Spatially resolved deconvolution of the fibrotic niche in lung fibrosis.

A defining pathological feature of human lung fibrosis is localized tissue heterogeneity, which challenges the interpretation of transcriptomic studies that typically lose spatial information. Here we investigate spatial gene expression in diagnostic tissue using digital profiling technology. We identify distinct, region-specific gene expression signatures as well as shared gene signatures. By integration with single-cell data, we spatially map the cellular composition within and distant from the fibrotic niche, demonstrating discrete changes in homeostatic and pathologic cell populations even in morphologically preserved lung, while through ligand-receptor analysis, we investigate cellular cross-talk within the fibrotic niche. We confirm findings through bioinformatic, tissue, and in vitro analyses, identifying that loss of NFKB inhibitor zeta in alveolar epithelial cells dysregulates the TGFß/IL-6 signaling axis, which may impair homeostatic responses to environmental stress. Thus, spatially resolved deconvolution advances understanding of cell composition and microenvironment in human lung fibrogenesis.

Microglial morphology in Alzheimer's disease and after Aß immunotherapy.

Microglia are the brain immune cells and their function is highly dependent on cell motility. It was hypothesised that morphological variability leads to differences in motility, ultimately impacting on the microglial function. Here, we assessed microglial morphology in 32 controls, 44 Alzheimer's disease (AD) cases and 16 AD cases from patients immunised against Aß42 (iAD) using 2D and 3D approaches. Our 2D assessment showed an increased number of microglia in iAD vs. AD (P = 0.032) and controls (P = 0.018). Ramified microglia were fewer in AD vs. controls (P = 0.041) but increased in iAD compared to AD (P < 0.001) and controls (P = 0.006). 3D reconstructions highlighted larger cell bodies in AD vs. controls (P = 0.049) and increased total process length in iAD vs. AD (P = 0.032), with negative correlations detected for pan-Aß load with total process length (P < 0.001) in AD and number of primary processes (P = 0.043) in iAD. In summary, reactive/amoeboid microglia are the most represented population in the aged human brain. AD does not affect the number of microglia, but the ramified population is decreased adopting a more reactive morphology. Aß removal by immunotherapy leads to increased ramified microglia, implying that the cells retain plasticity in an aged disease brain meriting further investigation.

TNF receptor agonists induce distinct receptor clusters to mediate differential agonistic activity.

Monoclonal antibodies (mAb) and natural ligands targeting costimulatory tumor necrosis factor receptors (TNFR) exhibit a wide range of agonistic activities and antitumor responses. The mechanisms underlying these differential agonistic activities remain poorly understood. Here, we employ a panel of experimental and clinically-relevant molecules targeting human CD40, 4-1BB and OX40 to examine this issue. Confocal and STORM microscopy reveal that strongly agonistic reagents induce clusters characterized by small area and high receptor density. Using antibody pairs differing only in isotype we show that hIgG2 confers significantly more receptor clustering than hIgG1 across all three receptors, explaining its greater agonistic activity, with receptor clustering shielding the receptor-agonist complex from further molecular access. Nevertheless, discrete receptor clustering patterns are observed with different hIgG2 mAb, with a unique rod-shaped assembly observed with the most agonistic mAb. These findings dispel the notion that larger receptor clusters elicit greater agonism, and instead point to receptor density and subsequent super-structure as key determinants.

Oligomeric Aß1-42 Induces an AMD-Like Phenotype and Accumulates in Lysosomes to Impair RPE Function.

Alzheimer's disease-associated amyloid beta (Aß) proteins accumulate in the outer retina with increasing age and in eyes of age-related macular degeneration (AMD) patients. To study Aß-induced retinopathy, wild-type mice were injected with nanomolar human oligomeric Aß1-42, which recapitulate the Aß burden reported in human donor eyes. In vitro studies investigated the cellular effects of Aß in endothelial and retinal pigment epithelial (RPE) cells. Results show subretinal Aß-induced focal AMD-like pathology within 2 weeks. Aß exposure caused endothelial cell migration, and morphological and barrier alterations to the RPE. Aß co-localized to late-endocytic compartments of RPE cells, which persisted despite attempts to clear it through upregulation of lysosomal cathepsin B, revealing a novel mechanism of lysosomal impairment in retinal degeneration. The rapid upregulation of cathepsin B was out of step with the prolonged accumulation of Aß within lysosomes, and contrasted with enzymatic responses to internalized photoreceptor outer segments (POS). Furthermore, RPE cells exposed to Aß were identified as deficient in cargo-carrying lysosomes at time points that are critical to POS degradation. These findings imply that Aß accumulation within late-endocytic compartments, as well as lysosomal deficiency, impairs RPE function over time, contributing to visual defects seen in aging and AMD eyes.

A novel ACE2 isoform is expressed in human respiratory epithelia and is upregulated in response to interferons and RNA respiratory virus infection.

Angiotensin-converting enzyme 2 (ACE2) is the main entry point in airway epithelial cells for SARS-CoV-2. ACE2 binding to the SARS-CoV-2 protein spike triggers viral fusion with the cell plasma membrane, resulting in viral RNA genome delivery into the host. Despite ACE2's critical role in SARS-CoV-2 infection, full understanding of ACE2 expression, including in response to viral infection, remains unclear. ACE2 was thought to encode five transcripts and one protein of 805 amino acids. In the present study, we identify a novel short isoform of ACE2 expressed in the airway epithelium, the main site of SARS-CoV-2 infection. Short ACE2 is substantially upregulated in response to interferon stimulation and rhinovirus infection, but not SARS-CoV-2 infection. This short isoform lacks SARS-CoV-2 spike high-affinity binding sites and, altogether, our data are consistent with a model where short ACE2 is unlikely to directly contribute to host susceptibility to SARS-CoV-2 infection.

Pericytes on placental capillaries in terminal villi preferentially cover endothelial junctions in regions furthest away from the trophoblast.

INTRODUCTION: Pericytes are a common feature in the placental microvasculature but their roles are not well understood. Pericytes may provide physical or endocrine support for endothelium and in some tissues mediate vasoconstriction. METHODS: This study uses serial block-face scanning electron microscopy (SBFSEM) to generate three-dimensional (3D) reconstructions of placental pericytes of the terminal villi and transmission electron microscopy (TEM) to study pericyte endothelial cell interactions. The proportion of endothelial cell junctions covered by pericytes was determined. RESULTS: The detailed 3D models of placental pericytes show pericyte structure at a new level of detail. Placental pericytes have many fingers extending from the cell body which can span multiple capillary branches. The proportion of endothelial cell-cell junctions covered by pericytes was significantly higher than pericyte coverage of capillary endothelium as a whole (endothelium: 14%, junctions: 43%, p < 0.0001). However, the proportion of endothelial cell-cell junctions covered by pericytes in regions adjacent to trophoblast was reduced compared to regions >3 µm away from trophoblast (27% vs 62% respectively, p < 0.001). No junctional complexes were observed connecting pericytes and endothelial cells but there were regions of cell membrane with features suggestive of intercellular adhesions. DISCUSSION: These data suggest that the localisation of pericytes on the villous capillary is not random but organised in relation to both endothelial junctions and the location of adjacent trophoblast. This further suggests that pericyte coverage may favour capillary permeability in regions that are most important for exchange, but limit capillary permeability in other regions.

The Role of Cholesterol on Triterpenoid Saponin-Induced Endolysosomal Escape of a Saporin-Based Immunotoxin.

Cholesterol seems to play a central role in the augmentation of saporin-based immunotoxin (IT) cytotoxicity by triterpenoid saponins. Endolysosomal escape has been proposed as one mechanism for the saponin-mediated enhancement of targeted toxins. We investigated the effects of lipid depletion followed by repletion on Saponinum album (SA)-induced endolysosomal escape of Alexa Fluor labelled saporin and the saporin-based immunotoxin OKT10-SAP, directed against CD38, in Daudi lymphoma cells. Lipid deprived cells showed reduced SA-induced endolysosomal escape at two concentrations of SA, as determined by a flow cytometric method. The repletion of membrane cholesterol by low density lipoprotein (LDL) restored SA-induced endolysosomal escape at a concentration of 5 µg/mL SA but not at 1 µg/mL SA. When LDL was used to restore the cholesterol levels in lipid deprived cells, the SA augmentation of OKT10-SAP cytotoxicity was partially restored at 1 µg/mL SA and fully restored at 5 µg/mL SA. These results suggest that different mechanisms of action might be involved for the two different concentrations of SA and that endosomal escape may not be the main mechanism for the augmentation of saporin IT cytotoxicity by SA at the sub-lytic concentration of 1 µg/mL SA.

An In-Vitro Cell Model of Intracellular Protein Aggregation Provides Insights into RPE Stress Associated with Retinopathy.

Impaired cargo trafficking and the aggregation of intracellular macromolecules are key features of neurodegeneration, and a hallmark of aged as well as diseased retinal pigment epithelial (RPE) cells in the eye. Here, photoreceptor outer segments (POS), which are internalized daily by RPE cells, were modified by UV-irradiation to create oxidatively modified POS (OxPOS). Oxidative modification was quantified by a protein carbonyl content assay. Human ARPE-19 cells were synchronously pulsed with POS or OxPOS to study whether oxidatively modified cargos can recapitulate features of RPE pathology associated with blinding diseases. Confocal immunofluorescence microscopy analysis showed that OxPOS was trafficked to LAMP1, LAMP2 lysosomes and to LC3b autophagy vacuoles. Whilst POS were eventually degraded, OxPOS cargos were sequestered in late compartments. Co-localization of OxPOS was also associated with swollen autolysosomes. Ultrastructural analysis revealed the presence of electron-dense OxPOS aggregates in RPE cells, which appeared to be largely resistant to degradation. Measurement of cellular autofluorescence, using parameters used to assess fundus autofluorescence (FAF) in age-related macular disease (AMD) patients, revealed that OxPOS contributed significantly to a key feature of aged and diseased RPE. This in vitro cell model therefore represents a versatile tool to study disease pathways linked with RPE damage and sight-loss.

Peri-arterial pathways for clearance of α-Synuclein and tau from the brain: Implications for the pathogenesis of dementias and for immunotherapy.

INTRODUCTION: Accumulation of amyloid beta (Aß), α-synuclein (αSyn), and tau in dementias indicates their age-related failure of elimination from the brain. Aß is eliminated along basement membranes in walls of cerebral arterioles and leptomeningeal arteries (intramural peri-arterial drainage [IPAD]); IPAD is impaired with age. We test the hypothesis that αSyn and tau are also eliminated from the normal brain along IPAD pathways. METHODS: Soluble αSyn or tau was injected into mouse hippocampus. Animals were perfused 5 minutes to 7 days post-injection. Blood vessels were identified by ROX-SE for light-sheet and immunolabeling for confocal microscopy. IPAD was quantified by measuring the proportion of arterioles with αSyn/tau. RESULTS: αSyn and tau are eliminated from the brain by IPAD but with different dynamics. DISCUSSION: Age-related failure of IPAD may play a role in the pathogenesis of synucleinopathies and tauopathies. αSyn persists within IPAD at 24 hours, which may affect immunotherapy for αSyn.

Anti-PD-1 immunotherapy leads to tuberculosis reactivation via dysregulation of TNF-α.

Previously, we developed a 3-dimensional cell culture model of human tuberculosis (TB) and demonstrated its potential to interrogate the host-pathogen interaction (Tezera et al., 2017a). Here, we use the model to investigate mechanisms whereby immune checkpoint therapy for cancer paradoxically activates TB infection. In patients, PD-1 is expressed in Mycobacterium tuberculosis (Mtb)-infected lung tissue but is absent in areas of immunopathology. In the microsphere model, PD-1 ligands are up-regulated by infection, and the PD-1/PD-L1 axis is further induced by hypoxia. Inhibition of PD-1 signalling increases Mtb growth, and augments cytokine secretion. TNF-α is responsible for accelerated Mtb growth, and TNF-α neutralisation reverses augmented Mtb growth caused by anti-PD-1 treatment. In human TB, pulmonary TNF-α immunoreactivity is increased and circulating PD-1 expression negatively correlates with sputum TNF-α concentrations. Together, our findings demonstrate that PD-1 regulates the immune response in TB, and inhibition of PD-1 accelerates Mtb growth via excessive TNF-α secretion.

A Flow Cytometric Method to Quantify the Endosomal Escape of a Protein Toxin to the Cytosol of Target Cells.

PURPOSE: The aim of this work was to develop a quantitative, flow cytometric method for tracking the endolysosomal escape of a fluorescently labelled saporin toxin. METHODS: Flow cytometric measurements of fluorescent pulse width and height were used to track the endocytic uptake into Daudi cells of a fluorescently labelled saporin toxin and the saporin based immunotoxin, OKT10-SAP. Subsequently, measurement of changes in pulse width were used to investigate the effect of a triterpenoid saponin on the endolysosomal escape of internalised toxin into the cytosol. Live cell confocal microscopy was used to validate the flow cytometry data. RESULTS: Increased endolysosomal escape of saporin and OKT10-SAP was observed by confocal microscopy in cells treated with saponin. Fluorescent pulse width measurements were also able to detect and quantify escape more sensitively than confocal microscopy. Saponin induced endolysosomal escape could be abrogated by treatment with chloroquine, an inhibitor of endolysosomal acidification. Chloroquine abrogation of escape was also mirrored by a concomitant abrogation of cytotoxicity. CONCLUSIONS: Poor endolysosomal escape is often a rate limiting step for the cytosolic delivery of protein toxins and other macromolecules. Pulse width analysis offers a simple method to semi-quantify the endolysosomal escape of this and similar molecules into the cytosol.

Oxidative Stress and Dysfunctional Intracellular Traffic Linked to an Unhealthy Diet Results in Impaired Cargo Transport in the Retinal Pigment Epithelium (RPE).

SCOPE: Oxidative stress and dysregulated intracellular trafficking are associated with an unhealthy diet which underlies pathology. Here, these effects on photoreceptor outer segment (POS) trafficking in the retinal pigment epithelium (RPE), a major pathway of disease underlying irreversible sight-loss, are studied. METHODS AND RESULTS: POS trafficking is studied in ARPE-19 cells using an algorithm-based quantification of confocal-immunofluorescence data supported by ultrastructural studies. It is shown that although POS are tightly regulated and trafficked via Rab5, Rab7 vesicles, LAMP1/2 lysosomes and LC3b-autophagosomes, there is also a considerable degree of variation and flexibility in this process. Treatment with H2 O2 and bafilomycin A1 reveals that oxidative stress and dysregulated autophagy target intracellular compartments and trafficking in strikingly different ways. These effects appear limited to POS-containing vesicles, suggesting a cargo-specific effect. CONCLUSION: The findings offer insights into how RPE cells cope with stress, and how mechanisms influencing POS transport/degradation can have different outcomes in the senescent retina. These shed new light on cellular processes underlying retinopathies such as age-related macular degeneration. The discoveries reveal how diet and nutrition can cause fundamental alterations at a cellular level, thus contributing to a better understanding of the diet-disease axis.

Augmentation of Saporin-Based Immunotoxins for Human Leukaemia and Lymphoma Cells by Triterpenoid Saponins: The Modifying Effects of Small Molecule Pharmacological Agents.

Triterpenoid saponins from Saponinum album (SA) significantly augment the cytotoxicity of saporin-based immunotoxins but the mechanism of augmentation is not fully understood. We investigated the effects of six small molecule pharmacological agents, which interfere with endocytic and other processes, on SA-mediated augmentation of saporin and saporin-based immunotoxins (ITs) directed against CD7, CD19, CD22 and CD38 on human lymphoma and leukaemia cell lines. Inhibition of clathrin-mediated endocytosis or endosomal acidification abolished the SA augmentation of saporin and of all four immunotoxins tested but the cytotoxicity of each IT or saporin alone was largely unaffected. The data support the hypothesis that endocytic processes are involved in the augmentative action of SA for saporin ITs targeted against a range of antigens expressed by leukaemia and lymphoma cells. In addition, the reactive oxygen species (ROS) scavenger tiron reduced the cytotoxicity of BU12-SAP and OKT10-SAP but had no effect on 4KB128-SAP or saporin cytotoxicity. Tiron also had no effect on SA-mediated augmentation of the saporin-based ITs or unconjugated saporin. These results suggest that ROS are not involved in the augmentation of saporin ITs and that ROS induction is target antigen-dependent and not directly due to the cytotoxic action of the toxin moiety.

A convenient protocol for establishing a human cell culture model of the outer retina.

The retinal pigment epithelium (RPE) plays a key role in the pathogenesis of several blinding retinopathies. Alterations to RPE structure and function are reported in Age-related Macular Degeneration, Stargardt and Best disease as well as pattern dystrophies. However, the precise role of RPE cells in disease aetiology remains incompletely understood. Many studies into RPE pathobiology have utilised animal models, which only recapitulate limited disease features. Some studies are also difficult to carry out in animals as the ocular space remains largely inaccessible to powerful microscopes. In contrast, in-vitro models provide an attractive alternative to investigating pathogenic RPE changes associated with age and disease. In this article we describe the step-by-step approach required to establish an experimentally versatile in-vitro culture model of the outer retina incorporating the RPE monolayer and supportive Bruch's membrane (BrM). We show that confluent monolayers of the spontaneously arisen human ARPE-19 cell-line cultured under optimal conditions reproduce key features of native RPE. These models can be used to study dynamic, intracellular and extracellular pathogenic changes using the latest developments in microscopy and imaging technology. We also discuss how RPE cells from human foetal and stem-cell derived sources can be incorporated alongside sophisticated BrM substitutes to replicate the aged/diseased outer retina in a dish. The work presented here will enable users to rapidly establish a realistic in-vitro model of the outer retina that is amenable to a high degree of experimental manipulation which will also serve as an attractive alternative to using animals. This in-vitro model therefore has the benefit of achieving the 3Rs objective of reducing and replacing the use of animals in research. As well as recapitulating salient structural and physiological features of native RPE, other advantages of this model include its simplicity, rapid set-up time and unlimited scope for detailed single-cell resolution and matrix studies.

Smartphone application for preventing depression: study protocol for a workplace randomised controlled trial.

INTRODUCTION: Depression is the leading cause of life years lost due to disability. Appropriate prevention has the potential to reduce the incidence of new cases of depression, however, traditional prevention approaches face significant scalability issues. Prevention programmes delivered by via smartphone applications provide a potential solution. The workplace is an ideal setting to roll out this form of intervention, particularly among industries that are unlikely to access traditional health initiatives and whose workplace characteristics create accessibility and portability issues. The study aims to evaluate the effectiveness of a smartphone application designed to prevent depression and improve well-being. The effectiveness of the app as a universal, selective and indicated prevention tool will also be evaluated. METHODS AND ANALYSIS: A multicentre randomised controlled trial, to determine the effectiveness of the intervention compared with an active mood monitoring control in reducing depressive symptoms (primary outcome) and the prevalence of depression at 3 months, with secondary outcomes assessing well-being and work performance. Employees from a range of industries will be invited to participate. Participants with likely current depression at baseline will be excluded. Following baseline assessment, participants, blinded to their allocation, will be randomised to receive one of two versions of the application: headgear (a 30-day mental health intervention) or a control application (mood monitoring for 30 days). Both versions of the app contain a risk calculator to provide a measure of future risk. Analyses will be conducted within an intention-to-treat framework using mixed modelling, with additional analyses conducted to compare the moderating effect of baseline risk level and depression symptom severity on the intervention's effectiveness. ETHICS AND DISSEMINATION: The current trial has received ethics approval from the University of New South Wales Human Research Ethics Committee (HC17021). Study results will be disseminated through peer-reviewed journals and conferences. TRIAL REGISTRATION NUMBER: ACTRN12617000548336; Results.

Collective Cell Behavior in Mechanosensing of Substrate Thickness.

Extracellular matrix stiffness has a profound effect on the behavior of many cell types. Adherent cells apply contractile forces to the material on which they adhere and sense the resistance of the material to deformation-its stiffness. This is dependent on both the elastic modulus and the thickness of the material, with the corollary that single cells are able to sense underlying stiff materials through soft hydrogel materials at low (<10 µm) thicknesses. Here, we hypothesized that cohesive colonies of cells exert more force and create more hydrogel deformation than single cells, therefore enabling them to mechanosense more deeply into underlying materials than single cells. To test this, we modulated the thickness of soft (1 kPa) elastic extracellular-matrix-functionalized polyacrylamide hydrogels adhered to glass substrates and allowed colonies of MG63 cells to form on their surfaces. Cell morphology and deformations of fluorescent fiducial-marker-labeled hydrogels were quantified by time-lapse fluorescence microscopy imaging. Single-cell spreading increased with respect to decreasing hydrogel thickness, with data fitting to an exponential model with half-maximal response at a thickness of 3.2 µm. By quantifying cell area within colonies of defined area, we similarly found that colony-cell spreading increased with decreasing hydrogel thickness but with a greater half-maximal response at 54 µm. Depth-sensing was dependent on Rho-associated protein kinase-mediated cellular contractility. Surface hydrogel deformations were significantly greater on thick hydrogels compared to thin hydrogels. In addition, deformations extended greater distances from the periphery of colonies on thick hydrogels compared to thin hydrogels. Our data suggest that by acting collectively, cells mechanosense rigid materials beneath elastic hydrogels at greater depths than individual cells. This raises the possibility that the collective action of cells in colonies or sheets may allow cells to sense structures of differing material properties at comparatively large distances.

Convective influx/glymphatic system: tracers injected into the CSF enter and leave the brain along separate periarterial basement membrane pathways.

Tracers injected into CSF pass into the brain alongside arteries and out again. This has been recently termed the "glymphatic system" that proposes tracers enter the brain along periarterial "spaces" and leave the brain along the walls of veins. The object of the present study is to test the hypothesis that: (1) tracers from the CSF enter the cerebral cortex along pial-glial basement membranes as there are no perivascular "spaces" around cortical arteries, (2) tracers leave the brain along smooth muscle cell basement membranes that form the Intramural Peri-Arterial Drainage (IPAD) pathways for the elimination of interstitial fluid and solutes from the brain. 2 µL of 100 µM soluble, fluorescent fixable amyloid ß (Aß) were injected into the CSF of the cisterna magna of 6-10 and 24-30 month-old male mice and their brains were examined 5 and 30 min later. At 5 min, immunocytochemistry and confocal microscopy revealed Aß on the outer aspects of cortical arteries colocalized with α-2 laminin in the pial-glial basement membranes. At 30 min, Aß was colocalised with collagen IV in smooth muscle cell basement membranes in the walls of cortical arteries corresponding to the IPAD pathways. No evidence for drainage along the walls of veins was found. Measurements of the depth of penetration of tracer were taken from 11 regions of the brain. Maximum depths of penetration of tracer into the brain were achieved in the pons and caudoputamen. Conclusions drawn from the present study are that tracers injected into the CSF enter and leave the brain along separate periarterial basement membrane pathways. The exit route is along IPAD pathways in which Aß accumulates in cerebral amyloid angiopathy (CAA) in Alzheimer's disease. Results from this study suggest that CSF may be a suitable route for delivery of therapies for neurological diseases, including CAA.

Ex-vivo models of the Retinal Pigment Epithelium (RPE) in long-term culture faithfully recapitulate key structural and physiological features of native RPE.

The Retinal Pigment Epithelium (RPE) forms the primary site of pathology in several blinding retinopathies. RPE cultures are being continuously refined so that dynamic disease processes in this important monolayer can be faithfully studied outside the eye over longer periods. The RPE substrate, which mimics the supportive Bruch's membrane (BrM), plays a key role in determining how well in-vitro cultures recapitulate native RPE cells. Here, we evaluate how two different types of BrM substrates; (1) a commercially-available polyester transwell membrane, and (2) a novel electrospun scaffold developed in our laboratory, could support the generation of realistic RPE tissues in culture. Our findings reveal that both substrates were capable of supporting long-lasting RPE monolayers with structural and functional specialisations of in-situ RPE cells. These cultures were used to study autofluorescence and barrier formation, as well as activities such as outer-segment internalisation/trafficking and directional secretion of key proteins; the impairment of which underlies retinal disease. Hence, both substrates fulfilled important criteria for generating authentic in-vitro cultures and act as powerful tools to study RPE pathophysiology. However, RPE grown on electrospun scaffolds may be better suited to studying complex RPE-BrM interactions such as the formation of drusen-like deposits associated with early retinal disease.

Membrane cholesterol is essential for triterpenoid saponin augmentation of a saporin-based immunotoxin directed against CD19 on human lymphoma cells.

Triterpenoid saponins from Saponinum Album (SA) exert potent lytic effects on eukaryotic cell plasma membranes and, when used at sub-lytic concentrations, significantly augment the cytotoxicity of saporin-based immunotoxins (IT). To help elucidate the mechanism(s) behind these two phenomena we investigated the role of cholesterol to both. Human Daudi lymphoma cells were lipid deprived using a combination of three different approaches. Following treatment, the total cellular lipid content was analyzed by electrospray ionization mass spectrometry (ESI-MS) and plasma membrane (PM) cholesterol content measured using the lipophilic fluorescent probe NR12S. Maximal lipid deprivation of cells resulted in a complete loss of sensitivity to lysis by SA. Similarly augmentation of the anti-CD19 immunotoxin (IT) BU12-SAPORIN by SA was lost but without a concomitant loss of intrinsic IT cytotoxicity. The lytic activity of SA was restored following incubation of lipid deprived Daudi cells with Synthecol or LDL. The augmentative effect of SA on IT cytotoxicity for Daudi cells was restored following repletion of PM cholesterol levels with LDL. NR12S fluorescence and ESI-MS analysis of cellular lipids demonstrated that restoration of SA lytic activity by Synthecol was entirely due to increased PM cholesterol levels. Restoration of cellular and PM cholesterol levels by LDL also restored the augmentative effect of SA for IT, an effect associated with repletion of PM cholesterol with minor changes in some phospholipid species. These results indicate that the lytic and IT augmentative properties of SA are cholesterol-dependent in contrast to intrinsic IT cytotoxicity that is at least partially cholesterol independent.

The Alzheimer's-related amyloid beta peptide is internalised by R28 neuroretinal cells and disrupts the microtubule associated protein 2 (MAP-2).

Age-related Macular Degeneration (AMD) is a common, irreversible blinding condition that leads to the loss of central vision. AMD has a complex aetiology with both genetic as well as environmental risks factors, and share many similarities with Alzheimer's disease. Recent findings have contributed significantly to unravelling its genetic architecture that is yet to be matched by molecular insights. Studies are made more challenging by observations that aged and AMD retinas accumulate the highly pathogenic Alzheimer's-related Amyloid beta (Aß) group of peptides, for which there appears to be no clear genetic basis. Analyses of human donor and animal eyes have identified retinal Aß aggregates in retinal ganglion cells (RGC), the inner nuclear layer, photoreceptors as well as the retinal pigment epithelium. Aß is also a major drusen constituent; found correlated with elevated drusen-load and age, with a propensity to aggregate in retinas of advanced AMD. Despite this evidence, how such a potent driver of neurodegeneration might impair the neuroretina remains incompletely understood, and studies into this important aspect of retinopathy remains limited. In order to address this we exploited R28 rat retinal cells which due to its heterogeneous nature, offers diverse neuroretinal cell-types in which to study the molecular pathology of Aß. R28 cells are also unaffected by problems associated with the commonly used RGC-5 immortalised cell-line, thus providing a well-established model in which to study dynamic Aß effects at single-cell resolution. Our findings show that R28 cells express key neuronal markers calbindin, protein kinase C and the microtubule associated protein-2 (MAP-2) by confocal immunofluorescence which has not been shown before, but also calretinin which has not been reported previously. For the first time, we reveal that retinal neurons rapidly internalised Aß1-42, the most cytotoxic and aggregate-prone amongst the Aß family. Furthermore, exposure to physiological amounts of Aß1-42 for 24 h correlated with impairment to neuronal MAP-2, a cytoskeletal protein which regulates microtubule dynamics in axons and dendrites. Disruption to MAP-2 was transient, and had recovered by 48 h, although internalised Aß persisted as discrete puncta for as long as 72 h. To assess whether Aß could realistically localise to living retinas to mediate such effects, we subretinally injected nanomolar levels of oligomeric Aß1-42 into wildtype mice. Confocal microscopy revealed the presence of focal Aß deposits in RGC, the inner nuclear and the outer plexiform layers 8 days later, recapitulating naturally-occurring patterns of Aß aggregation in aged retinas. Our novel findings describe how retinal neurons internalise Aß to transiently impair MAP-2 in a hitherto unreported manner. MAP-2 dysfunction is reported in AMD retinas, and is thought to be involved in remodelling and plasticity of post-mitotic neurons. Our insights suggest a molecular pathway by which this could occur in the senescent eye leading to complex diseases such as AMD.