Pranab K. Mukherjee

Neurotrophins enhance retinal pigment epithelial cell survival through neuroprotectin D1 signaling

Integrity of retinal pigment epithelial cells is necessary for photoreceptor survival and vision. The essential omega-3 fatty acid, docosahexaenoic acid, attains its highest concentration in the human body in photoreceptors and is assumed to be a target for lipid peroxidation during cell damage. We have previously shown, in contrast, that docosahexaenoic acid is also the precursor of neuroprotectin D1 (NPD1), which now we demonstrate, acts against apoptosis mediated by A2E, a byproduct of phototransduction that becomes toxic when it accumulates in aging retinal pigment epithelial (RPE) cells and in some inherited retinal degenerations. Furthermore, we show that neurotrophins, particularly pigment epithelium-derived factor, induce NPD1 synthesis and its polarized apical secretion. Moreover, docosahexaenoic acid (DHA) elicits a concentration-dependent and selective potentiation of pigment epithelial-derived factor-stimulated NPD1 synthesis and release through the apical RPE cell surface. The bioactivity of signaling activated by pigment epithelium-derived factor and DHA uncovered synergistic cytoprotection with concomitant NPD1 synthesis when cells were challenged with oxidative stress. Also, DHA and pigment epithelium-derived factor synergistically modify the expression of Bcl-2 family members, activating antiapoptotic proteins and decreasing proapoptotic proteins, and by attenuating caspase 3 activation during oxidative stress. Thus, our findings demonstrate that DHA-derived NPD1 protects against RPE cell damage mediated by aging/disease-induced A2E accumulation. Also, our results identify neurotrophins as regulators of NPD1 and of its polarized apical efflux from RPE cells. Taken together, these findings imply NPD1 may elicit autocrine actions on RPE cells and paracrine bioactivity in cells located in the proximity of the interphotoreceptor matrix.

Selective Survival Rescue in 15-Lipoxygenase-1-deficient Retinal Pigment Epithelial Cells by the Novel Docosahexaenoic Acid-derived Mediator, Neuroprotectin D1

The integrity of the retinal pigment epithelial (RPE) cell is essential for the survival of rod and cone photoreceptor cells. Several stressors, including reactive oxygen species, trigger apoptotic damage in RPE cells preceded by an anti-inflammatory, pro-survival response, the formation of neuroprotectin D1 (NPD1), an oxygenation product derived from the essential omega-3 fatty acid family member docosahexaenoic acid. To define the ability of NPD1 and other endogenous novel lipid mediators in cell survival, we generated a stable knockdown human RPE (ARPE-19) cell line using short hairpin RNA to target 15-lipoxygenase-1. The 15-lipoxygenase-1-deficient cells exhibited 30% of the protein expression, and 15-lipoxygenase-2 remained unchanged, as compared with an ARPE-19 cell line control established using nonspecific short hairpin RNA transfected cells. NPD1 synthesis was stimulated by tumor necrosis factor α/H2O2-mediated oxidative stress in nonspecific cells (controls), whereas in silenced cells, negligible amounts of NPD1, 12(S)- and 15(S)-hydroxyeicosatetraenoic acid, and lipoxin A4 were found under these conditions. Neither control nor the deficient cells showed an increase in 15-lipoxygenase-1 protein content after 16 h of oxidative stress, suggesting that the increased activity of 15-lipoxygenase-1 is due to activation of pre-existing proteins. 15-Lipoxygenase-silenced cells also displayed an exacerbated sensitivity to oxidative stress-induced apoptosis when compared with the control cells. NPD1 selectively and potently rescued 15-lipoxygenase-silenced cells from oxidative stress-induced apoptosis. These results demonstrate that 15-lipoxygenase-1 is activated by oxidative stress in ARPE-19 cells and that NPD1 is part of an early survival signaling in RPE cells.

Neuroprotectin D1/Protectin D1 Stereoselective and Specific Binding With Human Retinal Pigment Epithelial Cells and Neutrophils

Retinal pigment epithelial (RPE) cells, derived from the neuroectoderm, biosynthesize the novel lipid mediator neuroprotectin D1 (NPD1) from docosahexaenoic acid (DHA) in response to oxidative stress or to neurotrophins, and in turn, elicits cytoprotection. Here, we report the identification of a 16,17-epoxide-containing intermediate in the biosynthesis of NPD1 in ARPE-19 cells from 17S-hydro-(peroxy)-docosahexaenoic acid. We prepared and isolated tritium-labeled NPD1 ([(3)H]-NPD1) and demonstrate specific and high-affinity stereoselective binding to ARPE-19 cells (K(d)=31.3+/-13.1 pmol/mg of cell protein). The stereospecific NPD1 interactions with these cells in turn gave potent protection against oxidative stress-induced apoptosis, and other structurally related compounds were weak competitors of NPD1 specific binding. This [(3)H]-NPD1/PD1 also displayed specific and selective high affinity binding with isolated human neutrophils (K(d) approximately 25 nM). Neither resolvin E1 nor lipoxin A(4) competed for [(3)H]-NPD1/PD1 specific binding with human neutrophils. Together, these results provide evidence for stereoselective specific binding of NPD1/PD1 with retinal pigment epithelial cells as well as human neutrophils. Moreover, they suggest specific receptors for this novel mediator in both the immune and visual systems.

Photoreceptor outer segment phagocytosis attenuates oxidative stress-induced apoptosis with concomitant neuroprotectin D1 synthesis

Photoreceptor cell (rods and cones) renewal is accompanied by intermittent shedding of the distal tips of the outer segment followed by their phagocytosis in the retinal pigment epithelial (RPE) cells. This renewal is essential for vision, and it is thought that it fosters survival of photoreceptors and of RPE cells. However, no specific survival messenger/mediators have as yet been identified. We show here that photoreceptor outer segment (POS) phagocytosis markedly attenuates oxidative stress-induced apoptosis in ARPE-19 cells in culture. This phenomenon does not seem to be a generalized outcome of phagocytosis because nonbiological (polystyrene microsphere) phagocytosis did not elicit protection. The free docosahexaenoic acid (DHA) pool size and neuroprotectin D1 (NPD1) content increased during POS phagocytosis but not during microspheres phagocytosis. We have also explored other lipid mediators [lipoxin A4 and 15(S)- and 12(S)-hydroxyeicosatetraenoic acids] under these conditions and found them unchanged upon POS phagocytosis. Moreover, oxidative stress challenge to RPE cells undergoing POS phagocytosis further increased DHA and NPD1 content. Under these conditions, NPD1 was found within the RPE cells as well as in the culture medium, suggesting autocrine and paracrine bioactivity. Furthermore, using deuterium-labeled DHA, we show that as the availability of free DHA increases during oxidative stress, NPD1 synthesis is augmented in ARPE-19 cells. Our data suggest a distinct signaling that promotes survival of photoreceptor and RPE cells by enhancing the synthesis of NPD1 during phagocytosis. Taken together, NPD1 may be a mediator that promotes homeostatic regulation of cell integrity during photoreceptor cell renewal.

Mycobiota in gastrointestinal diseases

New insights gained through the use of state-of-the-art technologies, including next-generation sequencing, are starting to reveal that the association between the gastrointestinal tract and the resident mycobiota (fungal community) is complex and multifaceted, in which fungi are active participants influencing health and disease. Characterizing the human mycobiome (the fungi and their genome) in healthy individuals showed that the gastrointestinal tract contains 66 fungal genera and 184 fungal species, with Candida as the dominant fungal genera. Although fungi have been associated with a number of gastrointestinal diseases, characterization of the mycobiome has mainly been focused on patients with IBD and graft-versus-host disease. In this Review, we summarize the findings from studies investigating the relationship between the gut mycobiota and gastrointestinal diseases, which indicate that fungi contribute to the aggravation of the inflammatory response, leading to increased disease severity. A model explaining the mechanisms underlying the role of the mycobiota in gastrointestinal diseases is also presented. Our understanding of the contribution of the mycobiota to health and disease is still in its infancy and leaves a number of questions to be addressed. Answering these questions might lead to novel approaches to prevent and/or manage acute as well as chronic gastrointestinal disease.

Platelet-activating factor is a downstream messenger of kainate-induced activation of mitogen-activated protein kinases in primary hippocampal neurons†

Excitatory amino acids transduce physiological and pathological signals to neurons. Similarly, the neuroactive lipid platelet‐activating factor (PAF) has been implicated in modulating long‐term potentiation and neuronal survival. Excitatory amino acids and PAF have been shown to increase mitogen‐activated protein (MAP) kinases in different cell types. Here, we have investigated the similarities and differences between PAF and kainate in activating MAP kinases in primary hippocampal neurons in vitro. Extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase, and p38 kinases were activated by kainate or PAF in hippocampal neurons. This activation was blocked by the receptor antagonists CNQX and BN 50730 for kainate and PAF, respectively. The PAF receptor antagonist BN 50730 also blocked kainate activation. CNQX had no effect on PAF activation of the kinases, indicating that PAF is downstream of kainate activation. Coapplication of submaximal concentrations of PAF and kainate resulted in a less than additive activation, suggesting similar routes of activation by the two agonists. Both CNQX and BN 50730 blocked kainate‐induced neurotoxicity. These results indicate that PAF and kainate activate similar kinase pathways. Therefore, PAF acts downstream of the kainate subtype of glutamate receptors, and when excessive receptor activation takes place, this bioactive lipid may contribute to neuronal cell death. J. Neurosci. Res. 53:297–303, 1998.

Ataxin-1 poly-Q-induced proteotoxic stress and apoptosis are attenuated in neural cells by docosahexaenoic acid-derived neuroprotectin D1

Background: Neurodegenerative diseases involve proteotoxic stress and apoptosis. Results: NPD1 inhibits proteotoxic stress-induced apoptosis. Conclusion: NPD1 synthesis is an early response to proteotoxic stress. Significance: This might be one of the first survival defenses activated in neurodegenerations. Neurodegenerative diseases share two common features: enhanced oxidative stress and cellular inability to scavenge structurally damaged abnormal proteins. Pathogenesis of polyglutamine (poly(Q)) diseases involves increased protein misfolding, along with ubiquitin and chaperon protein-containing nuclear aggregates. In spinocerebellar ataxia, the brain and retina undergo degeneration. Neuroprotectin D1 (NPD1) is made on-demand in the nervous system and retinal pigment epithelial (RPE) cells in response to oxidative stress, which activates prosurvival signaling via regulation of gene expression and other processes. We hypothesized that protein misfolding-induced proteotoxic stress triggers NPD1 synthesis. We used ARPE-19 cells as a cellular model to assess stress due to ataxin-1 82Q protein expression and determine whether NPD1 prevents apoptosis. Ectopic ataxin-1 expression induced RPE cell apoptosis, which was abrogated by 100 nm docosahexaenoic acid, 10 ng/ml pigment epithelium-derived factor, or NPD1. Similarly, NPD1 was protective in neurons and primary human RPE cells. Furthermore, when ataxin-1 82Q was expressed in 15-lipoxygenase-1-deficient cells, apoptosis was greatly enhanced, and only NPD1 (50 nm) rescued cells from death. NPD1 reduced misfolded ataxin-1-induced accumulation of proapoptotic Bax in the cytoplasm, suggesting that NPD1 acts by preventing proapoptotic signaling pathways from occurring. Finally, NPD1 signaling interfered with ataxin-1/capicua repression of gene expression and decreased phosphorylated ataxin-1 in an Akt-independent manner, suggesting that NPD1 signaling modulates formation or stabilization of ataxin-1 complexes. These data suggest that 1) NPD1 synthesis is an early response induced by proteotoxic stress due to abnormally folded ataxin-1, and 2) NPD1 promotes cell survival through modulating stabilization of ataxin-1 functional complexes and pro-/antiapoptotic and inflammatory pathways.

Downregulation of COX-2 and JNK expression after induction of ischemic tolerance in the gerbil brain.

The response of the inducible isoform of the prostaglandin H2 synthase (COX-2) and the c-Jun N-terminal kinase (JNK) in post-ischemic neuronal damage was assessed in a model of ischemic tolerance in Mongolian Gerbils. After a single 6-min bilateral carotid occlusion, histological damage was evident in the CA1 region of hippocampus, correlated with a high expression of JNK and COX-2 mRNA. However, in the group of animals with a 2-min ischemia and the tolerance group, in which a 2-min bilateral carotid occlusion was followed 3 days later by a 6-min ischemia, no hippocampal or cortical damage was detected. Accordingly, the JNK and COX-2 mRNA levels remained unaffected. We suggest that the level of JNK and COX-2 expression may determine the outcome as either post-ischemic cell death or tolerance.

A2E selectively induces COX-2 in ARPE-19 and human neural cells

Purpose: To investigate the expression of cyclooxygenase (COX)-1, -2, and -3 RNA and protein in retinal pigment epithelial (ARPE-19) cells and in human neural (HN) cells exposed to the stress-inducing cytokines IL-1β and TNF-α, the oxidizing peroxide H2O2, the combination of TNF-α + H2O2, and the lipofuscin fluorophore A2E. Methods: Three-week-old ARPE-19 and HN cells were incubated with IL-1β (10 ng/ml), TNF-α (10 ng/ml), H2O2 (0.6 μM), TNF-α + H2O2 (10 ng/ml and 0.6 μM), or A2E (10 μM) for 8 hr, after which total RNA and whole cellular proteins were isolated. Cyclooxygenase-1, -2, and -3 RNA and protein levels were quantified using Northern and Western immunoassay. Results: IL-1β-, H2O2-, TNF-α-, TNF-α + H2O2-, or A2E-stressed ARPE-19 or HN cells displayed no significant upregulation in COX-1 or COX-3 RNA message abundance; however, significant upregulation was observed in COX-2 RNA message and protein abundance. A2E treatment of HN cells resulted in modest increases in COX-3 protein, an effect that was not observed in ARPE-19 cells. Conclusions: COX-2 RNA levels were induced in cytokine-, peroxide-, and A2E-stressed ARPE-19 and HN cells. Lack of induction of COX-3 RNA message by A2E, coupled with increases in COX-3 protein under identical treatment conditions, suggest that significant post-transcriptional or post-translational controls may regulate COX-3 gene expression in HN cells. Stress-induced upregulation of COX-2 gene expression in ARPE-19 and HN cells may play a mechanistic role in promoting proinflammatory and/or pro-oxidative pathology in these tissues.

Elovanoids are novel cell-specific lipid mediators necessary for neuroprotective signaling for photoreceptor cell integrity

Docosahexaenoic acid (DHA, 22:6 n-3) is abundant in the retina and is enzymatically converted into pro-homeostatic docosanoids. The DHA- or eicosapentaenoic acid (EPA)-derived 26 carbon fatty acid is a substrate of elongase ELOVL4, which is expressed in photoreceptor cells and generates very long chain (≥C28) polyunsaturated fatty acids including n-3 (VLC-PUFAs,n-3). While ELOVL4 mutations are linked to vision loss and neuronal dysfunctions, the roles of VLC-PUFAs remain unknown. Here we report a novel class of lipid mediators biosynthesized in human retinal pigment epithelial (RPE) cells that are oxygenated derivatives of VLC-PUFAs,n-3; we termed these mediators elovanoids (ELV). ELVs have structures reminiscent of docosanoids but with different physicochemical properties and alternatively-regulated biosynthetic pathways. The structures, stereochemistry, and bioactivity of ELVs were determined using synthetic materials produced by stereo-controlled chemical synthesis. ELVs enhance expression of pro-survival proteins in cells undergoing uncompensated oxidative stress. Our findings unveil a novel autocrine/paracrine pro-homeostatic RPE cell signaling that aims to sustain photoreceptor cell integrity and reveal potential therapeutic targets for retinal degenerations.