Rod-specific downregulation of omega-3 very-long-chain polyunsaturated fatty acid pathway in age-related macular degeneration.

Docosahexaenoic acid (DHA; 22:6) plays a key role in vision and is the precursor for very-long-chain polyunsaturated fatty acids (VLC-PUFAs). The release of 32- and 34-carbon VLC-PUFAs and DHA from sn-1 and sn-2 of phosphatidylcholine (PC) leads to the synthesis of cell-survival mediators, the elovanoids (ELVs) and neuroprotectin D1 (NPD1), respectively. Macula and periphery from age-related macular degeneration (AMD) donor retinas were assessed for the availability of DHA-related lipids by LC-MS/MS-based lipidomic analysis and MALDI-molecular imaging. We found reduced retina DHA and VLC-PUFA pathways to synthesize omega-3 ELVs from precursors that likely resulted in altered disks and photoreceptor loss. Additionally, we compared omega-3 (n-3) fatty acid with DHA (22:6) and omega-6 (n-6) fatty acid with arachidonic acid (AA; 20:4) pathways. n-3 PC(22:6/22:6, 44:12) and n-6 PC(20:4/20:4, 40:8) showed differences among male/female, macula/periphery, and normal/AMD retinas. Periphery of AMD retina males increased 44:12 abundance, while normal females increased 40:8 (all macula had an upward 40:8 tendency). We also showed that female AMD switched from n-3 to n-6 fatty acids; most changes in AMD occurred in the periphery of female AMD retinas. DHA and VLC-PUFA release from PCs leads to conversion in pro-survival NPD1 and ELVs. The loss of the neuroprotective precursors of ELVs in the retina periphery from AMD facilitates uncompensated stress and cell loss. In AMD, the female retina loses peripheral rods VLC-PUFAs to about 33% less than in males limiting ELV formation and its protective bioactivity.

NPD1 Plus RvD1 Mediated Ischemic Stroke Penumbra Protection Increases Expression of Pro-homeostatic Microglial and Astrocyte Genes.

Neuroprotection to attenuate or block the ischemic cascade and salvage neuronal damage has been extensively explored for treating ischemic stroke. However, despite increasing knowledge of the physiologic, mechanistic, and imaging characterizations of the ischemic penumbra, no effective neuroprotective therapy has been found. This study focuses on the neuroprotective bioactivity of docosanoid mediators: Neuroprotectin D1 (NPD1), Resolvin D1 (RvD1), and their combination in experimental stroke. Molecular targets of NPD1 and RvD1 are defined by following dose-response and therapeutic window. We demonstrated that treatment with NPD1, RvD1, and combination therapy provides high-grade neurobehavioral recovery and decreases ischemic core and penumbra volumes even when administered up to 6 h after stroke. The expression of the following genes was salient: (a) Cd163, an anti-inflammatory stroke-associated gene, was the most differentially expressed gene by NPD1+RvD1, displaying more than a 123-fold upregulation in the ipsilesional penumbra (Lisi et al., Neurosci Lett 645:106-112, 2017); (b) 100-fold upregulation takes place in astrocyte gene PTX3, a key regulator of neurogenesis and angiogenesis after cerebral ischemia (. Rodriguez-Grande et al., J Neuroinflammation 12:15, 2015); and (c) Tmem119 and P2y12, two markers of homeostatic microglia, were found to be enhanced by ten- and fivefold, respectively (Walker et al. Int J Mol Sci 21:678, 2020). Overall, we uncovered that protection after middle cerebral artery occlusion (MCAo) by the lipid mediators elicits expression of microglia and astrocyte-specific genes (Tmem119, Fcrls, Osmr, Msr1, Cd68, Cd163, Amigo2, Thbs1, and Tm4sf1) likely participating in enhancing homeostatic microglia, modulating neuroinflammation, promoting DAMP clearance, activating NPC differentiation and maturation, synapse integrity and contributing to cell survival.

Cerebrospinal Fluid Profile of Lipid Mediators in Alzheimer's Disease.

Alzheimer's disease (AD) develops into dementia over a period of several years, during which subjective cognitive impairment (SCI) and mild cognitive impairment (MCI) can be used as intermediary diagnoses of increasing severity. Chronic neuroinflammation resulting from insufficient resolution is involved in the pathogenesis of AD and is associated with cognitive impairment. Specialized pro-resolving lipid mediators (LMs) that promote the resolution of inflammation may be valuable markers in AD diagnosis and as therapeutic targets. Liquid chromatography-tandem mass spectrometry was used to analyze pro-resolving and pro-inflammatory LMs in cerebrospinal fluid (CSF) from patients with cognitive impairment ranging from subjective impairment to a diagnosis of AD and correlated to cognition, CSF tau, and ß-amyloid. Resolvin (Rv) D4, RvD1, neuroprotectin D1 (NPD1), maresin 1 (MaR1), and RvE4 were lower in AD and/or MCI compared to SCI. The pro-inflammatory LTB4 and 15-HETE were higher in AD and MCI, respectively, while PGD2, PGE2, and PGF2a were decreased in AD, compared to SCI. RvD4 was also negatively correlated to AD tangle biomarkers, and positive correlations to cognitive test scores were observed for both pro-resolving LMs and their precursor fatty acids. In this exploratory study of the lipidome in CSF of AD, MCI, and SCI, the results indicate a shift in the LM profile from pro-resolving to pro-inflammatory in progression to AD, suggesting that it may be of use as a biomarker when followed by confirmation by replication studies.

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.

Age-related changes in brain phospholipids and bioactive lipids in the APP knock-in mouse model of Alzheimer's disease.

Sustained brain chronic inflammation in Alzheimer's disease (AD) includes glial cell activation, an increase in cytokines and chemokines, and lipid mediators (LMs), concomitant with decreased pro-homeostatic mediators. The inflammatory response at the onset of pathology engages activation of pro-resolving, pro-homeostatic LMs followed by a gradual decrease. We used an APP knock-in (App KI) AD mouse that accumulates ß-amyloid (Aß) and presents cognitive deficits (at 2 and 6 months of age, respectively) to investigate LMs, their precursors, biosynthetic enzymes and receptors, glial activation, and inflammatory proteins in the cerebral cortex and hippocampus at 2-, 4-, 8- and 18-month-old in comparison with wild-type (WT) mice. We used LC-mass-spectrometry and MALDI molecular imaging to analyze LMs and phospholipids, and immunochemistry for proteins. Our results revealed an age-specific lipid and cytokine profile, and glial activation in the App KI mice. Despite an early onset of Aß pathology, pro-inflammatory and pro-resolving LMs were prominently increased only in the oldest age group. Furthermore, the LM biosynthetic enzymes increased, and their receptor expression decreased in the aged App KI mice. Arachidonic acid (AA)-containing phospholipid molecular species were elevated, correlating with decreased cPLA2 activity. MALDI molecular imaging depicted differential distribution of phospholipids according to genotype in hippocampal layers. Brain histology disclosed increased microglia proliferation starting from young age in the App KI mice, while astrocyte numbers were enhanced in older ages. Our results demonstrate that the brain lipidome is modified preferentially during aging as compared to amyloid pathology in the model studied here. However, alterations in phospholipids signal early pathological changes in membrane composition.

Membrane-type frizzled-related protein regulates lipidome and transcription for photoreceptor function.

Molecular decision-makers of photoreceptor (PRC) membrane organization and gene regulation are critical to understanding sight and retinal degenerations that lead to blindness. Using Mfrprd6 mice, which develop PRC degeneration, we uncovered that membrane-type frizzled-related protein (MFRP) participates in docosahexaenoic acid (DHA, 22:6) enrichment in a manner similar to adiponectin receptor 1 (AdipoR1). Untargeted imaging mass spectrometry demonstrates cell-specific reduction of phospholipids containing 22:6 and very long-chain polyunsaturated fatty acids (VLC-PUFAs) in Adipor1-/- and Mfrprd6 retinas. Gene expression of pro-inflammatory signaling pathways is increased and gene-encoding proteins for PRC function decrease in both mutants. Thus, we propose that both proteins are necessary for retinal lipidome membrane organization, visual function, and to the understanding of the early pathology of retinal degenerative diseases.

Elovanoids counteract oligomeric ß-amyloid-induced gene expression and protect photoreceptors.

The onset of neurodegenerative diseases activates inflammation that leads to progressive neuronal cell death and impairments in cognition (Alzheimer's disease) and sight (age-related macular degeneration [AMD]). How neuroinflammation can be counteracted is not known. In AMD, amyloid ß-peptide (Aß) accumulates in subretinal drusen. In the 5xFAD retina, we found early functional deficiencies (ERG) without photoreceptor cell (PRC) death and identified early insufficiency in biosynthetic pathways of prohomeostatic/neuroprotective mediators neuroprotectin D1 (NPD1) and elovanoids (ELVs). To mimic an inflammatory milieu in wild-type mouse, we triggered retinal pigment epithelium (RPE) damage/PRC death by subretinally injected oligomeric ß-amyloid (OAß) and observed that ELVs administration counteracted their effects, protecting these cells. In addition, ELVs prevented OAß-induced changes in gene expression engaged in senescence, inflammation, autophagy, extracellular matrix remodeling, and AMD. Moreover, as OAß targets the RPE, we used primary human RPE cell cultures and demonstrated that OAß caused cell damage, while ELVs protected and restored gene expression as in mouse. Our data show OAß activates senescence as reflected by enhanced expression of p16INK4a, MMP1, p53, p21, p27, and Il-6, and of senescence-associated phenotype secretome, followed by RPE and PRC demise, and that ELVs 32 and 34 blunt these events and elicit protection. In addition, ELVs counteracted OAß-induced expression of genes engaged in AMD, autophagy, and extracellular matrix remodeling. Overall, our data uncovered that ELVs downplay OAß-senescence program induction and inflammatory transcriptional events and protect RPE cells and PRC, and therefore have potential as a possible therapeutic avenue for AMD.

Elovanoids are a novel class of homeostatic lipid mediators that protect neural cell integrity upon injury.

We report the characterization of a novel class of lipid mediators termed elovanoids (ELVs) (ELV-N32 and ELV-N34), which are dihydroxylated derivatives of 32:6n3 and 34:6n3, respectively. The precursors of ELVs are made by elongation of a 22:6n3 fatty acid and catalyzed by ELOVL4 (elongation of very-long-chain fatty acids-4). The structure and stereochemistry of ELVs were established using synthetic compounds produced by stereocontrolled total synthesis. We report that ELV-mediated protection is induced in neuronal cultures undergoing either oxygen/glucose deprivation or N-methyl-d-aspartate receptor-mediated excitotoxicity, as well as in experimental ischemic stroke. The methyl ester or sodium salt of ELV-N32 and ELV-N34 resulted in reduced infarct volumes, promoted cell survival, and diminished neurovascular unit disruption when administered 1 hour following 2 hours of ischemia by middle cerebral artery occlusion. Together, our data reveal a novel prohomeostatic and neuroprotective lipid-signaling mechanism aiming to sustain neural cell integrity.

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.

OTX2 loss causes rod differentiation defect in CRX-associated congenital blindness.

Leber congenital amaurosis (LCA) encompasses a set of early-onset blinding diseases that are characterized by vision loss, involuntary eye movement, and nonrecordable electroretinogram (ERG). At least 19 genes are associated with LCA, which is typically recessive; however, mutations in homeodomain transcription factor CRX lead to an autosomal dominant form of LCA. The mechanism of CRX-associated LCA is not understood. Here, we identified a spontaneous mouse mutant with a frameshift mutation in Crx (CrxRip). We determined that CrxRip is a dominant mutation that results in congenital blindness with nonrecordable response by ERG and arrested photoreceptor differentiation with no associated degeneration. Expression of LCA-associated dominant CRX frameshift mutations in mouse retina mimicked the CrxRip phenotype, which was rescued by overexpression of WT CRX. Whole-transcriptome profiling using deep RNA sequencing revealed progressive and complete loss of rod differentiation factor NRL in CrxRip retinas. Expression of NRL partially restored rod development in CrxRip/+ mice. We show that the binding of homeobox transcription factor OTX2 at the Nrl promoter was obliterated in CrxRip mice and ectopic expression of OTX2 rescued the rod differentiation defect. Together, our data indicate that OTX2 maintains Nrl expression in developing rods to consolidate rod fate. Our studies provide insights into CRX mutation-associated congenital blindness and should assist in therapeutic design.

The transcription-splicing protein NonO/p54nrb and three NonO-interacting proteins bind to distal enhancer region and augment rhodopsin expression.

Phototransduction machinery in vertebrate photoreceptors is contained within the membrane discs of outer segments. Daily renewal of 10% of photoreceptor outer segments requires stringent control of gene expression. Rhodopsin constitutes over 90% of the protein in rod discs, and its altered expression or transport is associated with photoreceptor dysfunction and/or death. Two cis-regulatory sequences have been identified upstream of the rhodopsin transcription start site. While the proximal promoter binds to specific transcription factors, including NRL and CRX, the rhodopsin enhancer region (RER) reportedly contributes to precise and high-level expression of rhodopsin in vivo. Here, we report the identification of RER-bound proteins by mass spectrometry. We validate the binding of NonO (p54(nrb)), a protein implicated in coupling transcription to splicing, and three NonO-interacting proteins-hnRNP M, Ywhaz and Ppp1ca. NonO and its interactors can activate rhodopsin promoter in HEK293 cells and function synergistically with NRL and CRX. DNA-binding domain of NonO is critical for rhodopsin promoter activation. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) analysis demonstrates high occupancy of NonO at rhodopsin and a subset of phototransduction genes. Furthermore, shRNA knockdown of NonO in mouse retina leads to loss of rhodopsin expression and rod cell death, which can be partially rescued by a C-terminal NonO construct. RNA-seq analysis of the NonO shRNA-treated retina revealed splicing defects and altered expression of genes, specifically those associated with phototransduction. Our studies identify an important contribution of NonO and its interacting modulator proteins in enhancing rod-specific gene expression and controlling rod homeostasis.

Combinatorial regulation of photoreceptor differentiation factor, neural retina leucine zipper gene NRL, revealed by in vivo promoter analysis.

Development and homeostasis require stringent spatiotemporal control of gene expression patterns that are established, to a large extent, by combinatorial action of transcription regulatory proteins. The bZIP transcription factor NRL (neural retina leucine zipper) is critical for rod versus cone photoreceptor cell fate choice during retinal development and acts as a molecular switch to produce rods from postmitotic precursors. Loss of Nrl in mouse leads to a cone-only retina, whereas ectopic expression of Nrl in photoreceptor precursors generates rods. To decipher the transcriptional regulatory mechanisms upstream of Nrl, we identified putative cis-control elements in the Nrl promoter/enhancer region by examining cross-species sequence conservation. Using in vivo transfection of promoter-reporter constructs into the mouse retina, we show that a 0.9-kb sequence upstream of the Nrl transcription initiation site is sufficient to drive reporter gene expression in photoreceptors. We further define a 0.3-kb sequence including a proximal promoter (cluster A1) and an enhancer (cluster B) that can direct rod-specific expression in vivo. Electrophoretic mobility shift assays using mouse retinal nuclear extracts, in combination with specific antibodies, demonstrate the binding of retinoid-related orphan nuclear receptor ß (RORß), cone rod homeobox, orthodenticle homolog 2, and cyclic AMP response element-binding protein to predicted consensus elements within clusters A and B. Our studies demonstrate Nrl as a direct transcriptional target of RORß and suggest that combinatorial action of multiple regulatory factors modulates the expression of Nrl in developing and mature retina.