cRel and Wnt5a/Frizzled 5 Receptor-Mediated Inflammatory Regulation Reveal Novel Neuroprotectin D1 Targets for Neuroprotection.

Wnt5a triggers inflammatory responses and damage via NFkB/p65 in retinal pigment epithelial (RPE) cells undergoing uncompensated oxidative stress (UOS) and in experimental ischemic stroke. We found that Wnt5a-Clathrin-mediated uptake leads to NFkB/p65 activation and that Wnt5a is secreted in an exosome-independent fashion. We uncovered that docosahexaenoic acid (DHA) and its derivative, Neuroprotectin D1 (NPD1), upregulate c-Rel expression that, as a result, blunts Wnt5a abundance by competing with NFkB/p65 on the Wnt5a promoter A. Wnt5a increases in ischemic stroke penumbra and blood, while DHA reduces Wnt5a abundance with concomitant neuroprotection. Peptide inhibitor of Wnt5a binding, Box5, is also neuroprotective. DHA-decreased Wnt5a expression is concurrent with a drop in NFkB-driven inflammatory cytokine expression, revealing mechanisms after stroke, as in RPE cells exposed to UOS. Limiting the Wnt5a activity via Box5 reduces stroke size, suggesting neuroprotection pertinent to onset and progression of retinal degenerations and stroke consequences. NPD1 disrupts Wnt5a feedback loop at two sites: (1) decreasing FZD5, thus Wnt5a internalization, and (2) by enhancing cREL activity, which competes with p65/NFkB downstream endocytosis. As a result, Wnt5a expression is reduced, and so is its inflammatory signaling in RPE cells and neurons in ischemic stroke.

The Docosanoid Neuroprotectin D1 Induces TH-Positive Neuronal Survival in a Cellular Model of Parkinson's Disease.

Parkinson's disease (PD) does not manifest clinically until 80 % of striatal dopamine is reduced, thus most neuronal damage takes place before the patient presents clinical symptoms. Therefore, it is important to develop preventive strategies for this disease. In the experimental models of PD, 1-methyl-4-phenylpyridinium ion (MPP+) and rotenone induce toxicity in dopaminergic neurons. Neuroprotectin D1 (NPD1) displays neuroprotection in cells undergoing proteotoxic and oxidative stress. In the present report, we established an in vitro model using a primary neuronal culture from mesencephalic embryonic mouse tissue in which 17-20 % of neurons were TH-positive when differentiated in vitro. NPD1 (100 nM) rescued cells from apoptosis induced by MPP+ and rotenone, and the dendritic arbor of surviving neurons was examined using Sholl analysis. Rotenone, as well as MPP+ and its precursor 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), severely promoted retraction of dendritic arbor distal segments, thus decreasing the maximum branch order reached. On average, NPD1 counteracted the effects of MPP+ on the dendritic arborization, but failed to do so in the rotenone-treated neurons. However, rotenone did decrease the Sholl intersection number from radii 25 to 125 µm, and NPD1 did restore the pattern to control levels. Similarly, NPD1 partially reverted the dendrite retraction caused by MPP+ and MPTP. These results suggest that the apoptosis occurring in mesencephalic TH-positive neurons is not a direct consequence of mitochondrial dysfunction alone and that NPD1 signaling may be counteracting this damage. These findings lay the groundwork for the use of the in vitro model developed for future studies and for the search of specific molecular events that NPD1 targets to prevent early neurodegeneration in PD.

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

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.

Elovanoid-N34 modulates TXNRD1 key in protection against oxidative stress-related diseases.

The thioredoxin (TXN) system is an NADPH + H+/FAD redox-triggered effector that sustains homeostasis, bioenergetics, detoxifying drug networks, and cell survival in oxidative stress-related diseases. Elovanoid (ELV)-N34 is an endogenously formed lipid mediator in neural cells from omega-3 fatty acid precursors that modulate neuroinflammation and senescence gene programming when reduction-oxidation (redox) homeostasis is disrupted, enhancing cell survival. Limited proteolysis (LiP) screening of human retinal pigment epithelial (RPE) cells identified TXNRD1 isoforms 2, 3, or 5, the reductase of the TXN system, as an intracellular target of ELV-N34. TXNRD1 silencing confirmed that the ELV-N34 target was isoform 2 or 3. This lipid mediator induces TXNRD1 structure changes that modify the FAD interface domain, leading to its activity modulation. The addition of ELV-N34 decreased membrane and cytosolic TXNRD1 activity, suggesting localizations for the targeted reductase. These results show for the first time that the lipid mediator ELV-N34 directly modulates TXNRD1 activity, underling its protection in several pathologies when uncompensated oxidative stress (UOS) evolves.

New Retinal Pigment Epithelial Cell Model to Unravel Neuroprotection Sensors of Neurodegeneration in Retinal Disease.

Retinal pigment epithelial (RPE) cells sustain photoreceptor integrity, and when this function is disrupted, retinal degenerations ensue. Herein, we characterize a new cell line from human RPE that we termed ABC. These cells remarkably recapitulate human eye native cells. Distinctive from other epithelia, RPE cells originate from the neural crest and follow a neural development but are terminally differentiated into "epithelial" type, thus sharing characteristics with their neuronal lineages counterparts. Additionally, they form microvilli, tight junctions, and honeycomb packing and express distinctive markers. In these cells, outer segment phagocytosis, phagolysosome fate, phospholipid metabolism, and lipid mediator release can be studied. ABC cells display higher resistance to oxidative stress and are protected from senescence through mTOR inhibition, making them more stable in culture. The cells are responsive to Neuroprotectin D1 (NPD1), which downregulates inflammasomes and upregulates antioxidant and anti-inflammatory genes. ABC gene expression profile displays close proximity to native RPE lineage, making them a reliable cell system to unravel signaling in uncompensated oxidative stress (UOS) and retinal degenerative disease to define neuroprotection sites.

Elovanoids downregulate SARS-CoV-2 cell-entry, canonical mediators and enhance protective signaling in human alveolar cells.

The pro-homeostatic lipid mediators elovanoids (ELVs) attenuate cell binding and entrance of the SARS-CoV-2 receptor-binding domain (RBD) as well as of the SARS-CoV-2 virus in human primary alveoli cells in culture. We uncovered that very-long-chain polyunsaturated fatty acid precursors (VLC-PUFA, n-3) activate ELV biosynthesis in lung cells. Both ELVs and their precursors reduce the binding to RBD. ELVs downregulate angiotensin-converting enzyme 2 (ACE2) and enhance the expression of a set of protective proteins hindering cell surface virus binding and upregulating defensive proteins against lung damage. In addition, ELVs and their precursors decreased the signal of spike (S) protein found in SARS-CoV-2 infected cells, suggesting that the lipids curb viral infection. These findings open avenues for potential preventive and disease-modifiable therapeutic approaches for COVID-19.

Rescue and repair during photoreceptor cell renewal mediated by docosahexaenoic acid-derived neuroprotectin D1.

Retinal degenerative diseases result in retinal pigment epithelial (RPE) and photoreceptor cell loss. These cells are continuously exposed to the environment (light) and to potentially pro-oxidative conditions, as the retina's oxygen consumption is very high. There is also a high flux of docosahexaenoic acid (DHA), a PUFA that moves through the blood stream toward photoreceptors and between them and RPE cells. Photoreceptor outer segment shedding and phagocytosis intermittently renews photoreceptor membranes. DHA is converted through 15-lipoxygenase-1 into neuroprotectin D1 (NPD1), a potent mediator that evokes counteracting cell-protective, anti-inflammatory, pro-survival repair signaling, including the induction of anti-apoptotic proteins and inhibition of pro-apoptotic proteins. Thus, NPD1 triggers activation of signaling pathway/s that modulate/s pro-apoptotic signals, promoting cell survival. This review provides an overview of DHA in photoreceptors and describes the ability of RPE cells to synthesize NPD1 from DHA. It also describes the role of neurotrophins as agonists of NPD1 synthesis and how photoreceptor phagocytosis induces refractoriness to oxidative stress in RPE cells, with concomitant NPD1 synthesis.

Neuroprotectin D1 modulates the induction of pro-inflammatory signaling and promotes retinal pigment epithelial cell survival during oxidative stress.

Retinal pigment epithelial (RPE) cells are the most restrictive layer of the three components of the outer Blood-Retina Barrier, preventing the passage of biomolecules in relation to size and charge and thus preserving a controlled environment for the photoreceptors. The retinal pigment epithelium is a tight structure that, when disrupted as a cause or consequence of pathological conditions, deeply affects the neural retina. Since adult human RPE cells are not replicative cells, their preservation is of major interest for the biomedical field due to their loss in many retino-degenerative pathologies. There are several triggers that elicit reactive oxygen species (ROS) formation in normal and pathological circumstances. When the production of these species overwhelms the scavenging and detoxifying systems, their activity results in programmed cell death. Docosahexaenoic acid (DHA) is an essential lipid that is conspicuously accumulated in photoreceptors and RPE cells in the retina. DHA and its oxygenation product, neuroprotectin D1 (NPD1), are major players in the protection of these cells and the retina. NPD1 promotes the synthesis of anti-apoptotic proteins of certain members of the Bcl-2 family and blocks the expression of pro-inflammatory proteins like cyclooxygenase-2.

Neuroprotectin D1 upregulates Iduna expression and provides protection in cellular uncompensated oxidative stress and in experimental ischemic stroke.

Ring finger protein 146 (Iduna) facilitates DNA repair and protects against cell death induced by NMDA receptor-mediated glutamate excitotoxicity or by cerebral ischemia. Neuroprotectin D1 (NPD1), a docosahexaenoic acid (DHA)-derived lipid mediator, promotes cell survival under uncompensated oxidative stress (UOS). Our data demonstrate that NPD1 potently upregulates Iduna expression and provides remarkable cell protection against UOS. Iduna, which was increased by the lipid mediator, requires the presence of the poly(ADP-ribose) (PAR) sites. Moreover, astrocytes and neurons in the penumbra display an enhanced abundance of Iduna, followed by remarkable neurological protection when DHA, a precursor of NPD1, is systemically administered 1 h after 2 h of ischemic stroke. These findings provide a conceptual advancement for survival of neural cells undergoing challenges to homeostasis because a lipid mediator, made 'on demand,' modulates the abundance of a critically important protein for cell survival.

Adiponectin receptor 1 conserves docosahexaenoic acid and promotes photoreceptor cell survival.

The identification of pathways necessary for photoreceptor and retinal pigment epithelium (RPE) function is critical to uncover therapies for blindness. Here we report the discovery of adiponectin receptor 1 (AdipoR1) as a regulator of these cells' functions. Docosahexaenoic acid (DHA) is avidly retained in photoreceptors, while mechanisms controlling DHA uptake and retention are unknown. Thus, we demonstrate that AdipoR1 ablation results in DHA reduction. In situ hybridization reveals photoreceptor and RPE cell AdipoR1 expression, blunted in AdipoR1(-/-) mice. We also find decreased photoreceptor-specific phosphatidylcholine containing very long-chain polyunsaturated fatty acids and severely attenuated electroretinograms. These changes precede progressive photoreceptor degeneration in AdipoR1(-/-) mice. RPE-rich eyecup cultures from AdipoR1(-/-) reveal impaired DHA uptake. AdipoR1 overexpression in RPE cells enhances DHA uptake, whereas AdipoR1 silencing has the opposite effect. These results establish AdipoR1 as a regulatory switch of DHA uptake, retention, conservation and elongation in photoreceptors and RPE, thus preserving photoreceptor cell integrity.

Docosahexaenoic acid and its derivative neuroprotectin D1 display neuroprotective properties in the retina, brain and central nervous system.

The significance of the selective enrichment in omega-3 essential fatty acids (docosahexaenoyl - DHA - chains of membrane phospholipids, 22C and 6 double bonds) in the nervous system (e.g. synaptic membranes and dendrites) has remained, until recently, incompletely understood. While studying mechanisms of neuronal survival, we contributed to the discovery of a docosanoid synthesized by 15-lipoxygenase-1 from DHA, which we dubbed neuroprotectin D1 (NPD1;10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15E,19Z hexaenoic acid). NPD1 is a docosanoid because it is derived from a 22C precursor (DHA), unlike eicosanoids, which are derived from the 20C arachidonic acid family of essential fatty acids not enriched in the nervous system. We found that NPD1 is promptly made in response to oxidative stress, seizures and brain ischemia-reperfusion. NPD1 is neuroprotective in experimental brain damage, retinal pigment epithelial cells, and in human brain cells. Thus, NPD1 acts as a protective sentinel, one of the very first defenses activated when cell homeostasis is threatened by neurodegenerations. NPD1 also has been shown to have a specificity and potency that provides beneficial bioactivity during initiation and early progression of neuronal and retinal degenerations, epilepsy and stroke. In short, NPD1 regulation promotes homeostatic regulation of neural circuitry integrity.

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 alpha/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.