The cytokine IL-27 reduces inflammation and protects photoreceptors in a mouse model of retinal degeneration.

BACKGROUND: Retinal degenerative diseases are a group of conditions characterized by photoreceptor death and vision loss. Excessive inflammation and microglial activation contribute to the pathology of retinal degenerations and a major focus in the field is identifying more effective anti-inflammatory therapeutic strategies that promote photoreceptor survival. A major challenge to developing anti-inflammatory treatments is to selectively suppress detrimental inflammation while maintaining beneficial inflammatory responses. We recently demonstrated that endogenous levels of the IL-27 cytokine were upregulated in association with an experimental treatment that increased photoreceptor survival. IL-27 is a pleiotropic cytokine that regulates tissue reactions to infection, neuronal disease and tumors by inducing anti-apoptotic and anti-inflammatory genes and suppressing pro-inflammatory genes. IL-27 is neuroprotective in the brain, but its function during retinal degeneration has not been investigated. In this study, we investigated the effect of IL-27 in the rd10 mouse model of inherited photoreceptor degeneration. METHODS: Male and female rd10 mice were randomly divided into experimental (IL-27) and control (saline) groups and intravitreally injected at age post-natal day (P) 18. Retina function was analyzed by electroretinograms (ERGs), visual acuity by optomotor assay, photoreceptor death by TdT-mediated dUTP nick-end labeling (TUNEL) assay, microglia/macrophage were detected by immunodetection of IBA1 and inflammatory mediators by cytoplex and QPCR analysis. The distribution of IL-27 in the retina was determined by immunohistochemistry on retina cross-sections and primary Muller glia cultures. RESULTS: We demonstrate that recombinant IL-27 decreased photoreceptor death, increased retinal function and reduced inflammation in the rd10 mouse model of retinal degeneration. Furthermore, IL-27 injections led to lower levels of the pro-inflammatory proteins Ccl22, IL-18 and IL-12. IL-27 expression was localized to Muller glia and IL-27 receptors to microglia, which are key cell types that regulate photoreceptor survival. CONCLUSION: Our results identify for the first time anti-inflammatory and neuroprotective activities of IL-27 in a genetic model of retinal degeneration. These findings provide new insight into the therapeutic potential of anti-inflammatory cytokines as a treatment for degenerative diseases of the retina.

Assessment of outer retinal thickness and function in mice after experimental optic nerve trauma.

BACKGROUND: Optic nerve trauma caused by crush injury is frequently used for investigating experimental treatments that protect retinal ganglion cells (RGCs) and induce axonal regrowth. Retaining outer retinal light responses is essential for therapeutic rescue of RGCs after injury. However, whether optic nerve crush also damages the structure or function of photoreceptors has not been systematically investigated. In this study, we investigated whether outer retinal thickness and visual function are altered by optic nerve crush in the mouse. METHODS: Wildtype mice underwent optic nerve crush and intravitreal injection of a control solution in one eye with the fellow eye remaining uninjured. Two weeks after injury, the thickness of the ganglion cell region (GCL to IPL) and photoreceptor layer (bottom of the OPL to top of the RPE) were measured using OCT. Retinal function was assessed using flash ERGs. Immunodetection of RGCs was performed on retinal cryosections and RGCs and ONL nuclei rows were counted. Multiple comparison analyses were conducted using Analysis of Variance (ANOVA) with Tukey's post hoc test and P values less than 0.05 were considered statistically significant. RESULTS: Optic nerve crush injury induced RGC death as expected, demonstrated by thinning of the ganglion cell region and RGC loss. In contrast, outer retinal thickness, photopic and scotopic a-wave and b-wave amplitudes and photoreceptor nuclei counts, were equivalent between injured and uninjured eyes. CONCLUSIONS: Secondary degeneration of the outer retina was not detected after optic nerve injury in the presence of significant RGC death, suggesting that the retina has the capacity to compartmentalize damage. These findings also indicate that experimental treatments to preserve the GCL and rescue vision using this optic nerve injury model would not require additional strategies to preserve the ONL.

Quantitative proteomic analysis after neuroprotective MyD88 inhibition in the retinal degeneration 10 mouse.

Progressive photoreceptor death occurs in blinding diseases such as retinitis pigmentosa. Myeloid differentiation primary response protein 88 (MyD88) is a central adaptor protein for innate immune system Toll-like receptors (TLR) and induces cytokine secretion during retinal disease. We recently demonstrated that inhibiting MyD88 in mouse models of retinal degeneration led to increased photoreceptor survival, which was associated with altered cytokines and increased neuroprotective microglia. However, the identity of additional molecular changes associated with MyD88 inhibitor-induced neuroprotection is not known. In this study, we used isobaric tags for relative and absolute quantification (iTRAQ) labelling followed by LC-MS/MS for quantitative proteomic analysis on the rd10 mouse model of retinal degeneration to identify protein pathways changed by MyD88 inhibition. Quantitative proteomics using iTRAQ LC-MS/MS is a high-throughput method ideal for providing insight into molecular pathways during disease and experimental treatments. Forty-two proteins were differentially expressed in retinas from mice treated with MyD88 inhibitor compared with control. Notably, increased expression of multiple crystallins and chaperones that respond to cellular stress and have anti-apoptotic properties was identified in the MyD88-inhibited mice. These data suggest that inhibiting MyD88 enhances chaperone-mediated retinal protection pathways. Therefore, this study provides insight into molecular events contributing to photoreceptor protection from modulating inflammation.

Wnt signaling induces neurite outgrowth in mouse retinal ganglion cells.

Wingless-type (Wnt) signaling pathways mediate axonal growth and remodeling in the embryonic optic nerve, brain and spinal cord. Recent studies demonstrated that the canonical Wnt/ß-catenin signaling pathway also induces axonal regeneration after injury in the optic nerve of adult animals. However, the molecular mechanisms of Wnt-mediated axonal growth are not well understood. Additionally, because Wnt signaling is stimulated in neurons as well as neighboring non-neuronal cells, the cell type(s) responsible for Wnt-induced axonal regeneration are not known. The objectives of this study were to investigate potential mechanisms and target cells of Wnt3a stimulated neurite growth using primary retinal ganglion cell (RGC) cultures. We demonstrated that Wnt3a ligand induced dose-dependent increases in average neurite length and number of neurites in RGCs. QPCR analysis of candidate mediators showed that Wnt3a-dependent neurite growth was associated with lower expression of Ripk1 and Ripk3 genes. Additionally, inhibiting Ripk1 signaling with Necrostatin-1s led to increased neurite number per cell but not increased neurite length. Therefore, Ripk signaling may be involved in mediating the effects of Wnt3a on neurite number but Ripk activity does not seem to be required for Wnt3a-dependent regulation of neurite length. This study shows that RGCs are direct cellular targets of Wnt3a-induced axonal growth, and we identified a novel association between Wnt signaling and Rip kinases in neurite formation.

Human Tear Serotonin Levels Correlate with Symptoms and Signs of Dry Eye.

PURPOSE: Serotonin, a neurotransmitter known to be involved in nociceptor sensitization, is present in human tears. The purpose of this study was to correlate tear serotonin levels, as a marker of nociceptor sensitization, to facets of dry eye (DE), including symptoms and signs. DESIGN: Cross-sectional study. PARTICIPANTS: A total of 62 patients with normal eyelid and corneal anatomy were prospectively recruited from a Veterans Administration Ophthalmology Clinic over 11 months. METHODS: Dry eye symptoms (Ocular Surface Disease Index [OSDI]), signs (tear break-up time [TBUT], corneal staining, and Schirmer's score), and clinical descriptors of neuropathic ocular pain (NOP) (sensitivity to light or sensitivity to wind) were assessed. For tear analysis, each patient's tears were collected after instilling 50 µl of sterile saline to the lower cul-de-sac of each eye and using capillary action microcaps to collect the ocular wash. Tear serotonin levels were measured using enzyme-linked immunosorbent assay. MAIN OUTCOME MEASURES: Correlations between tear serotonin concentrations and DE symptoms and signs. RESULTS: The mean age of the population was 61±14 years, and 84% (n = 52) of the patients were male. Serotonin concentrations negatively correlated with Schirmer's scores (r = -0.28; P = 0.02) but did not correlate with other DE parameters, such as OSDI scores, sensitivity to light or wind, TBUT, and staining. According to our hypothesis, we divided patients into groups based on both DE symptoms and aqueous tear production; serotonin concentrations were significantly higher in DE group 1 (OSDI ≥6 and Schirmer's <8) compared with both DE group 2 (OSDI ≥6 and Schirmer's ≥8) and controls (OSDI <6 and Schirmer's ≥8). Patients in DE group 2 more frequently reported sensitivity to light (64%) and wind (67%) compared with DE group 1 (40% and 60%, respectively) and controls (8% and 17%, respectively). CONCLUSIONS: Patients with DE symptoms and aqueous tear deficiency had higher tear serotonin levels compared with those with DE symptoms but normal tear production and those without DE symptoms.

A novel protective role for the innate immunity Toll-Like Receptor 3 (TLR3) in the retina via Stat3.

The innate immune system and inflammatory pathways play key roles in numerous diseases of the central nervous system (CNS). Recent evidence indicates that innate immunity induces both pathogenesis and protection during neuronal injury. To test the possibility that the conflicting roles of innate immunity in the CNS depends on the cellular environment in which innate immunity is stimulated, we analyzed the effect of Toll-Like Receptor 3 (TLR3) activation on neuronal survival in the presence and absence of oxidative injury in a mouse model system. We demonstrated that activation of TLR3 by the double stranded RNA activator, Poly (I:C), during paraquat induced oxidative stress, significantly protected mouse photoreceptors, as measured by increased retinal structure, function, and improved visual acuity. In contrast, TLR3 activation without concurrent oxidative injury was neurotoxic. The neurotoxic and protective effects of Poly (I:C) stimulation were absent in TLR3 knockout animals, which indicates that protection by Poly (I:C) is dependent on the TLR3 signaling pathway. Furthermore, we identified the pro-survival transcription factor Stat3 as a necessary mechanism for protection. Knockdown of Stat3 using lentivirally delivered shRNA abolished the protective effects of TLR3 signaling in the retina during oxidative stress. Therefore, TLR3 activation in the context of oxidative stress triggers protective instead of pathogenic signaling, suggesting that TLR3 is a potential therapeutic target for neurodegeneration where oxidative stress is a significant contributor.

Repurposing development genes for axonal regeneration following injury: Examining the roles of Wnt signaling.

In this review, we explore the connections between developmental embryology and axonal regeneration. Genes that regulate embryogenesis and central nervous system (CNS) development are discussed for their therapeutic potential to induce axonal and cellular regeneration in adult tissues after neuronal injury. Despite substantial differences in the tissue environment in the developing CNS compared with the injured CNS, recent studies have identified multiple molecular pathways that promote axonal growth in both scenarios. We describe various molecular cues and signaling pathways involved in neural development, with an emphasis on the versatile Wnt signaling pathway. We discuss the capacity of developmental factors to initiate axonal regrowth in adult neural tissue within the challenging environment of the injured CNS. Our discussion explores the roles of Wnt signaling and also examines the potential of other embryonic genes including Pax, BMP, Ephrin, SOX, CNTF, PTEN, mTOR and STAT3 to contribute to axonal regeneration in various CNS injury model systems, including spinal cord and optic crush injuries in mice, Xenopus and zebrafish. Additionally, we describe potential contributions of Müller glia redifferentiation to neuronal regeneration after injury. Therefore, this review provides a comprehensive summary of the state of the field, and highlights promising research directions for the potential therapeutic applications of specific embryologic molecular pathways in axonal regeneration in adults.

Fluorescence Angiography with Dual Fluorescence for the Early Detection and Longitudinal Quantitation of Vascular Leakage in Retinopathy.

BACKGROUND: Diabetic retinopathy (DR) afflicts more than 93 million people worldwide and is a leading cause of vision loss in working adults. While DR therapies are available, early DR development may go undetected without treatment due to the lack of sufficiently sensitive tools. Therefore, early detection is critically important to enable efficient treatment before progression to vision-threatening complications. A major clinical manifestation of early DR is retinal vascular leakage that may progress from diffuse to more localized focal leakage, leading to increased retinal thickness and diabetic macular edema (DME). In preclinical research, a hallmark of DR in mouse models is diffuse retinal leakage without increased thickness or DME, which limits the utility of optical coherence tomography and fluorescein angiography (FA) for early detection. The Evans blue assay detects diffuse leakage but requires euthanasia, which precludes longitudinal studies in the same animals. METHODS: We developed a new modality of ratiometric fluorescence angiography with dual fluorescence (FA-DF) to reliably detect and longitudinally quantify diffuse retinal vascular leakage in mouse models of induced and spontaneous DR. RESULTS: These studies demonstrated the feasibility and sensitivity of FA-DF in detecting and quantifying retinal vascular leakage in the same mice over time during DR progression in association with chronic hyperglycemia and age. CONCLUSIONS: These proof-of-concept studies demonstrated the promise of FA-DF as a minimally invasive method to quantify DR leakage in preclinical mouse models longitudinally.

Context-Dependent Effects of the Ketogenic Diet on Retinal Ganglion Cell Survival and Axonal Regeneration After Optic Nerve Injury.

Purpose: There is increasing interest in nonpharmacologic approaches to protect retinal ganglion cells (RGCs) after injury and enhance the efficacy of therapeutic molecules. Accumulating evidence demonstrates neuroprotection by the high-fat low-carbohydrate ketogenic diet (KD) in humans and animal models of neurologic diseases. However, no studies to date have examined whether the KD protects RGCs and promotes axonal regrowth after traumatic injury to the optic nerve (ON) or whether it increases efficacy of experimental proregenerative molecules. In this study, we investigated whether the KD promoted RGC survival and axonal regeneration after ON injury in the presence and absence of neuroprotective Wnt3a ligand. Methods: Adult mice were placed on a KD or control diet before ON crush injury and remained on the diet until the end of the experiment. Nutritional ketosis was confirmed by measuring serum beta-hydroxybutyrate levels. Mice were intravitreally injected with Wnt3a ligand or phosphate-buffered saline (PBS), and RGC survival, function, axonal regeneration, and inflammatory responses were measured. Results: Mice fed the KD showed increased RGC survival and reduced inflammatory cells in PBS-injected mice. Also, mice fed the KD had increased RGC functional responses but not increased RGC numbers in the presence of Wnt3a, indicating that the KD did not enhance the prosurvival effect of Wnt3a. The KD did not promote axonal regeneration in the presence or absence of Wnt3a. Conclusions: The KD has a complex protective effect after ON injury and cotreatment with Wnt3a. This work sets the foundation for studies identifying underlying molecular mechanisms.

Identification of a Novel Axon Regeneration Role for Noncanonical Wnt Signaling in the Adult Retina after Injury.

Canonical and noncanonical Wnt signaling pathways are essential for development and maintenance of the CNS. Whereas the roles of canonical Wnt pathways in neuronal survival and axonal regeneration in adult CNS have been described, the functions of noncanonical Wnt pathways are not well understood. Furthermore, the role of noncanonical Wnt ligands in the adult retina has not been investigated. Noncanonical Wnt signaling shares receptors with canonical Wnt ligands but functions through calcium and c-Jun N-terminal kinase (JNK) signaling pathways. Noncanonical ligands, such as the prototypic ligand Wnt5a, have varying effects in the developing CNS, including inhibiting or promoting axonal growth. To identify a role for noncanonical Wnt signaling in the developed retina after injury, we characterized the effect of Wnt5a on neurite outgrowth in cultured retinal ganglion cell (RGC) neurons and on axonal regeneration in the injured optic nerve in the mouse. Endogenous Wnt5a was upregulated after injury and exogenous Wnt5a significantly enhanced neurite growth of primary RGCs and led to extensive axonal regeneration after optic nerve crush (ONC) injury. Wnt5a also significantly increased RGC survival. Furthermore, Wnt5a induced phosphorylation of CamKII and JNK and induced expression of their downstream pathway components. Therefore, these results demonstrate for the first time that Wnt5a promotes axonal growth and protects RGCs in the adult retina.

Exploring the role of interleukin-27 as a regulator of neuronal survival in central nervous system diseases.

Interleukin-27 is a pleiotropic cytokine that is involved in tissue responses to infection, cell stress, neuronal disease, and tumors. Recent studies in various tissues indicate that interleukin-27 has complex activating and inhibitory properties in innate and acquired immunity. The availability of recombinant interleukin-27 protein and mice with genetic deletions of interleukin-27, its receptors and signaling mediators have helped define the role of interleukin-27 in neurodegenerative diseases. Interleukin-27 has been well-characterized as an important regulator of T cell activation and differentiation that enhances or suppresses T cell responses in autoimmune conditions in the central nervous system. Evidence is also accumulating that interleukin-27 has neuroprotective activities in the retina and brain. Interleukin-27 is secreted from and binds to infiltrating microglia, macrophage, astrocytes, and even neurons and it promotes neuronal survival by regulating pro- and anti-inflammatory cytokines, neuroinflammatory pathways, oxidative stress, apoptosis, autophagy, and epigenetic modifications. However, interleukin-27 can have the opposite effect and induce inflammation and cell death in certain situations. In this review, we describe the current understanding of regulatory activities of interleukin-27 on cell survival and inflammation and discuss its mechanisms of action in the brain, spinal cord, and retina. We also review evidence for and against the therapeutic potential of interleukin-27 for dampening harmful neuroinflammatory responses in central nervous system diseases.

The Wnt signaling pathway protects retinal ganglion cell 5 (RGC-5) cells from elevated pressure.

The Wnt pathway is an essential signaling cascade that regulates survival and differentiation in the retina. We recently demonstrated that retinal ganglion cells (RGCs) have constitutively active Wnt signaling in vivo. However, the role of Wnt in RGC viability or function is unknown. In this study, we investigated whether Wnt protects the retinal ganglion cell line RGC-5 from elevated pressure, oxidative stress, and hypoxia injuries. Expression of RGC marker genes in the RGC-5 cultures was confirmed by immunocytochemistry and PCR. We demonstrated that the Wnt3a ligand significantly reduced pressure-induced caspase activity in RGC-5 cells (n = 5, P = 0.03) and decreased the number of TUNEL-positive cells (n = 5, P = 0.0014). Notably, Wnt3a-dependent protection was reversed by the Wnt signaling inhibitor Dkk1. In contrast, Wnt3a did not protect RGC-5 cells from oxidative stress or hypoxia. Furthermore, Wnt3a significantly increased growth factor expression in the presence of elevated pressure but not in the presence of oxidative stress and hypoxia. These results indicate that Wnt3a induces injury-specific survival pathways in RGC-5 cells, potentially by upregulating neuroprotective growth factors. Therefore, activation of the Wnt pathway by Wnt3a could be investigated further as a tool to develop novel molecular therapeutic strategies for the prevention of RGC death in retinal disease.

Recruitment and activation of caspase-8 by the Huntingtin-interacting protein Hip-1 and a novel partner Hippi.

In Huntington disease, polyglutamine expansion of the protein huntingtin (Htt) leads to selective neurodegenerative loss of medium spiny neurons throughout the striatum by an unknown apoptotic mechanism. Binding of Hip-1, a protein normally associated with Htt, is reduced by polyglutamine expansion. Free Hip-1 binds to a hitherto unknown polypeptide, Hippi (Hip-1 protein interactor), which has partial sequence homology to Hip-1 and similar tissue and subcellular distribution. The availability of free Hip-1 is modulated by polyglutamine length within Htt, with disease-associated polyglutamine expansion favouring the formation of pro-apoptotic Hippi-Hip-1 heterodimers. This heterodimer can recruit procaspase-8 into a complex of Hippi, Hip-1 and procaspase-8, and launch apoptosis through components of the 'extrinsic' cell-death pathway. We propose that Htt polyglutamine expansion liberates Hip-1 so that it can form a caspase-8 recruitment complex with Hippi. This novel non-receptor-mediated pathway for activating caspase-8 might contribute to neuronal death in Huntington disease.

Neuroprotection by the Ketogenic Diet: Evidence and Controversies.

The ketogenic diet (KD) is a high-fat low-carbohydrate diet that has been used for decades as a non-pharmacologic approach to treat metabolic disorders and refractory pediatric epilepsy. In recent years, enthusiasm for the KD has increased in the scientific community due to evidence that the diet reduces pathology and improves various outcome measures in animal models of neurodegenerative disorders, including multiple sclerosis, stroke, glaucoma, spinal cord injury, retinal degenerations, Parkinson's disease and Alzheimer's disease. Clinical trials also suggest that the KD improved quality of life in patients with multiple sclerosis and Alzheimer's disease. Furthermore, the major ketone bodies BHB and ACA have potential neuroprotective properties and are now known to have direct effects on specific inflammatory proteins, transcription factors, reactive oxygen species, mitochondria, epigenetic modifications and the composition of the gut microbiome. Neuroprotective benefits of the KD are likely due to a combination of these cellular processes and other potential mechanisms that are yet to be confirmed experimentally. This review provides a comprehensive summary of current evidence for the effectiveness of the KD in humans and preclinical models of various neurological disorders, describes molecular mechanisms that may contribute to its beneficial effects, and highlights key controversies and current gaps in knowledge.

Analysis of Dickkopf3 interactions with Wnt signaling receptors.

Wnt signaling regulates essential biological processes ranging from embryogenesis to neurodegeneration. Recently, we demonstrated that Dickkopf3 (Dkk3) is a pro-survival glycoprotein that positively modulates Wnt signaling. An important step in understanding the mechanism of action of Dkk3 is identifying its interacting proteins in the Wnt pathway. In this study, we used a series of biochemical and functional assays to investigate the interaction between Dkk3 and the Wnt pathway receptors Kremen1 (Krm1), Kremen 2 (Krm2) and low-density lipoprotein receptor-related protein 6 (LRP6). Here, we report that, contrary to previous studies, Dkk3 interacts with Krm1 and Krm2. However, Dkk3 did not interact with, or alter expression of, LRP6. Blocking protein glycosylation did not alter the interaction between Dkk3 and Krm proteins. Additionally, Krm2 abolished Dkk3-mediated potentiation of Wnt signaling. Therefore, our data establish that Krm proteins are novel binding partners of Dkk3 and suggest a mechanism by which Dkk3 potentiates Wnt signaling.

Lithium chloride regulates the proliferation of stem-like cells in retinoblastoma cell lines: a potential role for the canonical Wnt signaling pathway.

PURPOSE: Cancer stem cells are found in many tumor types and are believed to lead to regrowth of tumor mass due to their chemoresistance and self-renewal capacity. We previously demonstrated small subpopulations of cells in retinoblastoma tissue and cell lines that display cancer stem cell-like activities, including expression of stem cell markers, Hoechst dye exclusion, slow cycling, and self-renewal ability. Identifying factors regulating stem cell proliferation will be important for selectively targeting stem cells and controlling tumor growth. Wingless and Int1 (Wnt) signaling is an essential cellular communication pathway that regulates proliferation and differentiation of non-neoplastic stem/progenitor cells in the retina and other tissues, but its role in cancer stem cells in the retinal tumor retinoblastoma is unknown. In this study, we investigated whether the Wnt pathway activator lithium chloride (LiCl) regulates proliferation of retinoblastoma cancer stem-like cells. METHODS: The number of stem-like cells in Weri and Y79 retinoblastoma cell line cultures was measured by 5-bromo-2-deoxyuridine (BrdU) pulse-chase, immunohistochemistry, and quantitative polymerase chain reaction (PCR) for stem cell marker genes. The cell lines were sorted into stem-like and non-stem-like populations by fluorescence-activated cell sorting (FACS), using an antibody against the stem cell marker ATP-binding cassette, subfamily G, member 2 (ABCG2). Activated Wnt signaling was measured in the sorted cells by western blotting and immunolocalization of the central mediator beta-catenin. RESULTS: LiCl increased the number of stem-like cells, measured by BrdU retention and elevated expression of the stem cell marker genes Nanog, octamer transcription factor 3 and 4 (Oct3/4), Musashi 1 (Msi1), and ABCG2. Sorted ABCG2-positive stem-like cells had higher levels of beta-catenin than ABCG2-negative non-stem cells, suggesting elevated canonical Wnt signaling. Furthermore, stem cell marker gene expression increased after small interfering RNA (siRNA) knock-down of the Wnt inhibitor secreted frizzled-related protein 2 (SFRP2). CONCLUSIONS: These results indicate that the cancer stem-like cell population in retinoblastoma is regulated by canonical Wnt/beta-catenin signaling, which identifies the Wnt pathway as a potential mechanism for the control of stem cell renewal and tumor formation in retinoblastoma tumors in vivo.

Increased Neuroprotective Microglia and Photoreceptor Survival in the Retina from a Peptide Inhibitor of Myeloid Differentiation Factor 88 (MyD88).

Myeloid differentiation factor 88 (MyD88) is an adaptor protein for the Toll-like receptor (TLR) and interleukin 1 receptor (IL-1R) families of innate immunity receptors that mediate inflammatory responses to cellular injury. TLR/IL1R/MyD88 signaling is known to contribute to retinal degeneration, although how MyD88 regulates neuronal survival, and the effect of MyD88 on the inflammatory environment in the retina, is mostly unknown. In this study, we tested the hypothesis that blocking MyD88-mediated signaling early in retinal degeneration promotes transition of microglia towards a neuroprotective anti-inflammatory phenotype, resulting in enhanced photoreceptor survival. We also tested whether systemic delivery of a pharmacologic MyD88 inhibitor has therapeutic potential. The rd10 mouse model of retinal degeneration was injected intraperitoneally with increasing doses of a MyD88 blocking peptide or control peptide early in degeneration, and inflammatory responses and photoreceptor survival were measured at specific time points using flow cytometry, cytokine profiling, and electroretinograms. Our results demonstrated that rd10 mice injected with a low dose of MyD88 inhibitor peptide showed increased rod photoreceptor function and reduced apoptosis compared with control peptide and uninjected mice. MyD88 inhibition also resulted in fewer microglia/macrophage cells in the photoreceptor layer whereas total peripheral and retinal macrophage were not changed. Furthermore, increased number of cells expressing the Arg1 marker of neuroprotective microglia in the photoreceptor layer and higher MCP-1 and anti-inflammatory cytokine IL-27 were associated with photoreceptor survival. Therefore, these data suggest that the MyD88 inhibitor modified the retina environment to become less inflammatory, leading to improved photoreceptor function and survival.

The effect of extrinsic Wnt/ß-catenin signaling in Muller glia on retinal ganglion cell neurite growth.

Muller glia are the predominant glial cell type in the retina, and they structurally and metabolically support retinal neurons. Wnt/ß-catenin signaling pathways play essential roles in the central nervous system, including glial and neuronal differentiation, axonal growth, and neuronal regeneration. We previously demonstrated that Wnt signaling activation in retinal ganglion cells (RGC) induces axonal regeneration after injury. However, whether Wnt signaling within the adjacent Muller glia plays an axongenic role is not known. In this study, we characterized the effect of Wnt signaling in Muller glia on RGC neurite growth. Primary Muller glia and RGC cells were grown in transwell co-cultures and adenoviral constructs driving Wnt regulatory genes were used to activate and inhibit Wnt signaling specifically in primary Muller glia. Our results demonstrated that activation of Wnt signaling in Muller glia significantly increased RGC average neurite length and branch site number. In addition, the secretome of Muller glia after induction or inhibition of Wnt signaling was characterized using protein profiling of conditioned media by Q Exactive mass spectrometry. The Muller glia secretome after activation of Wnt signaling had distinct and more numerous proteins involved in regulation of axon extension, axon projection and cell adhesion. Furthermore, we showed highly redundant expression of Wnt signaling ligands in Muller glia and Frizzled receptors in RGCs and Muller glia. Therefore, this study provides new information about potential neurite growth promoting molecules in the Muller glia secretome, and identified Wnt-dependent target proteins that may mediate the axonal growth.

Association of the Indoor Environment With Dry Eye Metrics.

Importance: The ocular surface is continuously exposed to the environment. Although studies have focused on associations between outdoor environmental conditions and dry eye, information on associations between the indoor environment and dry eye is lacking. Objective: To determine associations between the indoor environment and dry eye. Design, Setting, and Participants: This prospective cross-sectional study sample of 97 veterans with a wide range of dry eye metrics was recruited from the Miami Veterans Affairs Healthcare eye clinic from October 19, 2017, to August 30, 2018. Dry eye metrics were first evaluated in the clinic, followed by indoor home environmental metrics within 1 week using a handheld particle counter. Data were analyzed from October 19, 2017, to August 30, 2018. Main Outcomes and Measures: Symptoms of dry eye were assessed with standardized questionnaires. Dry eye signs were assessed via standard examination. Indoor environmental metrics included temperature, humidity, and particulate matter mass and count. Results: Of the 97 participants included in the analysis, 81 (84%) were men, with a mean (SD) age of 58.2 (11.9) years. Dry eye symptoms were in the moderate range with a mean (SD) Ocular Surface Disease Index (OSDI) score of 31.2 (23.6). Humidity was associated with worse symptoms and signs, including OSDI score (r = 0.30 [95% CI, 0.07-0.49]; P = .01), inflammation (r = 0.32 [95% CI, 0.10-0.51]; P = .01), Schirmer score (r = -0.25 [95% CI, -0.45 to 0.02]; P = .03), eyelid vascularity (r = 0.27 [95% CI, 0.05-0.47]; P = .02), and meibomian gland dropout (r = 0.27 [95% CI, 0.05-0.47]; P = .02). In multivariate analyses, particulate matter of 2.5 µm or less (PM2.5) was associated with dry eye metrics when adjusted for demographic characteristics, comorbidities, medications, and interaction variables. For example, a 1-unit increase in instrumented PM2.5 level was associated with a 1.59 increase in the OSDI score (95% CI, 0.58-2.59; P = .002), a 0.39 reduction in Schirmer score (95% CI, -0.75 to -0.03; P = .04), a 0.07 increase in meibomian gland dropout (95% CI, 0.01-0.13; P = .02), and a 0.06 increase in inflammation (95% CI, 0.02-0.11; P = .009). Conclusions and Relevance: When adjusting for humidity, this study found that increased particulate matter exposure was associated with worse dry eye metrics. Humidity was positively associated with dry eye metrics, potentially because higher humidity increases microbial growth and particulate matter size and mass.

Multi-Omic Analyses of Growth Cones at Different Developmental Stages Provides Insight into Pathways in Adult Neuroregeneration.

Growth cones (GCs) are structures associated with growing neurons. GC membrane expansion, which necessitates protein-lipid interactions, is critical to axonal elongation in development and in adult neuritogenesis. We present a multi-omic analysis that integrates proteomics and lipidomics data for the identification of GC pathways, cell phenotypes, and lipid-protein interactions, with an analytic platform to facilitate the visualization of these data. We combine lipidomic data from GC and adult axonal regeneration following optic nerve crush. Our results reveal significant molecular variability in GCs across developmental ages that aligns with the upregulation and downregulation of lipid metabolic processes and correlates with distinct changes in the lipid composition of GC plasmalemma. We find that these processes also define the transition into a growth-permissive state in the adult central nervous system. The insight derived from these analyses will aid in promoting adult regeneration and functional innervation in devastating neurodegenerative diseases.