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Reactive microglia are a hallmark of age-related retinal degenerative diseases including age-related macular degeneration (AMD). These cells are capable of secreting neurotoxic substances that may aggravate inflammation that leads to loss of photoreceptors and impaired vision. Despite their role in driving detrimental inflammation, microglia also play supporting roles in the retina as they are a crucial cellular component of the regulatory innate immune system. In this study, we used the colony stimulating factor 1 receptor (CSF1R)-antagonist PLX3397 to investigate the effects of microglia depletion and repopulation in a mouse model of acute retinal degeneration that mimics some aspects of dry AMD. Our main goal was to investigate whether microglia depletion and repopulation affects the outcome of light-induced retinal degeneration. We found that microglia depletion effectively decreased the expression of several key pro-inflammatory factors but was unable to influence the extent of retinal degeneration as determined by optical coherence tomography (OCT) and histology. Interestingly, we found prominent cell debris accumulation in the outer retina under conditions of microglia depletion, presumably due to the lack of efficient phagocytosis that could not be compensated by the retinal pigment epithelium. Moreover, our in vivo experiments showed that renewal of retinal microglia by repopulation did also not prevent rapid microglia activation or preserve photoreceptor death under conditions of light damage. We conclude that microglia ablation strongly reduces the expression of pro-inflammatory factors but cannot prevent photoreceptor loss in the light-damage paradigm of retinal degeneration.

Abstract Background Dysfunctional humoral and cellular innate immunity are key components in the development and progression of age-related macular degeneration (AMD). Specifically, chronically activated microglia and their disturbed regulatory system contribute to retinal degeneration. Galectin-3, a β-galactose binding protein, is a potent driver of macrophage and microglia activation and has been implicated in neuroinflammation, including neurodegenerative diseases of the brain. Here, we hypothesized that genetic deficiency of galectin-3 or its modulation via TD139 dampens mononuclear phagocyte reactivity and delays retinal degeneration. Methods Galectin-3 expression in AMD patients was analyzed by immunohistochemical stainings. Galectin-3 knockout and BALB/cJ mice were exposed to white bright light with an intensity of 15,000 lux for 1 h and Cx3cr1GFP/+ mice to focal blue light of 50,000 lux for 10 min. BALB/cJ and Cx3cr1GFP/+ mice received intraperitoneal injections of 15 mg/kg TD139 or vehicle for five consecutive days, starting one day prior to light exposure. The effects of galectin-3 deficiency or inhibition on microglia were analyzed by immunohistochemical stainings and in situ hybridization of retinal sections and flat mounts. Pro-inflammatory cytokine levels in the retina and retinal pigment epithelium (RPE) were quantified by qRT-PCR and transcriptomic changes were analyzed by RNA-sequencing. Retinal thickness and structure were evaluated by optical coherence tomography. Results We found that galectin-3 expression was strongly upregulated in reactive retinal mononuclear phagocytes of AMD patients and in the two related mouse models of light-induced retinal degeneration. The experimental in vivo data further showed that specific targeting of galectin-3 by genetic knockout or administration of the small-molecule inhibitor TD139 reduced microglia reactivity and delayed retinal damage in both light damage conditions. Conclusion This study defines galectin-3 as a potent driver of retinal degeneration and highlights the protein as a drug target for ocular immunomodulatory therapies.

Defects in primary or motile cilia result in a variety of human pathologies, and retinal degeneration is frequently associated with these so-called ciliopathies. We found that homozygosity for a truncating variant in CEP162, a centrosome and microtubule-associated protein required for transition zone assembly during ciliogenesis and neuronal differentiation in the retina, caused late-onset retinitis pigmentosa in 2 unrelated families. The mutant CEP162-E646R*5 protein was expressed and properly localized to the mitotic spindle, but it was missing from the basal body in primary and photoreceptor cilia. This impaired recruitment of transition zone components to the basal body and corresponded to complete loss of CEP162 function at the ciliary compartment, reflected by delayed formation of dysmorphic cilia. In contrast, shRNA knockdown of Cep162 in the developing mouse retina increased cell death, which was rescued by expression of CEP162-E646R*5, indicating that the mutant retains its role for retinal neurogenesis. Human retinal degeneration thus resulted from specific loss of the ciliary function of CEP162.

Abstract Background Retinal degenerative diseases significantly contribute to visual impairment and blindness. Microglia reactivity is a hallmark of neurodegenerative diseases including retinal cell death and immunomodulation emerges as a therapeutic option. Indole-3-carbinol (I3C) is a natural ligand of aryl hydrocarbon receptor (AhR), with potent immunomodulatory properties. Here, we hypothesized that I3C may inhibit microglia reactivity and exert neuroprotective effects in the light-damaged murine retina mimicking important immunological aspects of retinal degeneration. Methods BV-2 microglia were treated in vitro with I3C followed by lipopolysaccharide (LPS) stimulation to analyze pro-inflammatory and anti-oxidant responses by quantitative real-time PCR (qRT-PCR) and Western blots. Nitric oxide (NO) secretion, caspase 3/7 levels, phagocytosis rates, migration, and morphology were analyzed in control and AhR knockdown cells. I3C or vehicle was systemically applied to light-treated BALB/cJ mice as an experimental model of retinal degeneration. Pro-inflammatory and anti-oxidant responses in the retina were examined by qRT-PCR, ELISA, and Western blots. Immunohistochemical staining of retinal flat mounts and cryosections were performed. The retinal thickness and structure were evaluated by in vivo imaging using spectral domain-optical coherence tomography (SD-OCT). Results The in vitro data showed that I3C potently diminished LPS-induced pro-inflammatory gene expression of I-NOS, IL-1ß, NLRP3, IL-6, and CCL2 and induced anti-oxidants gene levels of NQO1, HMOX1, and CAT1 in BV-2 cells. I3C also reduced LPS-induced NO secretion, phagocytosis, and migration as important functional microglia parameters. siRNA-mediated knockdown of AhR partially prevented the previously observed gene regulatory events. The in vivo experiments revealed that I3C treatment diminished light-damage induced I-NOS, IL-1ß, NLRP3, IL-6, and CCL2 transcripts and also reduced CCL2, I-NOS, IL-1ß, p-NFkBp65 protein levels in mice. Moreover, I3C increased anti-oxidant NQO1 and HMOX1 protein levels in light-exposed retinas. Finally, I3C therapy prevented the accumulation of amoeboid microglia in the subretinal space and protected from retinal degeneration. Conclusions The AhR ligand I3C potently counter-acts microgliosis and light-induced retinal damage, highlighting a potential treatment concept for retinal degeneration.

Age-related macular degeneration (AMD) is a major cause of blindness in the elderly. Here, the authors show that microglia-specific deletion of TSPO and chemical inhibition of TSPO prevent neuroinflammation and vascular damage in a mouse model of AMD.

Abstract Background Age-related macular degeneration (AMD) is a leading cause of visual impairment in the elderly. The neovascular (wet) form of AMD can be treated with intravitreal injections of different anti-vascular endothelial growth factor (VEGF) agents. Placental growth factor (PGF) is another member of the VEGF family of cytokines with pro-angiogenic and pro-inflammatory effects. Here, we aimed to compare single and combined inhibition of VEGF-A and PGF in the laser-induced mouse model of choroidal neovascularization (CNV) with a focus on the effects on retinal mononuclear phagocytes. Methods CNV was induced in C57BL/6J mice using a YAG-Laser. Immediately after laser damage antibodies against VEGF-A (aVEGF), anti-PGF (aPGF), aVEGF combined with aPGF, aflibercept, or IgG control were injected intravitreally in both eyes. Three and 7 days after laser damage, the vascular leakage was determined by fluorescence angiography. Lectin staining of retinal and RPE/choroidal flat mounts was used to monitor CNV. In situ mRNA co-expression of Iba1, VEGF and PGF were quantified using in situ hybridization. Retinal and RPE/choroidal protein levels of VEGF and PGF as well as the pro-inflammatory cytokines IL-6, IL1-beta, and TNF were determined by ELISA. Results Early (day 3) and intermediate (day 7) vascular leakage and CNV were significantly inhibited by PGF and VEGF-A co-inhibition, most effectively with the trap molecule aflibercept. While VEGF-A blockage alone had no effects, trapping PGF especially with aflibercept prevented the accumulation of reactive microglia and macrophages in laser lesions. The lesion-related mRNA expression and secretion of VEGF-A and PGF by mononuclear phagocytes were potently suppressed by PGF and partially by VEGF-A inhibition. Protein levels of IL-6 and IL1-beta were strongly reduced in all treatment groups. Conclusions Retinal inhibition of PGF in combination with VEGF-A prevents vascular leakage and CNV possibly via modulating their own expression in mononuclear phagocytes. PGF-related, optimized strategies to target inflammation-mediated angiogenesis may help to increase efficacy and reduce non-responders in the treatment of wet AMD patients.

Chronic activation of microglia is associated with retinal degeneration, which makes them a potential therapeutic target for retinal degenerative diseases including age-related macular degeneration (AMD). Interferon-beta (IFN-β) is a potent immune regulator, commonly used for the treatment of multiple sclerosis patients. We have previously shown that IFN-β prevents microgliosis and choroidal neovascularization in a laser model of wet AMD. Here, we hypothesized that microglia modulation via IFN-β may also dampen mononuclear phagocyte reactivity and thereby protect from retinal degeneration in a light-damage paradigm mimicking some features of dry AMD. BALB/cJ mice received intraperitoneal injections of 10,000 U IFN-β or vehicle every other day; starting at the day of exposure to 15,000 lux white light for 1 h. Systemic treatment with IFN-β partially enhanced IFN-α/β receptor (IFNAR) signaling in the retina and reduced the number of reactivated microglia in the subretinal space. However, four days after light damage neither decreased expression of complement factors nor rescue of retinal thickness was found. We conclude that IFNAR signaling modulate retinal microglia but cannot prevent strong retinal degeneration as elicited by acute white light damage.

Diabetic retinopathy is a vision-threatening disease affecting neurons and microvasculature of the retina. The development of this disease is associated with the action of inflammatory factors that are connected to the activation of microglial cells, the resident tissue macrophages of the CNS. In the quiescent state, microglial cells help maintain tissue homeostasis in the retina through phagocytosis and control of low-grade inflammation. However, prolonged tissue stress due to hyperglycemia primes microglia to become overly reactive with the concomitant production of pro-inflammatory cytokines and chemokines causing chronic inflammation. In this review, we provide evidence of microglial cell activation and pro-inflammatory molecules associated with the development and progression of diabetic retinopathy. We further highlight innovative animal models that can mimic the disease in humans and discuss strategies in modulating microglial-mediated inflammation as potential therapeutic approaches in managing the disease.

Significant association signals from genome-wide association studies (GWAS) point to genomic regions of interest. However, for most loci the causative genetic variant remains undefined. Determining expression quantitative trait loci (eQTL) in a disease relevant tissue is an excellent approach to zoom in on disease- or trait-associated association signals and hitherto on relevant disease mechanisms. To this end, we explored regulation of gene expression in healthy retina (n = 311) and generated the largest cis-eQTL data set available to date. Genotype- and RNA-Seq data underwent rigorous quality control protocols before FastQTL was applied to assess the influence of genetic markers on local (cis) gene expression. Our analysis identified 403,151 significant eQTL variants (eVariants) that regulate 3,007 genes (eGenes) (Q-Value < 0.05). A conditional analysis revealed 744 independent secondary eQTL signals for 598 of the 3,007 eGenes. Interestingly, 99,165 (24.71%) of all unique eVariants regulate the expression of more than one eGene. Filtering the dataset for eVariants regulating three or more eGenes revealed 96 potential regulatory clusters. Of these, 31 harbour 130 genes which are partially regulated by the same genetic signal. To correlate eQTL and association signals, GWAS data from twelve complex eye diseases or traits were included and resulted in identification of 80 eGenes with potential association. Remarkably, expression of 10 genes is regulated by eVariants associated with multiple eye diseases or traits. In conclusion, we generated a unique catalogue of gene expression regulation in healthy retinal tissue and applied this resource to identify potentially pleiotropic effects in highly prevalent human eye diseases. Our study provides an excellent basis to further explore mechanisms of various retinal disease etiologies.

Abstract This review highlights the role of three key immune pathways in the pathophysiology of major retinal degenerative diseases including diabetic retinopathy, age‐related macular degeneration, and rare retinal dystrophies. We first discuss the mechanisms how loss of retinal homeostasis evokes an unbalanced retinal immune reaction involving responses of local microglia and recruited macrophages, activity of the alternative complement system, and inflammasome assembly in the retinal pigment epithelium. Presenting these key mechanisms as complementary targets, we specifically emphasize the concept of immunomodulation as potential treatment strategy to prevent or delay vision loss. Promising molecules are ligands for phagocyte receptors, specific inhibitors of complement activation products, and inflammasome inhibitors. We comprehensively summarize the scientific evidence for this strategy from preclinical animal models, human ocular tissue analyses, and clinical trials evolving in the last few years.

Microglia activation plays an important role in immune responses in the CNS including the retina. Crocin, a plant-derived carotenoid, has been reported to possess anti-inflammatory, anti-apoptotic and anti-oxidative capacity in models of retinal damage and degeneration. If these neuroprotective effects could be mediated by direct modulation of microglial cells is unclear. Here, we examined the direct effects of crocin on key functions and pro-inflammatory gene expression in lipopolysaccharide (LPS)-activated BV-2 microglia. We found that crocin stimulation strongly promoted filopodia formation and markedly increased microglial phagocytosis, two important parameters relevant for physiological microglia functions. Moreover, crocin significantly reduced gene expression of the pro-inflammatory markers IL6, CCL2, and iNOS in LPS-challenged BV-2 cells and potently blocked NO production in these microglia. The observed immunomodulatory effects of crocin were not mediated by general inhibition of NFkB nuclear translocation. Our findings indicate that many of the anti-inflammatory effects of crocin demonstrated in animal models of neuronal degeneration could be mediated by its direct effects on microglia homeostasis.

Abstract Age‐related macular degeneration (AMD) is a major cause of blindness in the elderly population. Its pathophysiology is linked to reactive oxygen species (ROS) and activation of the complement system. Sialic acid polymers prevent ROS production of human mononuclear phagocytes via the inhibitory sialic acid‐binding immunoglobulin‐like lectin‐11 (SIGLEC11) receptor. Here, we show that low‐dose intravitreal injection of low molecular weight polysialic acid with average degree of polymerization 20 (polySia avDP20) in humanized transgenic mice expressing SIGLEC11 on mononuclear phagocytes reduced their reactivity and vascular leakage induced by laser coagulation. Furthermore, polySia avDP20 prevented deposition of the membrane attack complex in both SIGLEC11 transgenic and wild‐type animals. In vitro, polySia avDP20 showed two independent, but synergistic effects on the innate immune system. First, polySia avDP20 prevented tumor necrosis factor‐α, vascular endothelial growth factor A, and superoxide production by SIGLEC11‐positive phagocytes. Second, polySia avDP20 directly interfered with complement activation. Our data provide evidence that polySia avDP20 ameliorates laser‐induced damage in the retina and thus is a promising candidate to prevent AMD‐related inflammation and angiogenesis.

Microglia represent the primary resident immune cells of the central nervous system (CNS) and modulate local immune responses. Depending on their physiological functions, microglia can be classified into pro- (M1) and anti-inflammatory (M2) phenotype. Interleukin (IL)-10 is an important modulator of neuronal homeostasis, with anti-inflammatory and neuroprotective functions, and can be released by microglia. Here, we investigated how IL-10 deficiency affected the M1/2 polarization of primary microglia upon lipopolysaccharide (LPS) stimulation in vitro. Microglia phenotypes were analyzed via flow cytometry. Cytokine and chemokine secretion were examined by ELISA and bead-based multiplex LEGENDplexTM. Our results showed that genetic depletion of IL-10 led to elevated M1 like phenotype (CD86+ CD206−) under pro-inflammatory conditions associated with increased frequency of IL-6+, TNF-α+ cells and enhanced release of several pro-inflammatory chemokines. Absence of IL-10 led to an attenuated M2 like phenotype (CD86− CD206+) and a reduced secretion of TGF-β1 upon LPS stimulation. In conclusion, IL-10 deficiency may promote the polarization of microglia into M1-prone phenotype under pro-inflammatory conditions.

The retina is a complex tissue with multiple cell layers that are highly ordered. Its sophisticated structure makes it especially sensitive to external or internal perturbations that exceed the homeostatic range. This necessitates the continuous surveillance of the retina for the detection of noxious stimuli. This task is mainly performed by microglia cells, the resident tissue macrophages which confer neuroprotection against transient pathophysiological insults. However, under sustained pathological stimuli, microglial inflammatory responses become dysregulated, often worsening disease pathology. In this review, we provide an overview of recent studies that depict microglial responses in diverse retinal pathologies that have degeneration and chronic immune reactions as key pathophysiological components. We also discuss innovative immunomodulatory therapy strategies that dampen the detrimental immunological responses to improve disease outcome.

Abstract Age‐related macular degeneration (AMD) is a leading cause of vision loss among the elderly. AMD pathogenesis involves chronic activation of the innate immune system including complement factors and microglia/macrophage reactivity in the retina. Here, we show that lack of interferon‐β signaling in the retina accelerates mononuclear phagocyte reactivity and promotes choroidal neovascularization (CNV) in the laser model of neovascular AMD. Complete deletion of interferon‐α/β receptor (Ifnar) using Ifnar1−/− mice significantly enhanced early microglia and macrophage activation in lesion areas. This triggered subsequent vascular leakage and CNV at later stages. Similar findings were obtained in laser‐treated Cx3cr1CreER:Ifnar1fl/fl animals that allowed the tamoxifen‐induced conditional depletion of Ifnar in resident mononuclear phagocytes only. Conversely, systemic IFN‐β therapy of laser‐treated wild‐type animals effectively attenuated microgliosis and macrophage responses in the early stage of disease and significantly reduced CNV size in the late phase. Our results reveal a protective role of Ifnar signaling in retinal immune homeostasis and highlight a potential use for IFN‐β therapy in the eye to limit chronic inflammation and pathological angiogenesis in AMD.

Juvenile neuronal ceroid lipofuscinosis (jNCL) is a rare but fatal inherited lysosomal storage disorder mainly affecting children. The disease is caused by mutations in the CLN3 gene that lead to the accumulation of storage material in many tissues, prominent immune responses and neuronal degeneration. One of the first symptoms is vision loss followed by motor dysfunction and mental decline. The established Cln3Δex7/8 mouse model mimics many pathological features of the human disease except the retinal phenotype, which is very mild and occurs only very late in these mice. Here, we first carefully analyzed the retinal structure and microglia responses in these animals. While prominent autofluorescent spots were present in the fundus, only a moderate reduction of retinal thickness and no prominent microgliosis was seen in young CLN3-deficient mice. We next genetically introduced a light-sensitive RPE65 variant and established a light-damage paradigm that showed a high susceptibility of young Cln3Δex7/8 mice after exposure to 10,000 lux bright light for 30 min. Under these ‘low light’ conditions, CLN3-deficient mice showed a strong retinal degeneration, microglial activation, deposition of autofluorescent material and transcriptomic changes compared to wild-type animals. Finally, we treated the light-exposed Cln3Δex7/8 animals with the immunomodulatory compound minocycline, and thereby rescued the retinal phenotype and diminished microgliosis. Our findings indicate that exposure to specific light conditions accelerates CLN3-dependent retinal degeneration, and that immunomodulation by minocycline could be a possible treatment option to delay vision loss in jNCL patients. This article has an associated First Person interview with the first author of the paper.

HLA-B27 is the allele most frequently associated with human anterior uveitis. The majority of HLA-B27-positive [acute anterior uveitis (AAU)] patients develop clinically distinct symptoms with acute symptomatic onset of flare and a recurrent disease course characterized by a massive cellular ocular infiltrate during uveitis relapse. By contrast, uveitis in HLA-B27-negative [idiopathic anterior uveitis (IAU)] patients tends to develop a clinically less fulminant, more chronic, and typically asymptomatic disease course. To analyze systemic immune responses in the different uveitis entities, we analyzed peripheral blood cells by flow cytometry. In addition, as a pro-inflammatory biomarker serum, S100A8/A9 levels were quantified by ELISA from patients with AAU (n = 27) and IAU (n = 21), and in healthy controls (n = 30). Data were obtained either during active uveitis flare or after 3 months of inactivity. IAU patients showed a transiently increased frequency of CD56- and CD163-positive monocytes and of both granulocytic myeloid-derived suppressor cells and Th17 cells during active uveitis. By contrast, AAU patients showed an elevated frequency of monocytes, activated T cells, and elevated S100A8/A9 serum levels during clinically quiescent disease. The differentially regulated response of both innate and adaptive immune cells in the blood may be related to the clinically distinct characteristics of the two different uveitis entities.

A chronic pro-inflammatory environment is a hallmark of retinal degenerative diseases and neurological disorders that affect vision. Inflammatory responses during retinal pathophysiology are orchestrated by microglial cells which constitute the resident immune cell population. Following activation, microglia cells lose their ramified protrusions, proliferate and rapidly migrate to the damaged areas and resolve tissue damage. However, sustained presence of tissue stress primes microglia to become overreactive and results in the excessive production of pro-inflammatory mediators that favor retinal degenerative changes. Consequently, interventions aimed at overriding microglial pro-inflammatory and pro-oxidative properties may attenuate photoreceptor demise and preserve retinal integrity. We highlight the positive effects of ligands for the translocator protein 18 kDa (TSPO) and the cytokine interferon beta (IFN-β) in modulating microgliosis during retinal pathologies and discuss their plausible mechanisms of action.

Abstract Neovascular and inflammatory retinal diseases including wet age‐related macular degeneration (AMD) can cause severe vision loss among the elderly. Simultaneous neutralization of vascular endothelial growth factor‐A (VEGF‐A) and angiopoietin 2 (ANG2) is envisioned as a novel candidate approach to treat wet AMD with better efficacy. However, earlier published data from a genetic mouse model showed data aberrations (Regula et al, 2016). In this issue of EMBO Molecular Medicine, Foxton et al (2019) have provided compelling evidence replicating the data and confirming the overall concept that VEGF‐A/ANG2 combotherapy is effective in suppressing retinal neovascularization.

Abstract Neovascular age‐related macular degeneration (nvAMD) can cause severe vision loss among the elderly. Genetic risk factors for AMD include several variants related to the immune system and lipid metabolism. Obesity is a well‐known predisposing factor for nvAMD but how this metabolic disorder modulates angiogenesis in the posterior eye segment was largely unknown. In this issue of EMBO Molecular Medicine, Andriessen et al (2016) show that high‐fat diet‐induced obesity causes dysbiosis in the gut that drives retinal inflammation and pathological angiogenesis in a mouse model of laser‐induced choroidal neovascularization (CNV).

Although ChIP-chip is a powerful tool for genome-wide discovery of transcription factor target genes, the steps involving raw data analysis, identification of promoters, and correlation with binding sites are still laborious processes. Therefore, we report an integrated workflow for the analysis of promoter tiling arrays with the Genomatix ChipInspector system. We compare this tool with open-source software packages to identify PU.1 regulated genes in mouse macrophages. Our results suggest that ChipInspector data analysis, comparative genomics for binding site prediction, and pathway/network modeling significantly facilitate and enhance whole-genome promoter profiling to reveal in vivo sites of transcription factor-DNA interactions.

Malignant glioma belong to the most aggressive neoplasms in humans with no successful treatment available. Patients suffering from glioblastoma multiforme (GBM), the highest-grade glioma, have an average survival time of only around one year after diagnosis. Both microglia and peripheral macrophages/monocytes accumulate within and around glioma, but fail to exert effective anti-tumor activity and even support tumor growth. Here we use microarray analysis to compare the expression profiles of glioma-associated microglia/macrophages and naive control cells. Samples were generated from CD11b+ MACS-isolated cells from naïve and GL261-implanted C57BL/6 mouse brains. Around 1000 genes were more than 2-fold up- or downregulated in glioma-associated microglia/macrophages when compared to control cells. A comparison with published data sets of M1, M2a,b,c-polarized macrophages revealed a gene expression pattern that has only partial overlap with any of the M1 or M2 gene expression patterns. Samples for the qRT-PCR validation of selected M1 and M2a,b,c-specific genes were generated from two different glioma mouse models and isolated by flow cytometry to distinguish between resident microglia and invading macrophages. We confirmed in both models the unique glioma-associated microglia/macrophage phenotype including a mixture of M1 and M2a,b,c-specific genes. To validate the expression of these genes in human we MACS-isolated CD11b+ microglia/macrophages from GBM, lower grade brain tumors and control specimens. Apart from the M1/M2 gene analysis, we demonstrate that the expression of Gpnmb and Spp1 is highly upregulated in both murine and human glioma-associated microglia/macrophages. High expression of these genes has been associated with poor prognosis in human GBM, as indicated by patient survival data linked to gene expression data. We also show that microglia/macrophages are the predominant source of these transcripts in murine and human GBM. Our findings provide new potential targets for future anti-glioma therapy.

The zinc finger protein ZNF202 is a transcriptional repressor that binds to promoter elements predominantly found in genes involved in lipid metabolism. Here we demonstrate that ZNF202 mRNA expression is inversely correlated with ATP binding cassette A1 (ABCA1), ABCG1, and apolipoprotein E (apoE) in human monocytes. Upregulation of ABCA1, ABCG1, and apoE expression during monocyte differentiation and foam cell formation was accompanied by a simultaneous downregulation of both ZNF202 mRNA isoforms m1 and m3. Conversely, deloading of macrophage foam cells with HDL3 caused upregulation of ZNF202 mRNA. To further characterize the transcriptional regulation of the ZNF202 gene, comparative genomic sequence analysis and reporter gene assays were performed. The ZNF202 core promoter region resides within 247 bp upstream of the transcription initiation site and is highly active in THP-1 monocytes, yet downregulated upon macrophage differentiation. Using site-directed mutagenesis, we show that two highly conserved transcription factor binding sites, a GC-box and an Ets-binding motif, are required for ZNF202 gene expression. Furthermore, electrophoretic mobility shift assays demonstrate in vitro binding of PU.1 and GC-box binding proteins to the ZNF202 proximal promoter.We conclude that the inversely correlated transcriptional activity of ZNF202 and its target genes during macrophage differentiation may reflect a direct regulatory interdependence and thus provide further evidence for ZNF202 as an important gatekeeper of lipid efflux.

The molecular cloning and identification of mutations in ATP-binding cassette transporters in hereditary diseases have greatly expanded our knowledge of the normal physiology of intracellular lipid transport processes. In addition to the well-known ATP-binding cassette transporter A1 (ABCA1) molecule, ABC transporters belonging to the ABCG (White) subfamily (ABCG1, ABCG5, and ABCG8) have been shown to be critically involved in the regulation of lipid-trafficking mechanisms in macrophages, hepatocytes, and intestinal mucosa cells. ABCG1, the product of a sterol-induced gene, participates in cholesterol and phospholipid efflux. The ABCG5 and ABCG8 transporters, defective in β-sitosterolemia, are also now considered interesting targets in the control and influence of total body sterol homeostasis.In this review, advances referring to the regulation and function of ABCG half-size transporters are summarized and discussed. In addition, new implications for the transcriptional control, as well as the intracellular routing and localization, of these proteins are presented.

Cells from the human monocytic leukemia cell line THP-1 differentiate towards a macrophage-like phenotype when stimulated with phorbol 12-myristate-13-acetate (PMA), 1,25-dihydroxy-vitamin D3, and various other agents. We demonstrate here that the expression of the lysosomal enzyme acid sphingomyelinase (ASM; E.C. 3.1.4.12) is induced during this process and is strongly elevated in differentiated THP-1 cells, as well as in differentiated human mononuclear phagocytes. Using Northern blotting, RNase protection assay, and nuclear run-on analyses, we show that the up-regulation of ASM expression is regulated mainly at the level of transcription and that new protein synthesis is required for enhanced ASM activity. This cell-type specific induction by PMA treatment was further investigated with respect to transcriptional control. A series of 5′ deletion derivatives of the upstream regulatory region were used in transient transfection assays to identify promoter elements required for basal and inducible gene expression. A PMA responsive element was localized to a region between –319 and –219 bp upstream of the initiation codon and co-transfections with transcription factor expression plasmids for AP-2 and Sp1 resulted in augmented ASM promoter activity, which was abolished when the binding sites for these two factors were deleted. Using electrophoretic mobility shift assays and supershift assays we demonstrate that this region is specifically bound by Sp1 and AP-2. These factors are present in nuclear extracts prepared from both induced and uninduced THP-1 cells. However, the intensity of the complex formed appeared to increase when nuclear extracts from PMA-treated cells were used. From these studies, we conclude that a concerted action of the transcription factors AP-2 and Sp1 is essential for the up-regulation of ASM expression during the process of macrophage differentiation.—Langmann, T., C. Buechler, S. Ries, A. Schaeffler, C. Aslanidis, M. Schuierer, M. Weiler, K. Sandhoff, P. J. de Jong, and G. Schmitz. Transcription factors Sp1 and AP-2 mediate induction of acid sphingomyelinase during monocytic differentiation. J. Lipid Res. 1999. 40: 870–880.

Human lysosomal acid lipase (LAL) is a hydrolase required for the cleavage of cholesteryl esters and triglycerides derived from plasma lipoproteins. It is shown here that during monocyte to macrophage differentiation, the expression of LAL-mRNA is induced. This induction is dependent on protein kinase C activity and protein synthesis. The cell type-specific increase in LAL expression is further investigated in the THP-1 cell line with respect to transcriptional regulation. The human monocytic leukemia cell line THP-1 differentiates into macrophage-like cells when treated with phorbol esters. In order to determine the cis-acting elements necessary for both basal and phorbol 12-myristate-13 acetate (PMA)-enhanced promoter activity, we performed deletion analysis and reporter gene assays. A PMA responsive element has been identified between −182 bp and −107 bp upstream of the major transcription start site. Gel mobility shift assays demonstrated that binding of Sp1 and AP-2 to the LAL promoter is increased by PMA in THP-1 cells. Co-transfections with expression plasmids for Sp1 and AP-2 further emphasized the important role of these transcription factors in both basal and PMA-enhanced LAL expression. Our data suggest that differentiation dependent increase of lysosomal acid lipase (LAL) expression in THP-1 cells is mediated by a concerted action of Sp1 and AP-2. —Ries, S., C. Büchler, T. Langmann, P. Fehringer, C. Aslanidis, and G. Schmitz. Transcriptional regulation of lysosomal acid lipase in differentiating monocytes is mediated by transcription factors Sp1 and AP-2. J. Lipid Res. 1998. 39: 2125–2134.

Inherited retinal diseases are mainly caused by mutations in genes that are highly expressed in photoreceptors of the retina. The majority of these genes is under the control of the transcription factor Cone rod homeobox (Crx), that acts as a master transcription factor in photoreceptors. Using a genome-wide chromatin immunoprecipitation dataset that highlights all potential in vivo targets of Crx, we have identified a novel sterile alpha motif (SAM) domain containing protein, Samd7. mRNA Expression of Samd7 was confined to the late postnatal and adult mouse retina as well as the pineal gland. Using immunohistochemistry and Western blot, we could detect Samd7 protein in the outer nuclear layer of adult mouse retina. Ectopic over-expression in HEK293 cells demonstrated that Samd7 resides in the cytoplasm as well as the nucleus. In vitro electroporation of fluorescent reporters into living mouse retinal cultures revealed that transcription of the Samd7 gene depends on evolutionary conserved Crx motifs located in the first intron enhancer. Moreover, Crx knock-down with shRNA strongly reduced Samd7 reporter activity and endogenous Samd7 protein, indicating that Crx is required for retinal expression of Samd7. Finally, using co-transfections in luciferase reporter assays we found that Samd7 interferes with Crx-dependent transcription. Samd7 suppressed luciferase activity from a reporter plasmid with five Crx consensus repeats in a dose dependent manner and reduced Crx-mediated transactivation of regulatory sequences in the retinoschisin gene and the Samd7 gene itself. Taken together, we have identified a novel retinal SAM domain protein, Samd7, which could act as a transcriptional repressor involved in fine-tuning of Crx-regulated gene expression.

Neuronal ceroid lipofuscinosis (NCL) is a group of neurodegenerative lysosomal storage disorders characterized by vision loss, mental and motor deficits, and spontaneous seizures. Neuropathological analyses of autopsy material from NCL patients and animal models revealed brain atrophy closely associated with glial activity. Earlier reports also noticed loss of retinal cells and reactive gliosis in some forms of NCL. To study this phenomenon in detail, we analyzed the ocular phenotype of CLN6 (nclf) mice, an established mouse model for variant-late infantile NCL. Retinal morphometry, immunohistochemistry, optokinetic tracking, electroretinography, and mRNA expression were used to characterize retinal morphology and function as well as the responses of Müller cells and microglia. Our histological data showed a severe and progressive degeneration in the CLN6 (nclf) retina co-inciding with reactive Müller glia. Furthermore, a prominent phenotypic transformation of ramified microglia to phagocytic, bloated, and mislocalized microglial cells was identified in CLN6 (nclf) retinas. These events overlapped with a rapid loss of visual perception and retinal function. Based on the strong microglia reactivity we hypothesized that dietary supplementation with immuno-regulatory compounds, curcumin and docosahexaenoic acid (DHA), could ameliorate microgliosis and reduce retinal degeneration. Our analyses showed that treatment of three-week-old CLN6 (nclf) mice with either 5% DHA or 0.6% curcumin for 30 weeks resulted in a reduced number of amoeboid reactive microglia and partially improved retinal function. DHA-treatment also improved the morphology of CLN6 (nclf) retinas with a preserved thickness of the photoreceptor layer in most regions of the retina. Our results suggest that microglial reactivity closely accompanies disease progression in the CLN6 (nclf) retina and both processes can be attenuated with dietary supplemented immuno-modulating compounds.