Photoreceptor Cilia and Retinal Ciliopathies Bujakowska, Kinga M; Liu, Qin; Pierce, Eric A
Cold Spring Harbor perspectives in biology,
10/2017, Letnik:
9, Številka:
10
Journal Article
Recenzirano
Odprti dostop
Photoreceptors are sensory neurons designed to convert light stimuli into neurological responses. This process, called phototransduction, takes place in the outer segments (OS) of rod and cone ...photoreceptors. OS are specialized sensory cilia, with analogous structures to those present in other nonmotile cilia. Deficient morphogenesis and/or dysfunction of photoreceptor sensory cilia (PSC) caused by mutations in a variety of photoreceptor-specific and common cilia genes can lead to inherited retinal degenerations (IRDs). IRDs can manifest as isolated retinal diseases or syndromic diseases. In this review, we describe the structure and composition of PSC and different forms of ciliopathies with retinal involvement. We review the genetics of the IRDs, which are monogenic disorders but genetically diverse with regard to causality.
The mechanisms that connect complement system activation and basal deposit formation in early stages of age-related macular degeneration (AMD) are insufficiently understood, which complicates the ...design of efficient therapies to prevent disease progression. Using human fetal (hf) retinal pigment epithelial (RPE) cells, we have established an in vitro model to investigate the effect of complement C3a on RPE cells and its role in the formation of sub-RPE deposits. The results of these studies revealed that C3a produced after C3 activation is sufficient to induce the formation of sub-RPE deposits via complement-driven proteasome inhibition. C3a binds the C3a receptor (C3aR), stimulates deposition of collagens IV and VI underneath the RPE, and impairs the extracellular matrix (ECM) turnover by increased MMP-2 activity, all mediated by downregulation of the ubiquitin proteasome pathway (UPP). The formation of basal deposits can be prevented by the addition of a C3aR antagonist, which restores the UPP activity and ECM turnover. These findings indicate that the cell-based model can be used to test potential therapeutic agents in vitro. The data suggest that modulation of C3aR-mediated events could be a therapeutic approach for treatment of early AMD.
Several groups have reported the results of clinical trials of gene augmentation therapy for Leber congenital amaurosis (LCA) because of mutations in the RPE65 gene. These studies have used ...subretinal injection of adeno-associated virus (AAV) vectors to deliver the human RPE65 cDNA to the retinal pigment epithelial (RPE) cells of the treated eyes. In all of the studies reported to date, this approach has been shown to be both safe and effective. The successful clinical trials of gene augmentation therapy for retinal degeneration caused by mutations in the RPE65 gene sets the stage for broad application of gene therapy to treat retinal degenerative disorders.
In natural freshwater and sediments, mercuric mercury (Hg(II)) is largely associated with particulate minerals and organics, but it remains unclear under what conditions particulates may become a ...sink or a source for Hg(II) and whether the particulate-bound Hg(II) is bioavailable for microbial uptake and methylation. In this study, we investigated Hg(II) sorption–desorption characteristics on three organo-coated hematite particulates and a Hg-contaminated natural sediment and evaluated the potential of particulate-bound Hg(II) for microbial methylation. Mercury rapidly sorbed onto particulates, especially the cysteine-coated hematite and sediment, with little desorption observed (0.1–4%). However, the presence of Hg-binding ligands, such as low-molecular-weight thiols and humic acids, resulted in up to 60% of Hg(II) desorption from the Hg-laden hematite particulates but <6% from the sediment. Importantly, the particulate-bound Hg(II) was bioavailable for uptake and methylation by a sulfate-reducing bacterium Desulfovibrio desulfuricans ND132 under anaerobic incubations, and the methylation rate was 4–10 times higher than the desorption rate of Hg(II). These observations suggest direct contacts and interactions between bacterial cells and the particulate-bound Hg(II), resulting in rapid exchange or uptake of Hg(II) by the bacteria. The results highlight the importance of Hg(II) partitioning at particulate–water interfaces and the role of particulates as a significant source of Hg(II) for methylation in the environment.
Mouse models are powerful tools for the study of ocular diseases. Alterations in the morphology and function of the retinal pigment epithelium (RPE) are common features shared by many ocular ...disorders. We report a detailed protocol to collect, seed, culture and characterize RPE cells from mice. We describe a reproducible method that we previously developed to collect and culture murine RPE cells on Transwells as functional polarized monolayers. The collection of RPE cells takes ∼3 h, and the cultures mimic in vivo RPE cell features within 1 week. This protocol also describes methods to characterize the cells on Transwells within 1-2 weeks by transmission and scanning electron microscopy (TEM and SEM, respectively), immunostaining of vibratome sections and flat mounts, and measurement of transepithelial electrical resistance. The RPE cell cultures are suitable to study the biology of the RPE from wild-type and genetically modified strains of mice between the ages of 10 d and 12 months. The RPE cells can also be manipulated to investigate molecular mechanisms underlying the RPE pathology in the numerous mouse models of ocular disorders. Furthermore, modeling the RPE pathology in vitro represents a new approach to testing drugs that will help accelerate the development of therapies for vision-threatening disorders such as macular degeneration (MD).
Liquid elemental mercury (Hg0 L) pollution can remain in soils for decades and, over time, will undergo corrosion, a process in which the droplet surface oxidizes soil constituents to form more ...reactive phases, such as mercury oxide (HgO). While these reactive coatings may enhance Hg migration in the subsurface, little is known about the transformation potential of corroded Hg0 L in the presence of reduced inorganic sulfur species to form sparingly soluble HgS particles, a process that enables the long-term sequestration of mercury in soils and generally reduces its mobility and bioavailability. In this study, we investigated the dissolution of corroded Hg0 L in the presence of sulfide by quantifying rates of aqueous Hg release from corroded Hg0 L droplets under different sulfide concentrations (expressed as the S:Hg molar ratio). For droplets corroded in ambient air, no differences in soluble Hg release were observed among all sulfide exposure levels (S:Hg mole ratios ranging from 10–4 to 10). However, for droplets oxidized in the presence of a more reactive oxidant (hydrogen peroxide, H2O2), we observed a 10- to 25-fold increase in dissolved Hg when the oxidized droplets were exposed to low sulfide concentrations (S:Hg ratios from 10–4 to 10–1) relative to droplets exposed to high sulfide concentrations. These results suggest two critical factors that dictate the release of soluble Hg from Hg0 L in the presence of sulfide: the extent of surface corrosion of the Hg0 L droplet and sufficient sulfide concentration for the formation of HgS solids. The mobilization of Hg0 L in porous media, therefore, largely depends on aging conditions in the subsurface and chemical reactivity at the Hg0 L droplet interface.
Soils contain a tangle of minerals, water, nutrients, gases, plant roots, decaying organic matter, and microorganisms which work together to cycle nutrients and support terrestrial plant growth. Most ...soil microorganisms live in periodically interconnected communities closely associated with soil aggregates, i.e., small (<2 mm), strongly bound clusters of minerals and organic carbon that persist through mechanical disruptions and wetting events. Their spatial structure is important for biogeochemical cycling, and we cannot reliably predict soil biological activities and variability by studying bulk soils alone. To fully understand the biogeochemical processes at work in soils, it is necessary to understand the micrometer-scale interactions that occur between soil particles and their microbial inhabitants. Here, we review the current state of knowledge regarding soil aggregate microbial communities and identify areas of opportunity to study soil ecosystems at a scale relevant to individual cells. We present a framework for understanding aggregate communities as "microbial villages" that are periodically connected through wetting events, allowing for the transfer of genetic material, metabolites, and viruses. We describe both top-down (whole community) and bottom-up (reductionist) strategies for studying these communities. Understanding this requires combining "model system" approaches (e.g., developing mock community artificial aggregates), field observations of natural communities, and broader study of community interactions to include understudied community members, like viruses. Initial studies suggest that aggregate-based approaches are a critical next step for developing a predictive understanding of how geochemical and community interactions govern microbial community structure and nutrient cycling in soil.
Abstract
The design of efficient therapies for age-related macular degeneration (AMD) is limited by our understanding of the pathogenesis of basal deposits, which form between retinal pigment ...epithelium (RPE) and Bruch's membrane (BrM) early in disease, and involve activation of the complement system. To investigate the roles of BrM, RPE and complement in an AMD, we generated abnormal extracellular matrix (ECM) using CRISPR-edited ARPE-19 cells. We introduced to these cells the p.R345W mutation in EFEMP1, which causes early-onset macular degeneration. The abnormal ECM binds active complement C3 and causes the formation of basal deposits by normal human fetal (hf)RPE cells. Human fetal RPE (hfRPE) cells grown on abnormal ECM or BrM explants from AMD donors show chronic activation of the alternative complement pathway by excessive deposition of C3b. This process is exacerbated by impaired ECM turnover via increased matrix metalloproteinase-2 activity. The local cleavage of C3 via convertase-independent mechanisms can be a new therapeutic target for early AMD.
Microbial methylation and demethylation are two competing processes controlling the net production and bioaccumulation of neurotoxic methylmercury (MeHg) in natural ecosystems. Although mercury (Hg) ...methylation by anaerobic microorganisms and demethylation by aerobic Hg-resistant bacteria have both been extensively studied, little attention has been given to MeHg degradation by anaerobic bacteria, particularly the iron-reducing bacterium Geobacter bemidjiensis Bem. Here we report, for the first time, that the strain G. bemidjiensis Bem can mediate a suite of Hg transformations, including Hg(II) reduction, Hg(0) oxidation, MeHg production and degradation under anoxic conditions. Results suggest that G. bemidjiensis utilizes a reductive demethylation pathway to degrade MeHg, with elemental Hg(0) as the major reaction product, possibly due to the presence of genes encoding homologues of an organomercurial lyase (MerB) and a mercuric reductase (MerA). In addition, the cells can strongly sorb Hg(II) and MeHg, reduce or oxidize Hg, resulting in both time and concentration-dependent Hg species transformations. Moderate concentrations (10–500 μM) of Hg-binding ligands such as cysteine enhance Hg(II) methylation but inhibit MeHg degradation. These findings indicate a cycle of Hg methylation and demethylation among anaerobic bacteria, thereby influencing net MeHg production in anoxic water and sediments.
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