In the Eyes of the Beholder-New Knockout Mouse and Re-Evaluation of Phagocytosis versus Anti-Inflammatory Functions of MERTK.

Greg Lemke's laboratory was one of the pioneers of research into the TAM family of receptor tyrosine kinases (RTKs). Not only was cloned in his laboratory, but his group also extensively studied mice knocked out for individual or various combinations of the TAM RTKs , , and . Here we primarily focus on one of the paralogs-MERTK.

A novel quantification method for retinal pigment epithelium phagocytosis using a very-long-chain polyunsaturated fatty acids-based strategy.

Introduction: The vertebrate retinal pigment epithelium (RPE) lies adjacent to the photoreceptors and is responsible for the engulfment and degradation of shed photoreceptor outer segment fragments (POS) through receptor-mediated phagocytosis. Phagocytosis of POS is critical for maintaining photoreceptor function and is a key indicator of RPE functionality. Popular established methods to assess RPE phagocytosis rely mainly on quantifying POS proteins, especially their most abundant protein rhodopsin, or on fluorescent dye conjugation of bulk, unspecified POS components.

Clearance phagocytosis by the retinal pigment epithelial during photoreceptor outer segment renewal: Molecular mechanisms and relation to retinal inflammation.

Mammalian photoreceptor outer segment renewal is a highly coordinated process that hinges on timed cell signaling between photoreceptor neurons and the adjacent retinal pigment epithelial (RPE). It is a strictly rhythmic, synchronized process that underlies in part circadian regulation. We highlight findings from recently developed methods that quantify distinct phases of outer segment renewal in retinal tissue.

Inflammation of the retinal pigment epithelium drives early-onset photoreceptor degeneration in -associated retinitis pigmentosa.

Severe, early-onset photoreceptor (PR) degeneration associated with mutations is thought to result from failed phagocytosis by retinal pigment epithelium (RPE). Notwithstanding, the severity and onset of PR degeneration in mouse models of ablation are determined by the hypomorphic expression or the loss of the paralog . Here, we find that loss of and reduced expression/loss of led to RPE inflammation even before eye-opening.

Differences in Diurnal Rhythm of Rod Outer Segment Renewal between 129T2/SvEmsJ and C57BL/6J Mice.

In all mammalian species tested to date, rod photoreceptor outer segment renewal is a circadian process synchronized by light with a burst of outer segment fragment (POS) shedding and POS phagocytosis by the adjacent retinal pigment epithelium (RPE) every morning at light onset. Recent reports show that RPE phagocytosis also increases shortly after dark onset in C57BL/6 (C57) mice. Genetic differences between C57 mice and 129T2/SvEmsJ (129) mice may affect regulation of outer segment renewal.

Tissue-specific modifier alleles determine loss-of-function traits.

Knockout (KO) mouse models play critical roles in elucidating biological processes behind disease-associated or disease-resistant traits. As a presumed consequence of gene KO, mice display certain phenotypes. Based on insight into the molecular role of said gene in a biological process, it is inferred that the particular biological process causally underlies the trait.

Cell culture models to study retinal pigment epithelium-related pathogenesis in age-related macular degeneration.

Age-related macular degeneration (AMD) is a disease that affects the macula - the central part of the retina. It is a leading cause of irreversible vision loss in the elderly. AMD onset is marked by the presence of lipid- and protein-rich extracellular deposits beneath the retinal pigment epithelium (RPE), a monolayer of polarized, pigmented epithelial cells located between the photoreceptors and the choroidal blood supply.

Galectin-3 Promotes Müller Glia Clearance Phagocytosis MERTK and Reduces Harmful Müller Glia Activation in Inherited and Induced Retinal Degeneration.

Clearance phagocytosis is a documented function of Müller glia in the retina. However, the molecular mechanisms of Müller glia phagocytosis remain largely undefined. Here, we show that extracellular galectin-3 and protein S promote clearance phagocytosis by immortalized human MIO-M1 Müller cells in an additive, saturable manner.

Sex-specific multi-level 3D genome dynamics in the mouse brain.

The female mammalian brain exhibits sex hormone-driven plasticity during the reproductive period. Recent evidence implicates chromatin dynamics in gene regulation underlying this plasticity. However, whether ovarian hormones impact higher-order chromatin organization in post-mitotic neurons in vivo is unknown.

Probing Photoreceptor Outer Segment Phagocytosis by the RPE In Vivo: Models and Methodologies.

In the vertebrate retina, the light-sensitive photoreceptor rods and cones constantly undergo renewal by generating new portions of the outer segment and shedding their distal, spent tips. The neighboring RPE provides the critical function of engulfing the spent material by phagocytosis. RPE phagocytosis of shed rod outer segment fragments is a circadian process that occurs in a burst of activity shortly after daily light onset with low activity at other times, a rhythm that has been reported for many species and over 50 years.

Acute RhoA/Rho Kinase Inhibition Is Sufficient to Restore Phagocytic Capacity to Retinal Pigment Epithelium Lacking the Engulfment Receptor MerTK.

The diurnal phagocytosis of spent photoreceptor outer segment fragments (POS) by retinal pigment epithelial (RPE) cells is essential for visual function. POS internalization by RPE cells requires the assembly of F-actin phagocytic cups beneath surface-tethered POS and Mer tyrosine kinase (MerTK) signaling. The activation of the Rho family GTPase Rac1 is necessary for phagocytic cup formation, and Rac1 is activated normally in MerTK-deficient RPE.

Lack of the antioxidant enzyme methionine sulfoxide reductase A in mice impairs RPE phagocytosis and causes photoreceptor cone dysfunction.

Methionine sulfoxide reductase A (MsrA) is a widely expressed antioxidant enzyme that counteracts oxidative protein damage and contributes to protein regulation by reversing oxidation of protein methionine residues. In retinal pigment epithelial (RPE) cells in culture, MsrA overexpression increases phagocytic capacity by supporting mitochondrial ATP production. Here, we show elevated retinal protein carbonylation indicative of oxidation, decreased RPE mitochondrial membrane potential, and attenuated RPE phagocytosis in msra mice.

Diurnal Photoreceptor Outer Segment Renewal in Mice Is Independent of Galectin-3.

Purpose: Galectin-3 (gal-3) is a soluble glycoprotein that has been associated with diverse forms of phagocytosis, including some mediated by the engulfment receptor MerTK. Retinal pigment epithelium (RPE) in vivo uses MerTK (or the related Tyro3) for phagocytosis of shed outer segment fragments during diurnal outer segment renewal. Here, we test if gal-3 plays a role in outer segment renewal in mice and if exogenous gal-3 can promote MerTK-dependent engulfment of isolated outer segment fragments by primary RPE cells in culture.

Microglia Inhibition Delays Retinal Degeneration Due to MerTK Phagocytosis Receptor Deficiency.

Retinitis Pigmentosa (RP) is a group of inherited retinal diseases characterized by progressive loss of rod followed by cone photoreceptors. An especially early onset form of RP with blindness in teenage years is caused by mutations in , the gene encoding the clearance phagocytosis receptor Mer tyrosine kinase (MerTK). The cause for blindness in mutant MerTK-associated RP (mutMerTK-RP) is the failure of retinal pigment epithelial cells in diurnal phagocytosis of spent photoreceptor outer segment debris.

No Difference Between Age-Matched Male and Female C57BL/6J Mice in Photopic and Scotopic Electroretinogram a- and b-Wave Amplitudes or in Peak Diurnal Outer Segment Phagocytosis by the Retinal Pigment Epithelium.

Mice provide informative models of enormous utility for eye research. Sex as biological variable must be considered when conducting studies exploring mouse models. To determine if sex confounds neural retina or retinal pigment epithelium (RPE) activity in wild-type C57BL/6J mice, we compared male and female mice with respect to retinal light response and RPE phagocytosis.

Annexin A5 regulates surface αvβ5 integrin for retinal clearance phagocytosis.

Diurnal clearance phagocytosis by the retinal pigment epithelium (RPE) is a conserved efferocytosis process whose binding step is mediated by αvβ5 integrin receptors. Two related annexins, A5 (ANXA5) and A6 (ANXA6), share an αvβ5 integrin-binding motif. Here, we report that ANXA5, but not ANXA6, regulates the binding capacity for spent photoreceptor outer segment fragments or apoptotic cells by fibroblasts and RPE.

Non-invasive in vivo fluorescence imaging of apoptotic retinal photoreceptors.

Phosphatidylserine externalization is an early molecular signature for apoptosis. In many retinal degenerative diseases, photoreceptor neurons die by apoptosis. Here, we report utility of the phosphatidylserine-binding conjugate of Bis(zinc(II)-dipicolylamine (Zn-DPA) with Texas-red (PSVue-550) in transiently labeling apoptotic photoreceptors in living pigmented or albino rats and mice with retinal degeneration.

Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration.

Recessive Stargardt disease (STGD1) is an inherited blinding disorder caused by mutations in the gene. ABCA4 is a flippase in photoreceptor outer segments (OS) that translocates retinaldehyde conjugated to phosphatidylethanolamine across OS disc membranes. Loss of ABCA4 in mice and STGD1 patients causes buildup of lipofuscin in the retinal pigment epithelium (RPE) and degeneration of photoreceptors, leading to blindness.

Advanced Analysis of Photoreceptor Outer Segment Phagocytosis by RPE Cells in Culture.

Retinal pigment epithelial (RPE) cells are among the most actively phagocytic cells in nature. Primary RPE and stable RPE cell lines provide experimental model systems that possess the same phagocytic machinery as RPE in situ. Upon experimental challenge with isolated photoreceptor outer segment fragments (POS), these cells promptly and efficiently recognize, bind, internalize, and digest POS.

Expression and characterization of αvβ5 integrin on intestinal macrophages.

Macrophages play a crucial role in maintaining homeostasis in the intestine, but the underlying mechanisms have not yet been elucidated fully. Here, we show for the first time that mature intestinal macrophages in mouse intestine express high levels of αvβ5 integrin, which acts as a receptor for the uptake of apoptotic cells and can activate molecules involved in several aspects of tissue homeostasis such as angiogenesis and remodeling of the ECM. αvβ5 is not expressed by other immune cells in the intestine, is already present on intestinal macrophages soon after birth, and its expression is not dependent on the microbiota.

Quantified F-Actin Morphology Is Predictive of Phagocytic Capacity of Stem Cell-Derived Retinal Pigment Epithelium.

With stem cell-derived retinal pigment epithelial (RPE) replacement therapies in clinical testing, establishing potency of RPE prior to transplantation is imperative. Phagocytosis of photoreceptor outer segment fragments (POS) is a key indicator of RPE functionality. Comparing RPE derived from different donor human adult RPE stem cell lines, we found that cells were either high-phagocytic or low-phagocytic despite sharing phagocytic receptors and ligands, junctional ZO-1, and lack of epithelial-mesenchymal transition.

The Developmental Stage of Adult Human Stem Cell-Derived Retinal Pigment Epithelium Cells Influences Transplant Efficacy for Vision Rescue.

Age-related macular degeneration (AMD) is a common cause of central visual loss in the elderly. Retinal pigment epithelial (RPE) cell loss occurs early in the course of AMD and RPE cell transplantation holds promise to slow disease progression. We report that subretinal transplantation of RPE stem cell (RPESC)-derived RPE cells (RPESC-RPE) preserved vision in a rat model of RPE cell dysfunction.

Semaphorin4D-PlexinB1 Signaling Attenuates Photoreceptor Outer Segment Phagocytosis by Reducing Rac1 Activity of RPE Cells.

Semaphorins form a family of secreted and membrane-bound molecules that were identified originally as axonal guidance factors during neuronal development. Given their wide distribution in many including mature tissues, semaphorin 4D (sema4D) and its main functional receptor plexin B1 (plxnB1) are expected to fulfill additional functions that remain to be uncovered. A main characteristic of the plexin receptor family is its ability to reorganize the cytoskeleton.

Retinal Pre-Conditioning by CD59a Knockout Protects against Light-Induced Photoreceptor Degeneration.

Complement dysregulation plays a key role in the pathogenesis of age-related macular degeneration (AMD), but the specific mechanisms are incompletely understood. Complement also potentiates retinal degeneration in the murine light damage model. To test the retinal function of CD59a, a complement inhibitor, CD59a knockout (KO) mice were used for light damage (LD) experiments.

Dimerization deficiency of enigmatic retinitis pigmentosa-linked rhodopsin mutants.

Retinitis pigmentosa (RP) is a blinding disease often associated with mutations in rhodopsin, a light-sensing G protein-coupled receptor and phospholipid scramblase. Most RP-associated mutations affect rhodopsin's activity or transport to disc membranes. Intriguingly, some mutations produce apparently normal rhodopsins that nevertheless cause disease.