CD44 signaling in Müller cells impacts photoreceptor function and survival in healthy and diseased retinas.

Retinitis pigmentosa (RP), an inherited retinal disease, affects 1,5 million people worldwide. The initial mutation-driven photoreceptor degeneration leads to chronic inflammation, characterized by Müller cell activation and upregulation of CD44. CD44 is a cell surface transmembrane glycoprotein and the primary receptor for hyaluronic acid. It is involved in many pathological processes, but little is known about CD44's retinal functions. CD44 expression is also increased in Müller cells from our Pde6bSTOP/STOP RP mouse model. To gain a more detailed understanding of CD44's role in healthy and diseased retinas, we analyzed Cd44-/- and Cd44-/-Pde6bSTOP/STOP mice, respectively. The loss of CD44 led to enhanced photoreceptor degeneration, reduced retinal function, and increased inflammatory response. To understand the underlying mechanism, we performed proteomic analysis on isolated Müller cells from Cd44-/- and Cd44-/-Pde6bSTOP/STOP retinas and identified a significant downregulation of glutamate transporter 1 (SLC1A2). This downregulation was accompanied by higher glutamate levels, suggesting impaired glutamate homeostasis. These novel findings indicate that CD44 stimulates glutamate uptake via SLC1A2 in Müller cells, which in turn, supports photoreceptor survival and function.

The glucocorticoid receptor as a master regulator of the Müller cell response to diabetic conditions in mice.

Diabetic retinopathy (DR) is considered a primarily microvascular complication of diabetes. Müller glia cells are at the centre of the retinal neurovascular unit and play a critical role in DR. We therefore investigated Müller cell-specific signalling pathways that are altered in DR to identify novel targets for gene therapy. Using a multi-omics approach on purified Müller cells from diabetic db/db mice, we found the mRNA and protein expression of the glucocorticoid receptor (GR) to be significantly decreased, while its target gene cluster was down-regulated. Further, oPOSSUM TF analysis and ATAC- sequencing identified the GR as a master regulator of Müller cell response to diabetic conditions. Cortisol not only increased GR phosphorylation. It also induced changes in the expression of known GR target genes in retinal explants. Finally, retinal functionality was improved by AAV-mediated overexpression of GR in Müller cells. Our study demonstrates an important role of the glial GR in DR and implies that therapeutic approaches targeting this signalling pathway should be aimed at increasing GR expression rather than the addition of more ligand.

Retinal regions shape human and murine Müller cell proteome profile and functionality.

The human macula is a highly specialized retinal region with pit-like morphology and rich in cones. How Müller cells, the principal glial cell type in the retina, are adapted to this environment is still poorly understood. We compared proteomic data from cone- and rod-rich retinae from human and mice and identified different expression profiles of cone- and rod-associated Müller cells that converged on pathways representing extracellular matrix and cell adhesion. In particular, epiplakin (EPPK1), which is thought to play a role in intermediate filament organization, was highly expressed in macular Müller cells. Furthermore, EPPK1 knockout in a human Müller cell-derived cell line led to a decrease in traction forces as well as to changes in cell size, shape, and filopodia characteristics. We here identified EPPK1 as a central molecular player in the region-specific architecture of the human retina, which likely enables specific functions under the immense mechanical loads in vivo.

Expression and subcellular localization of USH1C/harmonin in human retina provides insights into pathomechanisms and therapy.

Usher syndrome (USH) is the most common form of hereditary deaf-blindness in humans. USH is a complex genetic disorder, assigned to three clinical subtypes differing in onset, course and severity, with USH1 being the most severe. Rodent USH1 models do not reflect the ocular phenotype observed in human patients to date; hence, little is known about the pathophysiology of USH1 in the human eye. One of the USH1 genes, USH1C, exhibits extensive alternative splicing and encodes numerous harmonin protein isoforms that function as scaffolds for organizing the USH interactome. RNA-seq analysis of human retinae uncovered harmonin_a1 as the most abundant transcript of USH1C. Bulk RNA-seq analysis and immunoblotting showed abundant expression of harmonin in Müller glia cells (MGCs) and retinal neurons. Furthermore, harmonin was localized in the terminal endfeet and apical microvilli of MGCs, presynaptic region (pedicle) of cones and outer segments (OS) of rods as well as at adhesive junctions between MGCs and photoreceptor cells (PRCs) in the outer limiting membrane (OLM). Our data provide evidence for the interaction of harmonin with OLM molecules in PRCs and MGCs and rhodopsin in PRCs. Subcellular expression and colocalization of harmonin correlate with the clinical phenotype observed in USH1C patients. We also demonstrate that primary cilia defects in USH1C patient-derived fibroblasts could be reverted by the delivery of harmonin_a1 transcript isoform. Our studies thus provide novel insights into PRC cell biology, USH1C pathophysiology and development of gene therapy treatment(s).

Release of VAMP5-positive extracellular vesicles by retinal Müller glia in vivo.

Cell-cell interactions in the central nervous system are based on the release of molecules mediating signal exchange and providing structural and trophic support through vesicular exocytosis and the formation of extracellular vesicles. The specific mechanisms employed by each cell type in the brain are incompletely understood. Here, we explored the means of communication used by Müller cells, a type of radial glial cells in the retina, which forms part of the central nervous system. Using immunohistochemical, electron microscopic, and molecular analyses, we provide evidence for the release of distinct extracellular vesicles from endfeet and microvilli of retinal Müller cells in adult mice in vivo. We identify VAMP5 as a Müller cell-specific SNARE component that is part of extracellular vesicles and responsive to ischemia, and we reveal differences between the secretomes of immunoaffinity-purified Müller cells and neurons in vitro. Our findings suggest extracellular vesicle-based communication as an important mediator of cellular interactions in the retina.

As in Real Estate, Location Matters: Cellular Expression of Complement Varies Between Macular and Peripheral Regions of the Retina and Supporting Tissues.

The cellular events that dictate the initiation of the complement pathway in ocular degeneration, such as age-related macular degeneration (AMD), is poorly understood. Using gene expression analysis (single cell and bulk), mass spectrometry, and immunohistochemistry, we dissected the role of multiple retinal and choroidal cell types in determining the complement homeostasis. Our scRNA-seq data show that the cellular response to early AMD is more robust in the choroid, particularly in fibroblasts, pericytes and endothelial cells. In late AMD, complement changes were more prominent in the retina especially with the expression of the classical pathway initiators. Notably, we found a spatial preference for these differences. Overall, this study provides insights into the heterogeneity of cellular responses for complement expression and the cooperation of neighboring cells to complete the pathway in healthy and AMD eyes. Further, our findings provide new cellular targets for therapies directed at complement.

The role of Müller cell glucocorticoid signaling in diabetic retinopathy.

Diabetic retinopathy (DR) is a sight-threatening complication associated with the highly prevalent diabetes disorder. Both the microvascular damage and neurodegeneration detected in the retina caused by chronic hyperglycemia have brought special attention to Müller cells, the major macroglia of the retina that are responsible for retinal homeostasis. Given the role of glucocorticoid signaling in anti-inflammatory responses and the almost exclusive expression of glucocorticoid receptors (GRs) in retinal Müller cells, administration of corticosteroid agonists as a potential treatment option has been widely studied. Although these approaches have been moderately efficacious in treating or de-escalating DR pathomechanisms, there are various side effects and gaps of knowledge with regard to introducing exogenous glucocorticoids to the diseased retina. In this paper, we provide a review of the literature concerning the available evidence for the role of Müller cell glucocorticoid signaling in DR and we discuss previously investigated approaches in modulating this system as possible treatment options. Furthermore, we propose a novel alternative to the available choices of treatment by using gene therapy as a tool to regulate the expression of GR in retinal Müller cells. Upregulating GR expression allows for induced glucocorticoid signaling with more enduring effects compared to injection of agonists. Hence, repetitive injections would no longer be required. Lastly, side effects of glucocorticoid therapy such as glucocorticoid resistance of GR following chronic exposure to excess ligands or agonists can be avoided.

Bovine polymerized hemoglobin versus Hextend resuscitation in a swine model of severe controlled hemorrhagic shock with delay to definitive care.

To compare the efficacy of low-volume resuscitation with bovine polymerized hemoglobin (HBOC-201) versus hetastarch (HEX) in an intermediate severity combat-relevant hemorrhagic shock swine model with a simulated delay to hospital care. Twenty-four anesthetized pigs were hemorrhaged 55% estimated blood volume in conjunction with a 5-min rectus abdominus crush. At 20 min, pigs were resuscitated with 10 mL/kg of HBOC-201 or HEX or nothing (NON); resuscitated pigs received additional infusions (5 mL/kg) at 30, 60, 120, or 180 min if hypotension or tachycardia persisted. Pigs were monitored for a 4-h "prehospital" period. At 4-h, hospital arrival was simulated: surgical sites were repaired, blood, or saline provided, and pigs were recovered from anesthesia. Pigs were monitored for 72 h and then killed for histological evaluation. One hundred percent (8/8) of HBOC-201-, 75% (6/8) of HEX-, and 25% (2/8) of NON-resuscitated pigs survived to 72 h (P = 0.007 overall, HBOC vs. HEX P > 0.05). Mean arterial pressure and mean pulmonary arterial pressure were highest in the HBOC-201 group (P < 0.001), and HR was lowest (P < 0.001). HBOC-201- and HEX-resuscitated pigs had comparable cardiac index and prehospital fluid requirements. HBOC-201 pigs had higher transcutaneous tissue oxygen tension, P < 0.001) and lower urine output (P < 0.001). At simulated hospital arrival, no HBOC-201 pigs required additional fluids or blood transfusion. In contrast, 100% of HEX pigs required blood transfusions (P < 0.01). In this swine model of controlled hemorrhage with low-volume resuscitation and delayed definitive care, HBOC-201 pigs had improved hemodynamics, transcutaneous tissue oxygen tension, and transfusion avoidance compared with HEX.