SummaryTelomerase adds telomeric repeats to chromosome ends to balance incomplete replication. Telomerase regulation is implicated in cancer, aging and other human diseases, but progress towards telomerase clinical manipulation is hampered by the lack of structural data. Here we present the cryo-electron microscopy structure of substrate-bound human telomerase holoenzyme at subnanometer resolution, describing two flexibly RNA-tethered lobes: the catalytic core with telomerase reverse transcriptase (TERT) and conserved motifs of telomerase RNA (hTR), and an H/ACA ribonucleoprotein (RNP). In the catalytic core, RNA encircles TERT, adopting a well-ordered tertiary structure with surprisingly limited protein-RNA interactions. The H/ACA RNP lobe comprises two sets of heterotetrameric H/ACA proteins and one Cajal body protein, TCAB1, representing a pioneering structure of a large eukaryotic family of ribosome and spliceosome biogenesis factors. Our findings provide a structural framework for understanding human telomerase disease mutations and represent an important step towards telomerase-related clinical therapeutics.

It is often difficult to find consistent changes in the retinal microvasculature due to large intersubject variability. However, with a novel application of AOSLO imaging, it is possible to visualize parafoveal capillaries and identify AV channels noninvasively. AV channels are disrupted in type 2 diabetes, even before the onset of diabetic retinopathy.

Using motion cues to enhance vessel contrast is a powerful tool for visualizing the capillary network, in the absence of contrast agents. The authors demonstrate a tool to study the microcirculation of healthy subjects noninvasively.

Adaptive Optics Scanning Laser Ophthalmoscopy was used to noninvasively acquire videos of single-file flow through live human retinal parafoveal capillaries. Videos were analyzed offline to investigate capillary flow dynamics. Certain capillaries accounted for a clear majority of leukocyte traffic (Leukocyte-Preferred-Paths, LPPs), while other capillaries primarily featured plasma gap flow (Plasma-Gap-Capillaries, PGCs). LPPs may serve as a protective mechanism to prevent inactivated leukocytes from entering exchange capillaries, and PGCs may serve as relief valves to minimize flow disruption due to the presence of a leukocyte in a neighboring LPP.

Stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion (STED) microscopy are two super-resolution optical microscopy approaches that have rapidly gained popularity in recent years. Both modalities offer super-resolution imaging capabilities with the potential for imaging in multiple colors, three-dimensions, and the possibility to image in live cells. In this review, we focus on the specific advantages and disadvantages of each technique in the context of each other. STORM has been reported to achieve higher spatial resolution when compared to STED, but a lengthy acquisition may be required. STED utilizes relatively higher laser intensities, but is able to generate a super-resolution image immediately after acquisition without the need for any additional data processing. Ultimately, the choice between STORM and STED will depend not only on the specific application, but also on the users' ability to understand and optimize the various parameters ranging from sample preparation to image acquisition, which determine the quality of the final image.

Purpose To establish adaptive optics scanning laser ophthalmoscopy (AOSLO) as a method to detect and characterize microscopic signs of diabetic retinopathy, in capillaries and cone photoreceptors in the parafovea. Methods Recently, AOSLO has enabled noninvasive assessment of photoreceptors, capillaries, and leukocytes in the retinas of live human subjects. Repeated application of AOSLO imaging along with comparison to fluorescein angiography was used to track individual capillaries near the foveal avascular zone (FAZ) from one eye affected with severe non-proliferative diabetic retinopathy. Fluorescein angiography was used to identify clinical signs of diabetic retinopathy, such as microaneurysms and intra-retinal microvascular abnormalities (IRMAs), and corresponding regions were imaged and assessed using the AOSLO. In addition, the structural integrity of photoreceptors and the spatial distribution of leukocytes around the parafoveal capillary network were quantitatively assessed. Results Capillaries and cone photoreceptors were visualized using the AOSLO, without the use of injected contrast agents. Although the majority of capillaries were stable over a period of 16 months, one capillary at the edge of the FAZ dropped out, leading to a small, but significant increase in FAZ size. Longitudinal assessment of the capillaries also showed microaneurysm formation and disappearance, as well as the formation of tiny capillary bends similar in appearance to IRMAs. The leukocytes in the capillary network were found to preferentially travel through the same routes in all four visits, suggesting that these channels are robust against small changes to the surrounding capillaries. In this eye, cone photoreceptor spacing was increased in the fovea when compared to normal data, but stable across all visits. Conclusions AOSLO imaging can be used to longitudinally track capillaries, leukocytes and photoreceptors in diabetic retinopathy. Capillary changes that can be detected include dropout of individual capillaries, as well as formation and disappearance of microaneurysms.

PurposeThe purpose of this study was to establish that retinal pigment epithelial (RPE) cells take up indocyanine green (ICG) dye following systemic injection and that adaptive optics enhanced indocyanine green ophthalmoscopy (AO-ICG) enables direct visualization of the RPE mosaic in the living human eye.MethodsA customized adaptive optics scanning light ophthalmoscope (AOSLO) was used to acquire high-resolution retinal fluorescence images of residual ICG dye in human subjects after intravenous injection at the standard clinical dose. Simultaneously, multimodal AOSLO images were also acquired, which included confocal reflectance, nonconfocal split detection, and darkfield. Imaging was performed in 6 eyes of three healthy subjects with no history of ocular or systemic diseases. In addition, histologic studies in mice were carried out.ResultsThe AO-ICG channel successfully resolved individual RPE cells in human subjects at various time points, including 20 minutes and 2 hours after dye administration. Adaptive optics-ICG images of RPE revealed detail which could be correlated with AO dark-field images of the same cells. Interestingly, there was a marked heterogeneity in the fluorescence of individual RPE cells. Confirmatory histologic studies in mice corroborated the specific uptake of ICG by the RPE layer at a late time point after systemic ICG injection.ConclusionsAdaptive optics-enhanced imaging of ICG dye provides a novel way to visualize and assess the RPE mosaic in the living human eye alongside images of the overlying photoreceptors and other cells.

Age-related macular degeneration (AMD), a leading contributor of vision loss, currently lacks comprehensive treatment. While AMD histopathology involves retinal pigment epithelium (RPE) injury associated with immune cell infiltration, the nature of immune cell responses to RPE injury remains undefined. We induced RPE injury pharmacologically and genetically in transgenic mouse models in which microglia and systemic monocytes were separately tagged, enabling a spatial and temporal dissection of the relative contributions of microglia vs. monocytes to post-injury changes. We found that myeloid cell responses to RPE injury occur in stages: (1) an early mobilization of endogenous microglia from the inner retina to the RPE layer, followed by (2) subsequent monocyte infiltration from the retinal vasculature into the inner retina that replenishes the local myeloid cell population in a CCR2-regulated manner. These altered distributions of myeloid cells post-injury were long-lived, with recruited monocytes acquiring the distribution, markers, and morphologies of neighboring endogenous microglia in a durable manner. These findings indicate the role played by infiltrating monocytes in maintaining myeloid cell homeostasis in the retina following AMD-relevant RPE injury and provide a foundation for understanding and therapeutically modulating immune aspects in retinal disease.