α-Synuclein modulates fibronectin expression in the trabecular meshwork independent of TGFß2.

α-Synuclein (α-Syn) is implicated in Parkinson's disease (PD), a neuromotor disorder with prominent visual symptoms. The underlying cause of motor dysfunction has been studied extensively, and is attributed to the death of dopaminergic neurons mediated in part by intracellular aggregation of α-Syn. The cause of visual symptoms, however, is less clear. Neuroretinal degeneration due to the presence of aggregated α-Syn has been reported, but the evidence is controversial. Other symptoms including those arising from primary open angle glaucoma (POAG) are believed to be the side-effects of medications prescribed for PD. Here, we explored the alternative hypothesis that dysfunction of α-Syn in the anterior eye alters the interaction between the actin cytoskeleton of trabecular meshwork (TM) cells with the extracellular matrix (ECM), impairing their ability to respond to physiological changes in intraocular pressure (IOP). A similar dysfunction in neurons is responsible for impaired neuritogenesis, a characteristic feature of PD. Using cadaveric human and bovine TM tissue and primary human TM cells as models, we report two main observations: 1) α-Syn is expressed in human and bovine TM cells, and significant amounts of monomeric and oligomeric α-Syn are present in the AH, and 2) primary human TM cells and human and bovine TM tissue endocytose extracellular recombinant monomeric and oligomeric α-Syn via the prion protein (PrPC), and upregulate fibronectin (FN) and α-smooth muscle actin (α-SMA), fibrogenic proteins implicated in POAG. Transforming growth factor ß2 (TGFß2), a fibrogenic cytokine implicated in ∼50% cases of POAG, is also increased, and so is RhoA-associated coiled-coil-containing protein kinase 1 (ROCK-1). However, silencing of α-Syn in primary human TM cells reduces FN, α-SMA, and ROCK-1 in the absence or presence of over-expressed active TGFß2, suggesting modulation of FN and ROCK-1 independent of, or upstream of TGFß2. These observations suggest that extracellular α-Syn modulates ECM proteins in the TM independently or via PrPC by activating the RhoA-ROCK pathway. These observations reveal a novel function of α-Syn in the anterior eye, and offer new therapeutic options.

Direct modulation of microglial function by electrical field.

Non-invasive electric stimulation (ES) employing a low-intensity electric current presents a potential therapeutic modality that can be applied for treating retinal and brain neurodegenerative disorders. As neurons are known to respond directly to ES, the effects of ES on glia cells are poorly studied. A key question is if ES directly mediates microglial function or modulates their activity merely via neuron-glial signaling. Here, we demonstrated the direct effects of ES on microglia in the BV-2 cells-an immortalized murine microglial cell line. The low current ES in a biphasic ramp waveform, but not that of rectangular or sine waveforms, significantly suppressed the motility and migration of BV-2 microglia in culture without causing cytotoxicity. This was associated with diminished cytoskeleton reorganization and microvilli formation in BV-2 cultures, as demonstrated by immunostaining of cytoskeletal proteins, F-actin and ß-tubulin, and scanning electron microscopy. Moreover, ES of a ramp waveform reduced microglial phagocytosis of fluorescent zymosan particles and suppressed lipopolysaccharide (LPS)-induced pro-inflammatory cytokine expression in BV-2 cells as shown by Proteome Profiler Mouse Cytokine Array. The results of quantitative PCR and immunostaining for cyclooxygenase-2, Interleukin 6, and Tumor Necrosis Factor-α corroborated the direct suppression of LPS-induced microglial responses by a ramp ES. Transcriptome profiling further demonstrated that ramp ES effectively suppressed nearly half of the LPS-induced genes, primarily relating to cellular motility, energy metabolism, and calcium signaling. Our results reveal a direct modulatory effect of ES on previously thought electrically "non-responsive" microglia and suggest a new avenue of employing ES for anti-inflammatory therapy.

Release of Iron-Loaded Ferritin in Sodium Iodate-Induced Model of Age Related Macular Degeneration: An In-Vitro and In-Vivo Study.

To evaluate the role of iron in sodium iodate (NaIO3)-induced model of age-related macular degeneration (AMD) in ARPE-19 cells in-vitro and in mouse models in-vivo. ARPE-19 cells, a human retinal pigment epithelial cell line, was exposed to 10 mM NaIO3 for 24 h, and the expression and localization of major iron modulating proteins was evaluated by Western blotting (WB) and immunostaining. Synthesis and maturation of cathepsin-D (cat-D), a lysosomal enzyme, was evaluated by quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) and WB, respectively. For in-vivo studies, C57BL/6 mice were injected with 40 mg/kg mouse body weight of NaIO3 intraperitoneally, and their retina was evaluated after 3 weeks as above. NaIO3 induced a 10-fold increase in ferritin in ARPE-19 cells, which co-localized with LC3II, an autophagosomal marker, and LAMP-1, a lysosomal marker. A similar increase in ferritin was noted in retinal lysates and retinal sections of NaIO3-injected mice by WB and immunostaining. Impaired synthesis and maturation of cat-D was also noted. Accumulated ferritin was loaded with iron, and released from retinal pigmented epithelial (RPE) cells in Perls' and LAMP-1 positive vesicles. NaIO3 impairs lysosomal degradation of ferritin by decreasing the transcription and maturation of cat-D in RPE cells. Iron-loaded ferritin accumulates in lysosomes and is released in lysosomal membrane-enclosed vesicles to the extracellular milieu. Accumulation of ferritin in RPE cells and fusion of ferritin-containing vesicles with adjacent photoreceptor cells is likely to create an iron overload, compromising their viability. Moreover, reduced activity of cat-D is likely to promote accumulation of other cellular debris in lysosomal vesicles, contributing to AMD-like pathology.

Upregulation of brain hepcidin in prion diseases.

Accumulation of redox-active iron in human sporadic Creutzfeldt-Jakob disease (sCJD) brain tissue and scrapie-infected mouse brains has been demonstrated previously. Here, we explored whether upregulation of local hepcidin secreted within the brain is the underlying cause of iron accumulation and associated toxicity. Using scrapie-infected mouse brains, we demonstrate transcriptional upregulation of hepcidin relative to controls. As a result, ferroportin (Fpn), the downstream effector of hepcidin and the only known iron export protein was downregulated, and ferritin, an iron storage protein was upregulated, suggesting increased intracellular iron. A similar transcriptional and translational upregulation of hepcidin, and decreased expression of Fpn and an increase in ferritin expression was observed in sCJD brain tissue. Further evaluation in human neuroblastoma cells (M17) exposed to synthetic mini-hepcidin showed downregulation of Fpn, upregulation of ferritin, and an increase in reactive oxygen species (ROS), resulting in cytotoxicity in a dose-dependent manner. Similar effects were noted in primary neurons isolated from mouse brain. As in M17 cells, primary neurons accumulated ferritin and ROS, and showed toxicity at five times lower concentration of mini-hepcidin. These observations suggest that upregulation of brain hepcidin plays a significant role in iron accumulation and associated neurotoxicity in human and animal prion disorders.

Upregulation of Local Hepcidin Contributes to Iron Accumulation in Alzheimer's Disease Brains.

BACKGROUND: Accumulation of iron is a consistent feature of Alzheimer's disease (AD) brains. The underlying cause, however, remains debatable. OBJECTIVE: To explore whether local hepcidin synthesized by brain cells contributes to iron accumulation in AD brains. METHODS: Brain tissue from the cingulate cortex of 33 cases of AD pre-assigned to Braak stage I-VI, 6 cases of non-dementia, and 15 cases of non-AD dementia were analyzed for transcriptional upregulation of hepcidin by RT-qPCR and RT-PCR. Change in the expression of ferritin, ferroportin (Fpn), microglial activation marker Iba1, IL-6, and TGFß2 was determined by western blotting. Total tissue iron was determined by colorimetry. RESULTS: Significant transcriptional upregulation of hepcidin was observed in Braak stage III-VI relative to Braak stage I and II, non-AD dementia, and non-dementia samples. Ferritin was increased in Braak stage V, and a significant increase in tissue iron was evident in Braak stage III-VI. The expression of Iba1 and IL-6 was also increased in Braak stage III-VI relative to Braak stage I and II and non-AD dementia samples. Amyloid-ß plaques were absent in most Braak stage I and II samples, and present in Braak stage III-VI samples with few exceptions. CONCLUSION: These observations suggest that upregulation of brain hepcidin is mediated by IL-6, a known transcriptional activator of hepcidin. The consequent downregulation of Fpn on neuronal and other cells results in accumulation of iron in AD brains. The increase in hepcidin is disease-specific, and increases with disease progression, implicating AD-specific pathology in the accumulation of iron.

TGFß2-Hepcidin Feed-Forward Loop in the Trabecular Meshwork Implicates Iron in Glaucomatous Pathology.

Purpose: Elevated levels of transforming-growth-factor (TGF)-ß2 in the trabecular meshwork (TM) and aqueous humor are associated with primary open-angle glaucoma (POAG). The underlying mechanism includes alteration of extracellular matrix homeostasis through Smad-dependent and independent signaling. Smad4, an essential co-Smad, upregulates hepcidin, the master regulator of iron homeostasis. Here, we explored whether TGF-ß2 upregulates hepcidin, implicating iron in the pathogenesis of POAG. Methods: Primary human TM cells and human and bovine ex vivo anterior segment organ cultures were exposed to bioactive TGF-ß2, hepcidin, heparin (a hepcidin antagonist), or N-acetyl carnosine (an antioxidant), and the change in the expression of hepcidin, ferroportin, ferritin, and TGF-ß2 was evaluated by semiquantitative RT-PCR, Western blotting, and immunohistochemistry. Increase in reactive oxygen species (ROS) was quantified with dihydroethidium, an ROS-sensitive dye. Results: Primary human TM cells and bovine TM tissue synthesize hepcidin locally, which is upregulated by bioactive TGF-ß2. Hepcidin downregulates ferroportin, its downstream target, increasing ferritin and iron-catalyzed ROS. This causes reciprocal upregulation of TGF-ß2 at the transcriptional and translational levels. Heparin downregulates hepcidin, and reduces TGF-ß2-mediated increase in ferritin and ROS. Notably, both heparin and N-acetyl carnosine reduce TGF-ß2-mediated reciprocal upregulation of TGF-ß2. Conclusions: The above observations suggest that TGF-ß2 and hepcidin form a self-sustained feed-forward loop through iron-catalyzed ROS. This loop is partially disrupted by a hepcidin antagonist and an anti-oxidant, implicating iron and ROS in TGF-ß2-mediated POAG. We propose that modification of currently available hepcidin antagonists for ocular use may prove beneficial for the therapeutic management of TGF-ß2-associated POAG.

Local synthesis of hepcidin in the anterior segment of the eye: A novel observation with physiological and pathological implications.

PURPOSE: The avascular cornea, trabecular meshwork (TM), and lens obtain iron, an essential biometal, from the aqueous humor (AH). The mechanism by which this exchange is regulated, however, is unclear. Recently we reported that non-pigmented ciliary epithelial cells express ferroportin (Fpn) (Ashok, 2018b), an iron export protein modulated by hepcidin, the master regulator of iron homeostasis secreted mainly by the liver. Here, we explored whether ciliary epithelial and other cells in the anterior segment synthesize hepcidin, suggesting local regulation of iron exchange at this site. METHODS: Human and bovine eyes were dissected to isolate the ciliary body (CB), corneal endothelial (CE), TM, lens epithelial (LE), and outer epithelial cell layer of the iris. Total mRNA and protein lysates were processed to evaluate the synthesis and expression of hepcidin, the iron regulatory peptide hormone, Fpn, the only known iron export protein, ceruloplasmin (Cp), a ferroxidase necessary for iron export, transferrin receptor (TfR), a major iron uptake protein, and ferritin, a major iron storage protein. A combination of techniques including reverse transcription polymerase chain reaction (RT-PCR) of total mRNA, Western blotting of protein lysates, and immunofluorescence of fixed tissue sections were used to accomplish these goals. RESULTS: RT-PCR of isolated tissue samples revealed hepcidin-specific mRNA in the CB, TM, CE, and LE of the bovine eye. Western blotting of protein lysates from these tissues showed reactivity for hepcidin, Fpn, ferritin, and TfR. Western blotting and immunohistochemistry of similar tissues isolated from cadaveric human eyes showed expression of hepcidin, Fpn, and Cp in these samples. Notably, Fpn and Cp were expressed on the basolateral membrane of non-pigmented ciliary epithelial cells, facing the AH. CONCLUSIONS: Synthesis and expression of hepcidin and Fpn in the ciliary epithelium suggests local regulation of iron transport from choroidal plexus in the ciliary body to the AH across the blood-aqueous barrier. Expression of hepcidin and Fpn in CE, TM, and LE cells indicates additional regulation of iron exchange between the AH and cornea, TM, and lens, suggesting autonomous regulation of iron homeostasis in the anterior segment. Physiological and pathological implications of these observations are discussed.

Prion protein modulates iron transport in the anterior segment: Implications for ocular iron homeostasis and prion transmission.

Iron is an essential biometal in the aqueous humor, the principal source of nutrients for the avascular cornea and the lens. Here, we explored whether the ciliary body (CB), the source of aqueous humor, transports iron, and if the prion protein (PrPC) facilitates this process as in the outer retina. Using a combination of human, bovine, and mouse eyes as models, we report the expression of iron export proteins ferroportin and ceruloplasmin, and major iron uptake and storage proteins transferrin, transferrin receptor, and ferritin in the ciliary epithelium, indicating active exchange of iron at this site. Ferroportin and transferrin receptor are also expressed in the corneal endothelium. However, the relative expression of iron export and uptake proteins suggests export from the ciliary epithelium and import by corneal endothelium. In addition, abundant expression of PrPC, a ferrireductase that facilitates iron transport, is noted in pigmented and non-pigmented epithelium of the CB, posterior pigmented epithelium of the iris, corneal endothelium and epithelium, and lens epithelium. Notably, majority of PrPC in the ciliary epithelium is cleaved at the ß-site as in retinal pigment epithelial cells, suggesting a role in iron transport. Most of the PrPC in the cornea, however, is full-length, and susceptible to aggregation by intracerebrally inoculated PrP-scrapie, an infectious conformation of PrPC responsible for human and animal prion disorders. Soluble PrPC is present in the aqueous and vitreous humor, a provocative observation with significant implications. Together, these observations suggest independent cycling of iron in the anterior segment, and a prominent role of PrPC in this process. Aggregation of PrPC in the cornea of PrP-scrapie-infected animals raises the alarming possibility of transmission of animal prions through corneal abrasions.

Prion protein modulates glucose homeostasis by altering intracellular iron.

The prion protein (PrPC), a mainly neuronal protein, is known to modulate glucose homeostasis in mouse models. We explored the underlying mechanism in mouse models and the human pancreatic ß-cell line 1.1B4. We report expression of PrPC on mouse pancreatic ß-cells, where it promoted uptake of iron through divalent-metal-transporters. Accordingly, pancreatic iron stores in PrP knockout mice (PrP-/-) were significantly lower than wild type (PrP+/+) controls. Silencing of PrPC in 1.1B4 cells resulted in significant depletion of intracellular (IC) iron, and remarkably, upregulation of glucose transporter GLUT2 and insulin. Iron overloading, on the other hand, resulted in downregulation of GLUT2 and insulin in a PrPC-dependent manner. Similar observations were noted in the brain, liver, and neuroretina of iron overloaded PrP+/+ but not PrP-/- mice, indicating PrPC-mediated modulation of insulin and glucose homeostasis through iron. Peripheral challenge with glucose and insulin revealed blunting of the response in iron-overloaded PrP+/+ relative to PrP-/- mice, suggesting that PrPC-mediated modulation of IC iron influences both secretion and sensitivity of peripheral organs to insulin. These observations have implications for Alzheimer's disease and diabetic retinopathy, known complications of type-2-diabetes associated with brain and ocular iron-dyshomeostasis.

Prion protein facilitates retinal iron uptake and is cleaved at the ß-site: Implications for retinal iron homeostasis in prion disorders.

Prion disease-associated retinal degeneration is attributed to PrP-scrapie (PrPSc), a misfolded isoform of prion protein (PrPC) that accumulates in the neuroretina. However, a lack of temporal and spatial correlation between PrPSc and cytotoxicity suggests the contribution of host factors. We report retinal iron dyshomeostasis as one such factor. PrPC is expressed on the basolateral membrane of retinal-pigment-epithelial (RPE) cells, where it mediates uptake of iron by the neuroretina. Accordingly, the neuroretina of PrP-knock-out mice is iron-deficient. In RPE19 cells, silencing of PrPC decreases ferritin while over-expression upregulates ferritin and divalent-metal-transporter-1 (DMT-1), indicating PrPC-mediated iron uptake through DMT-1. Polarization of RPE19 cells results in upregulation of ferritin by ~10-fold and ß-cleavage of PrPC, the latter likely to block further uptake of iron due to cleavage of the ferrireductase domain. A similar ß-cleavage of PrPC is observed in mouse retinal lysates. Scrapie infection causes PrPSc accumulation and microglial activation, and surprisingly, upregulation of transferrin despite increased levels of ferritin. Notably, detergent-insoluble ferritin accumulates in RPE cells and correlates temporally with microglial activation, not PrPSc accumulation, suggesting that impaired uptake of iron by PrPSc combined with inflammation results in retinal iron-dyshomeostasis, a potentially toxic host response contributing to prion disease-associated pathology.

Transport of Non-Transferrin Bound Iron to the Brain: Implications for Alzheimer's Disease.

A direct correlation between brain iron and Alzheimer's disease (AD) raises questions regarding the transport of non-transferrin-bound iron (NTBI), a toxic but less researched pool of circulating iron that is likely to increase due to pathological and/or iatrogenic systemic iron overload. Here, we compared the distribution of radiolabeled-NTBI (59Fe-NTBI) and transferrin-bound iron (59Fe-Tf) in mouse models of iron overload in the absence or presence of inflammation. Following a short pulse, most of the 59Fe-NTBI was taken up by the liver, followed by the kidney, pancreas, and heart. Notably, a strong signal of 59Fe-NTBI was detected in the brain ventricular system after 2 h, and the brain parenchyma after 24 h. 59Fe-Tf accumulated mainly in the femur and spleen, and was transported to the brain at a much slower rate than 59Fe-NTBI. In the kidney, 59Fe-NTBI was detected in the cortex after 2 h, and outer medulla after 24 hours. Most of the 59Fe-NTBI and 59Fe-Tf from the kidney was reabsorbed; negligible amount was excreted in the urine. Acute inflammation increased the uptake of 59Fe-NTBI by the kidney and brain from 2-24 hours. Chronic inflammation, on the other hand, resulted in sequestration of iron in the liver and kidney, reducing its transport to the brain. These observations provide direct evidence for the transport of NTBI to the brain, and reveal a complex interplay between inflammation and brain iron homeostasis. Further studies are necessary to determine whether transient increase in NTBI due to systemic iron overload is a risk factor for AD.