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1.
Nat Commun ; 14(1): 7300, 2023 11 11.
Article En | MEDLINE | ID: mdl-37949852

Anterior Uveitis (AU) is the inflammation of the anterior part of the eye, the iris and ciliary body and is strongly associated with HLA-B*27. We report AU exome sequencing results from eight independent cohorts consisting of 3,850 cases and 916,549 controls. We identify common genome-wide significant loci in HLA-B (OR = 3.37, p = 1.03e-196) and ERAP1 (OR = 0.86, p = 1.1e-08), and find IPMK (OR = 9.4, p = 4.42e-09) and IDO2 (OR = 3.61, p = 6.16e-08) as genome-wide significant genes based on the burden of rare coding variants. Dividing the cohort into HLA-B*27 positive and negative individuals, we find ERAP1 haplotype is strongly protective only for B*27-positive AU (OR = 0.73, p = 5.2e-10). Investigation of B*27-negative AU identifies a common signal near HLA-DPB1 (rs3117230, OR = 1.26, p = 2.7e-08), risk genes IPMK and IDO2, and several additional candidate risk genes, including ADGFR5, STXBP2, and ACHE. Taken together, we decipher the genetics underlying B*27-positive and -negative AU and identify rare and common genetic signals for both subtypes of disease.


Uveitis, Anterior , Humans , Uveitis, Anterior/genetics , Inflammation/genetics , Haplotypes , Genes, MHC Class I , HLA-B Antigens/genetics , Genetic Predisposition to Disease , Polymorphism, Single Nucleotide , Aminopeptidases/genetics , Minor Histocompatibility Antigens
2.
Neuron ; 111(18): 2831-2846.e10, 2023 09 20.
Article En | MEDLINE | ID: mdl-37453419

Intermittent fasting (IF) is a diet with salutary effects on cognitive aging, Alzheimer's disease (AD), and stroke. IF restricts a number of nutrient components, including glucose. 2-deoxyglucose (2-DG), a glucose analog, can be used to mimic glucose restriction. 2-DG induced transcription of the pro-plasticity factor, Bdnf, in the brain without ketosis. Accordingly, 2-DG enhanced memory in an AD model (5xFAD) and functional recovery in an ischemic stroke model. 2-DG increased Bdnf transcription via reduced N-linked glycosylation, consequent ER stress, and activity of ATF4 at an enhancer of the Bdnf gene, as well as other regulatory regions of plasticity/regeneration (e.g., Creb5, Cdc42bpa, Ppp3cc, and Atf3) genes. These findings demonstrate an unrecognized role for N-linked glycosylation as an adaptive sensor to reduced glucose availability. They further demonstrate that ER stress induced by 2-DG can, in the absence of ketosis, lead to the transcription of genes involved in plasticity and cognitive resilience as well as proteostasis.


Alzheimer Disease , Ketosis , Stroke , Humans , Deoxyglucose/pharmacology , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Brain-Derived Neurotrophic Factor/metabolism , Glucose/metabolism , Activating Transcription Factor 4/genetics , Activating Transcription Factor 4/metabolism
3.
J Neurosci ; 2022 Jul 29.
Article En | MEDLINE | ID: mdl-35906066

Genetic disorders which present during development make treatment strategies particularly challenging because there is a need to disentangle primary pathophysiology from downstream dysfunction caused at key developmental stages. To provide a deeper insight into this question, we studied a mouse model of X-linked juvenile retinoschisis (XLRS), an early-onset inherited condition caused by mutations in the Rs1 gene encoding retinoschisin (RS1) and characterized by cystic retinal lesions and early visual deficits. Using an unbiased approach in expressing the fast intracellular calcium indicator GCaMP6f in neuronal, glial, and vascular cells of the retina of RS1-deficient male mice, we found that initial cyst formation is paralleled by the appearance of aberrant spontaneous neuro-glial signals as early as postnatal day 15, when eyes normally open. These presented as glutamate-driven wavelets of neuronal activity and sporadic radial bursts of activity by Müller glia, spanning all retinal layers and disrupting light-induced signaling. This study confers a role to RS1 beyond its function as an adhesion molecule, identifies an early onset for dysfunction in the course of disease, establishing a potential window for disease diagnosis and therapeutic intervention.Significance StatementDevelopmental disorders make it difficult to distinguish pathophysiology due to ongoing disease from pathophysiology due to disrupted development. Here, we investigated a mouse model for X-linked retinoschisis (XLRS), a well-defined monogenic degenerative disease caused by mutations in the Rs1 gene, which codes for the protein retinoschisin. We evaluated the spontaneous activity of explanted retinas lacking retinoschisin at key stages of development using the unbiased approach of ubiquitously expressing GCaMP6f in all retinal neurons, vasculature and glia. In mice lacking RS1, we found an array of novel phenotypes which present around eye-opening, are linked to glutamatergic neurotransmission, and affect visual processing. These data identify novel pathophysiology linked to RS1, and define a window where treatments might be best targeted.

4.
Nat Commun ; 13(1): 159, 2022 01 10.
Article En | MEDLINE | ID: mdl-35013160

Abnormalities in brain glucose metabolism and accumulation of abnormal protein deposits called plaques and tangles are neuropathological hallmarks of Alzheimer's disease (AD), but their relationship to disease pathogenesis and to each other remains unclear. Here we show that succinylation, a metabolism-associated post-translational protein modification (PTM), provides a potential link between abnormal metabolism and AD pathology. We quantified the lysine succinylomes and proteomes from brains of individuals with AD, and healthy controls. In AD, succinylation of multiple mitochondrial proteins declined, and succinylation of small number of cytosolic proteins increased. The largest increases occurred at critical sites of amyloid precursor protein (APP) and microtubule-associated tau. We show that in vitro, succinylation of APP disrupted its normal proteolytic processing thereby promoting Aß accumulation and plaque formation and that succinylation of tau promoted its aggregation to tangles and impaired microtubule assembly. In transgenic mouse models of AD, elevated succinylation associated with soluble and insoluble APP derivatives and tau. These findings indicate that a metabolism-linked PTM may be associated with AD.


Alzheimer Disease/metabolism , Amyloid beta-Protein Precursor/metabolism , Plaque, Amyloid/metabolism , Protein Processing, Post-Translational , Succinic Acid/metabolism , tau Proteins/metabolism , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Amino Acid Sequence , Amyloid beta-Protein Precursor/genetics , Animals , Autopsy , Brain/metabolism , Brain/pathology , Case-Control Studies , Disease Models, Animal , Gene Expression Profiling , Humans , Mice , Mice, Transgenic , Mitochondria/genetics , Mitochondria/metabolism , Mitochondria/pathology , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Plaque, Amyloid/genetics , Plaque, Amyloid/pathology , Protein Aggregates , Proteolysis , Proteome/genetics , Proteome/metabolism , tau Proteins/genetics
5.
J Comp Neurol ; 530(8): 1302-1317, 2022 06.
Article En | MEDLINE | ID: mdl-34811744

Endothelial cells (ECs) are key players in the development and maintenance of the vascular tree, the establishment of the blood-brain barrier and control of blood flow. Disruption in ECs is an early and active component of vascular pathogenesis. However, our ability to selectively target ECs in the CNS for identification and manipulation is limited. Here, in the mouse retina, a tractable model of the CNS, we utilized a recently developed AAV-BR1 system to identify distinct classes of ECs along the vascular tree using a GFP reporter. We then developed an inducible EC-specific ectopic Connexin 43 (Cx43) expression system using AAV-BR1-CAG-DIO-Cx43-P2A-DsRed2 in combination with a mouse line carrying inducible CreERT2 in ECs. We targeted Cx43 because its loss has been implicated in microvascular impairment in numerous diseases such as diabetic retinopathy and vascular edema. GFP-labeled ECs were numerous, evenly distributed along the vascular tree and their morphology was polarized with respect to the direction of blood flow. After tamoxifen induction, ectopic Cx43 was specifically expressed in ECs. Similarly to endogenous Cx43, ectopic Cx43 was localized at the membrane contacts of ECs and it did not affect tight junction proteins. The ability to enhance gap junctions in ECs provides a precise and potentially powerful tool to treat microcirculation deficits, an early pathology in numerous diseases.


Connexin 43 , Diabetic Retinopathy , Animals , Connexin 43/genetics , Connexin 43/metabolism , Endothelial Cells , Gap Junctions/metabolism , Mice , Retina
6.
Proc Natl Acad Sci U S A ; 118(51)2021 12 21.
Article En | MEDLINE | ID: mdl-34903661

Local blood flow control within the central nervous system (CNS) is critical to proper function and is dependent on coordination between neurons, glia, and blood vessels. Macroglia, such as astrocytes and Müller cells, contribute to this neurovascular unit within the brain and retina, respectively. This study explored the role of microglia, the innate immune cell of the CNS, in retinal vasoregulation, and highlights changes during early diabetes. Structurally, microglia were found to contact retinal capillaries and neuronal synapses. In the brain and retinal explants, the addition of fractalkine, the sole ligand for monocyte receptor Cx3cr1, resulted in capillary constriction at regions of microglial contact. This vascular regulation was dependent on microglial Cx3cr1 involvement, since genetic and pharmacological inhibition of Cx3cr1 abolished fractalkine-induced constriction. Analysis of the microglial transcriptome identified several vasoactive genes, including angiotensinogen, a constituent of the renin-angiotensin system (RAS). Subsequent functional analysis showed that RAS blockade via candesartan abolished microglial-induced capillary constriction. Microglial regulation was explored in a rat streptozotocin (STZ) model of diabetic retinopathy. Retinal blood flow was reduced after 4 wk due to reduced capillary diameter and this was coincident with increased microglial association. Functional assessment showed loss of microglial-capillary response in STZ-treated animals and transcriptome analysis showed evidence of RAS pathway dysregulation in microglia. While candesartan treatment reversed capillary constriction in STZ-treated animals, blood flow remained decreased likely due to dilation of larger vessels. This work shows microglia actively participate in the neurovascular unit, with aberrant microglial-vascular function possibly contributing to the early vascular compromise during diabetic retinopathy.


Chemokine CX3CL1/metabolism , Diabetic Retinopathy/pathology , Microglia/physiology , Retina/pathology , Animals , Benzimidazoles/pharmacology , Biphenyl Compounds/pharmacology , Chemokine CX3CL1/pharmacology , Diabetic Retinopathy/chemically induced , Diabetic Retinopathy/metabolism , Gene Expression Profiling , Mice , Microglia/metabolism , Neurons/physiology , Pericytes/pathology , Rats , Renin-Angiotensin System/drug effects , Renin-Angiotensin System/genetics , Retina/metabolism , Retinal Vessels/drug effects , Retinal Vessels/pathology , Signal Transduction/drug effects , Streptozocin/pharmacology , Tetrazoles/pharmacology , Vasoconstriction/drug effects
7.
J Comp Neurol ; 529(6): 1121-1134, 2021 04 15.
Article En | MEDLINE | ID: mdl-32812219

Pericytes are a unique class of mural cells essential for angiogenesis, maintenance of the vasculature and are key players in microvascular pathology. However, their diversity and specific roles are poorly understood, limiting our insight into vascular physiology and the ability to develop effective therapies. Here, in the mouse retina, a tractable model of the CNS, we evaluated distinct classes of mural cells along the vascular tree for both structural characterization and physiological manipulation of blood flow. To accomplish this, we first tested three inducible mural cell-specific mouse lines using a sensitive Ai14 reporter and tamoxifen application either by a systemic injection, or by local administration in the form of eye drops. The specificity and pattern of cre activation varied significantly across the three lines, under either the PDGFRß or NG2 promoter (Pdgfrß-CreRha, Pdgfrß-CreCsln, and Cspg4-Cre). In particular, a mouse line with Cre under the NG2 promoter resulted in sparse TdTomato labeling of mural cells, allowing for an unambiguous characterization of anatomical features of individual sphincter cells and capillary pericytes. Furthermore, in one PDGFRß line, we found that focal eye drop application of tamoxifen led to an exclusive Cre-activation in pericytes, without affecting arterial mural cells. We then used this approach to boost capillary blood flow by selective expression of Halorhodopsin, a highly precise hyperpolarizing optogenetic actuator. The ability to exclusively target capillary pericytes may prove a precise and potentially powerful tool to treat microcirculation deficits, a common pathology in numerous diseases.


Blood Flow Velocity/physiology , Capillaries/physiology , Pericytes/physiology , Regional Blood Flow/physiology , Retina/physiology , Administration, Ophthalmic , Animals , Blood Flow Velocity/drug effects , Capillaries/chemistry , Capillaries/drug effects , Mice , Mice, Transgenic , Microscopy, Fluorescence, Multiphoton/methods , Pericytes/chemistry , Pericytes/drug effects , Regional Blood Flow/drug effects , Retina/chemistry , Retina/cytology , Retina/drug effects , Tamoxifen/administration & dosage
8.
Invest Ophthalmol Vis Sci ; 61(10): 44, 2020 08 03.
Article En | MEDLINE | ID: mdl-32841313

Purpose: Disruption in blood supply to active retinal circuits is the earliest hallmark of diabetic retinopathy (DR) and has been primarily attributed to vascular deficiency. However, accumulating evidence supports an early role for a disrupted neuronal function in blood flow impairment. Here, we tested the hypothesis that selectively stimulating cholinergic neurons could restore neurovascular signaling to preserve the capillary circulation in DR. Methods: We used wild type (wt) and choline acetyltransferase promoter (ChAT)-channelrhodopsin-2 (ChR2) mice expressing ChR2 exclusively in cholinergic cells. Mice were made diabetic by streptozotocin (STZ) injections. Two to 3 months after the last STZ injection, the rate of capillary blood flow was measured in vivo within each retinal vascular layer using high speed two-photon imaging. Measurements were done at baseline and following ChR2-driven activation of retinal cholinergic interneurons, the sole source of the vasodilating neurotransmitter acetylcholine. After recordings, retinas were collected and assessed for physiological and structural features. Results: In retinal explants from ChAT-ChR2 mice, we found that channelrhodopsin2 was selectively expressed in all cholinergic amacrine cells. Its direct activation by blue light led to dilation of adjacent retinal capillaries. In living diabetic ChAT-ChR2 animals, basal capillary blood flow was significantly higher than in diabetic mice without channelrhodopsin. However, optogenetic stimulation with blue light did not result in flickering light-induced functional hyperemia, suggesting a necessity for a concerted neurovascular interaction. Conclusions: These findings provide direct support to the utility and efficacy of an optogenetic approach for targeting selective retinal circuits to treat DR and its complications.


Amacrine Cells/physiology , Cholinergic Neurons/physiology , Diabetic Retinopathy/therapy , Optogenetics/methods , Amacrine Cells/pathology , Animals , Channelrhodopsins/metabolism , Channelrhodopsins/physiology , Cholinergic Neurons/pathology , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Regional Blood Flow , Retina/pathology , Retinal Vessels/pathology , Retinal Vessels/physiology
9.
Vis Neurosci ; 37: E005, 2020 08 11.
Article En | MEDLINE | ID: mdl-32778188

Diabetic retinopathy (DR) is a frequent complication of diabetes mellitus and an increasingly common cause of visual impairment. Blood vessel damage occurs as the disease progresses, leading to ischemia, neovascularization, blood-retina barrier (BRB) failure and eventual blindness. Although detection and treatment strategies have improved considerably over the past years, there is room for a better understanding of the pathophysiology of the diabetic retina. Indeed, it has been increasingly realized that DR is in fact a disease of the retina's neurovascular unit (NVU), the multi-cellular framework underlying functional hyperemia, coupling neuronal computations to blood flow. The accumulating evidence reveals that both neurochemical (synapses) and electrical (gap junctions) means of communications between retinal cells are affected at the onset of hyperglycemia, warranting a global assessment of cellular interactions and their role in DR. This is further supported by the recent data showing down-regulation of connexin 43 gap junctions along the vascular relay from capillary to feeding arteriole as one of the earliest indicators of experimental DR, with rippling consequences to the anatomical and physiological integrity of the retina. Here, recent advancements in our knowledge of mechanisms controlling the retinal neurovascular unit will be assessed, along with their implications for future treatment and diagnosis of DR.


Diabetic Retinopathy/physiopathology , Pericytes/physiology , Retinal Neurons/physiology , Animals , Blood-Retinal Barrier , Diabetic Retinopathy/metabolism , Humans , Receptors, Cholinergic/physiology , Receptors, Dopamine/physiology , Receptors, Neurotransmitter/physiology , Regional Blood Flow/physiology , Retinal Vessels/physiopathology
10.
Cell Discov ; 6: 39, 2020.
Article En | MEDLINE | ID: mdl-32566247

Functional hyperemia, or the matching of blood flow with activity, directs oxygen and nutrients to regionally firing neurons. The mechanisms responsible for this spatial accuracy remain unclear but are critical for brain function and establish the diagnostic resolution of BOLD-fMRI. Here, we described a mosaic of pericytes, the vasomotor capillary cells in the living retina. We then tested whether this net of pericytes and surrounding neuroglia predicted a connectivity map in response to sensory stimuli. Surprisingly, we found that these connections were not only selective across cell types, but also highly asymmetric spatially. First, pericytes connected predominantly to other neighboring pericytes and endothelial cells, and less to arteriolar smooth muscle cells, and not to surrounding neurons or glia. Second, focal, but not global stimulation evoked a directional vasomotor response by strengthening connections along the feeding vascular branch. This activity required local NO signaling and occurred by means of direct coupling via gap junctions. By contrast, bath application of NO or diabetes, a common microvascular pathology, not only weakened the vascular signaling but also abolished its directionality. We conclude that the exclusivity of neurovascular interactions may thus establish spatial accuracy of blood delivery with the precision of the neuronal receptive field size, and is disrupted early in diabetes.

11.
Int J Mol Sci ; 21(7)2020 Apr 05.
Article En | MEDLINE | ID: mdl-32260484

The nervous system demands an adequate oxygen and metabolite exchange, making pericytes (PCs), the only vasoactive cells on the capillaries, essential to neural function. Loss of PCs is a hallmark of multiple diseases, including diabetes, Alzheimer's, amyotrophic lateral sclerosis (ALS) and Parkinson's. Platelet-derived growth factor receptors (PDGFRs) have been shown to be critical to PC function and survival. However, how PDGFR-mediated PC activity affects vascular homeostasis is not fully understood. Here, we tested the hypothesis that imatinib, a chemotherapeutic agent and a potent PDGFR inhibitor, alters PC distribution and thus induces vascular atrophy. We performed a morphometric analysis of the vascular elements in sham control and imatinib-treated NG2-DsRed mice. Vascular morphology and the integrity of the blood-retina barrier (BRB) were evaluated using blood albumin labeling. We found that imatinib decreased the number of PCs and blood vessel (BV) coverage in all retinal vascular layers; this was accompanied by a shrinkage of BV diameters. Surprisingly, the total length of capillaries was not altered, suggesting a preferential effect of imatinib on PCs. Furthermore, blood-retina barrier disruption was not evident. In conclusion, our data suggest that imatinib could help in treating neurovascular diseases and serve as a model for PC loss, without BRB disruption.


Blood-Retinal Barrier/drug effects , Imatinib Mesylate/pharmacology , Pericytes/drug effects , Protein Kinase Inhibitors/pharmacology , Animals , Blood-Retinal Barrier/cytology , Mice , Mice, Inbred C57BL , Pericytes/metabolism , Receptors, Platelet-Derived Growth Factor/antagonists & inhibitors , Receptors, Platelet-Derived Growth Factor/metabolism
13.
Hum Mol Genet ; 28(18): 3072-3090, 2019 09 15.
Article En | MEDLINE | ID: mdl-31174210

X-linked juvenile retinoschisis (XLRS) is an early-onset inherited condition that affects primarily males and is characterized by cystic lesions of the inner retina, decreased visual acuity and contrast sensitivity and a selective reduction of the electroretinogram (ERG) b-wave. Although XLRS is genetically heterogeneous, all mouse models developed to date involve engineered or spontaneous null mutations. In the present study, we have studied three new Rs1 mutant mouse models: (1) a knockout with inserted lacZ reporter gene; (2) a C59S point mutant substitution and (3) an R141C point mutant substitution. Mice were studied from postnatal day (P15) to 28 weeks by spectral domain optical coherence tomography and ERG. Retinas of P21-22 mice were examined using biochemistry, single cell electrophysiology of retinal ganglion cells (RGCs) and by immunohistochemistry. Each model developed intraretinal schisis and reductions in the ERG that were greater for the b-wave than the a-wave. The phenotype of the C59S mutant appeared less severe than the other mutants by ERG at adult ages. RGC electrophysiology demonstrated elevated activity in the absence of a visual stimulus and reduced signal-to-noise ratios in response to light stimuli. Immunohistochemical analysis documented early abnormalities in all cells of the outer retina. Together, these results provide significant insight into the early events of XLRS pathophysiology, from phenotype differences between disease-causing variants to common mechanistic events that may play critical roles in disease presentation and progression.


Genes, X-Linked , Genetic Association Studies , Genetic Predisposition to Disease , Genotype , Phenotype , Retinoschisis/genetics , Retinoschisis/pathology , Animals , Biomarkers , Cell Adhesion Molecules/genetics , Disease Models, Animal , Electroretinography , Eye Proteins/genetics , Genetic Association Studies/methods , Immunohistochemistry , Mice , Mutation , Photic Stimulation , Retinoschisis/diagnosis , Severity of Illness Index , Tomography, Optical Coherence
14.
Cell ; 177(5): 1262-1279.e25, 2019 05 16.
Article En | MEDLINE | ID: mdl-31056284

Ferroptosis, a non-apoptotic form of programmed cell death, is triggered by oxidative stress in cancer, heat stress in plants, and hemorrhagic stroke. A homeostatic transcriptional response to ferroptotic stimuli is unknown. We show that neurons respond to ferroptotic stimuli by induction of selenoproteins, including antioxidant glutathione peroxidase 4 (GPX4). Pharmacological selenium (Se) augments GPX4 and other genes in this transcriptional program, the selenome, via coordinated activation of the transcription factors TFAP2c and Sp1 to protect neurons. Remarkably, a single dose of Se delivered into the brain drives antioxidant GPX4 expression, protects neurons, and improves behavior in a hemorrhagic stroke model. Altogether, we show that pharmacological Se supplementation effectively inhibits GPX4-dependent ferroptotic death as well as cell death induced by excitotoxicity or ER stress, which are GPX4 independent. Systemic administration of a brain-penetrant selenopeptide activates homeostatic transcription to inhibit cell death and improves function when delivered after hemorrhagic or ischemic stroke.


Brain Ischemia , Cell-Penetrating Peptides/pharmacology , Ferroptosis/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Intracranial Hemorrhages , Neurons , Phospholipid Hydroperoxide Glutathione Peroxidase/biosynthesis , Selenium/pharmacology , Stroke , Transcription, Genetic/drug effects , Animals , Brain Ischemia/drug therapy , Brain Ischemia/metabolism , Brain Ischemia/pathology , Disease Models, Animal , Endoplasmic Reticulum Stress/drug effects , Humans , Intracranial Hemorrhages/drug therapy , Intracranial Hemorrhages/metabolism , Intracranial Hemorrhages/pathology , Male , Mice , Neurons/metabolism , Neurons/pathology , Sp1 Transcription Factor/metabolism , Stroke/drug therapy , Stroke/metabolism , Stroke/pathology , Transcription Factor AP-2/metabolism
15.
J Comp Neurol ; 527(16): 2675-2693, 2019 11 01.
Article En | MEDLINE | ID: mdl-30950036

In the retina, diverse functions of neuronal gap junctions (GJs) have been established. However, the distribution and function of vascular GJs are less clear. Here in the mouse retina whole mounts, we combined structural immunohistochemical analysis and a functional assessment of cellular coupling with a GJ-permeable tracer Neurobiotin to determine distribution patterns of three major vascular connexins. We found that Cx43 was expressed in punctate fashion on astroglia, surrounding all types of blood vessels and in continuous string-like structures along endothelial cell contacts in specialized regions of the vascular tree. Specifically, these Cx43-positive strings originated at the finest capillaries and extended toward the feeding artery. As this structural arrangement promoted strong and exclusive coupling of pericytes and endothelial cells along the corresponding branch, we termed this region a "vascular relay." Cx40 expression was found predominantly along the endothelial cell contacts of the primary arteries and did not overlap with Cx43-positive strings. At their occupied territories, Cx43 and Cx40 clustered with tight junctions and, to a lesser extent, with adhesion contacts, both key elements of the blood-retina barrier. Finally, Cx37 puncta were associated with the entire surface of both mural and endothelial cells across all regions of the vascular tree. This combinatorial analysis of vascular connexins and identification of the vascular relay region will serve as a structural foundation for future studies of neurovascular signaling in health and disease.


Gap Junctions/metabolism , Retina/metabolism , Retinal Vessels/metabolism , Animals , Astrocytes/cytology , Astrocytes/metabolism , Cell Communication/physiology , Connexin 43/metabolism , Connexins/metabolism , Endothelial Cells/cytology , Endothelial Cells/metabolism , Mice, Transgenic , Retina/cytology , Vasomotor System/cytology , Vasomotor System/metabolism , Gap Junction alpha-5 Protein , Gap Junction alpha-4 Protein
16.
JCI Insight ; 52019 03 19.
Article En | MEDLINE | ID: mdl-30888334

Changes in neuronal activity alter blood flow to match energy demand with the supply of oxygen and nutrients. This functional hyperemia is maintained by interactions between neurons, vascular cells, and glia. However, how changing neuronal activity prevalent at the onset of neurodegenerative disease affects neurovascular elements is unclear. Here, in mice with photoreceptor degeneration, a model of neuron-specific dysfunction, we combined assessment of visual function, neurovascular unit structure, and the blood-retina barrier permeability. We found that the rod loss paralleled remodeling of the neurovascular unit, comprised of photoreceptors, retinal pigment epithelium, and Muller glia. When significant visual function was still present, blood flow became disrupted and blood-retina barrier began to fail, facilitating cone loss and vision decline. Thus, in contrast to the established view, vascular deficit in neuronal degeneration is not a late consequence of neuronal dysfunction, but is present early in the course of disease. These findings further establish the importance of vascular deficit and blood retina barrier function in neuron-specific loss, and highlight it as a target for early therapeutic intervention.


Blood-Retinal Barrier/metabolism , Cell Death , Retinal Cone Photoreceptor Cells/pathology , Retinal Rod Photoreceptor Cells/pathology , Retinal Vessels/pathology , Retinitis Pigmentosa/metabolism , Vision Disorders/metabolism , Animals , Blood-Retinal Barrier/pathology , Bystander Effect , Disease Models, Animal , Disease Progression , Ependymoglial Cells/pathology , Mice , Permeability , Photoreceptor Cells, Vertebrate/pathology , Regional Blood Flow , Retinal Pigment Epithelium/pathology , Retinal Vessels/physiopathology , Retinitis Pigmentosa/pathology , Retinitis Pigmentosa/physiopathology , Vision Disorders/pathology , Vision Disorders/physiopathology , Vision, Ocular
17.
Neuroscience ; 393: 61-72, 2018 11 21.
Article En | MEDLINE | ID: mdl-30312782

Cellular communication through chemical synapses is determined by the nature of the neurotransmitter and the composition of postsynaptic receptors. In the excitatory synapse between bipolar and ganglion cells of the retina, postsynaptic AMPA receptors mediate resting activity. During evoked response, however, more abundant and sustained levels of glutamate also activate GluN2B-containing NMDA receptors (NMDARs). This phasic recruitment of distinct glutamate receptors is essential for visual discrimination; however, the fidelity of this basic mechanism under elevated glutamate levels due to aberrant activity, a common pathophysiology, is not known. Here, in both male and female mice with retinal degeneration (rd10), a condition associated with elevated synaptic activity, we reveal that changes in synaptic input to ganglion cells altered both composition and activation of NMDARs. We found that, in contrast to wild type, the spontaneous activity of rd10 cells was largely NMDAR-dependent. Surprisingly, this activity was driven primarily by atypical activation of GluN2A -containing NMDARs, not GluN2B-NMDARs. Indeed, immunohistochemical analyses and Western blot showed greater levels of the GluN2A-NMDAR subunit expression in rd10 retina compared to wild type. Overall, these results demonstrate how aberrant signaling leads to pathway-specific alterations in NMDAR expression and function.


Receptors, N-Methyl-D-Aspartate/metabolism , Retina/metabolism , Retinal Degeneration/metabolism , Retinal Ganglion Cells/metabolism , Animals , Excitatory Postsynaptic Potentials/physiology , Ganglia, Invertebrate/metabolism , Glutamic Acid/metabolism , Mice , Synapses/physiology
18.
Sci Rep ; 8(1): 5797, 2018 04 11.
Article En | MEDLINE | ID: mdl-29643381

Pannexin 1 (Panx1) forms ATP-permeable membrane channels that play a key role in purinergic signaling in the nervous system in both normal and pathological conditions. In the retina, particularly high levels of Panx1 are found in retinal ganglion cells (RGCs), but the normal physiological function in these cells remains unclear. In this study, we used patch clamp recordings in the intact inner retina to show that evoked currents characteristic of Panx1 channel activity were detected only in RGCs, particularly in the OFF-type cells. The analysis of pattern electroretinogram (PERG) recordings indicated that Panx1 contributes to the electrical output of the retina. Consistently, PERG amplitudes were significantly impaired in the eyes with targeted ablation of the Panx1 gene in RGCs. Under ocular hypertension and ischemic conditions, however, high Panx1 activity permeated cell membranes and facilitated the selective loss of RGCs or stably transfected Neuro2A cells. Our results show that high expression of the Panx1 channel in RGCs is essential for visual function in the inner retina but makes these cells highly sensitive to mechanical and ischemic stresses. These findings are relevant to the pathophysiology of retinal disorders induced by increased intraocular pressure, such as glaucoma.


Connexins/metabolism , Electrophysiological Phenomena/drug effects , Nerve Tissue Proteins/metabolism , Retinal Ganglion Cells/drug effects , Retinal Ganglion Cells/physiology , Animals , Electroretinography , Evoked Potentials, Visual , Mice, Inbred C57BL , Mice, Knockout , Patch-Clamp Techniques
19.
J Neurosci ; 37(32): 7580-7594, 2017 08 09.
Article En | MEDLINE | ID: mdl-28674171

Adequate blood flow is essential to brain function, and its disruption is an early indicator in diseases, such as stroke and diabetes. However, the mechanisms contributing to this impairment remain unclear. To address this gap, in the diabetic and nondiabetic male mouse retina, we combined an unbiased longitudinal assessment of vasomotor activity along a genetically defined vascular network with pharmacological and immunohistochemical analyses of pericytes, the capillary vasomotor elements. In nondiabetic retina, focal stimulation of a pericyte produced a robust vasomotor response, which propagated along the blood vessel with increasing stimulus. In contrast, the magnitude, dynamic range, a measure of fine vascular diameter control, and propagation of vasomotor response were diminished in diabetic retinas from streptozotocin-treated mice. These functional changes were linked to several mechanisms. We found that density of pericytes and their sensitivity to stimulation were reduced in diabetes. The impaired response propagation from the stimulation site was associated with lower expression of connexin43, a major known gap junction unit in vascular cells. Indeed, selective block of gap junctions significantly reduced propagation but not initiation of vasomotor response in the nondiabetic retina. Our data establish the mechanisms for fine local regulation of capillary diameter by pericytes and a role for gap junctions in vascular network interactions. We show how disruption of this balance contributes to impaired vasomotor control in diabetes.SIGNIFICANCE STATEMENT Identification of mechanisms governing capillary blood flow in the CNS and how they are altered in disease provides novel insight into early states of neurological dysfunction. Here, we present physiological and anatomical evidence that both intact pericyte function as well as gap junction-mediated signaling across the vascular network are essential for proper capillary diameter control and vasomotor function. Changes to capillary blood flow precede other anatomical and functional hallmarks of diabetes establishing a significant window for prevention and treatment.


Connexin 43/metabolism , Diabetes Mellitus, Experimental/metabolism , Diabetic Retinopathy/metabolism , Gap Junctions/metabolism , Pericytes/metabolism , Retinal Vessels/metabolism , Animals , Blood Flow Velocity/physiology , Diabetes Mellitus, Experimental/pathology , Diabetic Retinopathy/pathology , Gap Junctions/pathology , Male , Mice , Mice, Inbred C57BL , Retinal Vessels/pathology
20.
Neurotherapeutics ; 13(2): 341-7, 2016 Apr.
Article En | MEDLINE | ID: mdl-26758692

Optogenetic techniques are a powerful tool for determining the role of individual functional components within complex neural circuits. By genetically targeting specific cell types, neural mechanisms can be actively manipulated to gain a better understanding of their origin and function, both in health and disease. The potential of optogenetics is not limited to answering biological questions, as it is also a promising therapeutic approach for neurological diseases. An important prerequisite for this approach is to have an identified target with a uniquely defined role within a given neural circuit. Here, we examine the retinal neurovascular unit, a circuit that incorporates neurons and vascular cells to control blood flow in the retina. We highlight the role of a specific cell type, the cholinergic amacrine cell, in modulating vascular cells, and demonstrate how this can be targeted and controlled with optogenetics. A better understanding of these mechanisms will not only extend our understanding of neurovascular interactions in the brain, but ultimately may also provide new targets to treat vision loss in a variety of retinal diseases.


Nervous System Diseases/therapy , Neural Pathways/physiopathology , Optogenetics , Animals , Humans , Nervous System Diseases/physiopathology , Neural Pathways/physiology , Optogenetics/methods
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