Progesterone Attenuates Microglial-Driven Retinal Degeneration and Stimulates Protective Fractalkine-CX3CR1 Signaling

Retinitis pigmentosa (RP) is a degenerative disease leading to photoreceptor cell loss. Mouse models of RP, such as the rd10 mouse (B6.CXBl-Pde6brd10/J), have enhanced our understanding of the disease, allowing for development of potential therapeutics. In 2011, our group first demonstrated that the synthetic progesterone analogue ‘Norgestrel’ is neuroprotective in two mouse models of retinal degeneration, including the rd10 mouse. We have since elucidated several mechanisms by which Norgestrel protects stressed photoreceptors, such as upregulating growth factors. This study consequently aimed to further characterize Norgestrel’s neuroprotective effects. Specifically, we sought to investigate the role that microglia might play; for microglial-derived inflammation has been shown to potentiate neurodegeneration. Dams of post-natal day (P) 10 rd10 pups were given a Norgestrel-supplemented diet (80mg/kg). Upon weaning, pups remained on Norgestrel. Tissue was harvested from P15-P50 rd10 mice on control or Norgestrel-supplemented diet. Norgestrel-diet administration provided significant retinal protection out to P40 in rd10 mice. Alterations in microglial activity coincided with significant protection, implicating microglial changes in Norgestrel-induced neuroprotection. Utilizing primary cultures of retinal microglia and 661W photoreceptor-like cells, we show that rd10 microglia drive neuronal cell death. We reveal a novel role of Norgestrel, acting directly on microglia to reduce pro-inflammatory activation and prevent neuronal cell death. Norgestrel effectively suppresses cytokine, chemokine and danger-associated molecular pattern molecule (DAMP) expression in the rd10 retina. Remarkably, Norgestrel upregulates fractalkine-CX3CR1 signaling 1 000-fold at the RNA level, in the rd10 mouse. Fractalkine-CX3CR1 signaling has been shown to protect neurons by regulating retinal microglial activation and migration. Ultimately, these results present Norgestrel as a promising treatment for RP, with dual actions as a neuroprotective and anti-inflammatory agent in the retina.     

一种视网膜锥体细胞退行性变大鼠基础生理值的测定

目的对视网膜锥体细胞退行性变（retinal cone degeneration,RCD）大鼠的主要脏器及血液、生化指标进行检测。方法随机选择BN（Brown Norway）-RCDF2代大鼠,视网膜电图鉴定,取静脉血检测生化指标,并对重要脏器进行准确称量。结果此大鼠脏器质量在雌雄之间除体重、大脑和肾差异有显著性外（P〈0.05）,其余均无差异;脏器系数脑干和小脑雌性显著大于雄性（P〈0.01）;生化指标碱性磷酸酶、葡萄糖、白蛋白、尿素氮、总蛋白雌性高于雄性,总胆固醇、天冬氨酸转氨酶、丙氨酸转氨酶、甘油三酯雌性低于雄性,其中雌性碱性磷酸酶活性显著高于雄性;雌性离子钙浓度显著高于雄性。结论BN-RCD大鼠基础生理值和生化参数均在正常范围内,其中一些参数在性别之间有差异,但与正常SD大鼠相比,天冬氨酸转氨酶、丙氨酸转氨酶、肌酐、钾离子差异显著,葡萄糖、甘油三酯、离子钙有差异。RCD大鼠为遗传性视网膜疾病的研究提供了一种新的突变模式动物。     

Correlation of Cytokine Levels and Microglial Cell Infiltration during Retinal Degeneration in RCS Rats

Microglial cells, which are immunocompetent cells, are involved in all diseases of the central nervous system. During their activation in various diseases, a variety of soluble factors are released. In the present study, the correlation between cytokine levels and microglial cell migration in the course of retinal degeneration of Royal College of Surgeons (RCS) rats was evaluated. MFG-E8 and CD11b were used to confirm the microglial cells. In the retina of RCS rats, the mRNA expression of seven genes (MFG-E8 and its integrins αυ and ß5, CD11b and the cytokines TNF-α, IL-1ß, and MCP-1) formed almost similar bimodal peak distributions, which were centred at P7 and P45 to P60. In contrast, in rdy rats, which comprised the control group, a unimodal peak distribution centred at P14 was observed. The gene expression accompanied the activation and migration of microglial cells from the inner to the outer layer of the retina during the process of degeneration. Principal component analysis and discriminant function analysis revealed that the expression of these seven genes, especially TNF-α and CD11b, positively correlated with retinal degeneration and microglial activity during retinal degeneration in RCS rats, but not in the control rats. Furthermore, linear regression analysis demonstrated a significant correlation between the expression of these genes and the activation of microglial cells in the dystrophic retina. Our findings suggest that the suppression of microglial cells and the blockade of their cytotoxic effects may constitute a novel therapeutic strategy for treating photoreceptor death in various retinal disorders.     

Programmed Cell Death in Retinal Degeneration: Targeting Apoptosis in Photoreceptors as Potential Therapy for Retinal Degeneration

Retinal degenerations are the major cause of incurable blindness characterized by loss of retinal photoreceptor cells. Several genes causing these genetic diseases have been identified, however the molecular characterization of a high percentage of patients affected by retinitis pigmentosa (RP), a common form of retinal degeneration, is still unknown. The high genetic heterogeneity of these diseases hampers the comprehension of the pathogenetic mechanism causing photoreceptor cell death. Therapies are not available yet and for this reason there is a lot of interest in understanding the etiology and the pathogenesis of these disorders at a cellular and molecular level. Some common features have been identified in different forms of RP. Apoptosis was reported to be the final outcome in all RP animal models and patients analyzed so far. We recently identified two apoptotic pathways co-activated in photoreceptors undergoing cell death in the retinal degeneration (rd1) mouse model of autosomal recessive RP. Our studies opened new perspectives together with many questions that require deeper analyses in order to take advantage of this knowledge and develop new therapeutic approaches. We believe that minimizing cell demise may represent a promising curing strategy that needs to be exploited for retinal degeneration.     

壳寡糖作用于巨噬细胞的机制初探

研究壳寡糖对免疫系统中巨噬细胞作用的具体机制.结合流式细胞仪和激光共聚焦实验检测壳寡糖与巨噬细胞相互作用,通过凝胶阻滞实验在体外验证壳寡糖的胞内定位.实验结果证明壳寡糖与巨噬细胞相互作用过程如下,先与细胞膜结合,然后进入细胞内,最后定位在细胞核的核酸(DNA/RNA)上.     

Retinal Ganglion Cells are Resistant to Photoreceptor Loss in Retinal Degeneration

The rapid and massive degeneration of photoreceptors in retinal degeneration might have a dramatic negative effect on retinal circuits downstream of photoreceptors. However, the impact of photoreceptor loss on the morphology and function of retinal ganglion cells (RGCs) is not fully understood, precluding the rational design of therapeutic interventions that can reverse the progressive loss of retinal function. The present study investigated the morphological changes in several identified RGCs in the retinal degeneration rd1 mouse model of retinitis pigmentosa (RP), using a combination of viral transfection, microinjection of neurobiotin and confocal microscopy. Individual RGCs were visualized with a high degree of detail using an adeno-associated virus (AAV) vector carrying the gene for enhanced green fluorescent protein (EGFP), allowed for large-scale surveys of the morphology of RGCs over a wide age range. Interestingly, we found that the RGCs of nine different types we encountered were especially resistant to photoreceptor degeneration, and retained their fine dendritic geometry well beyond the complete death of photoreceptors. In addition, the RGC-specific markers revealed a remarkable degree of stability in both morphology and numbers of two identified types of RGCs for up to 18 months of age. Collectively, our data suggest that ganglion cells, the only output cells of the retina, are well preserved morphologically, indicating the ganglion cell population might be an attractive target for treating vision loss.     

壳寡糖缓解甲萘醌诱导巨噬细胞损伤机制初探

目的:研究壳寡糖对甲萘醌诱导的巨噬细胞氧化损伤的保护作用.方法:通过MTT实验检测相应处理的细胞活力,并通过相应试剂盒检测细胞氧化还原体系中某些相关酶的活力及相应产物含量.结果:壳寡糖能够缓解甲萘醌诱导的细胞损伤,并且发现壳寡糖可以缓解甲萘醌导致的胞内超氧化物歧化酶(SOD),谷胱甘肽过氧化物酶(GSH-PX)活力的降...     

Argon Inhalation Attenuates Retinal Apoptosis after Ischemia/Reperfusion Injury in a Time- and Dose-Dependent Manner in Rats

Purpose

Retinal ischemia and reperfusion injuries (IRI) permanently affect neuronal tissue and function by apoptosis and inflammation due to the limited regenerative potential of neurons. Recently, evidence emerged that the noble gas Argon exerts protective properties, while lacking any detrimental or adverse effects. We hypothesized that Argon inhalation after IRI would exert antiapoptotic effects in the retina, thereby protecting retinal ganglion cells (RGC) of the rat''s eye.

Methods

IRI was performed on the left eyes of rats (n = 8) with or without inhaled Argon postconditioning (25, 50 and 75 Vol%) for 1 hour immediately or delayed after ischemia (i.e. 1.5 and 3 hours). Retinal tissue was harvested after 24 hours to analyze mRNA and protein expression of Bcl-2, Bax and Caspase-3, NF-κB. Densities of fluorogold-prelabeled RGCs were analyzed 7 days after injury in whole-mounts. Histological tissue samples were prepared for immunohistochemistry and blood was analyzed regarding systemic effects of Argon or IRI. Statistics were performed using One-Way ANOVA.

Results

IRI induced RGC loss was reduced by Argon 75 Vol% inhalation and was dose-dependently attenuated by lower concentrations, or by delayed Argon inhalation (1504±300 vs. 2761±257; p<0.001). Moreover, Argon inhibited Bax and Bcl-2 mRNA expression significantly (Bax: 1.64±0.30 vs. 0.78±0.29 and Bcl-2: 2.07±0.29 vs. 0.99±0.22; both p<0.01), as well as caspase-3 cleavage (1.91±0.46 vs. 1.05±0.36; p<0.001). Expression of NF-κB was attenuated significantly. Immunohistochemistry revealed an affection of Müller cells and astrocytes. In addition, IRI induced leukocytosis was reduced significantly after Argon inhalation at 75 Vol%.

Conclusion

Immediate and delayed Argon postconditioning protects IRI induced apoptotic loss of RGC in a time- and dose-dependent manner, possibly mediated by the inhibition of NF-κB. Further studies need to evaluate Argon''s possible role as a therapeutic option.     

Differential Light-induced Responses in Sectorial Inherited Retinal Degeneration

Retinitis pigmentosa (RP) is a group of genetically and clinically heterogeneous inherited degenerative retinopathies caused by abnormalities of photoreceptors or retinal pigment epithelium in the retina leading to progressive sight loss. Rhodopsin is the prototypical G-protein-coupled receptor located in the vertebrate retina and is responsible for dim light vision. Here, novel M39R and N55K variants were identified as causing an intriguing sector phenotype of RP in affected patients, with selective degeneration in the inferior retina. To gain insights into the molecular aspects associated with this sector RP phenotype, whose molecular mechanism remains elusive, the mutations were constructed by site-directed mutagenesis, expressed in heterologous systems, and studied by biochemical, spectroscopic, and functional assays. M39R and N55K opsins had variable degrees of chromophore regeneration when compared with WT opsin but showed no gross structural misfolding or altered trafficking. M39R showed a faster rate for transducin activation than WT rhodopsin with a faster metarhodopsinII decay, whereas N55K presented a reduced activation rate and an altered photobleaching pattern. N55K also showed an altered retinal release from the opsin binding pocket upon light exposure, affecting its optimal functional response. Our data suggest that these sector RP mutations cause different protein phenotypes that may be related to their different clinical progression. Overall, these findings illuminate the molecular mechanisms of sector RP associated with rhodopsin mutations.     

Taurine Provides Neuroprotection against Retinal Ganglion Cell Degeneration

Retinal ganglion cell (RGC) degeneration occurs in numerous retinal diseases leading to blindness, either as a primary process like in glaucoma, or secondary to photoreceptor loss. However, no commercial drug is yet directly targeting RGCs for their neuroprotection. In the 70s, taurine, a small sulfonic acid provided by nutrition, was found to be essential for the survival of photoreceptors, but this dependence was not related to any retinal disease. More recently, taurine deprivation was incriminated in the retinal toxicity of an antiepileptic drug. We demonstrate here that taurine can improve RGC survival in culture or in different animal models of RGC degeneration. Taurine effect on RGC survival was assessed in vitro on primary pure RCG cultures under serum-deprivation conditions, and on NMDA-treated retinal explants from adult rats. In vivo, taurine was administered through the drinking water in two glaucomatous animal models (DBA/2J mice and rats with vein occlusion) and in a model of Retinitis pigmentosa with secondary RGC degeneration (P23H rats). After a 6-day incubation, 1 mM taurine significantly enhanced RGCs survival (+68%), whereas control RGCs were cultured in a taurine-free medium, containing all natural amino-acids. This effect was found to rely on taurine-uptake by RGCs. Furthermore taurine (1 mM) partly prevented NMDA-induced RGC excitotoxicity. Finally, taurine supplementation increased RGC densities both in DBA/2J mice, in rats with vein occlusion and in P23H rats by contrast to controls drinking taurine-free water. This study indicates that enriched taurine nutrition can directly promote RGC survival through RGC intracellular pathways. It provides evidence that taurine can positively interfere with retinal degenerative diseases.     

Toll-like Receptor 2 Facilitates Oxidative Damage-Induced Retinal Degeneration

1. Download : Download high-res image (243KB)
2. Download : Download full-size image

     

Light Effects on Mitochondrial Photosensitizers in Relation to Retinal Degeneration

The retina captures and converts light between 400–760 nm into electrical signals that are sent to the brain by way of the optic nerve and in the process helps to translate these electrical signals into what is known as vision. The same light that allows vision to occur is nevertheless also potentially toxic to retinal cells in certain situations. The shorter wavelengths of light are known to interact with chromophores in photoreceptors and pigment epithelial cells to cause oxidative stress and severe damage. Indeed it is generally accepted that short wavelength light effects is one cause for loss of photoreceptor function in age-related macular degeneration. Recent studies have demonstrated that light may be a contributing factor for the death of retinal ganglion cells in certain situations. Light as impinging on the retina, especially the short wavelength form, affect mitochondrial chromophores and can result in neurone death. Importantly ganglion cell axons within the eye are laden with mitochondria and unlike the outer retina are not protected from short wavelength light by macular pigments. It has therefore been proposed that when ganglion cell function is already compromised, as in glaucoma, then light impinging on their mitochondria might be a contributor to their eventual demise.     

Retinal Gangliosides in RCS Mutant Rats

Abstract: The distribution of retinal gangliosides was studied in normal and mutant rats with retinal dystrophy at 30 and 180 days of age. The loss of photoreceptor cells in the retinal dystrophic RCS rats was not associated with a significant reduction in the relative distribution of any of the major retinal gangliosides. The loss of photoreceptors, however, caused a marked increase in total retinal ganglioside concentration. These findings suggest that photoreceptor cells contain a low concentration of gangliosides and that no major retinal ganglioside is localized or concentrated in these cells. The cellular localization and function of the most abundant retinal ganglioside, G_D3, is discussed.     

Tyro3 Modulates Mertk-Associated Retinal Degeneration

Inherited photoreceptor degenerations (IPDs) are the most genetically heterogeneous of Mendelian diseases. Many IPDs exhibit substantial phenotypic variability, but the basis is usually unknown. Mutations in MERTK cause recessive IPD phenotypes associated with the RP38 locus. We have identified a murine genetic modifier of Mertk-associated photoreceptor degeneration, the C57BL/6 (B6) allele of which acts as a suppressor. Photoreceptors degenerate rapidly in Mertk-deficient animals homozygous for the 129P2/Ola (129) modifier allele, whereas animals heterozygous for B6 and 129 modifier alleles exhibit an unusual intermixing of degenerating and preserved retinal regions, with females more severely affected than males. Mertk-deficient mice homozygous for the B6 modifier allele display degeneration only in the far periphery, even at 8 months of age, and have improved retinal function compared to animals homozygous for the 129 allele. We genetically mapped the modifier to an approximately 2-megabase critical interval that includes Tyro3, a paralog of Mertk. Tyro3 expression in the outer retina varies with modifier genotype in a manner characteristic of a cis-acting expression quantitative trait locus (eQTL), with the B6 allele conferring an approximately three-fold higher expression level. Loss of Tyro3 function accelerates the pace of photoreceptor degeneration in Mertk knockout mice, and TYRO3 protein is more abundant in the retinal pigment epithelium (RPE) adjacent to preserved central retinal regions of Mertk knockout mice homozygous for the B6 modifier allele. Endogenous human TYRO3 protein co-localizes with nascent photoreceptor outer segment (POS) phagosomes in a primary RPE cell culture assay, and expression of murine Tyro3 in cultured cells stimulates phagocytic ingestion of POS. Our findings demonstrate that Tyro3 gene dosage modulates Mertk-associated retinal degeneration, provide strong evidence for a direct role for TYRO3 in RPE phagocytosis, and suggest that an eQTL can modify a recessive IPD.     

Homeostatic Plasticity Shapes the Retinal Response to Photoreceptor Degeneration

1. Download : Download high-res image (165KB)
2. Download : Download full-size image