Danielle B. Gutierrez

Quantitative Autofluorescence and Cell Density Maps of the Human Retinal Pigment Epithelium

PURPOSE Lipofuscin (LF) accumulation within RPE cells is considered pathogenic in AMD. To test whether LF contributes to RPE cell loss in aging and to provide a cellular basis for fundus autofluorescence (AF) we created maps of human RPE cell number and histologic AF. METHODS Retinal pigment epithelium-Bruchs membrane flat mounts were prepared from 20 donor eyes (10 ≤ 51 and 10 > 80 years; postmortem: ≤4.2 hours; no retinal pathologies), preserving foveal position. Phalloidin-binding RPE cytoskeleton and LF-AF (488-nm excitation) were imaged at up to 90 predefined positions. Maps were assembled from 83,330 cells in 1470 locations. From Voronoi regions representing each cell, the number of neighbors, cell area, and total AF intensity normalized to an AF standard was determined. RESULTS Highly variable between individuals, RPE-AF increases significantly with age. A perifoveal ring of high AF mirrors rod photoreceptor topography and fundus-AF. Retinal pigment epithelium cell density peaks at the fovea, independent of age, yet no net RPE cell loss is detectable. The RPE monolayer undergoes considerable lifelong re-modeling. The relationship of cell size and AF, a surrogate for LF concentration, is orderly and linear in both groups. Autofluorescence topography differs distinctly from the topography of age-related rod loss. CONCLUSIONS Digital maps of quantitative AF, cell density, and packing geometry provide metrics for cellular-resolution clinical imaging and model systems. The uncoupling of RPE LF content, cell number, and photoreceptor topography in aging challenges LFs role in AMD.

Lack of correlation between the spatial distribution of A2E and lipofuscin fluorescence in the human retinal pigment epithelium.

PURPOSE The accumulation of lipofuscin in the RPE is a hallmark of aging in the eye. The best characterized component of lipofuscin is A2E, a bis-retinoid byproduct of the normal retinoid visual cycle, which exhibits a broad spectrum of cytotoxic effects in vitro. The purpose of our study was to correlate the distribution of lipofuscin and A2E across the human RPE. METHODS Lipofuscin fluorescence was imaged in flat-mounted RPE from human donors of various ages. The spatial distributions of A2E and its oxides were determined using matrix-assisted laser desorption-ionization imaging mass spectrometry (MALDI-IMS) on flat-mounted RPE tissue sections and retinal cross-sections. RESULTS Our data support the clinical observations of strong RPE fluorescence, increasing with age, in the central area of the RPE. However, there was no correlation between the distribution of A2E and lipofuscin, as the levels of A2E were highest in the far periphery and decreased toward the central region. High-resolution MALDI-IMS of retinal cross-sections confirmed the A2E localization data obtained in RPE flat-mounts. Singly- and doubly-oxidized A2E had distributions similar to A2E, but represented <10% of the A2E levels. CONCLUSIONS This report to our knowledge is the first description of the spatial distribution of A2E in the human RPE by imaging mass spectrometry. These data demonstrate that the accumulation of A2E is not responsible for the increase in lipofuscin fluorescence observed in the central RPE with aging.

Novel fatty acid acylation of lens integral membrane protein aquaporin-0.

Fatty acid acylation of proteins is a well-studied co- or posttranslational modification typically conferring membrane trafficking signals or membrane anchoring properties to proteins. Commonly observed examples of protein acylation include N-terminal myristoylation and palmitoylation of cysteine residues. In the present study, direct tissue profiling mass spectrometry of bovine and human lens sections revealed an abundant signal tentatively assigned as a lipid-modified form of aquaporin-0. LC/MS/MS proteomic analysis of hydrophobic tryptic peptides from lens membrane proteins revealed both N-terminal and C-terminal peptides modified by 238 and 264 Da which were subsequently assigned by accurate mass measurement as palmitoylation and oleoylation, respectively. Specific sites of modification were the N-terminal methionine residue and lysine 238 revealing, for the first time, an oleic acid modification via an amide linkage to a lysine residue. The specific fatty acids involved reflect their abundance in the lens fiber cell plasma membrane. Imaging mass spectrometry indicated abundant acylated AQP0 in the inner cortical region of both bovine and human lenses and acylated truncation products in the lens nucleus. Additional analyses revealed that the lipid-modified forms partitioned exclusively to a detergent-resistant membrane fraction, suggesting a role in membrane domain targeting.

Spatial analysis of human lens aquaporin-0 post-translational modifications by MALDI mass spectrometry tissue profiling

Aquaporin-0 (AQP0), the major integral membrane protein in lens fiber cells, becomes highly modified with increasing age. The functional consequences of these modifications are being revealed, and the next step is to determine how these modifications affect the ocular lens, which is directly related to their abundances and spatial distributions. The aim of this study was to utilize matrix-assisted laser desorption ionization (MALDI) direct tissue profiling methods, which produce spatially-resolved protein profiles, to map and quantify AQP0 post-translational modifications (PTMs). Direct tissue profiling was performed using frozen, equatorial human lens sections of various ages prepared by conditions optimized for MALDI mass spectrometry profiling of membrane proteins. Modified forms of AQP0 were identified and further investigated using liquid chromatography tandem mass spectrometry (LC-MS/MS). The distributions of unmodified, truncated, and oleoylated forms of AQP0 were examined with a maximum spatial resolution of 500 μm. Direct tissue profiling of intact human lens sections provided high quality, spatially-resolved, relative quantitative information of AQP0 and its modified forms indicating that 50% of AQP0 is truncated at a fiber cell age of 24 ± 1 year in all lenses examined. Furthermore, direct tissue profiling also revealed previously unidentified AQP0 modifications including N-terminal acetylation and carbamylation. N-terminal acetylation appears to provide a protective effect against N-terminal truncation.

Mass spectrometry provides accurate and sensitive quantitation of A2E.

Orange autofluorescence from lipofuscin in the lysosomes of the retinal pigment epithelium (RPE) is a hallmark of aging in the eye. One of the major components of lipofuscin is A2E, the levels of which increase with age and in pathologic conditions, such as Stargardt disease or age-related macular degeneration. In vitro studies have suggested that A2E is highly phototoxic and, more specifically, that A2E and its oxidized derivatives contribute to RPE damage and subsequent photoreceptor cell death. To date, absorption spectroscopy has been the primary method to identify and quantitate A2E. Here, a new mass spectrometric method was developed for the specific detection of low levels of A2E and compared to a traditional method of analysis. The new mass spectrometric method allows the detection and quantitation of approximately 10,000-fold less A2E than absorption spectroscopy and the detection and quantitation of low levels of oxidized A2E, with localization of the oxidation sites. This study suggests that identification and quantitation of A2E from tissue extracts by chromatographic absorption spectroscopy overestimates the amount of A2E. This mass spectrometric approach makes it possible to detect low levels of A2E and its oxidized metabolites with greater accuracy than traditional methods, thereby facilitating a more exact analysis of bis-retinoids in animal models of inherited retinal degeneration as well as in normal and diseased human eyes.

Ecophysiological Examination of the Lake Erie Microcystis Bloom in 2014: Linkages between Biology and the Water Supply Shutdown of Toledo, OH

Annual cyanobacterial blooms dominated by Microcystis have occurred in western Lake Erie (U.S./Canada) during summer months since 1995. The production of toxins by bloom-forming cyanobacteria can lead to drinking water crises, such as the one experienced by the city of Toledo in August of 2014, when the city was rendered without drinking water for >2 days. It is important to understand the conditions and environmental cues that were driving this specific bloom to provide a scientific framework for management of future bloom events. To this end, samples were collected and metatranscriptomes generated coincident with the collection of environmental metrics for eight sites located in the western basin of Lake Erie, including a station proximal to the water intake for the city of Toledo. These data were used to generate a basin-wide ecophysiological fingerprint of Lake Erie Microcystis populations in August 2014 for comparison to previous bloom communities. Our observations and analyses indicate that, at the time of sample collection, Microcystis populations were under dual nitrogen (N) and phosphorus (P) stress, as genes involved in scavenging of these nutrients were being actively transcribed. Targeted analysis of urea transport and hydrolysis suggests a potentially important role for exogenous urea as a nitrogen source during the 2014 event. Finally, simulation data suggest a wind event caused microcystin-rich water from Maumee Bay to be transported east along the southern shoreline past the Toledo water intake. Coupled with a significant cyanophage infection, these results reveal that a combination of biological and environmental factors led to the disruption of the Toledo water supply. This scenario was not atypical of reoccurring Lake Erie blooms and thus may reoccur in the future.

Molecule-Specific Imaging and Quantitation of A2E in the RPE

Lipofuscin accumulates in the aging retinal pigment epithelium (RPE). It is identified by its fluorescence; however, lipofuscin is a complex mixture, and fluorescence is not specific enough to identify its individual components. Utilizing matrix-assisted laser desorption-ionization imaging, we have recently determined the spatial distribution of lipofuscin components across the RPE. One of the most abundant signals was that of the bis-retinoid A2E, a byproduct of the visual cycle. To better understand the accumulation of A2E, we studied wild-type (wt), Rpe65 −/− , and Abca4 −/− mice. A2E was not found in Rpe65 −/− animals. In wt animals, A2E was most abundant in the center of the RPE and diminished toward the periphery. In contrast, the A2E signal was more intense and uniformly distributed in Abca4 −/− mice. The oxidized forms of A2E were also spatially localized. Furthermore, a highly sensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was utilized to quantitate A2E. A2E oxidation sites were determined both in organic extracts and directly from the tissue. The ability to image a specific retinoid and its modified products from fresh tissue suggests wide applicability in the research and potential treatments of retinal degeneration.

Imaging mass spectrometry of the visual system: Advancing the molecular understanding of retina degenerations

Visual sensation is fundamental for quality of life, and loss of vision to retinal degeneration is a debilitating condition. The eye is the only part of the central nervous system that can be noninvasively observed with optical imaging. In the clinics, various spectroscopic methods provide high spatial resolution images of the fundus and the developing degenerative lesions. However, the currently utilized tools are not specific enough to establish the molecular underpinnings of retinal diseases. In contrast, mass spectrometric imaging (MSI) is a powerful tool to identify molecularly specific disease indicators and classification markers. This technique is particularly well suited to the eye, where molecular information can be correlated with clinical data collected via noninvasive diagnostic imaging modalities. Recent studies during the last few recent years have uncovered a plethora of new spatially defined molecular information on several vision‐threatening diseases, including age‐related macular degeneration, Stargardt disease, glaucoma, cataract, as well as lipid disorders. Even though MS inside the eye cannot be performed noninvasively, by linking diagnostic and molecular information, these studies are the first step toward the development of smart ophthalmic diagnostic and surgical tools. Here, we provide an overview of current approaches applying MSI technology to ocular pathology.

Integrated, High-Throughput, Multiomics Platform Enables Data-Driven Construction of Cellular Responses and Reveals Global Drug Mechanisms of Action

An understanding of how cells respond to perturbation is essential for biological applications; however, most approaches for profiling cellular response are limited in scope to pre-established targets. Global analysis of molecular mechanism will advance our understanding of the complex networks constituting cellular perturbation and lead to advancements in areas, such as infectious disease pathogenesis, developmental biology, pathophysiology, pharmacology, and toxicology. We have developed a high-throughput multiomics platform for comprehensive, de novo characterization of cellular mechanisms of action. Platform validation using cisplatin as a test compound demonstrates quantification of over 10 000 unique, significant molecular changes in less than 30 days. These data provide excellent coverage of known cisplatin-induced molecular changes and previously unrecognized insights into cisplatin resistance. This proof-of-principle study demonstrates the value of this platform as a resource to understand complex cellular responses in a high-throughput manner.

Euglenophycin is produced in at least six species of euglenoid algae and six of seven strains of Euglena sanguinea

Euglena sanguinea is known to produce the alkaloid toxin euglenophycin and is known to cause fish kills and inhibit mammalian tissue and microalgal culture growth. An analysis of over 30 species of euglenoids for accumulation of euglenophycin identified six additional species producing the toxin; and six of the seven E. sanguinea strains produced the toxin. A phylogenetic assessment of these species confirmed most taxa were in the Euglenaceae, whereas synthesis capability apparently has been lost in the Phacus, Eutreptiella, and Discoplastis branches.