cell/cytokine physiology. The revision of our knowledge on cell/cytokine physiology, which has been acquired ex vivo at non physiological atmospheric (20–21%) O₂ concentrations representing a hyperoxic state for most primate cells, has thus become imperious. J. Cell. Physiol. 219: 271–275, 2009. © 2009 Wiley‐Liss, Inc.     

Neural stem cells in aging and disease

Aging in the central nervous system is associated with progressive loss of function which is exacerbated by neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The two primary cell replacement strategies involve transplantation of exogenous tissue, and activation of proliferation of endogenous cells. Transplanted tissue is used to either directly replace lost tissue, or to implant genetically engineered cells that secrete factors which promote survival and/or proliferation. However, successful application of any cell replacement therapy requires knowledge of the complex relationships between neural stem cells and the more restricted neural and glial progenitor cells. This review focuses on recent advances in the field of stem cell biology of the central nervous system, with an emphasis on cellular and molecular approaches to replacing cells lost in neurodegenerative disorders.     

Adenosine receptors in the central nervous system: relationship to the central actions of methylxanthines

Adenosine has a significant role in many functions of the central nervous system. Behaviorally, adenosine and adenosine analogs have marked depressant effects. Electrophysiologically, adenosine reduces spontaneous neuronal activity and inhibits transsynaptic potentials $\text{via}$ interaction with extracellular receptors. Biochemically, adenosine inhibits adenylate cyclase $\text{via}$ a “high” affinity receptor, and activates adenylate cyclase $\text{via}$ a “low” affinity receptor. These receptors, called “A₁” and “A₂” respectively, show differing profiles for activation by adenosine analogs. Radioactive N⁶-cyclohexyladenosine binds selectively to the “high” affinity receptor. One major class of antagonists is known at adenosine receptors: the alkylxanthines, including caffeine and theophylline. Radioactive 1,3-diethyl-8-phenylxanthine, a particularly potent antagonist, appears to bind to both low and high affinity adenosine receptors. Behavioral, electrophysiological, and biochemical effects of alkylxanthines are consistent with the hypothesis that the central stimulatory actions of caffeine and theophylline are due in large part to antagonism of central adenosine receptors.