Jorgelina M. Calandria

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 retinas 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.

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.

CORRELATION BETWEEN CORTISOL LEVEL AND SEROTONIN UPTAKE IN PATIENTS WITH CHRONIC STRESS AND DEPRESSION

In a recent study (Tafet, Toister-Achituv, & Shinitzky, 2001), we demonstrated that cortisol induces an increase in the expression of the gene coding for the serotonin transporter, associated with a subsequent elevation in the uptake of serotonin. This stimulatory effect, produced upon incubation with cortisol in vitro, was observed in peripheral blood lymphocytes from normal subjects. In the present work we investigated the cortisol-induced increase in serotonin uptake in lymphocytes from hypercortisolemic patients, including subjects with major depressive disorder (n = 8), and subjects with generalized anxiety disorder (n = 12), in comparison with a control group of normal healthy subjects (n = 8). A significant increase in serotonin uptake (+37%+14, M + SD) was observed in the control group, whereas neither the generalized anxiety disorder nor the major depression group exhibited changes in serotonin uptake upon incubation with cortisol. It is likely that under chronic stress or depression, the capacity for increase in serotonin transporter has reached its limit due to the chronically elevated blood cortisol level. The physiological and diagnostic implications of this observation are discussed.

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.

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.

NPD1-mediated stereoselective regulation of BIRC3 expression through cREL is decisive for neural cell survival.

Neuroprotectin D1 (NPD1), a docosahexaenoic acid (DHA)-derived mediator, induces cell survival in uncompensated oxidative stress (OS), neurodegenerations or ischemic stroke. The molecular principles underlying this protection remain unresolved. We report here that, in retinal pigment epithelial cells, NPD1 induces nuclear translocation and cREL synthesis that, in turn, mediates BIRC3 transcription. NPD1 activates NF-κB by an alternate route to canonical signaling, so the opposing effects of TNFR1 and NPD1 on BIRC3 expression are not due to interaction/s between NF-κB pathways. RelB expression follows a similar pattern as BIRC3, indicating that NPD1 also is required to activate cREL-mediated RelB expression. These results suggest that cREL, which follows a periodic pattern augmented by the lipid mediator, regulates a cluster of NPD1-dependent genes after cREL nuclear translocation. BIRC3 silencing prevents NPD1 induction of survival against OS. Moreover, brain NPD1 biosynthesis and selective neuronal BIRC3 abundance are increased by DHA after experimental ischemic stroke followed by remarkable neurological recovery. Thus, NPD1 bioactivity governs key counter-regulatory gene transcription decisive for retinal and brain neural cell integrity when confronted with potential disruptions of homeostasis.

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 1u2009h after 2u2009h 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.

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 80xa0% 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 17–20xa0% of neurons were TH-positive when differentiated in vitro. NPD1 (100xa0nM) 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 125xa0µm, 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.

Altered protein mannosylation in developing cerebral cortex by streptomycin.

Our research objective was to characterize the biochemical effect of streptomycin during postnatal rat cerebral cortex development using a sensitive method that preserves the in situ topological relationship. We found a decrease in the mannosylation of asparagine-linked oligosaccharides without affecting polypeptide synthesis, DNA synthesis or glucose and mannose disappearance from the medium in mini-tissue units derived from P5. In addition, the rate of Dolp-GlcNAc2Man9Glc3 synthesis and the oligosaccharide protein transferase activity did not change in the presence of the aminoglycoside. These findings strongly suggested that the alteration of protein mannosylation occurred downstream of G3 transfer to nascent polypeptides. Further, the mini-tissue units may be useful for the assessment of neurological toxicity of antibacterial agents.