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Significance Age-related bioenergetic insufficiency increases the vulnerability of retinal ganglion cells to intraocular pressure during glaucoma pathogenesis. This paper addresses these relationships and provides a deeper understanding of this common neurodegeneration. We demonstrate an intraocular pressure-dependent decline in retinal pyruvate levels coupled to dysregulated glucose metabolism, and detected mTOR activation at the mechanistic nexus of neurodegeneration and metabolism. Supporting this, oral supplementation of pyruvate or mTOR inhibition by rapamycin strongly protects from neurodegeneration. Bioenergetic enhancement thus provides a readily clinically translatable strategy for neurodegenerative disease. This study provides important avenues for neuroprotection against glaucoma by targeting key metabolic pathways that may be mirrored in other neurodegenerative diseases in which metabolic dysregulation may play a key role., Intraocular pressure-sensitive retinal ganglion cell degeneration is a hallmark of glaucoma, the leading cause of irreversible blindness. Here, we used RNA-sequencing and metabolomics to examine early glaucoma in DBA/2J mice. We demonstrate gene expression changes that significantly impact pathways mediating the metabolism and transport of glucose and pyruvate. Subsequent metabolic studies characterized an intraocular pressure (IOP)-dependent decline in retinal pyruvate levels coupled to dysregulated glucose metabolism prior to detectable optic nerve degeneration. Remarkably, retinal glucose levels were elevated 50-fold, consistent with decreased glycolysis but possibly including glycogen mobilization and other metabolic changes. Oral supplementation of the glycolytic product pyruvate strongly protected from neurodegeneration in both rat and mouse models of glaucoma. Investigating further, we detected mTOR activation at the mechanistic nexus of neurodegeneration and metabolism. Rapamycin-induced inhibition of mTOR robustly prevented glaucomatous neurodegeneration, supporting a damaging role for IOP-induced mTOR activation in perturbing metabolism and promoting glaucoma. Together, these findings support the use of treatments that limit metabolic disturbances and provide bioenergetic support. Such treatments provide a readily translatable strategy that warrants investigation in clinical trials.

Glaucoma is the leading cause of irreversible vision loss. Ocular hypertension is a major risk factor for glaucoma and recent work has demonstrated critical early neuroinflammatory insults occur in the optic nerve head following ocular hypertension. Microglia and infiltrating monocytes are likely candidates to drive these neuroinflammatory insults. However, the exact molecular identity / transcriptomic profile of microglia following ocular hypertensive insults is unknown. To elucidate the molecular identity of microglia after long-term exposure to ocular hypertension, we used a mouse model of glaucoma (DBA/2 J). We performed RNA-sequencing of microglia mRNA from the optic nerve head at a time point following ocular hypertensive insults, but preceding detectable neurodegeneration (with microglia identified as being CD45lo/CD11b+/CD11c−). Furthermore, RNA-sequencing was performed on optic nerve head microglia from mice treated with radiation therapy, a potent therapy preventing neuroinflammatory insults. Transcriptomic profiling of optic nerve head microglia mRNA identifies metabolic priming with marked changes in mitochondrial gene expression, and changes to phagocytosis, inflammatory, and sensome pathways. The data predict that many functions of microglia that help maintain tissue homeostasis are affected. Comparative analysis of these data with data from previously published whole optic nerve head tissue or monocyte-only samples from DBA/2 J mice demonstrate that many of the neuroinflammatory signatures in these data sets arise from infiltrating monocytes and not reactive microglia. Finally, our data demonstrate that prophylactic radiation therapy of DBA/2 J mice potently abolishes these microglia metabolic transcriptomic changes at the same time points. Together, our data provide a unique resource for the community to help drive further hypothesis generation and testing in glaucoma.

Importance Open-angle glaucoma may continue to progress despite significant lowering of intraocular pressure (IOP). Preclinical research has suggested that enhancing mitochondrial function and energy production may enhance retinal ganglion cell survival in animal models of glaucoma, but there is scant information on its effectiveness in a clinical setting. Objective To test the hypothesis that a combination of nicotinamide and pyruvate can improve retinal ganglion cell function in human glaucoma as measured with standard automated perimetry. Design, setting, and participants In this phase 2, randomized, double-blind, placebo-controlled clinical trial at a single academic institution, 197 patients were assessed for eligibility. Of these, 42 patients with treated open-angle glaucoma and moderate visual field loss in at least 1 eye were selected for inclusion and randomized. A total of 32 completed the study and were included in the final analysis. The mean (SD) age was 64.6 (9.8) years. Twenty-one participants (66%) were female. Participant race and ethnicity data were collected via self-report to ensure the distribution reflected that observed in clinical practice in the US but are not reported here to protect patient privacy. Recruitment took place in April 2019 and patients were monitored through December 2020. Data were analyzed from January to May 2021. Interventions Ascending oral doses of nicotinamide (1000 to 3000 mg) and pyruvate (1500 to 3000 mg) vs placebo (2:1 randomization). Main outcomes and measures Number of visual field test locations improving beyond normal variability in the study eye. Secondary end points were the rates of change of visual field global indices (mean deviation [MD], pattern standard deviation [PSD], and visual field index [VFI]). Results Twenty-two of 29 participants (76%) randomized to the intervention group and 12 of 13 participants (92%) randomized to placebo received their allocation, and 32 participants (32 eyes; ratio 21:11) completed the study (21 from the intervention group and 11 from the placebo group). Median (IQR) follow-up time was 2.2 (2.0-2.4) months. No serious adverse events were reported during the study. The number of improving test locations was significantly higher in the treatment group than in the placebo group (median [IQR], 15 [6-25] vs 7 [6-11]; P = .005). Rates of change of PSD suggested improvement with treatment compared with placebo (median, -0.06 vs 0.02 dB per week; 95% CI, 0.02 to 0.24; P = .02) but not MD (0.04 vs -0.002 dB per week; 95% CI, -0.27 to 0.09; P = .35) or VFI (0.09 vs -0.02% per week; 95% CI, -0.53 to 0.36; P = .71). Conclusions and relevance A combination of nicotinamide and pyruvate yielded significant short-term improvement in visual function, supporting prior experimental research suggesting a role for these agents in neuroprotection for individuals with glaucoma and confirming the need for long-term studies to establish their usefulness in slowing progression. Trial registration ClinicalTrials.gov Identifier: NCT03797469.

Chronically elevated intraocular pressure (IOP) is the major risk factor of primary open-angle glaucoma, a leading cause of blindness. Dysfunction of the trabecular meshwork (TM), which controls the outflow of aqueous humor (AqH) from the anterior chamber, is the major cause of elevated IOP. Here, we demonstrate that mice deficient in the Krüppel-like zinc finger transcriptional factor GLI-similar-1 (GLIS1) develop chronically elevated IOP. Magnetic resonance imaging and histopathological analysis reveal that deficiency in GLIS1 expression induces progressive degeneration of the TM, leading to inefficient AqH drainage from the anterior chamber and elevated IOP. Transcriptome and cistrome analyses identified several glaucoma- and extracellular matrix-associated genes as direct transcriptional targets of GLIS1. We also identified a significant association between GLIS1 variant rs941125 and glaucoma in humans (P = 4.73 × 10−6), further supporting a role for GLIS1 into glaucoma etiology. Our study identifies GLIS1 as a critical regulator of TM function and maintenance, AqH dynamics, and IOP., Dysfunction of the trabecular meshwork (TM) is the chief cause of elevated intraocular pressure, the major risk factor of glaucoma. Here, the authors identify the transcription factor, GLIS1, as a critical regulator of TM maintenance and intraocular pressure, and as a glaucoma risk gene.

The final step in proline biosynthesis is catalyzed by three pyrroline-5-carboxylate reductases, PYCR1, PYCR2, and PYCR3, which convert pyrroline-5-carboxylate (P5C) to proline. Mutations in human PYCR1 and ALDH18A1 (P5C Synthetase) cause Cutis Laxa (CL), whereas mutations in PYCR2 cause hypomyelinating leukodystrophy 10 (HLD10). Here, we investigated the genetics of Pycr1 and Pycr2 in mice. A null allele of Pycr1 did not show integument or CL-related phenotypes. We also studied a novel chemically-induced mutation in Pycr2. Mice with recessive loss-of-function mutations in Pycr2 showed phenotypes consistent with neurological and neuromuscular disorders, including weight loss, kyphosis, and hind-limb clasping. The peripheral nervous system was largely unaffected, with only mild axonal atrophy in peripheral nerves. A severe loss of subcutaneous fat in Pycr2 mutant mice is reminiscent of a CL-like phenotype, but primary features such as elastin abnormalities were not observed. Aged Pycr2 mutant mice had reduced white blood cell counts and altered lipid metabolism, suggesting a generalized metabolic disorder. PYCR1 and -2 have similar enzymatic and cellular activities, and consistent with previous studies, both were localized in the mitochondria in fibroblasts. Both PYCR1 and -2 were able to complement the loss of Pro3, the yeast enzyme that converts P5C to proline, confirming their activity as P5C reductases. In mice, Pycr1; Pycr2 double mutants were sub-viable and unhealthy compared to either single mutant, indicating the genes are largely functionally redundant. Proline levels were not reduced, and precursors were not increased in serum from Pycr2 mutant mice or in lysates from skin fibroblast cultures, but placing Pycr2 mutant mice on a proline-free diet worsened the phenotype. Thus, Pycr1 and -2 have redundant functions in proline biosynthesis, and their loss makes proline a semi-essential amino acid. These findings have implications for understanding the genetics of CL and HLD10, and for modeling these disorders in mice.

Variants in the LIM homeobox transcription factor 1-beta (LMX1B) gene predispose individuals to elevated intraocular pressure (IOP), a key risk factor for glaucoma. However, the effect of LMX1B mutations varies widely between individuals. To better understand the mechanisms underlying LMX1B-related phenotypes and individual differences, we backcrossed the Lmx1bV265D (also known as Lmx1bIcst) allele onto the C57BL/6J (B6), 129/Sj (129), C3A/BLiA-Pde6b+/J (C3H) and DBA/2J-Gpnmb+ (D2-G) mouse strain backgrounds. Strain background had a significant effect on the onset and severity of ocular phenotypes in Lmx1bV265D/+ mutant mice. Mice of the B6 background were the most susceptible to developing abnormal IOP distribution, severe anterior segment developmental anomalies (including malformed eccentric pupils, iridocorneal strands and corneal abnormalities) and glaucomatous nerve damage. By contrast, Lmx1bV265D mice of the 129 background were the most resistant to developing anterior segment abnormalities, had less severe IOP elevation than B6 mutants at young ages and showed no detectable nerve damage. To identify genetic modifiers of susceptibility to Lmx1bV265D-induced glaucoma-associated phenotypes, we performed a mapping cross between mice of the B6 (susceptible) and 129 (resistant) backgrounds. We identified a modifier locus on Chromosome 18, with the 129 allele(s) substantially lessening severity of ocular phenotypes, as confirmed by congenic analysis. By demonstrating a clear effect of genetic background in modulating Lmx1b-induced phenotypes, providing a panel of strains with different phenotypic severities and identifying a modifier locus, this study lays a foundation for better understanding the roles of LMX1B in glaucoma with the goal of developing new treatments.

Background The risk of glaucoma increases significantly with age and exposure to elevated intraocular pressure, two factors linked with neuroinflammation. The complement cascade is a complex immune process with many bioactive end-products, including mediators of inflammation. Complement cascade activation has been shown in glaucoma patients and models of glaucoma. However, the function of complement-mediated inflammation in glaucoma is largely untested. Here, the complement peptide C3a receptor 1 was genetically disrupted in DBA/2J mice, an ocular hypertensive model of glaucoma, to test its contribution to neurodegeneration. Methods A null allele of C3ar1 was backcrossed into DBA/2J mice. Development of iris disease, ocular hypertension, optic nerve degeneration, retinal ganglion cell activity, loss of RGCs, and myeloid cell infiltration in C3ar1-deficient and sufficient DBA/2J mice were compared across multiple ages. RNA sequencing was performed on microglia from primary culture to determine global effects of C3ar1 on microglia gene expression. Results Deficiency in C3ar1 lowered the risk of degeneration in ocular hypertensive mice without affecting intraocular pressure elevation at 10.5 months of age. Differences were found in the percentage of mice affected, but not in individual characteristics of disease progression. The protective effect of C3ar1 deficiency was then overcome by additional aging and ocular hypertensive injury. Microglia and other myeloid-derived cells were the primary cells identified that express C3ar1. In the absence of C3ar1, microglial expression of genes associated with neuroinflammation and other immune functions were differentially expressed compared to WT. A network analysis of these data suggested that the IL10 signaling pathway is a major interaction partner of C3AR1 signaling in microglia. Conclusions C3AR1 was identified as a damaging neuroinflammatory factor. These data help suggest complement activation causes glaucomatous neurodegeneration through multiple mechanisms, including inflammation. Microglia and infiltrating myeloid cells expressed high levels of C3ar1 and are the primary candidates to mediate its effects. C3AR1 appeared to be a major regulator of microglia reactivity and neuroinflammatory function due to its interaction with IL10 signaling and other immune related pathways. Targeting myeloid-derived cells and C3AR1 signaling with therapies is expected to add to or improve neuroprotective therapeutic strategies.

Variants in the LIM homeobox transcription factor 1-beta (LMX1B) gene predispose individuals to elevated intraocular pressure (IOP), a key risk factor for glaucoma. However, the effect of LMX1B mutations varies widely between individuals. To better understand the mechanisms underlying LMX1B-related phenotypes and individual differences, we backcrossed the Lmx1bV265D (also known as Lmx1bIcst) allele onto the C57BL/6J (B6), 129/Sj (129), C3A/BLiA-Pde6b+/J (C3H) and DBA/2J-Gpnmb+ (D2-G) mouse strain backgrounds. Strain background had a significant effect on the onset and severity of ocular phenotypes in Lmx1bV265D/+ mutant mice. Mice of the B6 background were the most susceptible to developing abnormal IOP distribution, severe anterior segment developmental anomalies (including malformed eccentric pupils, iridocorneal strands and corneal abnormalities) and glaucomatous nerve damage. By contrast, Lmx1bV265D mice of the 129 background were the most resistant to developing anterior segment abnormalities, had less severe IOP elevation than B6 mutants at young ages and showed no detectable nerve damage. To identify genetic modifiers of susceptibility to Lmx1bV265D-induced glaucoma-associated phenotypes, we performed a mapping cross between mice of the B6 (susceptible) and 129 (resistant) backgrounds. We identified a modifier locus on Chromosome 18, with the 129 allele(s) substantially lessening severity of ocular phenotypes, as confirmed by congenic analysis. By demonstrating a clear effect of genetic background in modulating Lmx1b-induced phenotypes, providing a panel of strains with different phenotypic severities and identifying a modifier locus, this study lays a foundation for better understanding the roles of LMX1B in glaucoma with the goal of developing new treatments., Summary: We observed that Lmx1b mutant mice of different strain backgrounds vary in onset and severity of glaucoma-related phenotypes and identified a modifier locus on Chr 18. These results could increase understanding of the mechanisms underlying glaucoma.

BackgroundThe risk of glaucoma increases significantly with age and exposure to elevated intraocular pressure, two factors linked with neuroinflammation. The complement cascade is a complex immune process with many bioactive end-products, including mediators of inflammation called anaphylatoxins. Complement cascade activation has been shown in glaucoma patients and models of glaucoma. However, the function of anaphylatoxin signaling in glaucoma is largely untested. Here,theC3AR1 anaphylatoxin receptor was genetically disrupted in DBA/2J mice, an ocular hypertensive model of glaucoma, to test its contribution to neurodegeneration. Methods A null allele of C3ar1 was backcrossed into DBA/2J mice. Development of iris disease, ocular hypertension, optic nerve degeneration, retinal ganglion cell activity, loss of RGCs, and myeloid cell infiltrationin C3ar1 deficient andsufficient DBA/2J mice were compared across multiple ages. RNA sequencing was performed on microglia from primary culture to determine global effects of C3ar1 on microglia gene expression. ResultsDeficiency in C3ar1 lowered the risk ofdegeneration in ocular hypertensive mice without affectingintraocular pressure elevation at 10.5 months of age. Differences were found in the percentage of mice affected, but not in individual characteristics of disease progression.The protective effect of C3ar1 deficiency was then overcome by additional aging and ocular hypertensive injury. Microglia and other myeloid derived cells were the primary cells identified that express C3ar1.In the absence of C3ar1, microglial expression of genes associated with neuroinflammation and other immune functions were differentially expressed compared to WT. A network analysis of these data suggested that the IL10 signaling pathway is a major interaction partner of C3AR1 signaling in microglia.ConclusionsC3AR1 was identified as a damaging neuroinflammatory factor. These datahelp suggest complement activation causesglaucomatous neurodegeneration through multiple mechanisms, including inflammation. Microglia and infiltrating myeloid cells expressed high levels ofC3ar1 and are the primary candidates to mediate its effects. C3AR1 appeared to be a major regulator ofmicrogliareactivity and neuroinflammatory function due to its interaction with IL10 signaling and other immune related pathways.Targeting myeloid-derived cells and anaphylatoxin signaling with therapiesis expected to improveneuroprotective therapeutic strategies.

Primary open-angle glaucoma (POAG) is a leading cause of irreversible vision loss, yet much of the genetic risk remains unaccounted for, especially in African-Americans who have a higher risk for developing POAG. We conduct a multiethnic genome-wide association study (GWAS) of POAG in the GERA cohort, with replication in the UK Biobank (UKB), and vice versa, GWAS in UKB with replication in GERA. We identify 24 loci (P, Primary open-angle glaucoma (POAG) leads to progressive vision loss. Here, Choquet et al. perform genome-wide association analysis for POAG in a multi-ethnic cohort, identify a total of nine novel genetic loci and show relevant function of FMNL2 and LMX1B using cell line and mouse experiments.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.

Primary congenital glaucoma (PCG) is a leading cause of blindness in children worldwide and is caused by developmental defects in 2 aqueous humor outflow structures, Schlemm’s canal (SC) and the trabecular meshwork. We previously identified loss-of-function mutations in the angiopoietin (ANGPT) receptor TEK in families with PCG and showed that ANGPT/TEK signaling is essential for SC development. Here, we describe roles for the major ANGPT ligands in the development of the aqueous outflow pathway. We determined that ANGPT1 is essential for SC development, and that Angpt1-knockout mice form a severely hypomorphic canal with elevated intraocular pressure. By contrast, ANGPT2 was dispensable, although mice deficient in both Angpt1 and Angpt2 completely lacked SC, indicating that ANGPT2 compensates for the loss of ANGPT1. In addition, we identified 3 human subjects with rare ANGPT1 variants within an international cohort of 284 PCG patients. Loss of function in 2 of the 3 patient alleles was observed by functional analysis of ANGPT1 variants in a combined in silico, in vitro, and in vivo approach, supporting a causative role for ANGPT1 in disease. By linking ANGPT1 with PCG, these results highlight the importance of ANGPT/TEK signaling in glaucoma pathogenesis and identify a candidate target for therapeutic development.

Glaucoma is a leading cause of blindness, affecting up to 70 million people worldwide. High intraocular pressure (IOP) is a major risk factor for glaucoma. It is well established that inefficient aqueous humor (AqH) outflow resulting from structural or functional alterations in ocular drainage tissues causes high IOP, but the genes and pathways involved are poorly understood. We previously demonstrated that mutations in the gene encoding the serine protease PRSS56 induces ocular angle closure and high IOP in mice and identified reduced ocular axial length as a potential contributing factor. Here, we show that Prss56−/− mice also exhibit an abnormal iridocorneal angle configuration characterized by a posterior shift of ocular drainage structures relative to the ciliary body and iris. Notably, we show that retina-derived PRSS56 is required between postnatal days 13 and 18 for proper iridocorneal configuration and that abnormal positioning of the ocular drainage tissues is not dependent on ocular size reduction in Prss56−/− mice. Furthermore, we demonstrate that the genetic context modulates the severity of IOP elevation in Prss56 mutant mice and describe a progressive degeneration of ocular drainage tissues that likely contributes to the exacerbation of the high IOP phenotype observed on the C3H/HeJ genetic background. Finally, we identify five rare PRSS56 variants associated with human primary congenital glaucoma, a condition characterized by abnormal development of the ocular drainage structures. Collectively, our findings point to a role for PRSS56 in the development and maintenance of ocular drainage tissues and IOP homeostasis, and provide new insights into glaucoma pathogenesis., Summary: This study reports a novel role of PRSS56 in the proper developmental positioning of ocular drainage tissues, establishing it as an important genetic factor involved in iridocorneal angle configuration and intraocular pressure homeostasis.

Background Glaucoma is characterized by the progressive dysfunction and loss of retinal ganglion cells. Recent work in animal models suggests that a critical neuroinflammatory event damages retinal ganglion cell axons in the optic nerve head during ocular hypertensive injury. We previously demonstrated that monocyte-like cells enter the optic nerve head in an ocular hypertensive mouse model of glaucoma (DBA/2 J), but their roles, if any, in mediating axon damage remain unclear. Methods To understand the function of these infiltrating monocyte-like cells, we used RNA-sequencing to profile their transcriptomes. Based on their pro-inflammatory molecular signatures, we hypothesized and confirmed that monocyte-platelet interactions occur in glaucomatous tissue. Furthermore, to test monocyte function we used two approaches to inhibit their entry into the optic nerve head: (1) treatment with DS-SILY, a peptidoglycan that acts as a barrier to platelet adhesion to the vessel wall and to monocytes, and (2) genetic targeting of Itgam (CD11b, an immune cell receptor that enables immune cell extravasation). Results Monocyte specific RNA-sequencing identified novel neuroinflammatory pathways early in glaucoma pathogenesis. Targeting these processes pharmacologically (DS-SILY) or genetically (Itgam / CD11b knockout) reduced monocyte entry and provided neuroprotection in DBA/2 J eyes. Conclusions These data demonstrate a key role of monocyte-like cell extravasation in glaucoma and demonstrate that modulating neuroinflammatory processes can significantly lessen optic nerve injury. Electronic supplementary material The online version of this article (10.1186/s13024-018-0303-3) contains supplementary material, which is available to authorized users.

Angle-closure glaucoma (ACG) is a severe form of glaucoma affecting up to 16 million people worldwide. In ACG, physical blockage of the ocular drainage tissue by the peripheral iris impedes the drainage of aqueous humor resulting in elevated intraocular pressure (IOP) and subsequent optic nerve damage. Despite the high prevalence of ACG, the precise mechanism(s) underlying pathogenesis are only partially understood. We have previously demonstrated that a mutation in the gene encoding the serine protease PRSS56 causes an ACG phenotype in mice. Notably, Prss56 mutant mice exhibit a reduced ocular axial length and a lens occupying a larger ocular volume compared to WT mice, recapitulating characteristic features of human AGC. Our findings utilizing mouse genetic models demonstrate that loss of PRSS56 function results in altered configuration of ocular angle structures characterized by a posterior shift in the positioning of the ocular drainage tissue relative to the ciliary body and iris during development, leading to a physical blockage of drainage structure (angle closure) and high IOP. Utilizing a previously employed genetic strategy of rescuing mutant Prss56 mediated reduction in ocular size by inactivation of EGR1 (Egr1;Prss56 double mutants) we determined the influence of ocular size on developmental positioning of the ocular angle tissues. Our findings suggest that abnormal positioning of the drainage structure as a result of loss of PRSS56 function is uncoupled from its effect on ocular axial length reduction. Furthermore, we demonstrate that the IOP elevation observed in Prss56 mutant mice is genetic context-dependent and identify a dominant modifier locus on Chromosome 2 of the C3H/HeJ genome conferring susceptibility to high IOP. Overall, our findings reveal a novel role for PRSS56 in the proper configuration of the iridocorneal angle and provide new insight into the developmental pathways implicated in glaucoma pathogenesis.

The cJun N-terminal kinases (JNKs; JNK1, JNK2, and JNK3) promote degenerative processes after neuronal injury and in disease. JNK2 and JNK3 have been shown to promote retinal ganglion cell (RGC) death after optic nerve injury. In their absence, long-term survival of RGC somas is significantly increased after mechanical optic nerve injury. In glaucoma, because optic nerve damage is thought to be a major cause of RGC death, JNKs are an important potential target for therapeutic intervention. To assess the role of JNK2 and JNK3 in an ocular hypertensive model of glaucoma, null alleles of Jnk2 and Jnk3 were backcrossed into the DBA/2J (D2) mouse. JNK activation occurred in RGCs following increased intraocular pressure in D2 mice. However, deficiency of both Jnk2 and Jnk3 together did not lessen optic nerve damage or RGC death. These results differentiate the molecular pathways controlling cell death in ocular hypertensive glaucoma compared with mechanical optic nerve injury. It is further shown that JUN, a pro-death component of the JNK pathway in RGCs, can be activated in glaucoma in the absence of JNK2 and JNK3. This implicates JNK1 in glaucomatous RGC death. Unexpectedly, at younger ages, Jnk2-deficient mice were more likely to develop features of glaucomatous neurodegeneration than D2 mice expressing Jnk2. This appears to be due to a neuroprotective effect of JNK2 and not due to a change in intraocular pressure. The Jnk2-deficient context also unmasked a lesser role for Jnk3 in glaucoma. Jnk2 and Jnk3 double knockout mice had a modestly increased risk of neurodegeneration compared with mice only deficient in Jnk2. Overall, these findings are consistent with pleiotropic effects of JNK isoforms in glaucoma and suggest caution is warranted when using JNK inhibitors to treat chronic neurodegenerative conditions.

A mismatch between optical power and ocular axial length results in refractive errors. Uncorrected refractive errors constitute the most common cause of vision loss and second leading cause of blindness worldwide. Although the retina is known to play a critical role in regulating ocular growth and refractive development, the precise factors and mechanisms involved are poorly defined. We have previously identified a role for the secreted serine protease PRSS56 in ocular size determination and PRSS56 variants have been implicated in the etiology of both hyperopia and myopia, highlighting its importance in refractive development. Here, we use a combination of genetic mouse models to demonstrate that Prss56 mutations leading to reduced ocular size and hyperopia act via a loss of function mechanism. Using a conditional gene targeting strategy, we show that PRSS56 derived from Müller glia contributes to ocular growth, implicating a new retinal cell type in ocular size determination. Importantly, we demonstrate that persistent activity of PRSS56 is required during distinct developmental stages spanning the pre- and post-eye opening periods to ensure optimal ocular growth. Thus, our mouse data provide evidence for the existence of a molecule contributing to both the prenatal and postnatal stages of human ocular growth. Finally, we demonstrate that genetic inactivation of Prss56 rescues axial elongation in a mouse model of myopia caused by a null mutation in Egr1. Overall, our findings identify PRSS56 as a potential therapeutic target for modulating ocular growth aimed at preventing or slowing down myopia, which is reaching epidemic proportions., Author summary Refractive errors mainly occur when changes in ocular size (ocular axial length) prevent light from focusing directly on the retina. Myopia (nearsightedness) is the most common form of refractive errors in which the focused image falls in front of the retina. The recent unprecedented rise in the incidence of myopia has significant implications as individuals with high myopia are at an increased risk of developing irreversible blinding conditions, including retinal detachment, macular degeneration, and glaucoma. Ocular axial growth is a key determinant of normal refractive development. Although the retina has been established as a central player involved in the regulation of ocular growth, the specific retinal cell type(s) and molecular pathways involved are poorly defined. Here, we have utilized genetic mouse models to provide significant insight into spatial and temporal requirements of the retinal factor PRSS56 in ocular size determination. Importantly, we have uncovered a previously unrecognized role for retinal Müller glia in ocular growth and demonstrated that Prss56 inactivation has translational potential to rescue axial length elongation in a mouse model of myopia. Collectively, our findings suggest that therapeutic strategies targeting PRSS56 to modulate ocular growth could have important clinical implications to prevent or slowdown the progression of myopia and associated blinding conditions in humans.

While all forms of glaucoma are characterized by a specific pattern of retinal ganglion cell death, they are clinically divided into several distinct subclasses, including normal tension glaucoma, primary open angle glaucoma, congenital glaucoma, and secondary glaucoma. For each type of glaucoma there are likely numerous molecular pathways that control susceptibility to the disease. Given this complexity, a single animal model will never precisely model all aspects of all the different types of human glaucoma. Therefore, multiple animal models have been utilized to study glaucoma but more are needed. Because of the powerful genetic tools available to use in the laboratory mouse, it has proven to be a highly useful mammalian system for studying the pathophysiology of human disease. The similarity between human and mouse eyes coupled with the ability to use a combination of advanced cell biological and genetic tools in mice have led to a large increase in the number of studies using mice to model specific glaucoma phenotypes. Over the last decade, numerous new mouse models and genetic tools have emerged, providing important insight into the cell biology and genetics of glaucoma. In this review, we describe available mouse genetic models that can be used to study glaucoma-relevant disease/pathobiology. Furthermore, we discuss how these models have been used to gain insights into ocular hypertension (a major risk factor for glaucoma) and glaucomatous retinal ganglion cell death. Finally, the potential for developing new mouse models and using advanced genetic tools and resources for studying glaucoma are discussed.

Electrical vagus nerve stimulation is a treatment alternative for many epileptic and depressed patients whose symptoms are not well managed with pharmaceutical therapy. However, the fixed stimulus, open loop dosing mechanism limits its efficacy and precludes major advances in the quality of therapy. A real-time, responsive form of vagus nerve stimulation is needed to control nerve activation according to therapeutic need. This personalized approach to therapy will improve efficacy and reduce the number and severity of side effects. We present autonomous neural control, a responsive, biofeedback-driven approach that uses the degree of measured nerve activation to control stimulus delivery. We demonstrate autonomous neural control in rats, showing that it rapidly learns how to most efficiently activate any desired proportion of vagal A, B, and/or C fibers over time. This system will maximize efficacy by minimizing patient response variability and by minimizing therapeutic failures resulting from longitudinal decreases in nerve activation with increasing durations of treatment. The value of autonomous neural control equally applies to other applications of electrical nerve stimulation.

Anterior chamber depth (ACD) is a key anatomical risk factor for primary angle closure glaucoma (PACG). We conducted a genome-wide association study (GWAS) on ACD to discover novel genes for PACG on a total of 5,308 population-based individuals of Asian descent. Genome-wide significant association was observed at a sequence variant within ABCC5 (rs1401999; per-allele effect size = −0.045 mm, P = 8.17×10−9). This locus was associated with an increase in risk of PACG in a separate case-control study of 4,276 PACG cases and 18,801 controls (per-allele OR = 1.13 [95% CI: 1.06–1.22], P = 0.00046). The association was strengthened when a sub-group of controls with open angles were included in the analysis (per-allele OR = 1.30, P = 7.45×10−9; 3,458 cases vs. 3,831 controls). Our findings suggest that the increase in PACG risk could in part be mediated by genetic sequence variants influencing anterior chamber dimensions., Author Summary The anterior chamber is the space within the eye which is bound by the cornea, and the anterior surfaces of the iris and lens. Anterior chamber depth (ACD) is the distance measured along the eye's optical axis, from the cornea to the lens surface. ACD is an important risk factor for primary angle closure glaucoma (PACG), a major cause of irreversible blindness worldwide, and in particular, individuals of Asian ethnicity. In order to identify the genes that underlie PACG susceptibility, we conducted a two-staged study. We first conducted a large scale genetic study on a total of 5,308 population-based individuals of Asian descent to identify the genetic variants that influence ACD. This was followed by testing for associations between the identified genetic variant and PACG in another independent collection of 4,276 PACG cases and 18,801 controls. We found that a genetic variant within ABCC5 was associated with an increased risk of having PACG. Our findings suggest that the increase in PACG risk could in part be mediated by genetic sequence variants that influence the anterior chamber dimensions of the eye.

Mitochondrial dysfunction may be an important, if not essential, component of human glaucoma. Using transcriptomics followed by molecular and neurobiological techniques, we have recently demonstrated that mitochondrial dysfunction within retinal ganglion cells is an early feature in the DBA/2J mouse model of inherited glaucoma. Guided by these findings, we discovered that the retinal level of nicotinamide adenine dinucleotide (NAD, a key molecule for mitochondrial health) declines in an age-dependent manner. We hypothesized that this decline in NAD renders retinal ganglion cells susceptible to damage during periods of elevated intraocular pressure. To replete NAD levels in this glaucoma, we administered nicotinamide (the amide of vitamin B3). At the lowest dose tested, nicotinamide robustly protected from glaucoma (~70% of eyes had no detectable glaucomatous neurodegeneration). At this dose, nicotinamide had no influence on intraocular pressure and so its affect was neuroprotective. At the highest dose tested, 93% of eyes had no detectable glaucoma. This represents a ~10-fold decrease in the risk of developing glaucoma. At this dose, intraocular pressure still became elevated but there was a reduction in the degree of elevation showing an additional benefit. Thus, nicotinamide is unexpectedly potent at preventing this glaucoma and is an attractive option for glaucoma therapeutics. Our findings demonstrate the promise for both preventing and treating glaucoma via interventions that bolster metabolism during increasing age and during periods of elevated intraocular pressure. Nicotinamide prevents age-related declines in NAD (a decline that occurs in different genetic contexts and species). NAD precursors are reported to protect from a variety of neurodegenerative conditions. Thus, nicotinamide may provide a much needed neuroprotective treatment against human glaucoma. This manuscript summarizes human data implicating mitochondria in glaucoma, and argues for studies to further assess the safety and efficacy of nicotinamide in human glaucoma care.

Various immune response pathways are altered during early, predegenerative stages of glaucoma; however, whether the early immune responses occur secondarily to or independently of neuronal dysfunction is unclear. To investigate this relationship, we used the Wlds allele, which protects from axon dysfunction. We demonstrate that DBA/2J.Wlds mice develop high intraocular pressure (IOP) but are protected from retinal ganglion cell (RGC) dysfunction and neuroglial changes that otherwise occur early in DBA/2J glaucoma. Despite this, immune pathways are still altered in DBA/2J.Wlds mice. This suggests that immune changes are not secondary to RGC dysfunction or altered neuroglial interactions, but may be directly induced by the increased strain imposed by high IOP. One early immune response following IOP elevation is up-regulation of complement C3 in astrocytes of DBA/2J and DBA/2J.Wlds mice. Unexpectedly, because the disruption of other complement components, such as C1Q, is protective in glaucoma, C3 deficiency significantly increased the number of DBA/2J eyes with nerve damage and RGC loss at an early time point after IOP elevation. Transcriptional profiling of C3-deficient cultured astrocytes implicated EGFR signaling as a hub in C3-dependent responses. Treatment with AG1478, an EGFR inhibitor, also significantly increased the number of DBA/2J eyes with glaucoma at the same early time point. These findings suggest that C3 protects from early glaucomatous damage, a process that may involve EGFR signaling and other immune responses in the optic nerve head. Therefore, therapies that target specific components of the complement cascade, rather than global inhibition, may be more applicable for treating human glaucoma.

Background We previously reported a profound long-term neuroprotection subsequent to a single radiation-therapy in the DBA/2J mouse model of glaucoma. This neuroprotection prevents entry of monocyte-like immune cells into the optic nerve head during glaucoma. Gene expression studies in radiation-treated mice implicated Glycam1 in this protection. Glycam1 encodes a proteoglycan ligand for L-selectin and is an excellent candidate to modulate immune cell entry into the eye. Here, we experimentally test the hypothesis that radiation-induced over-expression of Glycam1 is a key component of the neuroprotection. Methods We generated a null allele of Glycam1 on a DBA/2J background. Gene and protein expression of Glycam1, monocyte entry into the optic nerve head, retinal ganglion cell death, and axon loss in the optic nerve were assessed. Results Radiation therapy potently inhibits monocyte entry into the optic nerve head and prevents retinal ganglion cell death and axon loss. DBA/2J mice carrying a null allele of Glycam1 show increased monocyte entry and increased retinal ganglion cell death and axon loss following radiation therapy, but the majority of optic nerves were still protected by radiation therapy. Conclusions Although GlyCAM1 is an L-selectin ligand, its roles in immunity are not yet fully defined. The current study demonstrates a partial role for GlyCAM1 in radiation-mediated protection. Furthermore, our results clearly show that GlyCAM1 levels modulate immune cell entry from the vasculature into neural tissues. As Glycam1 deficiency has a more profound effect on cell entry than on neurodegeneration, further experiments are needed to precisely define the role of monocyte entry in DBA/2J glaucoma. Nevertheless, GlyCAM1’s function as a negative regulator of extravasation may lead to novel therapeutic strategies for an array of common conditions involving inflammation. Electronic supplementary material The online version of this article (doi:10.1186/s12974-017-0868-8) contains supplementary material, which is available to authorized users.

Glaucoma is a complex neurodegenerative disease characterized by progressive visual dysfunction leading to vision loss. Retinal ganglion cells are the primary affected neuronal population, with a critical insult damaging their axons in the optic nerve head. This insult is typically secondary to harmfully high levels of intraocular pressure. We have previously determined that early mitochondrial abnormalities within retinal ganglion cells lead to neuronal dysfunction, with age-related declines in NAD (NAD+ and NADH) rendering retinal ganglion cell mitochondria vulnerable to intraocular pressure-dependent stresses. The Wallerian degeneration slow allele, WldS, decreases the vulnerability of retinal ganglion cells in eyes with elevated intraocular pressure, but the exact mechanism(s) of protection from glaucoma are not determined. Here we demonstrate that WldS increases retinal NAD levels. Coupled with nicotinamide administration (an NAD precursor), it robustly protects from glaucomatous neurodegeneration in a mouse model of glaucoma (94% of eyes having no glaucoma, more than WldS or nicotinamide alone). Importantly, nicotinamide and WldS protect somal, synaptic, and axonal compartments, prevent loss of anterograde axoplasmic transport, and protect from visual dysfunction as assessed by pattern electroretinogram. Boosting NAD production generally benefits major compartments of retinal ganglion cells, and may be of value in other complex, age-related, axonopathies where multiple neuronal compartments are ultimately affected.

The endothelin system is implicated in various human and animal glaucomas. Targeting the endothelin system has great promise as a treatment for human glaucoma, but the cell types involved and the exact mechanisms of action are not clearly elucidated. Here, we report a detailed characterization of the endothelin system in specific cell types of the optic nerve head (ONH) during glaucoma in DBA/2J mice. First, we show that key components of the endothelin system are expressed in multiple cell types. We discover that endothelin 2 (EDN2) is expressed in astrocytes as well as microglia/monocytes in the ONH. The endothelin receptor type A (Ednra) is expressed in vascular endothelial cells, while the endothelin receptor type B (Ednrb) receptor is expressed in ONH astrocytes. Second, we show that Macitentan treatment protects from glaucoma. Macitentan is a novel, orally administered, dual endothelin receptor antagonist with greater affinity, efficacy and safety than previous antagonists. Finally, we test the combinatorial effect of targeting both the endothelin and complement systems as a treatment for glaucoma. Similar to endothelin, the complement system is implicated in a variety of human and animal glaucomas, and has great promise as a treatment target. We discovered that combined targeting of the endothelin (Bosentan) and complement (C1qa mutation) systems is profoundly protective. Remarkably, 80% of DBA/2J eyes subjected to this combined inhibition developed no detectable glaucoma. This opens an exciting new avenue for neuroprotection in glaucoma.

Injury to retinal ganglion cell (RGC) axons triggers rapid activation of Jun N-terminal kinase (JNK) signaling, a major prodeath pathway in injured RGCs. Of the multiple kinases that can activate JNK, dual leucine kinase (Dlk) is known to regulate both apoptosis and Wallerian degeneration triggered by axonal insult. Here we tested the importance of Dlk in regulating somal and axonal degeneration of RGCs following axonal injury. Removal of DLK from the developing optic cup did not grossly affect developmental RGC death or inner plexiform layer organization. In the adult, Dlk deficiency significantly delayed axonal-injury induced RGC death. The activation of JUN was also attenuated in Dlk deficient retinas. Dlk deficiency attenuated the activation of the somal pool of JNK but did not prevent activation of the axonal pool of JNK after axonal injury, indicating that JNK activation in different cellular compartments of an RGC following axonal injury is regulated by distinct upstream kinases. In contrast to its robust influence on somal degeneration, Dlk deficiency did not alter RGC axonal degeneration after axonal injury as assessed using physiological readouts of optic nerve function.

Purpose Glaucoma is a complex disease with major risk factors including advancing age and increased intraocular pressure (IOP). Dissecting these earliest events will likely identify new avenues for therapeutics. Previously, we performed transcriptional profiling in DBA/2J (D2) mice, a widely used mouse model relevant to glaucoma. Here, we use these data to identify and test regulators of early gene expression changes in DBA/2J glaucoma. Methods Upstream regulator analysis (URA) in Ingenuity Pathway Analysis was performed to identify potential master regulators of differentially expressed genes. The function of one putative regulator, mesenchyme homeobox 2 (Meox2), was tested using a combination of genetic, biochemical, and immunofluorescence approaches. Results URA identified Meox2 as a potential regulator of early gene expression changes in the optic nerve head (ONH) of DBA/2J mice. Meox2 haploinsufficiency did not affect the characteristic diseases of the iris or IOP elevation seen in DBA/2J mice but did cause a significant increase in the numbers of eyes with axon damage compared to controls. While young mice appeared normal, aged Meox2 haploinsufficient DBA/2J mice showed a 44% reduction in MEOX2 protein levels. This correlated with modulation of age- and disease-specific vascular and myeloid alterations. Conclusions Our data support a model whereby Meox2 controls IOP-dependent vascular remodeling and neuroinflammation to promote axon survival. Promoting these earliest responses prior to IOP elevation may be a viable neuroprotective strategy to delay or prevent human glaucoma.

At present, no animal models fully embody exfoliation syndrome or exfoliation glaucoma. Both genetic and environmental factors appear critical for disease manifestation, and both must be considered when generating animal models. Because mice provide a powerful mammalian platform for modeling complex disease, this paper focuses on mouse models of exfoliation syndrome and exfoliation glaucoma.

Glaucoma is a neurodegenerative disease affecting 70million people worldwide. For some time, analysis of human glaucoma and animal models suggested that RGC axonal injury in the optic nerve head (where RGC axons exit the eye) is an important early event in glaucomatous neurodegeneration. During the last decade advances in molecular biology and genome manipulation have allowed this hypothesis to be tested more critically, at least in animal models. Data indicate that RGC axon degeneration precedes soma death. Preventing soma death using mouse models that are mutant for BAX, a proapoptotic gene, is not sufficient to prevent the degeneration of RGC axons. This indicates that different degeneration processes occur in different compartments of the RGC during glaucoma. Furthermore, the Wallerian degeneration slow allele (Wld(s)) slows or prevents RGC axon degeneration in rodent models of glaucoma. These experiments and many others, now strongly support the hypothesis that axon degeneration is a critical pathological event in glaucomatous neurodegeneration. However, the events that lead from a glaucomatous insult (e.g. elevated intraocular pressure) to axon damage in glaucoma are not well defined. For developing new therapies, it will be necessary to clearly define and order the molecular events that lead from glaucomatous insults to axon degeneration.

Primary open angle glaucoma (POAG) is a genetically and phenotypically complex disease that is a leading cause of blindness worldwide. Previously we completed a genome-wide scan for early-onset POAG that identified a locus on 9q22 (GLC1J). To identify potential causative variants underlying GLC1J, we used targeted DNA capture followed by high throughput sequencing of individuals from four GLC1J pedigrees, followed by Sanger sequencing to screen candidate variants in additional pedigrees. A mutation likely to cause early-onset glaucoma was not identified, however COL15A1 variants were found in the youngest affected members of 7 of 15 pedigrees with variable disease onset. In addition, the most common COL15A1 variant, R163H, influenced the age of onset in adult POAG cases. RNA in situ hybridization of mouse eyes shows that Col15a1 is expressed in the multiple ocular structures including ciliary body, astrocytes of the optic nerve and cells in the ganglion cell layer. Sanger sequencing of COL18A1, a related multiplexin collagen, identified a rare variant, A1381T, in members of three additional pedigrees with early-onset disease. These results suggest genetic variation in COL15A1 and COL18A1 can modify the age of onset of both early and late onset POAG.

Glaucoma is one of the leading causes of blindness in the world. Although there is no cure for glaucoma, pharmaceutical or surgical interventions are known to delay the progression of this debilitating disease. In recent years, implantation of glaucoma drainage devices (GDD) have increased due to their ability to manage IOP better than other therapeutic approaches. However, only 50% of the implanted devices remain functional after 5 years often due to biofouling. Here, we report our latest progress towards developing self-clearing GDDs using integrated magnetic microactuators. Our hypothesis is that these magnetic microdevices can provide local mechanical perturbations to prophylactically remove biological accumulation. To reduce the cost and increase the throughput of fabrication, we utilize a maskless photolithography setup and commercially available liquid crystal polymer foils to create prototype devices. The mechanical response of the devices is reported and compared with the theoretical values.

Background Glaucoma is a complex, multifactorial disease characterised by the loss of retinal ganglion cells and their axons leading to a decrease in visual function. The earliest events that damage retinal ganglion cells in glaucoma are currently unknown. Retinal ganglion cell death appears to be compartmentalised, with soma, dendrite and axon changes potentially occurring through different mechanisms. There is mounting evidence from other neurodegenerative diseases suggesting that neuronal dendrites undergo a prolonged period of atrophy, including the pruning of synapses, prior to cell loss. In addition, recent evidence has shown the role of the complement cascade in synaptic pruning in glaucoma and other diseases. Results Using a genetic (DBA/2J mouse) and an inducible (rat microbead) model of glaucoma we first demonstrate that there is loss of retinal ganglion cell synapses and dendrites at time points that precede axon or soma loss. We next determine the role of complement component 1 (C1) in early synaptic loss and dendritic atrophy during glaucoma. Using a genetic knockout of C1qa (D2.C1qa -/- mouse) or pharmacological inhibition of C1 (in the rat bead model) we show that inhibition of C1 is sufficient to preserve dendritic and synaptic architecture. Conclusions This study further supports assessing the potential for complement-modulating therapeutics for the prevention of retinal ganglion cell degeneration in glaucoma.

Background C1q represents the initiating protein of the classical complement cascade, however recent findings indicate pathway independent roles such as developmental pruning of retinal ganglion cell (RGC) axons. Furthermore, chronic neuroinflammation, including increased expression of C1q and activation of microglia and astrocytes, appears to be a common finding among many neurodegenerative disease models. Here we compare the effects of a retinal ischemia/reperfusion (I/R) injury on glial activation and neurodegeneration in wild type (WT) and C1qa-deficient mice in the retina and superior colliculus (SC). Retinal I/R was induced in mice through elevation of intraocular pressure to 120 mmHg for 60 min followed by reperfusion. Glial cell activation and population changes were assessed using immunofluorescence. Neuroprotection was determined using histological measurements of retinal layer thickness, RGC counts, and visual function by flash electroretinography (ERG). Results Retinal I/R injury significantly upregulated C1q expression in the retina as early as 72 h and within 7 days in the superficial SC, and was sustained as long as 28 days. Accompanying increased C1q expression was activation of microglia and astrocytes as well as a significantly increased glial population density observed in the retina and SC. Microglial activation and changes in density were completely ablated in C1qa-deficient mice, interestingly however there was no effect on astrocytes. Furthermore, loss of C1qa significantly rescued I/R-induced loss of RGCs and protected against retinal layer thinning in comparison to WT mice. ERG assessment revealed early preservation of b-wave amplitude deficits from retinal I/R injury due to C1qa-deficiency that was lost by day 28. Conclusions Our results for the first time demonstrate the spatiotemporal changes in the neuroinflammatory response following retinal I/R injury at both local and distal sites of injury. In addition, we have shown a role for C1q as a primary mediator of microglial activation and pathological damage. This suggests developmental mechanisms of C1q may be re-engaged during injury response, modulation of which may be beneficial for neuroprotection. Electronic supplementary material The online version of this article (doi:10.1186/s13024-016-0089-0) contains supplementary material, which is available to authorized users.

Glaucoma is a complex neurodegenerative disorder that is expected to affect 80 million people by the end of this decade. Retinal ganglion cells (RGCs) are the most affected cell type and progressively degenerate over the course of the disease. RGC axons exit the eye and enter the optic nerve by passing through the optic nerve head (ONH). The ONH is an important site of initial damage in glaucoma. Higher intraocular pressure (IOP) is an important risk factor for glaucoma, but the molecular links between elevated IOP and axon damage in the ONH are poorly defined. In this review and focusing primarily on the ONH, we discuss recent studies that have contributed to understanding the etiology and pathogenesis of glaucoma. We also identify areas that require further investigation and focus on mechanisms identified in other neurodegenerations that may contribute to RGC dysfunction and demise in glaucoma.

Elevated intraocular pressure (IOP) is a causative risk factor for the development and progression of glaucoma. Glaucomatous mutations in myocilin (MYOC) damage the trabecular meshwork and elevate IOP in humans and in mice. Animal models of glaucoma are important to discover and better understand molecular pathogenic pathways and to test new glaucoma therapeutics. Although a number of different animal models of glaucoma have been developed and characterized, there are no true models of human primary open angle glaucoma (POAG). The overall goal of this work is to develop the first inducible mouse model of POAG using a human POAG relevant transgene (i.e. mutant MYOC) expression in mouse eyes to elevate IOP and cause pressure-induced damage to the optic nerve. Four mouse strains (A/J, BALB/cJ, C57BL/6J, and C3H/HeJ) were used in this study. Ad5.MYOC.Y437H (5 × 10(7) pfu) was injected intravitreally into one eye, with the uninjected contralateral eye serving as the control eye. Conscious IOP measurements were taken using a TonoLab rebound tonometer. Optic nerve damage was determined by scoring PPD stained optic nerve cross sections. Retinal ganglion cell and superior colliculus damage was assessed by Nissl stain cell counts. Intravitreal administration of viral vector Ad5.MYOC.Y437H caused a prolonged, reproducible, and statistically significant IOP elevation in BALB/cJ, A/J, and C57BL/6J mice. IOPs increased to approximately 25 mm Hg for 8 weeks (p < 0.0001). In contrast, the C3H/HeJ mouse strain was resistant to Ad5.MYOC.Y437H induced IOP elevation for the 8-week time period. IOPs were stable (12-15 mm Hg) in the uninjected control eyes. We also determined whether there were any strain differences in pressure-induced optic nerve damage. Even though IOP was similarly elevated in three of the strains tested (BALB/cJ, C57BL/6J, and A/J) only the A/J strain had considerable and significant optic nerve damage at the end of 8 weeks with optic nerve damage score of 2.64 ± 0.19 (n = 18, p < 0.001) in the injected eye. There was no statistical difference in retinal ganglion cell death or superior colliculus damage at the 8-week time point in any of the strains tested. These results demonstrate strain dependent responses to Ad5.MYOC.Y437H-induced ocular hypertension and pressure-induced optic nerve damage.

Glaucoma is a neurodegenerative disease characterized by the apoptotic death of retinal ganglion cells (RGCs). The primary insult to RGCs in glaucoma is thought to occur to their axons as they exit the eye in the optic nerve head. However, pathological signaling pathways that exert central roles in triggering RGC death following axonal injury remain unidentified. It is likely that the first changes to occur following axonal injury are signal relay events that transduce the injury signal from the axon to the cell body. Here we focus on the c-Jun N-terminal kinase (JNK1–3) family, a signaling pathway implicated in axonal injury signaling and neurodegenerative apoptosis, and likely to function as a central node in axonal injury-induced RGC death. We show that JNK signaling is activated immediately after axonal injury in RGC axons at the site of injury. Following its early activation, sustained JNK signaling is observed in axonally-injured RGCs in the form of JUN phosphorylation and upregulation. Using mice lacking specific Jnk isoforms, we show that Jnk2 and Jnk3 are the isoforms activated in injured axons. Combined deficiency of Jnk2 and Jnk3 provides robust long-term protection against axonal injury-induced RGC death and prevents downregulation of the RGC marker, BRN3B, and phosphorylation of JUN. Finally, using Jun deficient mice, we show that JUN-dependent pathways are important for axonal injury-induced RGC death. Together these data demonstrate that JNK signaling is the major early pathway triggering RGC death after axonal injury and may directly link axon injury to transcriptional activity that controls RGC death.

This paper presents an ultra-thin and flexible polymer-based capacitive pressure sensor for intraocular pressure (IOP) monitoring in a mouse eye. Due to the size limitation of the anterior chamber in the mouse eye, a volume of approximately 700 × 700 × 150 μm(3) on a small substrate is available for the MEMS capacitive pressure sensor. Moreover, the sensor would ideally be easily injectable into place. Further complicating the sensing is the need to operate the device on the curved surface of the anterior chamber with minimum damage to the eye tissue. Therefore, a thin and flexible substrate is required. We fabricate sensors by exploiting Parylene as a biocompatible structural material in a suitable form factor and 25 μm thick liquid crystal polymer (LCP) as a soft and flexible host substrate. Using our approach, the flexibility and small form factor necessary for a mouse eye implant is achieved, along with the sensitivity required to monitor IOP fluctuations. This device will allow better study of glaucoma through close monitoring in mice after integration with other components.