Systems approach in studies of glaucoma pathways
Neuronal loss in ocular pathologies is facilitated by a combination of genetic factors environmental stressors and altered crosstalk between neurons, glia and inflammatory cells. Such alterations result in the homeostatic imbalance, facilitate neurotoxic environment, inflammation and inhibit regeneration of injured neurons, thus contributing to a disease progression.
Astrocytes are the major glial type that control homeostasis, detoxify and modulate microenvironment in the optic nerve and retina in response to external and internal stimuli. Chronic activation of these cells in retina and optic nerve has been associated with axonal damage and loss of retinal ganglion cells in glaucoma and optic neuritis. To study the role of astrocytes in these ocular pathologies, we utilized systems biology approach and analyzed changes in astrocytic transcriptome and proteome triggered by these pathologies. These complex data has been analyzed for differentially activated pathways and signaling networks potentially implicated in pathophysiology of these diseases. In order to understand the molecular mechanisms implicated in both pathologies, we chose the strategy to reconstruct cellular and epigenetic networks affected by the disease process. We detected several glial cellular pathways potentially relevant to retinal ganglion cell loss in humans and are validating them in rodent models for glaucoma and multiple sclerosis.
Pathways implicated in glaucoma
Primary open angle glaucoma (POAG) is an ocular neuropathy, a complex multifactorial disease where irreversible damage to the axons in the optic nerve causes blindness via progressive loss of ganglion cell in the retina. Retinal ganglion cell (RGC) survival depends at large on the support from glial cells, particularly astrocytes. In pathological conditions, though, astrocytes reactivate, become dysfunctional and exert toxicity to RGCs. We seek to elucidate cellular changes in RGCs as well as in glial cells that contribute to development of the glaucomatous degeneration.
We utilize informatics to analyze global alterations to cellular pathways detected by gene expression and proteomic profiling of both cell types during the disease progression. Our network analysis of human optic nerve head astrocytes (In collaboration with Dr. Hernandez lab, Northwestern Univ.) we determined that four major network hubs (highlighted with blue on the figure) regulate the disease-mediated network with p-value< 0.002: transcription factors SP-1, AP1/c-Fos/JunD, NF-kB and vitaminD receptor (VDR). The parallel activation of these factors is a unique signature profile for reactive glaucomatous astrocytes and initiates a transcriptional program that likely contributes to ONHA neurotoxicity through activation of the JNK/MAPK-10 pathway, pro-inflammatory pathways including complement, IL-6, clusterin, and downregulation of pro-survival signaling via integrins, PDGF and STAT1. We concluded that concurrent activation of NFkB and AP-1 likely facilitates re-activation, while further maintenance of this phenotype is accomplished via activation of genes such as AKR1C1.
Given astroglial NF-kB alone contributes to activation of multiple stress- and inflammation-related processes (see figure below), we further scrutinized the role of this factor in a transgenic mouse model to test a hypothesis of it’s pivotal role in mediating astrocytic neurotoxicity in glaucoma. In collaboration with Dr. Bethea’s laboratory at U. Miami we demonstrated significant neuroprotection in ocular hypertensive mice with targeted suppression of NF-kB in astrocytes. This finding is a direct confirmation of astroglial toxicity in glaucoma, and a demonstration of feasibility of our approach to analysis of complex diseases.
Astrocytic pathways implicated in optic neuritis
Optic Neuritis (ON) is one of the most frequently presenting symptoms of multiple sclerosis (MS), a demyelinating disease of undetermined etiology. Resident microglia and astrocytes of the optic nerve were shown to contribute to the disease. Since pathological inflammatory processes are regulated by the NF-kB family of transcription factors, we studied the role of astroglial NF-kB in the pathophysiology of EAE using transgenic mouse line (collaboration with Dr. Bethea), in which NF-kB is suppressed in astrocytes by overexpressing a dominant negative form of the NF-kB super-repressor (IkBa-dn). We have shown that blocking astroglial-NF-kB (aNF-kB) significantly reduces disease severity and improves functional recovery following induction of EAE.
Transgenic mice with EAE had reduced inflammation, less inhibited regeneration and re-myelination and negligible neuronal loss and disease progression as compared to WT littermates. Neuroprotection in diseased transgenic mice correlates with synthesis of immunoregulatory molecules, increased CD45+ cell infiltration in the CNS. These results led us to propose the following hypotheses: 1) that inactivation of NF-kB in astrocytes modifies the CNS micro-environment to become neuroprotective, possibly through the recruitment of leukocytes that are immunoregulatory and “anti-inflammatory”, and 2) inhibiting aNF-kB creates a neuroprotective environment that suppresses apoptosis and is permissive for regenerative sprouting, and possibly remyelination. We are currently testing these hypotheses using both in vivo and in vitro approaches.