Retinal biomarkers of Alzheimer’s disease: insights from transgenic mouse models

In this paper, we use the retina as a window into the central nervous system and in particular to assess changes in the retinal tissue associated with the Alzheimer’s disease. We imaged the retina of wild-type (WT) and transgenic mouse model (TMM) of Alzheimer’s disease with optical coherence tomography and classify retinas into the WT and TMM groups using support vector machines with the radial basis function kernel. Predictions reached an accuracy over 80% at the age of 4 months and over 90% at the age of 8 months. Texture analysis of computed fundus reference images suggests a more heterogeneous organization of the retina in transgenic mice at the age of 8 months in comparison to controls.This study was supported by the Neuroscience Mantero Belard Prize 2015 (Santa Casa da Misericórdia)(MB-1049-2015), by The Portuguese Foundation for Science and Technology (PEst-UID/NEU/04539/2013), by FEDER-COMPETE (POCI-01-0145-FEDER-007440) and Centro 2020 Regional Operational Programme (CENTRO-01-0145-FEDER-000008: BrainHealth 2020).info:eu-repo/semantics/publishedVersio

Disruption of a neural microcircuit in the rod pathway of the mammalian retina by diabetes mellitus

Diabetes leads to dysfunction of the neural retina before and independent of classical microvascular diabetic retinopathy, but previous studies have failed to demonstrate which neurons and circuits are affected at the earliest stages. Here, using patch-clamp recording and two-photon Ca2+ imaging in rat retinal slices, we investigated diabetes-evoked changes in a microcircuit consisting of rod bipolar cells and their dyad postsynaptic targets, AII and A17 amacrine cells, which play an essential role in processing scotopic visual signals. AII amacrines forward their signals to ON- and OFF-cone bipolar cells and A17 amacrines provide GABAergic feedback inhibition to rod bipolar cells. Whereas Ca2+-permeable AMPA receptors mediate input from rod bipolar cells to both AII and A17 amacrines, diabetes changes the synaptic receptors on A17, but not AII amacrine cells. This was expressed as a change in pharmacological properties and single-channel conductance of the synaptic receptors, consistent with an upregulation of the AMPA receptor GluA2 subunit and reduced Ca2+ permeability. In addition, two-photon imaging revealed reduced agonist-evoked influx of Ca2+ in dendritic varicosities of A17 amacrine cells from diabetic compared with normal animals. Because Ca2+-permeable receptors in A17 amacrine cells mediate synaptic release of GABA, the reduced Ca2+ permeability of these receptors in diabetic animals leads to reduced release of GABA, followed by disinhibition and increased release of glutamate from rod bipolar cells. This perturbation of neuron and microcircuit dynamics can explain the decreased dynamic range and sensitivity of scotopic vision that has been observed in diabetes.publishedVersio

Diabetic hyperglycemia reduces Ca2+ permeability of extrasynaptic AMPA receptors in AII amacrine cells

There is increasing evidence that diabetic retinopathy is a primary neuropathological disorder that precedes the microvascular pathology associated with later stages of the disease. Recently, we found evidence for altered functional properties of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in A17, but not AII, amacrine cells in the mammalian retina, and the observed changes were consistent with an upregulation of the GluA2 subunit, a key determinant of functional properties of AMPA receptors, including Ca2+ permeability and current-voltage (I-V) rectification properties. Here, we have investigated functional changes of extrasynaptic AMPA receptors in AII amacrine cells evoked by diabetes. With patch-clamp recording of nucleated patches from retinal slices, we measured Ca2+ permeability and I–V rectification in rats with ∼3 wk of streptozotocin-induced diabetes and age-matched, noninjected controls. Under bi-ionic conditions (extracellular Ca2+ concentration = 30 mM, intracellular Cs+ concentration = 171 mM), the reversal potential (Erev) of AMPA-evoked currents indicated a significant reduction of Ca2+ permeability in diabetic animals [Erev = −17.7 mV, relative permeability of Ca2+ compared with Cs+ (PCa/PCs) = 1.39] compared with normal animals (Erev = −7.7 mV, PCa/PCs = 2.35). Insulin treatment prevented the reduction of Ca2+ permeability. I–V rectification was examined by calculating a rectification index (RI) as the ratio of the AMPA-evoked conductance at +40 and −60 mV. The degree of inward rectification in patches from diabetic animals (RI = 0.48) was significantly reduced compared with that in normal animals (RI = 0.30). These results suggest that diabetes evokes a change in the functional properties of extrasynaptic AMPA receptors of AII amacrine cells. These changes could be representative for extrasynaptic AMPA receptors elsewhere in AII amacrine cells and suggest that synaptic and extrasynaptic AMPA receptors are differentially regulated.acceptedVersio

Elevated glucose changes the expression of ionotropic glutamate receptor subunits and impairs calcium homeostasis in retinal neural cells

PURPOSE. Altered glutamatergic neurotransmission and calcium homeostasis may contribute to retinal neural cell dysfunction and apoptosis in diabetic retinopathy (DR). The purpose of this study was to determine the effect of high glucose on the protein content of -amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainate glutamate receptor subunits, particularly the GluR2 subunit, because it controls Ca2 permeability of AMPA receptor-associated channels. The effect of high glucose on the concentration of cytosolic free calcium ([Ca2 ]i) was also investigated. METHODS. The protein content of GluR1, GluR2, GluR6/7, and KA2 subunits was assessed by Western blot. Cobalt staining was used to identify cells containing calcium/cobalt-permeable AMPA receptors. The [Ca2 ]i changes evoked by KCl or kainate were recorded by live-cell confocal microscopy in R28 cells and in primary cultures of rat retina, loaded with fluo-4. RESULTS. In primary cultures, high glucose significantly decreased the protein content of GluR1 and GluR6/7 subunits and increased the protein content of GluR2 and KA2 subunits. High glucose decreased the number of cobalt-positive cells, suggesting a decrease in calcium permeability through AMPA receptor-associated channels. In high-glucose–treated cells, changes in [Ca2 ]i were greater than in control cells, and the recovery to basal levels was delayed. However, in the absence of Na , to prevent the activation of voltage-sensitive calcium channels, the [Ca2 ]i changes evoked by kainate in the presence of cyclothiazide, which inhibits AMPA receptor desensitization, were significantly lower in high-glucose–treated cells than in control cultures, further indicating that AMPA receptors were less permeable to calcium. Mannitol, used as an osmotic control, did not cause significant changes compared with the control. CONCLUSIONS. The results suggest that elevated glucose may alter glutamate neurotransmission and calcium homeostasis in the retina, which may have implications for the mechanisms of vision loss in DR.Foundation for Science and Technology, Portugal and FEDER (Grant POCTI/CBO/38545/01), The Juvenile Diabetes Research Foundation, The American Diabetes Association and the Pennsylvania Lions Sight Conservation and Eye Eye Research Foundation

Longitudinal normative OCT retinal thickness data for wild-type mice, and characterization of changes in the 3×Tg-AD mice model of Alzheimer's disease

Mice are widely used as models for many diseases, including eye and neurodegenerative diseases. However, there is a lack of normative data for retinal thickness over time, especially at young ages. In this work, we present a normative thickness database from one to four-months-old, for nine layers/layer-aggregates, including the total retinal thickness, obtained from the segmentation of spectral-domain optical coherence tomography (SD-OCT) data from the C57BL6/129S mouse strain. Based on fifty-seven mice, this normative database provides an opportunity to study the ageing of control mice and characterize disease models' ageing, such as the triple transgenic mouse model of Alzheimer's disease (3×Tg-AD) used in this work. We report thickness measurements, the differences in thickness per layer, demonstrate a nasal-temporal asymmetry, and the variation of thickness as a function to the distance to the optic disc center. Significant differences were found between the transgenic group's thickness and the normative database for the entire period covered in this study. Even though it is well accepted that retinal nerve fiber layer (RNFL) thinning is a hallmark of neurodegeneration, our results show a thicker RNFL-GCL (RNFL-Ganglion cell layer) aggregate for the 3×Tg-AD mice until four-months-old.This study was supported by The Portuguese Foundation for Science and Technology (FCT) through PTDC/ EMD-EMD/28039/2017, UIDB/04950/2020, Pest-UID/ NEU/04539/2019, and by FEDER-COMPETE through POCI-01-0145-FEDER-028039.info:eu-repo/semantics/publishedVersio

Diabetes changes the levels of ionotropic glutamate receptors in the rat retina

Purpose: Diabetic retinopathy (DR) is a leading cause of vision loss and blindness among adults between the age 20 to 74. Changes in ionotropic glutamate receptor subunit composition can affect retinal glutamatergic neurotransmission and, therefore, contribute to visual impairment. The purpose of this study was to investigate whether diabetes leads to changes in ionotropic glutamate receptor subunit expression at the protein and mRNA level in the rat retina. Methods: Changes in the expression of ionotropic glutamate receptor subunits were investigated at the mRNA and protein levels in retinas of streptozotocin (STZ)-induced diabetic and age-matched control rats. Animals were euthanized one, four and 12 weeks after the onset of diabetes. Retinal protein extracts were prepared, and the receptor subunit levels were assessed by western blotting. Transcript levels were assessed by real-time quantitative PCR. Results: Transcript levels of most ionotropic glutamate receptor subunits were not significantly changed in the retinas of diabetic rats, as compared to age-matched controls but protein levels of α-amino-3-hydroxyl-5-methyl-4-isoxazole- propionate (AMPA), kainate, and N-methyl-D-aspartic acid receptors (NMDA) receptors were found to be altered. Conclusions: The results provide evidence that diabetes affects the retinal content of ionotropic glutamate receptor subunits at the protein level. The possible implications of these changes on retinal physiology and visual impairment in DR are discusse

Microglia dysfunction caused by the loss of Rhoa disrupts neuronal physiology and leads to neurodegeneration

© 2020 The Author(s). Creative Commons Attribution (CC BY 4.0)Nervous tissue homeostasis requires the regulation of microglia activity. Using conditional gene targeting in mice, we demonstrate that genetic ablation of the small GTPase Rhoa in adult microglia is sufficient to trigger spontaneous microglia activation, producing a neurological phenotype (including synapse and neuron loss, impairment of long-term potentiation [LTP], formation of β-amyloid plaques, and memory deficits). Mechanistically, loss of Rhoa in microglia triggers Src activation and Src-mediated tumor necrosis factor (TNF) production, leading to excitotoxic glutamate secretion. Inhibiting Src in microglia Rhoa-deficient mice attenuates microglia dysregulation and the ensuing neurological phenotype. We also find that the Rhoa/Src signaling pathway is disrupted in microglia of the APP/PS1 mouse model of Alzheimer disease and that low doses of Aβ oligomers trigger microglia neurotoxic polarization through the disruption of Rhoa-to-Src signaling. Overall, our results indicate that disturbing Rho GTPase signaling in microglia can directly cause neurodegeneration.The authors acknowledge the support of the following i3S Scientific Platforms: Animal Facility, Translational Cytometry Unit (TraCy), BioSciences Screening (BS) and Advanced Light Microscopy (ALM), and members of the national infrastructure PPBI-Portuguese Platform of BioImaging (supported by POCI-01–0145-FEDER-022122). FCT Portugal ( PTDC/MED-NEU/31318/2017-031318 ) supported work in the J.B.R. lab. FCT Portugal , PEst ( UID/NEU/04539/2013 ), COMPETE-FEDER ( POCI-01-0145-FEDER-007440 ), Centro 2020 Regional Operational Programme ( CENTRO-01-0145-FEDER-000008 : BrainHealth 2020), and Strategic Project UIDB/04539/2020 and UIDP/04539/2020 (CIBB) supported work in the A.F.A. lab. C.C.P. and R.S. hold employment contracts financed by national funds through FCT (Fundação para a Ciência e a Tecnologia, I.P.) in the context of the program contract described in paragraphs 4, 5, and 6 of article 23 of law no. 57/2016, of August 29th, as amended by law no. 57/2017 of July 19th.info:eu-repo/semantics/publishedVersio