Targeting the vascular-specific phosphatase PTPRB protects against retinal ganglion cell loss in a pre-clinical model of glaucoma

Elevated intraocular pressure (IOP) due to insufficient aqueous humor outflow through the trabecular meshwork and Schlemm’s canal (SC) is the most important risk factor for glaucoma, a leading cause of blindness worldwide. We previously reported loss of function mutations in the receptor tyrosine kinase TEK or its ligand ANGPT1 cause primary congenital glaucoma in humans and mice due to failure of SC development. Here, we describe a novel approach to enhance canal formation in these animals by deleting a single allele of the gene encoding the phosphatase PTPRB during development. Compared to Tek haploinsufficient mice, which exhibit elevated IOP and loss of retinal ganglion cells, Tek+/-;Ptprb+/- mice have elevated TEK phosphorylation, which allows normal SC development and prevents ocular hypertension and RGC loss. These studies provide evidence that PTPRB is an important regulator of TEK signaling in the aqueous humor outflow pathway and identify a new therapeutic target for treatment of glaucoma.

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Cell atlas of aqueous humor outflow pathways in eyes of humans and four model species provides insight into glaucoma pathogenesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2001250117 ◽

2020 ◽

Vol 117 (19) ◽

pp. 10339-10349 ◽

Cited By ~ 7

Author(s):

Tavé van Zyl ◽

Wenjun Yan ◽

Alexi McAdams ◽

Yi-Rong Peng ◽

Karthik Shekhar ◽

...

Keyword(s):

Single Cell ◽

Aqueous Humor ◽

Ganglion Cells ◽

Anterior Segment ◽

Experimental Studies ◽

Cynomolgus Macaque ◽

Cell Types ◽

Model Systems ◽

Retinal Ganglion ◽

Aqueous Humor Outflow

Increased intraocular pressure (IOP) represents a major risk factor for glaucoma, a prevalent eye disease characterized by death of retinal ganglion cells; lowering IOP is the only proven treatment strategy to delay disease progression. The main determinant of IOP is the equilibrium between production and drainage of aqueous humor, with compromised drainage generally viewed as the primary contributor to dangerous IOP elevations. Drainage occurs through two pathways in the anterior segment of the eye called conventional and uveoscleral. To gain insights into the cell types that comprise these pathways, we used high-throughput single-cell RNA sequencing (scRNAseq). From ∼24,000 single-cell transcriptomes, we identified 19 cell types with molecular markers for each and used histological methods to localize each type. We then performed similar analyses on four organisms used for experimental studies of IOP dynamics and glaucoma: cynomolgus macaque (Macaca fascicularis), rhesus macaque (Macaca mulatta), pig (Sus scrofa), and mouse (Mus musculus). Many human cell types had counterparts in these models, but differences in cell types and gene expression were evident. Finally, we identified the cell types that express genes implicated in glaucoma in all five species. Together, our results provide foundations for investigating the pathogenesis of glaucoma and for using model systems to assess mechanisms and potential interventions.

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Cellular crosstalk regulates the aqueous humor outflow pathway and provides new targets for glaucoma therapies

Nature Communications ◽

10.1038/s41467-021-26346-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Benjamin R. Thomson ◽

Pan Liu ◽

Tuncer Onay ◽

Jing Du ◽

Stuart W. Tompson ◽

...

Keyword(s):

Aqueous Humor ◽

Open Angle Glaucoma ◽

Severe Disease ◽

Congenital Glaucoma ◽

Loss Of Function ◽

Developmental Defects ◽

Aqueous Humor Outflow ◽

Risk Alleles ◽

Adult Mice ◽

Outflow Pathway

AbstractPrimary congenital glaucoma (PCG) is a severe disease characterized by developmental defects in the trabecular meshwork (TM) and Schlemm’s canal (SC), comprising the conventional aqueous humor outflow pathway of the eye. Recently, heterozygous loss of function variants in TEK and ANGPT1 or compound variants in TEK/SVEP1 were identified in children with PCG. Moreover, common variants in ANGPT1and SVEP1 have been identified as risk alleles for primary open angle glaucoma (POAG) in GWAS studies. Here, we show tissue-specific deletion of Angpt1 or Svep1 from the TM causes PCG in mice with severe defects in the adjacent SC. Single-cell transcriptomic analysis of normal and glaucomatous Angpt1 deficient eyes allowed us to identify distinct TM and SC cell populations and discover additional TM-SC signaling pathways. Furthermore, confirming the importance of angiopoietin signaling in SC, delivery of a recombinant ANGPT1-mimetic promotes developmental SC expansion in healthy and Angpt1 deficient eyes, blunts intraocular pressure (IOP) elevation and RGC loss in a mouse model of PCG and lowers IOP in healthy adult mice. Our data highlight the central role of ANGPT1-TEK signaling and TM-SC crosstalk in IOP homeostasis and provide new candidates for SC-targeted glaucoma therapy.

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Cell Atlas of Aqueous Humor Outflow Pathways in Eyes of Humans and Four Model Species Provides Insights into Glaucoma Pathogenesis

10.1101/2020.02.04.933911 ◽

2020 ◽

Cited By ~ 1

Author(s):

Tavé van Zyl ◽

Wenjun Yan ◽

Alexi McAdams ◽

Yi-Rong Peng ◽

Karthik Shekhar ◽

...

Keyword(s):

Single Cell ◽

Aqueous Humor ◽

Ganglion Cells ◽

Anterior Segment ◽

Experimental Studies ◽

Cynomolgus Macaque ◽

Cell Types ◽

Model Systems ◽

Retinal Ganglion ◽

Aqueous Humor Outflow

ABSTRACTIncreased intraocular pressure (IOP) represents a major risk factor for glaucoma, a prevalent eye disease characterized by death of retinal ganglion cells that carry information from the eye to the brain; lowering IOP is the only proven treatment strategy to delay disease progression. The main determinant of IOP is the equilibrium between production and drainage of aqueous humor, with compromised drainage generally viewed as the primary contributor to dangerous IOP elevations. Drainage occurs through two pathways in the anterior segment of the eye, called conventional and uveoscleral. To gain insights into the cell types that comprise these pathways, we used high-throughput single cell RNA sequencing (scRNA-seq). From ∼24,000 single cell transcriptomes, we identified 19 cell types with molecular markers for each and used histological methods to localize each type. We then performed similar analyses on four organisms used for experimental studies of IOP dynamics and glaucoma: cynomolgus macaque (Macaca fascicularis), rhesus macaque (Macaca mulatta), pig (Sus scrofa) and mouse (Mus musculus). Many human cell types had counterparts in these models, but differences in cell types and gene expression were evident. Finally, we identified the cell types that express genes implicated in glaucoma in all five species. Together, our results provide foundations for investigating the pathogenesis of glaucoma, and for using model systems to assess mechanisms and potential interventions.

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A Chronic Ocular-Hypertensive Rat Model induced by Injection of the Sclerosant Agent Polidocanol in the Aqueous Humor Outflow Pathway

International Journal of Molecular Sciences ◽

10.3390/ijms20133209 ◽

2019 ◽

Vol 20 (13) ◽

pp. 3209 ◽

Cited By ~ 2

Author(s):

Román Blanco ◽

Gema Martinez-Navarrete ◽

Consuelo Pérez-Rico ◽

Francisco J. Valiente-Soriano ◽

Marcelino Avilés-Trigueros ◽

...

Keyword(s):

Aqueous Humor ◽

Rat Model ◽

Ganglion Cells ◽

Ex Vivo ◽

Cell Loss ◽

Fiber Layer ◽

Full Field ◽

Aqueous Humor Outflow ◽

Outflow Pathway

Background: To induce a moderate chronic ocular hypertension (OHT) by injecting polidocanol, a foamed sclerosant drug, in the aqueous humor outflow pathway. Methods: Intraocular pressure (IOP) was monitored for up to 6 months. Pattern and full-field electroretinogram (PERG and ERG) were recorded and retinal ganglion cells (RGC) and retinal nerve fiber layer (RNFL) thickness were assessed in vivo with optical coherence tomography (OCT) and ex vivo using Brn3a immunohistochemistry. Results: In the first 3 weeks post-injection, a significant IOP elevation was observed in the treated eyes (18.47 ± 3.36 mmHg) when compared with the control fellow eyes (12.52 ± 2.84 mmHg) (p < 0.05). At 8 weeks, 65% (11/17) of intervention eyes had developed an IOP increase >25% over the baseline. PERG responses were seen to be significantly reduced in the hypertensive eyes (2.25 ± 0.24 µV) compared to control eyes (1.44 ± 0.19 µV) (p < 0.01) at week 3, whereas the ERG components (photoreceptor a-wave and bipolar cell b-wave) remained unaltered. By week 24, RNFL thinning and cell loss in the ganglion cell layer was first detected (2/13, 15.3%) as assessed by OCT and light microscopy. Conclusions: This novel OHT rat model, with moderate levels of chronically elevated IOP, and abnormal PERG shows selective functional impairment of RGC.

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Autonomic Regulation of the Eye

Oxford Research Encyclopedia of Neuroscience ◽

10.1093/acrefore/9780190264086.013.276 ◽

2019 ◽

Cited By ~ 1

Author(s):

Paul J. May ◽

Anton Reiner ◽

Paul D. Gamlin

Keyword(s):

Blood Flow ◽

Nervous System ◽

Retinal Ganglion Cells ◽

Aqueous Humor ◽

Motor Neurons ◽

Parasympathetic Nervous System ◽

Ganglion Cells ◽

Reflex Response ◽

Retinal Ganglion ◽

Choroidal Blood Flow

The functions of the eye are regulated by and dependent upon the autonomic nervous system. The parasympathetic nervous system controls constriction of the iris and accommodation of the lens via a pathway with preganglionic motor neurons in the Edinger-Westphal nucleus and postganglionic motor neurons in the ciliary ganglion. The parasympathetic nervous system regulates choroidal blood flow and the production of aqueous humor through a pathway with preganglionic motor neurons in the superior salivatory nucleus and postganglionic motor neurons in the pterygopalatine (sphenopalatine) ganglion. The sympathetic nervous system controls dilation of the iris and may modulate the outflow of aqueous humor from the eye. The sympathetic preganglionic motor neurons lie in the intermediolateral cell column at the first level of the thoracic cord, and the postganglionic motor neurons are found in the superior cervical ganglion. The central pathways controlling different autonomic functions in the eye are found in a variety of locations within the central nervous system. The reflex response of the iris to changes in luminance levels begins with melanopsin-containing retinal ganglion cells in the retina that project to the olivary pretectal nucleus. This nucleus then projects upon the Edinger-Westphal preganglionic motoneurons. The dark response that produces maximal pupillary dilation involves the sympathetic pathways to the iris. Pupil size is also regulated by many other factors, but the pathways to the parasympathetic and sympathetic preganglionic motoneurons that underlie this are not well understood. Lens accommodation is controlled by premotor neurons located in the supraoculomotor area. These also regulate the pupil, and control vergence angle by modulating the activity of medial rectus, and presumably lateral rectus, motoneurons. Pathways from the frontal eye fields and cerebellum help regulate their activity. Blood flow in the choroid is regulated with respect to systemic blood pressure through pathways through the nucleus of the tractus solitarius. It is also regulated with respect to luminance levels, which likely involves the suprachiasmatic nucleus, which receives inputs from melanopsin-containing retinal ganglion cells, and other areas of the hypothalamus that project upon the parasympathetic preganglionic neurons of the superior salivatory nucleus that mediate choroidal vasodilation.

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mTOR-dependent dysregulation of autophagy contributes to the retinal ganglion cell loss in streptozotocin-induced diabetic retinopathy

10.21203/rs.3.rs-56279/v1 ◽

2020 ◽

Author(s):

Sanjar Batirovich Madrakhimov ◽

Jin Young Yang ◽

Jin Ha Kim ◽

Jung Woo Han ◽

Tae Kwann Park

Keyword(s):

Diabetic Retinopathy ◽

Ganglion Cell ◽

Ganglion Cells ◽

Neuronal Cell ◽

Cell Loss ◽

Retinal Ganglion ◽

Diabetic Mice ◽

Tissue Samples ◽

Glucose Transporter 1 ◽

Apoptosis Pathway

Abstract Background: Neurodegeneration, an early event in the pathogenesis of diabetic retinopathy (DR), precedes clinically detectable microvascular damage. Autophagy dysregulation is considered a potential cause of neuronal cell loss, however underlying mechanisms remain unclear. The mechanistic target of rapamycin (mTOR) integrates diverse environmental signals to coordinate biological processes, including autophagy. Here, we investigated the role of mTOR signaling in neuronal cell death in diabetic retinopathy. Methods: Diabetes was induced by a single intraperitoneal injection of streptozotocin and tissue samples were harvested at 1, 2, 3, 4, and 6 months of diabetes. Early-stage of diabetic retinopathy was investigated in 1-month-diabetic mice treated with phlorizin or rapamycin. The effect of autophagy modulation on retinal ganglion cells was investigated in 3-months-diabetic mice treated with phlorizin or MHY1485. Tissue samples obtained from treated/untreated diabetic mice and age-matched controls were used for Western blot and histologic analysis.Results: mTOR-related proteins and glucose transporter 1 (GLUT1) was upregulated at 1 month and downregulated in the following period up to 6 months. Diabetes-induced neurodegeneration was characterized by an increase of apoptotic marker – cleaved caspase 3, a decrease of the total number of cells, and NeuN immunoreactivity in the ganglion cell layer (GCL), as well as an increase of autophagic protein. Insulin-independent glycemic control restored the mTOR pathway activity and GLUT1 expression, along with a decrease of autophagic and apoptotic proteins in 3-months-diabetic mice neuroretina. However, blockade of autophagy using MHY1485 resulted in a more protective effect on ganglion cells compared with phlorizin treatment. Conclusion: Collectively, our study describes the mechanisms of neurodegeneration through the hyperglycemia/ mTOR/ autophagy/ apoptosis pathway.

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The loss of microglia activities facilitates glaucoma progression in association with CYP1B1 gene mutation (p.Gly61Glu)

PLoS ONE ◽

10.1371/journal.pone.0241902 ◽

2020 ◽

Vol 15 (11) ◽

pp. e0241902

Author(s):

Amani Alghamdi ◽

Wadha Aldossary ◽

Sarah Albahkali ◽

Batoul Alotaibi ◽

Bahauddeen M. Alrfaei

Keyword(s):

Stem Cells ◽

Optic Nerve ◽

Ganglion Cells ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Annexin V ◽

Congenital Glaucoma ◽

Retinal Ganglion ◽

Primary Congenital Glaucoma ◽

Metabolic Activities

Background Glaucoma represents the second main cause of irreversible loss of eyesight worldwide. Progression of the disease is due to changes around the optic nerve, eye structure and optic nerve environment. Focusing on primary congenital glaucoma, which is not completely understood, we report an evaluation of an untested mutation (c.182G>A, p.Gly61Glu) within the CYP1B1 gene in the context of microglia, astrocytes and mesenchymal stem cells. We investigated the behaviours of these cells, which are needed to maintain eye homeostasis, in response to the CYP1B1 mutation. Methods and results CRISPR technology was used to edit normal CYP1B1 genes within normal astrocytes, microglia and stem cells in vitro. Increased metabolic activities were found in microglia and astrocytes 24 hours after CYP1B1 manipulation. However, these activities dropped by 40% after 72 hrs. In addition, the nicotinamide adenine dinucleotide phosphate (NADP)/NADPH reducing equivalent process decreased by 50% on average after 72 hrs of manipulation. The cytokines measured in mutated microglia showed progressive activation leading to apoptosis, which was confirmed with annexin-V. The cytokines evaluated in mutant astrocytes were abnormal in comparison to those in the control. Conclusions The results suggest a progressive inflammation that was induced by mutations (p.Gly61Glu) on CYP1B1. Furthermore, the mutations enhanced the microglia’s loss of activity. We are the first to show the direct impact of the mutation on microglia. This progressive inflammation might be responsible for primary congenital glaucoma complications, which could be avoided via an anti-inflammatory regimen. This finding also reveals that progressive inflammation affects recovery failure after surgeries to relieve glaucoma. Moreover, microglia are important for the survival of ganglion cells, along with the clearing of pathogens and inflammation. The reduction of their activities may jeopardise homeostasis within the optic nerve environment and complicate the protection of optic nerve components (such as retinal ganglion and glial cells).

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Loss of function mutations in the gene encoding latent transforming growth factor beta binding protein 2, LTBP2, cause primary congenital glaucoma

Human Molecular Genetics ◽

10.1093/hmg/ddp338 ◽

2009 ◽

Vol 18 (20) ◽

pp. 3969-3977 ◽

Cited By ~ 62

Author(s):

Mehrnaz Narooie-Nejad ◽

Seyed Hassan Paylakhi ◽

Seyedmehdi Shojaee ◽

Zeinab Fazlali ◽

Mozhgan Rezaei Kanavi ◽

...

Keyword(s):

Growth Factor ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

Binding Protein ◽

Congenital Glaucoma ◽

Loss Of Function ◽

Gene Encoding ◽

Primary Congenital Glaucoma ◽

Growth Factor Beta

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Quantification and image-derived phenotyping of retinal ganglion cell nuclei in the nee mouse model of congenital glaucoma

10.1101/2021.04.13.439698 ◽

2021 ◽

Author(s):

Carly J. van der Heide ◽

Kacie J. Meyer ◽

Hannah E. Mercer ◽

Adam Hedberg-Buenz ◽

Michael G. Anderson

Keyword(s):

Mouse Model ◽

Ganglion Cell ◽

Retinal Ganglion Cell ◽

Time Course ◽

Cell Loss ◽

Average Density ◽

Congenital Glaucoma ◽

Retinal Ganglion ◽

Cell Nuclei ◽

Significant Difference

ABSTRACTThe nee mouse model exhibits characteristic features of congenital glaucoma, a common cause of childhood blindness. The current study of nee mice had two components. First, the time course of neurodegeneration in nee retinal flat-mounts was studied over time using a retinal ganglion cell (RGC)-marker, BRN3A; a pan-nuclear marker, TO-PRO-3; and H&E staining. Based on segmentation of nuclei using ImageJ and RetFM-J, this analysis identified a rapid loss of BRN3A+ nuclei from 4–15 weeks of age, with the first statistically significant difference in average density compared to age-matched controls detected in 8-week-old cohorts (49% reduction in nee). Consistent with a model of glaucoma, no reductions in BRN3A− nuclei were detected, but the combined analysis indicated that some RGCs lost BRN3A marker expression prior to actual cell loss. These results have a practical application in the design of experiments using nee mice to study mechanisms or potential therapies for congenital glaucoma. The second component of the study pertains to a discovery-based analysis of the large amount of image data with 748,782 segmented retinal nuclei. Using the automatedly collected region of interest feature data captured by ImageJ, we tested whether RGC density of glaucomatous mice was significantly correlated to average nuclear area, perimeter, Feret diameter, or MinFeret diameter. These results pointed to two events influencing nuclear size. For variations in RGC density above approximately 3,000 nuclei/mm2 apparent spreading was observed, in which BRN3A− nuclei—regardless of genotype—became slightly larger as RGC density decreased. This same spreading occurred in BRN3A+ nuclei of wild-type mice. For variation in RGC density below 3,000 nuclei/mm2, which only occurred in glaucomatous nee mutants, BRN3A+ nuclei became smaller as disease was progressively severe. These observations have relevance to defining RGCs of relatively higher sensitivity to glaucomatous cell death and the nuclear dynamics occurring during their demise.

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A novel approach to the functional classification of retinal ganglion cells

10.1101/2021.05.09.443323 ◽

2021 ◽

Author(s):

Gerrit Hilgen ◽

Evgenia Kartsaki ◽

Viktoriia Kartysh ◽

Bruno Cessac ◽

Evelyne Sernagor

Keyword(s):

Large Scale ◽

Ganglion Cells ◽

Brain Regions ◽

Designer Drugs ◽

Retinal Ganglion ◽

Novel Approach ◽

Functional Features ◽

Novel Strategy ◽

Post Hoc ◽

Multiple Clusters

Retinal neurons come in remarkable diversity based on structure, function and genetic identity. Classifying these cells is a challenging task, requiring multimodal methodology. Here, we introduce a novel approach for retinal ganglion cell (RGC) classification, based on pharmacogenetics combined with immunohistochemistry and large-scale retinal electrophysiology. Our novel strategy allows grouping of cells sharing gene expression and understanding how these cell classes respond to basic and complex visual scenes. Our approach consists of increasing the firing level of RGCs co-expressing a certain gene (Scnn1a or Grik4) using excitatory DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) and then correlate the location of these cells with post hoc immunostaining, to unequivocally characterize anatomical and functional features of these two groups. We grouped these isolated RGC responses into multiple clusters based on the similarity of the spike trains. With our approach, and accompanied by immunohistochemistry, we were able to extend the pre-existing list of Grik4 expressing RGC types to a total of 8 RGC types and, for the first time, we provide a phenotypical description of 14 Scnn1a-expressing RGCs. The insights and methods gained here can guide RGC classification but also neuronal classification challenges in other brain regions.

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