Tissue- and Species-Specific Patterns of RNA metabolism in Post-Mortem Mammalian Retina and Retinal Pigment Epithelium.

Accurate analysis of gene expression in human tissues using RNA sequencing is dependent on the quality of source material. One major source of variation in mRNA quality is post-mortem time. While it is known that individual transcripts show differential post-mortem stability, few studies have directly and comprehensively analyzed mRNA stability following death, and in particular the extent to which tissue- and species-specific factors influence post-mortem mRNA stability are poorly understood. This knowledge is particularly important for ocular tissues studies, where tissues obtained post-mortem are frequently used for research or therapeutic applications. To directly investigate this question, we profiled mRNA levels in both neuroretina and retinal pigment epithelium (RPE) from mouse and baboon over a series of post-mortem intervals. We found substantial changes in gene expression as early as 15 minutes in the mouse and as early as three hours in the baboon eye tissues. Importantly, our findings demonstrate both tissue- and species- specific patterns of RNA metabolism, by identifying a set of genes that are either rapidly degraded or very stable in both species and/or tissues. Taken together, the data from this study lay the foundation for understanding RNA regulation post-mortem and provide novel insights into RNA metabolism in the tissues of the mammalian eye

Separation of phospho- and non-phosphopeptides using reverse phase column chromatography

AbstractPeptides containing phosphoserine, phosphothreonine or phosphotyrosine and their parent non-phosphorylated forms were chromatographed using standard C18 reverse phase chromatography in the presence of a water/acetonitrile gradient supplemented with different counter ions. We obtained the best separation of phosphorylated from non-phosphorylated peptides in the presence of heptafluorobutyric acid, with differences in retention times as large as ∼ 20 min. The chromatographic method was reliable in separation of the same peptides phosphorylated at different positions, acidic or basic phospho-Ser/Thr-peptides or phospho-Tyr-containing peptides. The described separation conditions are useful in studying the kinetics of phosphorylation/ dephosphorylation and in analysis of phosphorylation sites in vivo

Three-dimensional architecture of murine rod outer segments determined by cryoelectron tomography

The rod outer segment (ROS) of photoreceptor cells houses all components necessary for phototransduction, a set of biochemical reactions that amplify and propagate a light signal. Theoretical approaches to quantify this process require precise information about the physical boundaries of the ROS. Dimensions of internal structures within the ROS of mammalian species have yet to be determined with the precision required for quantitative considerations. Cryoelectron tomography was utilized to obtain reliable three-dimensional morphological information about this important structure from murine retina. Vitrification of samples permitted imaging of the ROS in a minimally perturbed manner and the preservation of substructures. Tomograms revealed the characteristic highly organized arrangement of disc membranes stacked on top of one another with a surrounding plasma membrane. Distances among the various membrane components of the ROS were measured to define the space available for phototransduction to occur. Reconstruction of segments of the ROS from single-axis tilt series images provided a glimpse into the three-dimensional architecture of this highly differentiated neuron. The reconstructions revealed spacers that likely maintain the proper distance between adjacent discs and between discs and the plasma membrane. Spacers were found distributed throughout the discs, including regions that are distant from the rim region of discs

Characterisation and OSL dating of modern fluvial sediments in the lower Vistula River: testing the zeroing assumption

In this study recent sediments of the lower Vistula River were investigated to determine the relationship between the structure and texture specific features and the possibility of their zeroing. The samples of recent fluvial deposits were collected from the lower Vistula River at two sites in Toruń and Ciechocinek. Sand bars newly emerged from the river were selected for testing. The coarse quartz grains were separated for OSL measurements. The single-aliquot regenerative (SAR) technique was applied for measuring equivalent doses from multigrain aliquots. The obtained dose estimates were found to be very low, proving the reliability of OSL zeroing assumption. The dose rates were estimated by gamma-ray spectrometry, demonstrating homogeneity of the radiation field. These results related to fossil sediments of that type confirm their suitability for the OSL dating method

Function of mammalian M-cones depends on the level of CRALBP in Müller cells

Cone photoreceptors mediate daytime vision in vertebrates. The rapid and efficient regeneration of their visual pigments following photoactivation is critical for the cones to remain photoresponsive in bright and rapidly changing light conditions. Cone pigment regeneration depends on the recycling of visual chromophore, which takes place via the canonical visual cycle in the retinal pigment epithelium (RPE) and the Müller cell-driven intraretinal visual cycle. The molecular mechanisms that enable the neural retina to regenerate visual chromophore for cones have not been fully elucidated. However, one known component of the two visual cycles is the cellular retinaldehyde-binding protein (CRALBP), which is expressed both in the RPE and in Müller cells. To understand the significance of CRALBP in cone pigment regeneration, we examined the function of cones in mice heterozygous for Rlbp1, the gene encoding CRALBP. We found that CRALBP expression was reduced by ∼50% in both the RPE and retina of Rlbp1+/- mice. Electroretinography (ERG) showed that the dark adaptation of rods and cones is unaltered in Rlbp1+/- mice, indicating a normal RPE visual cycle. However, pharmacologic blockade of the RPE visual cycle revealed suppressed cone dark adaptation in Rlbp1+/- mice in comparison with controls. We conclude that the expression level of CRALPB specifically in the Müller cells modulates the efficiency of the retina visual cycle. Finally, blocking the RPE visual cycle also suppressed further cone dark adaptation in Rlbp1-/- mice, revealing a shunt in the classical RPE visual cycle that bypasses CRALBP and allows partial but unexpectedly rapid cone dark adaptation

Apo-opsin exists in equilibrium between a predominant inactive and a rare highly active state

Bleaching adaptation in rod photoreceptors is mediated by apo-opsin, which activates phototransduction with effective activity 1

Noninvasive two-photon imaging reveals retinyl ester storage structures in the eye

Visual sensation in vertebrates is triggered when light strikes retinal photoreceptor cells causing photoisomerization of the rhodopsin chromophore 11-cis-retinal to all-trans-retinal. The regeneration of preillumination conditions of the photoreceptor cells requires formation of 11-cis-retinal in the adjacent retinal pigment epithelium (RPE). Using the intrinsic fluorescence of all-trans-retinyl esters, noninvasive two-photon microscopy revealed previously uncharacterized structures (6.9 ± 1.1 μm in length and 0.8 ± 0.2 μm in diameter) distinct from other cellular organelles, termed the retinyl ester storage particles (RESTs), or retinosomes. These structures form autonomous all-trans-retinyl ester-rich intracellular compartments distinct from other organelles and colocalize with adipose differentiation-related protein. As demonstrated by in vivo experiments using wild-type mice, the RESTs participate in 11-cis-retinal formation. RESTs accumulate in Rpe65 (−/−) mice incapable of carrying out the enzymatic isomerization, and correspondingly, are absent in the eyes of Lrat (−/−) mice deficient in retinyl ester synthesis. These results indicate that RESTs located close to the RPE plasma membrane are essential components in 11-cis-retinal production

A mixture of U.S. Food and Drug Administration-approved monoaminergic drugs protects the retina from light damage in diverse models of night blindness

Purpose: The purpose of this study was to test the extent of light damage in different models of night blindness and apply these paradigms in testing the therapeutic efficacy of combination therapy by drugs acting on the Gi, Gs, and Gq protein-coupled receptors. Methods: Acute bright light exposure was used to test susceptibility to light damage in mice lacking the following crucial phototransduction proteins: rod transducin (GNAT1), cone transducin (GNAT2), visual arrestin 1 (ARR1), and rhodopsin kinase 1 (GRK1). Mice were intraperitoneally injected with either vehicle or drug combination consisting of metoprolol (β1-receptor antagonist), bromocriptine (dopamine family-2 receptor agonist) and tamsulosin (α1-receptor antagonist) before bright light exposure. Light damage was primarily assessed with optical coherence tomography and inspection of cone population in retinal whole mounts. Retinal inflammation was assessed in a subset of experiments using autofluorescence imaging by scanning laser ophthalmoscopy and by postmortem inspection of microglia and astrocyte activity. Results: The Gnat1-/- mice showed slightly increased susceptibility to rod light damage, whereas the Gnat2-/- mice were very resistant. The Arr1-/- and Grk1-/- mice were sensitive for both rod and cone light damage and showed robust retinal inflammation 7 days after bright light exposure. Pretreatment with metoprolol + bromocriptine + tamsulosin rescued the retina in all genetic backgrounds, starting at doses of 0.2 mg/kg metoprolol, 0.02 mg/kg bromocriptine, and 0.01 mg/kg tamsulosin in the Gnat1-/- mice. The therapeutic drug doses increased in parallel with light-damage severity. Conclusions: Our results suggest that congenital stationary night blindness and Oguchi disease patients can be at an elevated risk of the toxic effects of bright light. Furthermore, systems pharmacology drug regimens that stimulate Gi signaling and attenuate Gs and Gq signaling present a promising disease-modifying therapy for photoreceptor degenerative diseases

Alpha-Synuclein Disrupted Dopamine Homeostasis Leads to Dopaminergic Neuron Degeneration in Caenorhabditis elegans

This is the publisher's version, also available electronically from http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0009312Disruption of dopamine homeostasis may lead to dopaminergic neuron degeneration, a proposed explanation for the specific vulnerability of dopaminergic neurons in Parkinson's disease. While expression of human α-synuclein in C. elegans results in dopaminergic neuron degeneration, the effects of α-synuclein on dopamine homeostasis and its contribution to dopaminergic neuron degeneration in C. elegans have not been reported. Here, we examined the effects of α-synuclein overexpression on worm dopamine homeostasis. We found that α-synuclein expression results in upregulation of dopamine synthesis and content, and redistribution of dopaminergic synaptic vesicles, which significantly contribute to dopaminergic neuron degeneration. These results provide in vivo evidence supporting a critical role for dopamine homeostasis in supporting dopaminergic neuron integrity