Granulocyte-macrophage colony-stimulating factor mRNA and Neuroprotective Immunity in Parkinson's disease.

Restoring numbers and function of regulatory T cells (Tregs) is a novel therapeutic strategy for neurodegenerative disorders. Whether Treg function is boosted by adoptive cell transfer, pharmaceuticals, or immune modulators, the final result is a robust anti-inflammatory and neuronal sparing response. Herein, a newly developed lipid nanoparticle (LNP) containing mRNA encoding granulocyte-macrophage colony-stimulating factor (Gm-csf mRNA) was developed to peripherally induce Tregs and used for treatment in preclinical Parkinson's disease (PD) models. Administration of Gm-csf mRNA to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice and rats overexpressing alpha-synuclein produced dose-dependent increases in plasma GM-CSF levels and peripheral CD4+CD25+FoxP3+ Treg populations. This upregulation paralleled nigrostriatal neuroprotection, upregulated immunosuppression-associated mRNAs that led to the detection of a treatment-induced CD4+ T cell population, and decreased reactive microgliosis. The current findings strengthen prior works utilizing immune modulation by harnessing Gm-csf mRNA to augment adaptive immune function by employing a new delivery platform to treat PD and potentially other neurodegenerative disorders.

mRNA-encoded, constitutively active STINGV155M is a potent genetic adjuvant of antigen-specific CD8+ T cell response.

mRNA vaccines induce potent immune responses in preclinical models and clinical studies. Adjuvants are used to stimulate specific components of the immune system to increase immunogenicity of vaccines. We utilized a constitutively active mutation (V155M) of the stimulator of interferon (IFN) genes (STING), which had been described in a patient with STING-associated vasculopathy with onset in infancy (SAVI), to act as a genetic adjuvant for use with our lipid nanoparticle (LNP)-encapsulated mRNA vaccines. mRNA-encoded constitutively active STINGV155M was most effective at maximizing CD8+ T cell responses at an antigen/adjuvant mass ratio of 5:1. STINGV155M appears to enhance development of antigen-specific T cells by activating type I IFN responses via the nuclear factor κB (NF-κB) and IFN-stimulated response element (ISRE) pathways. mRNA-encoded STINGV155M increased the efficacy of mRNA vaccines encoding the E6 and E7 oncoproteins of human papillomavirus (HPV), leading to reduced HPV+ TC-1 tumor growth and prolonged survival in vaccinated mice. This proof-of-concept study demonstrated the utility of an mRNA-encoded genetic adjuvant.

PGC-1α Induces Human RPE Oxidative Metabolism and Antioxidant Capacity.

PURPOSE: Oxidative stress and metabolic dysregulation of the RPE have been implicated in AMD; however, the molecular regulation of RPE metabolism remains unclear. The transcriptional coactivator, peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) is a powerful mediator of mitochondrial function. This study examines the ability of PGC-1α to regulate RPE metabolic program and oxidative stress response. METHODS: Primary human fetal RPE (hfRPE) and ARPE-19 were matured in vitro using standard culture conditions. Mitochondrial mass of RPE was measured using MitoTracker staining and citrate synthase activity. Expression of PGC-1 isoforms, RPE-specific genes, oxidative metabolism proteins, and antioxidant enzymes was analyzed by quantitative PCR and Western blot. Mitochondrial respiration and fatty-acid oxidation were monitored using the Seahorse extracellular flux analyzer. Expression of PGC-1α was increased using adenoviral delivery. ARPE-19 were exposed to hydrogen peroxide to induce oxidative stress. Reactive oxygen species were measured by CM-H2DCFDA fluorescence. Cell death was analyzed by LDH release. RESULTS: Maturation of ARPE-19 and hfRPE was associated with significant increase in mitochondrial mass, expression of oxidative phosphorylation (OXPHOS) genes, and PGC-1α gene expression. Overexpression of PGC-1α increased expression of OXPHOS and fatty-acid ß-oxidation genes, ultimately leading to the potent induction of mitochondrial respiration and fatty-acid oxidation. PGC-1α gain of function also strongly induced numerous antioxidant genes and, importantly, protected RPE from oxidant-mediated cell death without altering RPE functions. CONCLUSIONS: This study provides important insights into the metabolic changes associated with RPE functional maturation and identifies PGC-1α as a potent driver of RPE mitochondrial function and antioxidant capacity.

Cp/Heph mutant mice have iron-induced neurodegeneration diminished by deferiprone.

Brain iron accumulates in several neurodegenerative diseases and can cause oxidative damage, but mechanisms of brain iron homeostasis are incompletely understood. Patients with mutations in the cellular iron-exporting ferroxidase ceruloplasmin (Cp) have brain iron accumulation causing neurodegeneration. Here, we assessed the brains of mice with combined mutation of Cp and its homolog hephaestin. Compared to single mutants, brain iron accumulation was accelerated in double mutants in the cerebellum, substantia nigra, and hippocampus. Iron accumulated within glia, while neurons were iron deficient. There was loss of both neurons and glia. Mice developed ataxia and tremor, and most died by 9 months. Treatment with the oral iron chelator deferiprone diminished brain iron levels, protected against neuron loss, and extended lifespan. Ferroxidases play important, partially overlapping roles in brain iron homeostasis by facilitating iron export from glia, making iron available to neurons. Above: Iron (Fe) normally moves from capillaries to glia to neurons. It is exported from the glia by ferroportin (Fpn) with ferroxidases ceruloplasmin (Cp) and/or Hephaestin (Heph). Below: In mice with mutation of Cp and Heph, iron accumulates in glia, while neurons have low iron levels. Both neurons and glia degenerate and mice become ataxic unless given an iron chelator.

Iron upregulates melanogenesis in cultured retinal pigment epithelial cells.

The purpose of our studies was to examine the relationship between iron and melanogenesis in retinal pigment epithelial cells, as prior observations had suggested that iron may promote melanogenesis. This relationship has potential clinical importance, as both iron overload and hyperpigmentation are associated with age-related macular degeneration (AMD). Human fetal retinal pigment epithelial cells and ARPE-19 cells were treated with iron in the form of ferric ammonium citrate, after which quantitative RT-PCR and electron microscopy were performed. Melanogenesis genes tyrosinase, tyrosinase-related protein 1, Hermansky-Pudlak Syndrome 3, premelanosome protein and dopachrome tautomerase were upregulated, as was the melanogenesis-controlling transcription factor, microphthalmia-associated transcription factor (MITF). Iron-treated cells had increased pigmentation and melanosome number. Multiple transcription factors upstream of MITF were upregulated by iron.

Ferroxidase hephaestin's cell-autonomous role in the retinal pigment epithelium.

Hephaestin (Heph) is a ferroxidase protein that converts ferrous to ferric iron to facilitate cellular iron export by ferroportin. Many tissues express either Heph or its homologue, ceruloplasmin (Cp), but the retina expresses both. In mice, a combined systemic mutation of Heph and systemic knockout of Cp (Cp(-/-), Heph(sla/sla)) causes retinal iron accumulation and retinal degeneration, with features of human age-related macular degeneration; however, the role of Heph and Cp in the individual retinal cells is unclear. Herein, we used conditional knockout mice to study Heph's role in retinal pigment epithelial (RPE) and photoreceptor cells. Loss of both Heph and Cp from RPE cells alone results in RPE cell iron accumulation and degeneration. We found, however, that RPE iron accumulation in these conditional knockout mice is not as great as in systemic knockout mice. Photoreceptor-specific Heph knockout indicates that the additional iron in the RPE cells does not result from loss of ferroxidases in the photoreceptors, and Cp and Heph play minor roles in photoreceptors. Instead, loss of ferroxidases in other retinal cells causes retinal iron accumulation and transfer of iron to the RPE cells. Cp and Heph are necessary for iron export from the retina but are not essential for iron import into the retina. Thus, our studies, revise how we think about iron import and export from the retina.

The Oral Iron Chelator Deferiprone Protects Against Retinal Degeneration Induced through Diverse Mechanisms.

PURPOSE: To investigate the effect of the iron chelator deferiprone (DFP) on sodium iodate (NaIO3)-induced retinal degeneration and on the hereditary retinal degeneration caused by the rd6 mutation. METHODS: Retinas from NaIO3-treated C57BL/6J mice, with or without DFP cotreatment, were analyzed by histology, immunofluorescence, and quantitative PCR to investigate the effect of DFP on retinal degeneration. To facilitate photoreceptor quantification, we developed a new function of MATLAB to perform this task in a semiautomated fashion. Additionally, rd6 mice treated with or without DFP were analyzed by histology to assess possible protection. RESULTS: In NaIO3-treated mice, DFP protected against retinal degeneration and significantly decreased expression of the oxidative stress-related gene heme oxygenase-1 and the complement gene C3. DFP treatment partially protected against NaIO3-induced reduction in the levels of mRNAs encoded by visual cycle genes rhodopsin (Rho) and retinal pigment epithelium-specific 65 kDa protein (Rpe65), consistent with the morphological data indicating preservation of photoreceptors and RPE, respectively. DFP treatment also protected photoreceptors in rd6 mice. CONCLUSIONS: The oral iron chelator DFP provides significant protection against retinal degeneration induced through different modalities. This suggests that iron chelation could be useful as a treatment for retinal degeneration even when the main etiology does not appear to be iron dysregulation. TRANSLATIONAL RELEVANCE: These data provide proof of principle that the oral iron chelator DFP can protect the retina against diverse insults. Further testing of DFP in additional animal retinal degeneration models at a range of doses is warranted.

The Oral Iron Chelator Deferiprone Protects Against Retinal Degeneration Induced through Diverse Mechanisms.

PURPOSE: To investigate the effect of the iron chelator deferiprone (DFP) on sodium iodate (NaIO3)-induced retinal degeneration and on the hereditary retinal degeneration caused by the rd6 mutation. METHODS: Retinas from NaIO3-treated C57BL/6J mice, with or without DFP cotreatment, were analyzed by histology, immunofluorescence, and quantitative PCR to investigate the effect of DFP on retinal degeneration. To facilitate photoreceptor quantification, we developed a new function of MATLAB to perform this task in a semiautomated fashion. Additionally, rd6 mice treated with or without DFP were analyzed by histology to assess possible protection. RESULTS: In NaIO3-treated mice, DFP protected against retinal degeneration and significantly decreased expression of the oxidative stress-related gene heme oxygenase-1 and the complement gene C3. DFP treatment partially protected against NaIO3-induced reduction in the levels of mRNAs encoded by visual cycle genes rhodopsin (Rho) and retinal pigment epithelium-specific 65 kDa protein (Rpe65), consistent with the morphological data indicating preservation of photoreceptors and RPE, respectively. DFP treatment also protected photoreceptors in rd6 mice. CONCLUSIONS: The oral iron chelator DFP provides significant protection against retinal degeneration induced through different modalities. This suggests that iron chelation could be useful as a treatment for retinal degeneration even when the main etiology does not appear to be iron dysregulation. TRANSLATIONAL RELEVANCE: These data provide proof of principle that the oral iron chelator DFP can protect the retina against diverse insults. Further testing of DFP in additional animal retinal degeneration models at a range of doses is warranted.

Bmp6 regulates retinal iron homeostasis and has altered expression in age-related macular degeneration.

Iron-induced oxidative stress causes hereditary macular degeneration in patients with aceruloplasminemia. Similarly, retinal iron accumulation in age-related macular degeneration (AMD) may exacerbate the disease. The cause of retinal iron accumulation in AMD is poorly understood. Given that bone morphogenetic protein 6 (Bmp6) is a major regulator of systemic iron, we examined the role of Bmp6 in retinal iron regulation and in AMD pathogenesis. Bmp6 was detected in the retinal pigment epithelium (RPE), a major site of pathology in AMD. In cultured RPE cells, Bmp6 was down-regulated by oxidative stress and up-regulated by iron. Intraocular Bmp6 protein injection in mice up-regulated retinal hepcidin, an iron regulatory hormone, and altered retinal labile iron levels. Bmp6(-/-) mice had age-dependent retinal iron accumulation and degeneration. Postmortem RPE from patients with early AMD exhibited decreased Bmp6 levels. Because oxidative stress is associated with AMD pathogenesis and down-regulates Bmp6 in cultured RPE cells, the diminished Bmp6 levels observed in RPE cells in early AMD may contribute to iron build-up in AMD. This may in turn propagate a vicious cycle of oxidative stress and iron accumulation, exacerbating AMD and other diseases with hereditary or acquired iron excess.

The oral iron chelator deferiprone protects against iron overload-induced retinal degeneration.

PURPOSE: Iron-induced oxidative stress may exacerbate age-related macular degeneration (AMD). Ceruloplasmin/Hephaestin double-knockout (DKO) mice with age-dependent retinal iron accumulation and some features of AMD were used to test retinal protection by the oral iron chelator deferiprone (DFP). METHODS: Cultured retinal pigment epithelial (ARPE-19) cells and mice were treated with DFP. Transferrin receptor mRNA (Tfrc), an indicator of iron levels, was quantified by qPCR. In mice, retinal oxidative stress was assessed by mass spectrometry, and degeneration by histology and electroretinography. RESULTS: DFP at 60 µM decreased labile iron in ARPE-19 cells, increasing Tfrc and protecting 70% of cells against a lethal dose of H(2)O(2). DFP 1 mg/mL in drinking water increased retinal Tfrc mRNA 2.7-fold after 11 days and also increased transferrin receptor protein. In DKOs, DFP over 8 months decreased retinal iron levels to 72% of untreated mice, diminished retinal oxidative stress to 70% of the untreated level, and markedly ameliorated retinal degeneration. DFP was not retina toxic in wild-type (WT) or DKO mice, as assessed by histology and electroretinography. CONCLUSIONS: Oral DFP was not toxic to the mouse retina. It diminished retinal iron levels and oxidative stress and protected DKO mice against iron overload-induced retinal degeneration. Further testing of DFP for retinal disease involving oxidative stress is warranted.

Age-dependent retinal iron accumulation and degeneration in hepcidin knockout mice.

PURPOSE: Iron dysregulation can cause retinal disease, yet retinal iron regulatory mechanisms are incompletely understood. The peptide hormone hepcidin (Hepc) limits iron uptake from the intestine by triggering degradation of the iron transporter ferroportin (Fpn). Given that Hepc is expressed in the retina and Fpn is expressed in cells constituting the blood-retinal barrier, the authors tested whether the retina may produce Hepc to limit retinal iron import. METHODS: Retinas of Hepc(-/-) mice were analyzed by histology, autofluorescence spectral analysis, atomic absorption spectrophotometry, Perls' iron stain, and immunofluorescence to assess iron-handling proteins. Retinal Hepc mRNA was evaluated through qPCR after intravitreal iron injection. Mechanisms of retinal Hepc upregulation were tested by Western blot analysis. A retinal capillary endothelial cell culture system was used to assess the effect of exogenous Hepc on Fpn. RESULTS: Hepc(-/-) mice experienced age-dependent increases in retinal iron followed by retinal degeneration with autofluorescent RPE, photoreceptor death, and subretinal neovascularization. Hepc(-/-) mice had increased Fpn immunoreactivity in vascular endothelial cells. Conversely, in cultured retinal capillary endothelial cells, exogenous Hepc decreased both Fpn levels and iron transport. The retina can sense increased iron levels, upregulating Hepc after phosphorylation of extracellular signal regulated kinases. CONCLUSIONS: These findings indicate that Hepc is essential for retinal iron regulation. In the absence of Hepc, retinal degeneration occurs. Increases in Hepc mRNA levels after intravitreal iron injection combined with Hepc-mediated decreases in iron export from cultured retinal capillary endothelial cells suggest that the retina may use Hepc for its tissue-specific iron regulation.

Iron chelation protects the retinal pigment epithelial cell line ARPE-19 against cell death triggered by diverse stimuli.

PURPOSE: Cell death can be induced by exogenous reactive oxygen species (ROS). Endogenous ROS can also play a role in cell death triggered by agents that are not themselves ROS. One of the most potent ROS-generating systems is the iron-catalyzed Fenton reaction. Herein, the authors tested whether iron plays an important role in cell death induced by diverse stimuli in retinal pigment epithelial (RPE) cells. METHODS: The ability of the iron chelator salicylaldehyde isonicotinoyl hydrazone (SIH) to chelate intracellular labile iron was tested in the human cell line ARPE-19. The ability of SIH to protect against RPE cell death induced by hydrogen peroxide, staurosporine, anti-Fas, and exposure to A2E plus blue light was determined. ROS production by staurosporine was assessed in the presence and absence of SIH. The protective activity of SIH was compared with that of other iron chelators and an antioxidant. RESULTS: Acute exposure to SIH was nontoxic and at least partially protective against cell death induced by all tested agents. On a molar basis, SIH was more protective against hydrogen peroxide than other iron chelators and an antioxidant. SIH decreased levels of staurosporine-induced ROS. CONCLUSIONS: Iron chelation with SIH can decrease levels of ROS and protect RPE cells against cell death induced by diverse stimuli. These results suggest a central role for iron in cell death pathways, potentially involving the generation of oxidative stress. SIH or related iron chelators may prove useful for protection against diseases involving RPE death, such as AMD.

Iron homeostasis and toxicity in retinal degeneration.

Iron is essential for many metabolic processes but can also cause damage. As a potent generator of hydroxyl radical, the most reactive of the free radicals, iron can cause considerable oxidative stress. Since iron is absorbed through diet but not excreted except through menstruation, total body iron levels buildup with age. Macular iron levels increase with age, in both men and women. This iron has the potential to contribute to retinal degeneration. Here we present an overview of the evidence suggesting that iron may contribute to retinal degenerations. Intraocular iron foreign bodies cause retinal degeneration. Retinal iron buildup resulting from hereditary iron homeostasis disorders aceruloplasminemia, Friedreich's ataxia, and panthothenate kinase-associated neurodegeneration cause retinal degeneration. Mice with targeted mutation of the iron exporter ceruloplasmin have age-dependent retinal iron overload and a resulting retinal degeneration with features of age-related macular degeneration (AMD). Post mortem retinas from patients with AMD have more iron and the iron carrier transferrin than age-matched controls. Over the past 10 years much has been learned about the intricate network of proteins involved in iron handling. Many of these, including transferrin, transferrin receptor, divalent metal transporter-1, ferritin, ferroportin, ceruloplasmin, hephaestin, iron-regulatory protein, and histocompatibility leukocyte antigen class I-like protein involved in iron homeostasis (HFE) have been found in the retina. Some of these proteins have been found in the cornea and lens as well. Levels of the iron carrier transferrin are high in the aqueous and vitreous humors. The functions of these proteins in other tissues, combined with studies on cultured ocular tissues, genetically engineered mice, and eye exams on patients with hereditary iron diseases provide clues regarding their ocular functions. Iron may play a role in a broad range of ocular diseases, including glaucoma, cataract, AMD, and conditions causing intraocular hemorrhage. While iron deficiency must be prevented, the therapeutic potential of limiting iron-induced ocular oxidative damage is high. Systemic, local, or topical iron chelation with an expanding repertoire of drugs has clinical potential.

Serum and forskolin cooperate to promote G1 progression in Schwann cells by differentially regulating cyclin D1, cyclin E1, and p27Kip expression.

Proliferation of Schwann cells in vitro, unlike most mammalian cells, is not induced by serum alone but additionally requires cAMP elevation and mitogenic stimulation. How these agents cooperate to promote progression through the G1 phase of the cell cycle is unclear. We studied the integrative effects of these compounds on receptor-mediated signaling pathways and regulators of G1 progression. We show that serum alone induces strong cyclical expression of cyclin D1 and E1, 6 and 12 h after addition, respectively. Serum also promotes strong but transient erbB2, ERK, and Akt phosphorylation, but Schwann cells remain arrested in G1 due to high levels of the inhibitor, p27(Kip). Forskolin with serum promotes G1 progression in 22% of Schwann cells between 18 and 24 h by inducing a steady decline in p27(Kip) levels that reaches a nadir at 12 h coinciding with peak cyclin E1 expression. Forskolin also delays neuregulin-induced loss of erbB2 receptors allowing strong acute activation of PI3K, sustained erbB2 phosphorylation and G1 progression in 31% of Schwann cells. We find that the ability of forskolin to decrease p27(Kip) is associated with its ability to decrease Krox-20 expression that is induced by serum and further increased by neuregulin. Our results explain why serum is required but insufficient to stimulate proliferation and identify two routes by which forskolin promotes proliferation in the presence of serum and neuregulin. These findings provide insights into how G1 progression and, cell cycle arrest leading to myelination are regulated in Schwann cells.

The iron carrier transferrin is upregulated in retinas from patients with age-related macular degeneration.

PURPOSE: Iron can cause oxidative stress, and elevated iron levels have been associated with several neurodegenerative diseases including age-related macular degeneration (AMD). Transferrin, an iron transport protein, is expressed at high levels in the retina. The purpose of this study was to assess transferrin involvement in AMD by determining the expression profile of transferrin in retinas with AMD compared with retinas without evidence of disease. METHODS: Postmortem retinas were obtained from AMD and non-AMD eyes. Expression of transferrin was assessed in a microarray dataset from 33 retinas of unaffected donors and 12 retinas of patients with AMD (six with neovascular AMD and six with non-neovascular AMD). Quantitative real-time RT-PCR (QPCR) was used to confirm the microarray results. Transferrin protein expression was assessed by semiquantitative Western blot analysis and immunohistochemistry. RESULTS: In comparison to unaffected retinas, mean transferrin mRNA levels, as measured by microarray analysis were elevated 3.5- and 2.1-fold in non-neovascular and neovascular AMD retinas, respectively. Semiquantitative Western blot analysis demonstrated a 2.1-fold increase in transferrin protein in AMD eyes. Immunohistochemistry showed more intense and widespread transferrin label in AMD maculas, particularly in large drusen, Müller cells, and photoreceptors. CONCLUSIONS: These data demonstrate that transferrin expression is increased in the retinas of patients with AMD relative to those of healthy control patients of comparable age. Along with previous studies that have demonstrated elevated iron levels in AMD retinas, early onset drusen formation in a patient with retinal iron overload resulting from aceruloplasminemia, and retinal degeneration with some features of macular degeneration in the iron-overloaded retinas of ceruloplasmin/hephestin knockout mice, the present study suggests that altered iron homeostasis is associated with AMD.

Paxillin binds schwannomin and regulates its density-dependent localization and effect on cell morphology.

Neurofibromatosis type 2 is an autosomal dominant disorder characterized by tumors, predominantly schwannomas, in the nervous system. It is caused by mutations in the gene NF2, encoding the growth regulator schwannomin (also known as merlin). Mutations occur throughout the 17-exon gene, with most resulting in protein truncation and undetectable amounts of schwannomin protein. Pathogenic mutations that result in production of defective schwannomin include in-frame deletions of exon 2 and three independent missense mutations within this same exon. Mice with conditional deletion of exon 2 in Schwann cells develop schwannomas, which confirms the crucial nature of exon 2 for growth control. Here we report that the molecular adaptor paxillin binds directly to schwannomin at residues 50-70, which are encoded by exon 2. This interaction mediates the membrane localization of schwannomin to the plasma membrane, where it associates with beta 1 integrin and erbB2. It defines a pathogenic mechanism for the development of NF2 in humans with mutations in exon 2 of NF2.