Human palmitoyl protein thioesterase: evidence for lysosomal targeting of the enzyme and disturbed cellular routing in infantile neuronal ceroid lipofuscinosis.

Palmitoyl protein thioesterase (PPT) is an enzyme that removes palmitate residues from various S-acylated proteins in vitro. We recently identified mutations in the human PPT gene in patients suffering from a neurodegenerative disease in childhood, infantile neuronal ceroid lipofuscinosis (INCL), with dramatic manifestations limited to the neurons of neocortical origin. Here we have expressed the human PPT cDNA in COS-1 cells and demonstrate the lysosomal targeting of the enzyme via the mannose 6-phosphate receptor-mediated pathway. The enzyme was also secreted into the growth medium and could be endocytosed by recipient cells. We further demonstrate the disturbed intracellular routing of PPT carrying the worldwide most common INCL mutation, Arg122Trp, to lysosomes. The results provide evidence that INCL represents a novel lysosomal enzyme deficiency. Further, the defect in the PPT gene causing a neurodegenerative disorder suggests that depalmitoylation of the still uncharacterized substrate(s) for PPT is critical for postnatal development or maintenance of cortical neurons.     

RAGE signaling contributes to neuroinflammation in infantile neuronal ceroid lipofuscinosis

Palmitoyl-protein thioesterase-1 (PPT1) deficiency causes infantile neuronal ceroid lipofuscinosis (INCL), a devastating childhood neurodegenerative storage disorder. We previously reported that neuronal apoptosis in INCL is mediated by endoplasmic reticulum-stress. ER-stress disrupts Ca²⁺-homeostasis and stimulates the expression of Ca²⁺-binding proteins. We report here that in the PPT1-deficient human and mouse brain the levels of S100B, a Ca²⁺-binding protein, and its receptor, RAGE (receptor for advanced glycation end-products) are elevated. We further demonstrate that activation of RAGE signaling in astroglial cells mediates pro-inflammatory cytokine production, which is inhibited by SiRNA-mediated suppression of RAGE expression. We propose that RAGE signaling contributes to neuroinflammation in INCL.     

Trefoil factor family peptide 2 acts pro-proliferative and pro-apoptotic in the murine retina

Although expression of trefoil factor family (TFF) peptides has been reported in the brain, nothing is known about TFF expression in the retina. The aim of this study was to test whether TFF peptides are expressed in the murine retina and have any function here. In contrast to most tissues studied, where TFF1 and TFF3 are the predominant peptides, TFF2 is the only peptide expressed in the murine retina. Immunohistochemical studies on murine retinal sections indicate that cells of the ganglion cell layer are the retinal source for murine TFF2 (Tff2). In organotypic murine retina cell cultures recombinant TFF2 exerted a strong pro-apoptotic and pro-proliferative rather than an anti-apoptotic and anti-proliferating effect described in most human cancer cell lines investigated so far. In blockage experiments we were able to demonstrate that the pro-apoptotic effect of TFF2 is caspase-dependent. Western blot analysis of TFF2 treated retinal wholemount homogenates revealed significant reductions in the phosphorylation level of ERK and STAT3 proteins compared to basal conditions, suggesting that in the developing murine retina survival mechanism are down-regulated upon TFF2 administration. Our results suggest that during retinal cell death periods, requiring a tightly regulated balance between cell survival and cell death, TFF2 acts pro-proliferative and pro-apoptotic at least in developing mouse retinae cultured in vivo.     

视网膜微血管内皮细胞在糖尿病视网膜病变中的研究现状

糖尿病视网膜疾病是导致成年人失明的主要因素,是糖尿病的一种令人恐惧的并发症,高血糖被认为是促进其发展的主要原因。高血糖不断地破坏视网膜的微血管系统最终导致视网膜的许多代谢,结构和功能的紊乱。视网膜微血管内皮细胞在微脉管系统中形成树枝状供应视网膜神经,这些内皮细胞的解剖和生理符合重要视觉保护的营养需求[1]。一方面,内皮组织务必确保氧的供应和代谢活跃的视网膜营养供应;另一方面,内皮细胞有助于血-视网膜屏障将循环产生的毒素分子,白细胞促炎性物质排出体外来保护视网膜,这种特性也可能会引起疾病,比如:视网膜血管的渗漏和新生血管,炎性物质转移,因此,视网膜内皮细胞在视网膜缺血性病变,血管炎中起到重要作用,包括糖尿病视网膜病变和视网膜炎症或感染尤其是后葡萄膜炎。使用基因表达和蛋白质组学分析等研究方法,有助于了解这些疾病的发病机制。为了进一步开展对糖尿病视网膜疾病的研究,有必要就目前有关糖尿病视网膜病变患者微血管内皮细胞的研究进展予以综述,旨在为糖尿病视网膜病变的深入研究提供参考依据。     

Murine glutamine synthetase: Cloning, developmental regulation, and glucocorticoid inducibility

We have cloned the murine glutamine synthetase (GS) gene and measured GS enzyme activity and mRNA in five tissues (retina, brain, liver, kidney, and skeletal muscle) during perinatal development. Retinal GS enzyme activity increases 200-fold between Day 1 and Day 21 and is accompanied by an increase in the level of GS mRNA; developmental regulation in other tissues is much less dramatic. Based on Southern blotting analysis, a single GS gene gives rise to the tissue-specific patterns of GS mRNA expression. The increase in murine retinal GS observed during perinatal development is similar in magnitude to that observed in the chicken retina just prior to hatching. In the embryonic chicken retina, glucocorticoid hormones mediate a large increase in the level of GS mRNA. However, although glucocorticoids induce a 12-fold increase in GS mRNA in murine skeletal muscle, expression of the retinal enzyme and mRNA is only modestly glucocorticoid-inducible in the mouse. Therefore, despite the hormonal responsiveness of the murine GS gene, it is not likely that glucocorticoids are important physiological modulators of the developmental rise in murine retinal GS.     

Unexpected diversity and photoperiod dependence of the zebrafish melanopsin system

Animals have evolved specialized photoreceptors in the retina and in extraocular tissues that allow them to measure light changes in their environment. In mammals, the retina is the only structure that detects light and relays this information to the brain. The classical photoreceptors, rods and cones, are responsible for vision through activation of rhodopsin and cone opsins. Melanopsin, another photopigment first discovered in Xenopus melanophores (Opn4x), is expressed in a small subset of retinal ganglion cells (RGCs) in the mammalian retina, where it mediates non-image forming functions such as circadian photoentrainment and sleep. While mammals have a single melanopsin gene (opn4), zebrafish show remarkable diversity with two opn4x-related and three opn4-related genes expressed in distinct patterns in multiple neuronal cell types of the developing retina, including bipolar interneurons. The intronless opn4.1 gene is transcribed in photoreceptors as well as in horizontal cells and produces functional photopigment. Four genes are also expressed in the zebrafish embryonic brain, but not in the photoreceptive pineal gland. We discovered that photoperiod length influences expression of two of the opn4-related genes in retinal layers involved in signaling light information to RGCs. Moreover, both genes are expressed in a robust diurnal rhythm but with different phases in relation to the light-dark cycle. The results suggest that melanopsin has an expanded role in modulating the retinal circuitry of fish.     

Distinct patterns of expression of the RB gene family in mouse and human retina

Although RB1 function is disrupted in the majority of human cancers, an undefined cell of developing human retina is uniquely sensitive to cancer induction when the RB1 tumor suppressor gene is lost. Murine retinoblastoma is initiated only when two of the RB family of genes, RB1 and p107 or p130, are inactivated. Although whole embryonic retina shows RB family gene expression by several techniques, when E14 developing retina was depleted of the earliest differentiating cells, ganglion cells, the remaining proliferating murine embryonic retinal progenitor cells clearly did not express RB1 or p130, while the longer splice form of p107 was expressed. Each retinal cell type expressed some member of the RB family at some stage of differentiation. Rod photoreceptors stained for the RB1 protein product, pRB, and p107 in only a brief window of postnatal murine development, with no detectable staining for any of the RB family proteins in adult human and mouse rod photoreceptors. Adult mouse and human Muller glia, ganglion and rare horizontal cells, and adult human, but not adult mouse, cone photoreceptors stained for pRB. The RB gene family is dynamically and variably expressed through retinal development in specific retinal cells.     

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.     

Role of matrix metalloproteinase-2 and -9 in the development of diabetic retinopathy

Diabetic retinopathy represents the most common causes of vision loss in patients affected by diabetes mellitus. The cause of vision loss in diabetic retinopathy is complex and remains incompletely understood. One of the earliest changes in the development of retinopathy is the accelerated apoptosis of retinal microvascular cells and the formation of acellular capillaries by unknown mechanism. Results of a recent research suggest an important role of matrix metalloproteinases (MMPs) in the development of diabetic retinopathy. MMPs are a large family of proteinases that remodel extracellular matrix components, and under pathological condition, its induction is considered as a negative regulator of cell survival; and in diabetes, latent MMPs are activated in the retina and its capillary cells, and activation of MMP-2 and -9 induces apoptosis of retinal capillary cells. This review will focus on the MMP-2 and MMP-9 in the diabetic retina with special reference to oxidative stress, mitochondria dysfunction, inflammation and angiogenesis, as well as summarizing the current information linking these proteins to pathogenesis of diabetic retinopathy.     

Changes in Retinal Glial Cells with Age and during Development of Age-Related Macular Degeneration

Age is the major risk factor in the age-related macular degeneration (AMD) which is a complex multifactor neurodegenerative disease of the retina and the main cause of irreversible vision loss in people over 60 years old. The major role in AMD pathogenesis belongs to structure-functional changes in the retinal pigment epithelium cells, while the onset and progression of AMD are commonly believed to be caused by the immune system dysfunctions. The role of retinal glial cells (Muller cells, astrocytes, and microglia) in AMD pathogenesis is studied much less. These cells maintain neurons and retinal vessels through the synthesis of neurotrophic and angiogenic factors, as well as perform supporting, separating, trophic, secretory, and immune functions. It is known that retinal glia experiences morphological and functional changes with age. Age-related impairments in the functional activity of glial cells are closely related to the changes in the expression of trophic factors that affect the status of all cell types in the retina. In this review, we summarized available literature data on the role of retinal macro- and microglia and on the contribution of these cells to AMD pathogenesis.     

mRNA expression of the murine glycoprotein (transmembrane) nmb (Gpnmb) gene is linked to the developing retinal pigment epithelium and iris

The murine homologue of the human glycoprotein (transmembrane) NMB (GPNMB) gene was identified by subtractive cloning from in vitro cultured murine primary osteoblast cells and subsequent RACE-PCR. GPNMB is a highly glycosylated type I transmembrane protein that shares significant sequence homology to several melanosomal proteins. Increasing expression of Gpnmb mRNA was observed during differentiation of murine primary osteoblast cell cultures. To address the potential functions of GPNMB we analysed its mRNA-expression during murine embryonic development. In early development Gpnmb mRNA is detected at high levels in the outer layer of the retina. Later in development expression gets restricted to the retinal pigment epithelium and iris. At the cytoplasmic domain of GPNMB, a conserved di-leucin-based endosomal/melanosomal-sorting signal (ExxPLL) was located, present as well in several known melanosomal proteins. To analyse the subcellular localization we used EGFP-tagged GPNMB transfected in COS7 and HEK293 cells. In both non-pigmented cell lines, the EGFP-GPNMB fusion protein was localized to vesicular, endosomal like structures. Sequence homology to known melanosomal proteins, mRNA expression and subcellular localization are suggestive for GPNMB as an intracellular, endosomal/melanosomal compartment specific protein important for melanin biosynthesis and the development of the retinal pigment epithelium and iris. As the gene coding for human GPNMB was localized to chromosome 7p15, a locus involved in the human inherited disease cystoid macular edema, also known as dominant cystoid macular dystrophy (OMIM 153880) we highly suggest that GPNMB is a candidate gene for this human inherited disease.     

The Wnt signaling pathway in retinal degenerations

The retina is a complex tissue composed of multiple interconnected cell layers, highly specialized for transforming light and color into electrical signals perceived by the brain. Damage or death of the primary light-sensing cells, the photoreceptors, results in devastating effects on vision. Despite the identification of numerous mutations that cause inherited retinal degenerations, the cellular and molecular mechanisms leading from the primary mutations to photoreceptor apoptosis are not understood. Wnt signaling has essential regulatory functions in a wide variety of critical developmental processes. Our research and others' have suggested that the Wnt pathway may be involved in retinal degeneration. Wnt ligands regulate developmental death of Drosophila photoreceptors, dysregulated Wnt signaling is involved in neuronal degeneration elsewhere in the central nervous system and Wnts control the expression of pro-survival growth factors in mammalian tissues. Additionally, altered expression of Wnt pathway genes, including the anti-apoptotic Wnt signaling regulator Dickkopf 3 (Dkk3), were observed during photoreceptor loss. This review examines the evidence and develops a model proposing a pro-survival role for Wnt signaling during photoreceptor injury. Because manipulating Wnt signaling has been demonstrated to have therapeutic potential for the treatment of Alzheimers disease, understanding the involvement of Wnts in photoreceptor death will determine whether targeting the Wnt pathway should also be considered as a possible therapeutic strategy for retinal degenerations.     

Lysosomal ceroid depletion by drugs: therapeutic implications for a hereditary neurodegenerative disease of childhood

Neuronal ceroid lipofuscinoses (NCLs) are the most common hereditary neurodegenerative diseases of childhood. The infantile form, INCL, is caused by lysosomal palmitoyl-protein thioesterase (PPT) deficiency, which impairs the cleavage of thioester linkages in palmitoylated proteins, preventing their hydrolysis by lysosomal proteinases. Consequent accumulation of these lipid-modified proteins (constituents of ceroid) in lysosomes leads to INCL. Because thioester linkages are susceptible to nucleophilic attack, drugs with this property may have therapeutic potential for INCL. We report here that two such drugs, phosphocysteamine and N-acetylcysteine, disrupt thioester linkages in a model thioester compound, [14C]palmitoyl approximately CoA. Most importantly, in lymphoblasts derived from INCL patients, phosphocysteamine, a known lysosomotrophic drug, mediates the depletion of lysosomal ceroids, prevents their re-accumulation and inhibits apoptosis. Our results define a novel pharmacological approach to lysosomal ceroid depletion and raise the possibility that nucleophilic drugs such as phosphocysteamine hold therapeutic potential for INCL.     

Angiotensin and diabetic retinopathy

Diabetic retinopathy develops in patients with both type 1 and type 2 diabetes and is the major cause of vision loss and blindness in the working population. In diabetes, damage to the retina occurs in the vasculature, neurons and glia resulting in pathological angiogenesis, vascular leakage and a loss in retinal function. The renin-angiotensin system is a causative factor in diabetic microvascular complications inducing a variety of tissue responses including vasoconstriction, inflammation, oxidative stress, cell hypertrophy and proliferation, angiogenesis and fibrosis. All components of the renin-angiotensin system including the angiotensin type 1 and angiotensin type 2 receptors have been identified in the retina of humans and rodents. There is evidence from both clinical and experimental models of diabetic retinopathy and hypoxic-induced retinal angiogenesis that the renin-angiotensin system is up-regulated. In these situations, retinal dysfunction has been linked to angiotensin-mediated induction of growth factors including vascular endothelial growth factor, platelet-derived growth factor and connective tissue growth factor. Evidence to date indicates that blockade of the renin-angiotensin system can confer retinoprotection in experimental models of diabetic retinopathy and ischemic retinopathy. This review examines the role of the renin-angiotensin system in diabetic retinopathy and the potential of its blockade as a treatment strategy for this vision-threatening disease.