CCN2/CTGF promotor activity in the developing and adult mouse eye.

CCN2/CTGF is a matricellular protein that is known to enhance transforming growth factor-ß signaling and to induce a myofibroblast-like phenotype in a variety of cell types. Here, we investigated Ccn2/Ctgf promotor activity during development and in the adult mouse eye, using CTGFLacZ/+ mice in which the ß-galactosidase reporter gene LacZ had been inserted into the open reading frame of Ccn2/Ctgf. Promotor activity was assessed by staining for ß-galactosidase activity and by immunolabeling using antibodies against ß-galactosidase. Co-immunostaining using antibodies against glutamine synthetase, glial fibrillary acidic protein, choline acetyltransferase, and CD31 was applied to identify specific cell types. Ccn2/Ctgf promotor activity was intense in neural crest-derived cells differentiating to corneal stroma and endothelium, and to the stroma of choroid, iris, ciliary body, and the trabecular meshwork during development. In the adult eye, a persistent and very strong promotor activity was present in the trabecular meshwork outflow pathways. In addition, endothelial cells of Schlemm's canal, and of retinal and choroidal vessels, retinal astrocytes, Müller glia, and starburst amacrine cells were stained. Very strong promoter activity was seen in the astrocytes of the glial lamina at the optic nerve head. We conclude that CCN2/CTGF signaling is involved in the processes that govern neural crest morphogenesis during ocular development. In the adult eye, CCN2/CTGF likely plays an important role for the trabecular meshwork outflow pathways and the glial lamina of the optic nerve head.

Decorin-An Antagonist of TGF-ß in Astrocytes of the Optic Nerve.

During the pathogenesis of glaucoma, optic nerve (ON) axons become continuously damaged at the optic nerve head (ONH). This often is associated with reactive astrocytes and increased transforming growth factor (TGF-ß) 2 levels. In this study we tested the hypothesis if the presence or absence of decorin (DCN), a small leucine-rich proteoglycan and a natural inhibitor of several members of the TGF family, would affect the expression of the TGF-ßs and connective tissue growth factor (CTGF/CCN2) in human ONH astrocytes and murine ON astrocytes. We found that DCN is present in the mouse ON and is expressed by human ONH and murine ON astrocytes. DCN expression and synthesis was significantly reduced after 24 h treatment with 3 nM CTGF/CCN2, while treatment with 4 pM TGF-ß2 only reduced expression of DCN significantly. Conversely, DCN treatment significantly reduced the expression of TGF-ß1, TGF-ß2 and CTGF/CCN2 vis-a-vis untreated controls. Furthermore, DCN treatment significantly reduced expression of fibronectin (FN) and collagen IV (COL IV). Notably, combined treatment with DCN and triciribine, a small molecule inhibitor of protein kinase B (AKT), attenuated effects of DCN on CTGF/CCN2, TGF-ß1, and TGF-ß2 mRNA expression. We conclude (1) that DCN is an important regulator of TGF-ß and CTGF/CCN2 expression in astrocytes of the ON and ONH, (2) that DCN thereby regulates the expression of extracellular matrix (ECM) components and (3) that DCN executes its negative regulatory effects on TGF-ß and CTGF/CCN2 via the pAKT/AKT signaling pathway in ON astrocytes.

Activation of Apoptosis in a ßB1-CTGF Transgenic Mouse Model.

To reveal the pathomechanisms of glaucoma, a common cause of blindness, suitable animal models are needed. As previously shown, retinal ganglion cell and optic nerve degeneration occur in ßB1-CTGF mice. Here, we aimed to determine possible apoptotic mechanisms and degeneration of different retinal cells. Hence, retinae were processed for immunohistology (n = 5-9/group) and quantitative real-time PCR analysis (n = 5-7/group) in 5- and 10-week-old ßB1-CTGF and wildtype controls. We noted significantly more cleaved caspase 3+ cells in ßB1-CTGF retinae at 5 (p = 0.005) and 10 weeks (p = 0.02), and a significant upregulation of Casp3 and Bax/Bcl2 mRNA levels (p < 0.05). Furthermore, more terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL+) cells were detected in transgenic mice at 5 (p = 0.03) and 10 weeks (p = 0.02). Neurofilament H staining (p = 0.01) as well as Nefh (p = 0.02) and Tubb3 (p = 0.009) mRNA levels were significantly decreased at 10 weeks. GABAergic synapse intensity was lower at 5 weeks, while no alterations were noted at 10 weeks. The glutamatergic synapse intensity was decreased at 5 (p = 0.007) and 10 weeks (p = 0.01). No changes were observed for bipolar cells, photoreceptors, and macroglia. We conclude that apoptotic processes and synapse loss precede neuronal death in this model. This slow progression rate makes the ßB1-CTGF mice a suitable model to study primary open-angle glaucoma.

Endogenous Wnt/ß-catenin signaling in Müller cells protects retinal ganglion cells from excitotoxic damage.

Purpose: To analyze whether activation of endogenous wingless (Wnt)/ß-catenin signaling in Müller cells is involved in protection of retinal ganglion cells (RGCs) following excitotoxic damage. Methods: Transgenic mice with a tamoxifen-dependent ß-catenin deficiency in Müller cells were injected with N-methyl-D-aspartate (NMDA) into the vitreous cavity of one eye to induce excitotoxic damage of the RGCs, while the contralateral eye received PBS only. Retinal damage was quantified by counting the total number of RGC axons in cross sections of optic nerves and measuring the thickness of the retinal layers on meridional sections. Then, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was performed to identify apoptotic cells in retinas of both genotypes. Western blot analyses to assess the level of retinal ß-catenin and real-time RT-PCR to quantify the retinal expression of neuroprotective factors were performed. Results: Following NMDA injection of wild-type mice, a statistically significant increase in retinal ß-catenin protein levels was observed compared to PBS-injected controls, an effect that was blocked in mice with a Müller cell-specific ß-catenin deficiency. Furthermore, in mice with a ß-catenin deficiency in Müller cells, NMDA injection led to a statistically significant decrease in RGC axons as well as a substantial increase in TUNEL-positive cells in the RGC layer compared to the NMDA-treated controls. Moreover, in the retinas of the control mice a NMDA-mediated statistically significant induction of leukemia inhibitory factor (Lif) mRNA was detected, an effect that was substantially reduced in mice with a ß-catenin deficiency in Müller cells. Conclusions: Endogenous Wnt/ß-catenin signaling in Müller cells protects RGCs against excitotoxic damage, an effect that is most likely mediated via the induction of neuroprotective factors, such as Lif.

Loss of retinal ganglion cells in a new genetic mouse model for primary open-angle glaucoma.

Primary open-angle glaucoma (POAG) is one of the most common causes for blindness worldwide. Although an elevated intraocular pressure (IOP) is the main risk factor, the exact pathology remained indistinguishable. Therefore, it is necessary to have appropriate models to investigate these mechanisms. Here, we analysed a transgenic glaucoma mouse model (ßB1-CTGF) to elucidate new possible mechanisms of the disease. Therefore, IOP was measured in ßB1-CTGF and wildtype mice at 5, 10 and 15 weeks of age. At 5 and 10 weeks, the IOP in both groups were comparable (P > 0.05). After 15 weeks, a significant elevated IOP was measured in ßB1-CTGF mice (P < 0.001). At 15 weeks, electroretinogram measurements were performed and both the a- and b-wave amplitudes were significantly decreased in ßB1-CTGF retinae (both P < 0.01). Significantly fewer Brn-3a+ retinal ganglion cells (RGCs) were observed in the ßB1-CTGF group on flatmounts (P = 0.02), cross-sections (P < 0.001) and also via quantitative real-time PCR (P = 0.02). Additionally, significantly more cleaved caspase 3+ RGCs were seen in the ßB1-CTGF group (P = 0.002). Furthermore, a decrease in recoverin+ cells was observable in the ßB1-CTGF animals (P = 0.004). Accordingly, a significant down-regulation of Recoverin mRNA levels were noted (P < 0.001). Gfap expression, on the other hand, was higher in ßB1-CTGF retinae (P = 0.023). Additionally, more glutamine synthetase signal was noted (P = 0.04). Although no alterations were observed regarding photoreceptors via immunohistology, a significant decrease of Rhodopsin (P = 0.003) and Opsin mRNA (P = 0.03) was noted. We therefore assume that the ßB1-CTGF mouse could serve as an excellent model for better understanding the pathomechanisms in POAG.

Intracameral Delivery of Layer-by-Layer Coated siRNA Nanoparticles for Glaucoma Therapy.

Glaucoma is the second leading cause of blindness worldwide, often associated with elevated intraocular pressure. Connective tissue growth factor (CTGF) is a mediator of pathological effects in the trabecular meshwork (TM) and Schlemm's canal (SC). A novel, causative therapeutic concept which involves the intracameral delivery of small interfering RNA against CTGF is proposed. Layer-by-layer coated nanoparticles of 200-260 nm with a final layer of hyaluronan (HA) are developed. The HA-coating should provide the nanoparticles sufficient mobility in the extracellular matrix and allow for binding to TM and SC cells via CD44. By screening primary TM and SC cells in vitro, in vivo, and ex vivo, the validity of the concept is confirmed. CD44 expression is elevated in glaucomatous versus healthy cells by about two- to sixfold. CD44 is significantly involved in the cellular uptake of HA-coated nanoparticles. Ex vivo organ culture of porcine, murine, and human eyes demonstrates up to threefold higher accumulation of HA compared to control nanoparticles and much better penetration into the target tissue. Gene silencing in primary human TM cells results in a significant reduction of CTGF expression. Thus, HA-coated nanoparticles combined with RNA interference may provide a potential strategy for glaucoma therapy.

Consensus recommendations for trabecular meshwork cell isolation, characterization and culture.

Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.

Synonymous variants in HTRA1 implicated in AMD susceptibility impair its capacity to regulate TGF-ß signaling.

High-temperature requirement A1 (HTRA1) is a secreted serine protease reported to play a role in the development of several cancers and neurodegenerative diseases. Still, the mechanism underlying the disease processes largely remains undetermined. In age-related macular degeneration (AMD), a common cause of vision impairment and blindness in industrialized societies, two synonymous polymorphisms (rs1049331:C>T, and rs2293870:G>T) in exon 1 of the HTRA1 gene were associated with a high risk to develop disease. Here, we show that the two polymorphisms result in a protein with altered thermophoretic properties upon heat-induced unfolding, trypsin accessibility and secretion behavior, suggesting unique structural features of the AMD-risk-associated HTRA1 protein. Applying MicroScale Thermophoresis and protease digestion analysis, we demonstrate direct binding and proteolysis of transforming growth factor ß1 (TGF-ß1) by normal HTRA1 but not the AMD-risk-associated isoform. As a consequence, both HTRA1 isoforms strongly differed in their ability to control TGF-ß mediated signaling, as revealed by reporter assays targeting the TGF-ß1-induced serpin peptidase inhibitor (SERPINE1, alias PAI-1) promoter. In addition, structurally altered HTRA1 led to an impaired autocrine TGF-ß signaling in microglia, as measured by a strong down-regulation of downstream effectors of the TGF-ß cascade such as phosphorylated SMAD2 and PAI-1 expression. Taken together, our findings demonstrate the effects of two synonymous HTRA1 variants on protein structure and protein interaction with TGF-ß1. As a consequence, this leads to an impairment of TGF-ß signaling and microglial regulation. Functional implications of the altered properties on AMD pathogenesis remain to be clarified.

SMAD7 deficiency stimulates Müller progenitor cell proliferation during the development of the mammalian retina.

The transforming growth factor-ß (TGF-ß) pathway contributes to maintain the quiescence of adult neural stem and progenitor cells in the brain. In the retina, Müller cells are discussed to represent a glial cell population with progenitor-like characteristics. Here, we aimed to investigate if elevated TGF-ß signaling modulates the proliferation of Müller cells during retinal development. We generated mutant mice with a systemic, heterozygous up-regulation of TGF-ß signaling by deleting its inhibitor SMAD7. We investigated apoptosis, proliferation, and differentiation of Müller cells in the developing retina. We show that a heterozygous deletion of SMAD7 results in an increased proliferation of Müller cell progenitors in the central retina at postnatal day 4, the time window when Müller cells differentiate in the mouse retina. This in turn results in a thickened retina and inner nuclear layer and a higher number of differentiated Müller cells in the more developed retina. Müller cells in mutant mice contain higher amounts of nestin than those of control animals which indicates that the increase in TGF-ß signaling activity during retinal development contribute to maintain some progenitor-like characteristics in Müller cells even after their differentiation period. We conclude that TGF-ß signaling influences Müller cell proliferation and differentiation during retinal development.

Altered mechanobiology of Schlemm's canal endothelial cells in glaucoma.

Increased flow resistance is responsible for the elevated intraocular pressure characteristic of glaucoma, but the cause of this resistance increase is not known. We tested the hypothesis that altered biomechanical behavior of Schlemm's canal (SC) cells contributes to this dysfunction. We used atomic force microscopy, optical magnetic twisting cytometry, and a unique cell perfusion apparatus to examine cultured endothelial cells isolated from the inner wall of SC of healthy and glaucomatous human eyes. Here we establish the existence of a reduced tendency for pore formation in the glaucomatous SC cell--likely accounting for increased outflow resistance--that positively correlates with elevated subcortical cell stiffness, along with an enhanced sensitivity to the mechanical microenvironment including altered expression of several key genes, particularly connective tissue growth factor. Rather than being seen as a simple mechanical barrier to filtration, the endothelium of SC is seen instead as a dynamic material whose response to mechanical strain leads to pore formation and thereby modulates the resistance to aqueous humor outflow. In the glaucomatous eye, this process becomes impaired. Together, these observations support the idea of SC cell stiffness--and its biomechanical effects on pore formation--as a therapeutic target in glaucoma.

The role of astrocytes in optic nerve head fibrosis in glaucoma.

Glaucoma is defined as a progressive optic neuropathy and is characterized by an irreversible loss of retinal ganglion cells. The main risk factor to develop glaucoma is an increased intraocular pressure (IOP). During the course of glaucoma structural changes in the optic nerve head (ONH) take place which lead to the characteristic excavation or cupping of the ONH. In this review we will focus on mechanisms and processes involved in structural alterations of the extracellular matrix in the lamina cribrosa (LC) of the ONH, which are associated with astrocytes. In glaucoma, a disordered deposition of elastic and collagen fibers and a typical pronounced thickening of the connective tissue septae surrounding the nerve fibers can be observed in the LC region. The remodeling process of the LC and the loss of ON axons are associated with a conversion of astrocytes from quiescent to a reactivated state. The extracellular matrix changes in the LC are thought to be due to a disturbed homeostatic balance of growth factors and the reactivated astrocytes are part of this process. Reactivated astrocytes, remodeling of the ECM within the LC and an elevated IOP are taking part in the retinal ganglion cell loss in glaucoma.

Hypertensive retinopathy in a transgenic angiotensin-based model.

Severe hypertension destroys eyesight. The RAS (renin-angiotensin system) may contribute to this. This study relied on an established angiotensin, AngII (angiotensin II)-elevated dTGR (double-transgenic rat) model and same-background SD (Sprague-Dawley) rat controls. In dTGRs, plasma levels of AngII were increased. We determined the general retinal phenotype and observed degeneration of ganglion cells that we defined as vascular degeneration. We also inspected relevant gene expression and lastly observed alterations in the outer blood-retinal barrier. We found that both scotopic a-wave and b-wave as well as oscillatory potential amplitude were significantly decreased in dTGRs, compared with SD rat controls. However, the b/a-wave ratio remained unchanged. Fluorescence angiography of the peripheral retina indicated that exudates, or fluorescein leakage, from peripheral vessels were increased in dTGRs compared with controls. Immunohistological analysis of blood vessels in retina whole-mount preparations showed structural alterations in the retina of dTGRs. We then determined the general retinal phenotype. We observed the degeneration of ganglion cells, defined vascular degenerations and finally found differential expression of RAS-related genes and angiogenic genes. We found the expression of both human angiotensinogen and human renin in the hypertensive retina. Although the renin gene expression was not altered, the AngII levels in the retina were increased 4-fold in the dTGR retina compared with that in SD rats, a finding with mechanistic implications. We suggest that alterations in the outer blood-retinal barrier could foster an area of visual-related research based on our findings. Finally, we introduce the dTGR model of retinal disease.

Ligand-functionalized nanoparticles target endothelial cells in retinal capillaries after systemic application.

To date, diseases affecting vascular structures in the posterior eye are mostly treated by laser photocoagulation and multiple intraocular injections, procedures that destroy healthy tissue and can cause vision-threatening complications. To overcome these drawbacks, we investigate the feasibility of receptor-mediated nanoparticle targeting to capillary endothelial cells in the retina after i.v. application. Cell-binding studies using microvascular endothelial cells showed receptor-specific binding and cellular uptake of cyclo(RGDfC)-modified quantum dots via the αvß3 integrin receptor. Conversely, Mueller cells and astrocytes, representing off-target cells located in the retina, revealed only negligible interaction with nanoparticles. In vivo experiments, using nude mice as the model organism, demonstrated a strong binding of the ligand-modified quantum dots in the choriocapillaris and intraretinal capillaries upon i.v. injection and 1-h circulation time. Nontargeted nanoparticles, in contrast, did not accumulate to a significant amount in the target tissue. The presented strategy of targeting integrin receptors in the retina could be of utmost value for future intervention in pathologies of the posterior eye, which are to date only accessible with difficulty.

The regulation of connective tissue growth factor expression influences the viability of human trabecular meshwork cells.

Connective tissue growth factor (CTGF) induces extracellular matrix (ECM) synthesis and contractility in human trabecular meshwork (HTM) cells. Both processes are involved in the pathogenesis of primary open-angle glaucoma. To date, little is known about regulation and function of CTGF expression in the trabecular meshwork (TM). Therefore, we analysed the effects of different aqueous humour proteins and stressors on CTGF expression in HTM cells. HTM cells from three different donors were treated with endothelin-1, insulin-like growth factor (IGF)-1, angiotensin-II, H2 O2 and heat shock and were analysed by immunohistochemistry, real-time RT-PCR and Western blotting. Viability after H2 O2 treatment was measured in CTGF silenced HTM-N cells and their controls. Latrunculin A reduced expression of CTGF by about 50% compared to untreated HTM cells, whereas endothelin-1, IGF-1, angiotensin-II, heat shock and oxidative stress led to a significant increase. Silencing of CTGF resulted in a delayed expression of αB-crystallin and in reduced cell viability in comparison to the controls after oxidative stress. Conversely, CTGF treatment led to a higher cell viability rate after H2 O2 treatment. CTGF expression is induced by factors that have been linked to glaucoma. An increased level of CTGF appears to protect TM cells against damage induced by stress. The beneficial effect of CTGF for viability of TM cells is likely associated with the effects on increased ECM synthesis and higher contractility of the TM, thereby contributing to reduced aqueous humour outflow facility causing increased intraocular pressure.

Heterozygous modulation of TGF-ß signaling does not influence Müller glia cell reactivity or proliferation following NMDA-induced damage.

The stimulation of progenitor or stem cells proliferation in the retina could be a therapeutic avenue for the treatment of various ocular neurodegenerative disorders. Müller glia cells have been discussed to represent a progenitor cell population in the adult retina. In the brain, TGF-ß signaling regulates the fate of stem cells; however, its role in the vertebrate retina is unclear. We therefore investigated whether manipulation of the TGF-ß signaling pathway is sufficient to promote Müller glia cell proliferation and subsequently their trans-differentiation into retinal neurons. To this end, we used mice with heterozygous deficiency of the essential TGF-ß receptor type II or of the inhibitory protein SMAD7, in order to down- or up-regulate the activity of TGF-ß signaling, respectively. Excitotoxic damage was applied by intravitreal N-methyl-D-aspartate injection, and BrdU pulse experiments were used to label proliferative cells. Although we successfully stimulated Müller glia cell reactivity, our findings indicate that a moderate modulation of TGF-ß signaling is not sufficient to provoke Müller glia cell proliferation. Hence, TGF-ß signaling in the retina might not be the essential causative factor to maintain mammalian Müller cells in a quiescent, non-proliferative state that prevents a stem cell-like function.

Anatomical study of pelvic nerves in relation to seminal vesicles, prostate and urethral sphincter: immunohistochemical staining, computerized planimetry and 3-dimensional reconstruction.

PURPOSE: Studies of male pelvic neuroanatomy are mandatory to improve functional outcome after radical prostatectomy. We performed a topographical investigation of nerves on the course from the seminal vesicles along the prostate toward the striated urethral sphincter. MATERIALS AND METHODS: Serial whole mount sections (1 mm intervals) of pelvic blocks of human adult male autopsy cadavers were investigated after immunohistochemical nerve staining. Computerized nerve quantification and planimetry of the total nerve surface area were performed within defined regions (ventral, ventrolateral, dorsolateral and dorsal) at the levels of the seminal vesicles and prostate, and at the striated urethral sphincter. The distance between the seminal vesicles and the nerves was measured. For improved topographical understanding 3-dimensional reconstructions were created. Differences between 3 independent variables were tested with the nonparametric Kruskal-Wallis test. RESULTS: We studied a total of 969 whole mount sections of 5 cadavers. Nerves were arranged in a vertical plate lateral to the seminal vesicles. Mean ± SD distance to the seminal vesicles was 1.68 ± 0.84, 1.50 ± 0.12 and 1.76 ± 0.37 mm at the tip, middle and base, respectively. Periprostatic nerves were mainly found dorsolaterally. At the striated urethral sphincter 38.9% of nerves had shifted to the dorsal region. The total nerve surface area decreased significantly from the seminal vesicle tip (50.2 mm(2)) to the striated urethral sphincter level (13.3 mm(2)) (p = 0.0004). CONCLUSIONS: Our findings underline that during nerve sparing prostatectomy nerve damage might occur during mobilization of the entire seminal vesicles, apical dissection and posterior reconstruction of the rhabdosphincter. Nerve planimetry revealed that 75% of the nerves from the seminal vesicles do not reach the striated urethral sphincter level and seem to innervate structures other than the corpora cavernosa.

Optineurin associates with the podocyte Golgi complex to maintain its structure.

Optineurin, a cytosolic protein associated with the actin cytoskeleton, microtubules, and the Golgi complex, appears to have an important function in neurons, as mutations in its gene are causative for neurodegenerative diseases such as primary open-angle glaucoma and amyotrophic lateral sclerosis. Here, we report that optineurin is localized in podocytes of the kidney and induced upon injury following treatment with puromycin aminonucleoside. In cultured human podocytes, optineurin localizes to the Golgi complex. Optineurin depletion by RNA interference causes Golgi fragmentation. Moreover, if the Golgi complex is fragmented following microtubule destabilization induced by nocodazole treatment, optineurin dissociates from Golgi vesicles. Furthermore, optineurin colocalizes with vinculin-labeled focal contacts of cultured podocytes and with lysosome-like structures. Optineurin is essential for the survival of cultured podocytes, as optineurin depletion causes cell death. Thus, optineurin appears to play an important role in the maintenance of the podocyte Golgi complex and in the trafficking of vesicles to focal contacts and lysosomes.

Heterozygote Wdr36-deficient mice do not develop glaucoma.

There is an ongoing controversy regarding the role of WDR36 sequence variants in the pathogenesis of primary open-angle glaucoma (POAG). WDR36 is a nucleolar protein involved in the maturation of 18S rRNA. The function of WDR36 is essential as homozygous Wdr36-deficient mouse embryos die before reaching the blastocyst stage. Here we provide a detailed analysis of the phenotype of heterozygous Wdr36-deficient mice. Loss of one Wdr36 allele causes a substantial reduction in the expression of Wdr36 mRNA. In the eyes of Wdr36(+/-) animals, the structure of the tissues involved in aqueous humor circulation and of the optic nerve head are not different from that of control littermates. In addition, one-year-old Wdr36(+/-) animals do not differ from wild-type animals with regards to intraocular pressure and number of optic nerve axons. The susceptibility of retinal ganglion cells to excitotoxic damage induced by NMDA is similar in Wdr36(+/-) and wild-type animals. Moreover, the amount of optic nerve axonal damage induced by high IOP is not different between Wdr36(+/-) and wild-type mice. Transgenic overexpression of mutated Del605-607 Wdr36 in Wdr36(+/-) animals does not cause changes in the number of optic nerve axons or susceptibility to excitotoxic damage. In addition, analysis of 18S rRNA maturation in Del605-607 Wdr36(+/-) or Wdr36(+/-) mice does not show obvious differences in rRNA processing or in the amounts of precursor forms when compared to wild-type animals. Our data obtained in Wdr36(+/-) mice do not support the assumption of a causative role for WDR36 in the pathogenesis of POAG.

In a novel autoimmune and high-pressure glaucoma model a complex immune response is induced.

Background: The neurodegenerative processes leading to glaucoma are complex. In addition to elevated intraocular pressure (IOP), an involvement of immunological mechanisms is most likely. In the new multifactorial glaucoma model, a combination of high IOP and optic nerve antigen (ONA) immunization leads to an enhanced loss of retinal ganglion cells accompanied by a higher number of microglia/macrophages in the inner retina. Here, we aimed to evaluate the immune response in this new model, especially the complement activation and the number of T-cells, for the first time. Further, the microglia/macrophage response was examined in more detail. Methods: Six-week-old wildtype (WT+ONA) and ßB1-connective tissue growth factor high-pressure mice (CTGF+ONA) were immunized with 1 mg ONA. A wildtype control (WT) and a CTGF group (CTGF) received NaCl instead. Six weeks after immunization, retinae from all four groups were processed for immunohistology, RT-qPCR, and flow cytometry, while serum was used for microarray analyses. Results: We noticed elevated numbers of C1q+ cells (classical complement pathway) in CTGF and CTGF+ONA retinae as well as an upregulation of C1qa, C1qb, and C1qc mRNA levels in these groups. While the complement C3 was only increased in CTGF and CTGF+ONA retinae, enhanced numbers of the terminal membrane attack complex were noted in all three glaucoma groups. Flow cytometry and RT-qPCR analyses revealed an enhancement of different microglia/macrophages markers, including CD11b, especially in CTGF and CTGF+ONA retinae. Interestingly, increased retinal mRNA as well as serum levels of the tumor necrosis factor α were found throughout the different glaucoma groups. Lastly, more T-cells could be observed in the ganglion cell layer of the new CTGF+ONA model. Conclusion: These results emphasize an involvement of the complement system, microglia/macrophages, and T-cells in glaucomatous disease. Moreover, in the new multifactorial glaucoma model, increased IOP in combination with autoimmune processes seem to enforce an additional T-cell response, leading to a more persistent pathology. Hence, this new model mimics the pathomechanisms occurring in human glaucoma more accurately and could therefore be a helpful tool to find new therapeutic approaches for patients in the future.

Connective tissue growth factor causes glaucoma by modifying the actin cytoskeleton of the trabecular meshwork.

The most critical risk factor for optic nerve damage in cases of primary open-angle glaucoma (POAG) is an increased intraocular pressure (IOP) caused by a resistance to aqueous humor outflow in the trabecular meshwork (TM). The molecular pathogenesis of this increase in outflow resistance in POAG has not yet been identified, but it may involve transforming growth factor TGF-ß2, which is found in higher amounts in the aqueous humor of patients with POAG. Connective tissue growth factor (CTGF) is a TGF-ß2 target gene with high constitutive TM expression. In this study, we show that either adenoviral-mediated or transgenic CTGF overexpression in the mouse eye increases IOP and leads to optic nerve damage. CTGF induces TM fibronectin and α-SMA in animals, whereas actin stress fibers and contractility are both induced in cultured TM cells. Depletion of CTGF by RNA interference leads to a marked attenuation of the actin cytoskeleton. Rho kinase inhibitors cause a reversible decline in the IOP of CTGF-overexpressing mice to levels seen in control littermates. Overall, the effects of CTGF on IOP appear to be caused by a modification of the TM actin cytoskeleton. CTGF-overexpressing mice provide a model that mimics the essential functional and structural aspects of POAG and offer a molecular mechanism to explain the increase of its most critical risk factor.