Franklin D. Echevarria

Stressor-dependent Alterations in Glycoprotein 130: Implications for Glial Cell Reactivity, Cytokine Signaling and Ganglion Cell Health in Glaucoma

OBJECTIVE The interleukin-6 (IL-6) family of cytokines is associated with retinal ganglion cell (RGC) survival and glial reactivity in glaucoma. The purpose of this study was to evaluate glaucoma-related changes in glycoprotein-130 (gp130), the common signal transducer of the IL-6 family of cytokines, as they relate to RGC health, glial reactivity and expression of IL-6 cytokine family members. METHODS For all experiments, we examined healthy retina (young C57), aged retina (aged C57), retina predisposed to glaucoma (young DBA/2) and retina with IOP-induced glaucoma (aged DBA/2). We determined retinal gene expression of gp130 and IL-6 family members, using quantitative PCR, and protein expression of gp130, using multiplex ELISA. For protein localization and cell-specific expression, we performed co-immunolabeling for gp130 and cell type-specific markers. We used quantitative microscopy to measure layer-specific expression of gp130 and its relationships to astrocyte and Müller glia reactivity and RGC axonal transport, as determined by uptake and transport of cholera toxin β-subunit (CTB). RESULTS Gene expression of gp130 was elevated with all glaucoma-related stressors, but only normal aging increased protein levels. In healthy retina, gp130 localized primarily to the inner retina, where it was expressed by astrocytes, Müller cells and RGCs. Layer-specific analysis of gp130 expression revealed increased expression in aging retina and decreased expression in glaucomatous retina that was eccentricity-dependent. These glaucoma-related changes in gp130 expression correlated with the level of GFAP and glutamine synthetase expression, as well as axonal transport in RGCs. The relationships between gp130, glial reactivity and RGC health could impact signaling by many IL-6 family cytokines, which exhibited overall increased expression in a stressor-dependent manner. CONCLUSIONS Glaucoma-related stressors, including normal aging, glaucoma predisposition and IOP-induced glaucoma, differentially alter expression of gp130 and these alterations have direct implications for astrocyte and Müller glia reactivity, RGC health and cytokine signaling.

Interleukin-6: A Constitutive Modulator of Glycoprotein 130, Neuroinflammatory and Cell Survival Signaling in Retina

Objective The interleukin-6 (IL-6) family of cytokines and their signal transducer glycoprotein (gp130) are implicated in inflammatory and cell survival functions in glaucoma. There are several avenues for interdependent modulation of IL-6 family members and gp130 signaling. Here we investigated whether IL-6 modulates gp130 and related neuroinflammatory, cell survival and regulatory signaling in both healthy and glaucomatous retina. Methods In naïve and glaucomatous (Microbead Occlusion Model), wildtype (WT) and IL-6 knockout (IL-6−/−) mice, we examined gp130 protein expression and localization, using western blot and immunohistochemistry. Gene targets related to IL-6 and gp130 signaling and pertinent to neuroinflammation (TNFα, IL-1β), cell health (Bax, Bcl-xl) and STAT3 regulation (Socs3) were quantified using qRTPCR. Results In the naïve retina, IL-6−/− retina contained significantly less gp130 compared to WT retina. This IL-6-related decrease in gp130 was accompanied by a reduction in mRNA expression of TNFα, Socs3 and Bax. After 4 weeks of microbead-induced ocular hypertension, both microbead- and saline-injected (control) eyes of IL-6−/− mice exhibited higher expression of TNFα, compared to WT mice. IL-1β expression was also reduced specifically in IL-6−/− retina with microbead-induced glaucoma. While saline and microbead injection increased Bcl-xl and Socs3 mRNA in both WT and IL-6−/− mice, IL-6−/− deficiency led to smaller increases for both Bcl-xl and Socs3. Conclusions Our findings support a role for IL-6 in setting baseline parameters for neuroinflammatory, cell health and gp130 regulatory signaling that can impact the nature and magnitude of retinal responses to glaucoma-related stressors.

Interleukin-6 Deficiency Attenuates Retinal Ganglion Cell Axonopathy and Glaucoma-Related Vision Loss

The pleotropic cytokine interleukin-6 (IL-6) is implicated in retinal ganglion cell (RGC) survival and degeneration, including that associated with glaucoma. IL-6 protects RGCs from pressure-induced apoptosis in vitro. However, it is unknown how IL-6 impacts glaucomatous degeneration in vivo. To study how IL-6 influences glaucomatous RGC axonopathy, accompanying glial reactivity, and resultant deficits in visual function, we performed neural tracing, histological, and neurobehavioral assessments in wildtype (B6;129SF2/J; WT) and IL-6 knock-out mice (B6;129S2-IL6tm1kopf/J; IL-6-/-) after 8 weeks of unilateral or bilateral microbead-induced glaucoma (microbead occlusion model). IOP increased by 20% following microbead injection in both genotypes (p < 0.05). However, deficits in wound healing at the site of corneal injection were noted. In WT mice, elevated IOP produced degenerating axon profiles and decreased axon density in the optic nerve by 15% (p < 0.01). In IL-6-/- mice, axon density in the optic nerve did not differ between microbead- and saline-injected mice (p > 0.05) and degenerating axon profiles were minimal. Preservation of RGC axons was reflected in visual function, where visual acuity decreased significantly in a time-dependent manner with microbead-induced IOP elevation in WT (p < 0.001), but not IL-6-/- mice (p > 0.05). Despite this preservation of RGC axons and visual acuity, both microbead-injected WT and IL-6-/- mice exhibited a 50% decrease in anterograde CTB transport to the superior colliculus, as compared to saline-injected controls (p < 0.01). Assessment of glial reactivity revealed no genotype- or IOP-dependent changes in retinal astrocytes. IOP elevation decreased microglia density and percent retinal area covered in WT mice (p < 0.05), while IL-6-/- mice exhibited only a decrease in density (p < 0.05). Together, our findings indicate that two defining features of RGC axonopathy—axon transport deficits and structural degeneration of axons—likely occur via independent mechanisms. Our data suggest that IL-6 is part of a mechanism that specifically leads to structural degeneration of axons. Furthermore, its absence is sufficient to prevent both structural degeneration of the optic nerve and vision loss. Overall, our work supports the proposition that functional deficits in axon transport represent a therapeutic window for RGC axonopathy and identify IL-6 signaling as a strong target for such a therapeutic.

Pressure-Induced Alterations in PEDF and PEDF-R Expression: Implications for Neuroprotective Signaling in Glaucoma.

Introduction Alterations in neuron-glia signaling are implicated in glaucoma, a neurodegenerative disease characterized by retinal ganglion cell (RGC) death. Pigment epithelium derived factor (PEDF) is a secreted protein with potential neuroprotective qualities in retinal disease, including chronic ocular hypertension. Here we sought to determine whether moderate, short-term elevations in IOP alter PEDF signaling and whether pressure-induced PEDF signaling directly impacts RGC apoptosis. Methods In retina from naïve mice and mice with unilateral, microbead-induced glaucoma, we examined expression and cell type-specific localization of PEDF and its receptor (PEDF-R), using quantitative PCR and immunohistochemistry. Using primary cultures of purified RGCs and Müller cells, we examined cell type-specific expression of PEDF in response to 48 hours of elevated hydrostatic pressure, using multiplex ELISA and immunocytochemistry. We also measured pressure-induced apoptosis of RGCs in the presence or absence of atglistatin, a potent and selective inhibitor of PEDF-R, and recombinant PEDF, using TUNEL assays. Results PEDF and PEDF-R are constitutively expressed in naïve retina, primarily in the ganglion cell and nerve fiber layers. Elevated IOP increases PEDF and PEDF-R expression, particularly associated with RGCs and Müller cells. Elevated pressure in vitro increased PEDF secretion by 6-fold in RGCs and trended towards an increase in expression by Müller cells, as compared to ambient pressure. This was accompanied by changes in the subcellular localization of PEDF-R in both cell types. Inhibition of PEDF signaling with atglistatin increased pressure-induced apoptosis in RGCs and treatment with recombinant PEDF inhibited pressure-induced apoptosis, both in a dose-dependent manner. Conclusion Our findings suggest that moderate, short-term elevations in IOP promote PEDF signaling via up-regulation of both PEDF and PEDF-R. Based on in vivo and in vitro studies, this PEDF signaling likely arises from both Müller cells and RGCs, and has the potential to directly inhibit RGC apoptosis.

Loss of Dopamine D2 Receptors Increases Parvalbumin-Positive Interneurons in the Anterior Cingulate Cortex

Disruption to dopamine homeostasis during brain development has been implicated in a variety of neuropsychiatric disorders, including depression and schizophrenia. Inappropriate expression or activity of GABAergic interneurons are common features of many of these disorders. We discovered a persistent upregulation of GAD67+ and parvalbumin+ neurons within the anterior cingulate cortex of dopamine D2 receptor knockout mice, while other GABAergic interneuron markers were unaffected. Interneuron distribution and number were not altered in the striatum or in the dopamine-poor somatosensory cortex. The changes were already present by postnatal day 14, indicating a developmental etiology. D2eGFP BAC transgenic mice demonstrated the presence of D2 receptor expression within a subset of parvalbumin-expressing cortical interneurons, suggesting the possibility of a direct cellular mechanism through which D2 receptor stimulation regulates interneuron differentiation or survival. D2 receptor knockout mice also exhibited decreased depressive-like behavior compared with wild-type controls in the tail suspension test. These data indicate that dopamine signaling modulates interneuron number and emotional behavior and that developmental D2 receptor loss or blockade could reveal a potential mechanism for the prodromal basis of neuropsychiatric disorders.

Constitutive and Stress-induced Expression of CCL5 Machinery in Rodent Retina

Signaling by inflammatory cytokines and chemokines is associated with neurodegeneration in disease and injury. Here we examined expression of the β-chemokine CCL5 and its receptors in the mouse retina and evaluated its relevance in glaucoma, a common optic neuropathy associated with sensitivity to intraocular pressure (IOP). Using quantitative PCR, fluorescent in situ hybridization, immunohistochemistry and quantitative image analysis, we found CCL5 mRNA and protein was constitutively expressed in the inner retina and synaptic layers. CCL5 appeared to associate with Müller cells and RGCs as well as synaptic connections between horizontal cells and bipolar cells in the OPL and amacrine cells, bipolar cells and RGCs in the IPL. Although all three high-affinity receptors (CCR5, CCR3, CCR1) for CCL5 were expressed constitutively, CCR5 expression was significantly higher than CCR3, which was also markedly greater than CCR1. Localization patterns for constitutive CCR5, CCR3 and CCR1 expression differed, particularly with respect to expression in inner retinal neurons. Stress-related expression of CCL5 was primarily altered in aged DBA/2 mice with elevated IOP. In contrast, changes in expression and localization of both CCR3 and CCR5 were evident not only in aged DBA/2 mice, but also in age-matched control mice and young DBA/2 mice. These groups do not exhibit elevated IOP, but possess either the aging stress (control mice) or the genetic predisposition to glaucoma (DBA/2 mice). Together, these data indicate that CCL5 and its high-affinity receptors are constitutively expressed in murine retina and differentially induced by stressors associated with glaucomatous optic neuropathy. Localization patterns further indicate that CCL5 signaling may be relevant for modulation of synapses in both health and disease, particularly in the inner plexiform layer.