Astrocyte Reactivity: A Biomarker for Retinal Ganglion Cell Health in Retinal Neurodegeneration.

Retinal ganglion cell (RGC) loss in glaucoma is sectorial in nature and preceded by deficits in axonal transport. Neuroinflammation plays an important role in the pathophysiology of glaucoma in the retina, optic nerve and visual centers of the brain, where it similarly appears to be regulated spatially. In a murine model, we examined the spatial characteristics of astrocyte reactivity (migration/proliferation, hypertrophy and GFAP expression) in healthy retina, retina with two glaucoma-related risk factors (aging and genetic predisposition) and glaucomatous retina and established relationships between these reactivity indices and the spatial organization of astrocytes as well as RGC health. Astrocyte reactivity was quantified by morphological techniques and RGC health was determined by uptake and transport of the neural tracer cholera toxin beta subunit (CTB). We found that: (1) astrocyte reactivity occurs in microdomains throughout glaucomatous retina as well as retina with risk factors for glaucoma, (2) these astrocyte microdomains are primarily differentiated by the degree of retinal area covered by the astrocytes within them and (3) percent retinal area covered by astrocytes is highly predictive of RGC health. Our findings suggest that microdomains of astrocyte reactivity are biomarkers for functional decline of RGCs. Based on current and emerging imaging technologies, diagnostic assessment of astrocytes in the nerve fiber layer could succeed in translating axonal transport deficits to a feasible clinical application.

Spatial regulation of interleukin-6 signaling in response to neurodegenerative stressors in the retina.

Neuroinflammation, defined as the induction of immune-related processes within the central nervous system, is recognized as a component of many neurodegenerative disorders, including glaucomatous degeneration of retinal ganglion cells (RGCs). Previous work in vitro identified IL-6 as a potential neuroprotective factor for RGCs, particularly those challenged by glaucoma-related stressors. Here we examined the temporal and spatial characteristics of IL-6 signaling in response to two stressors related to RGC neurodegeneration: age and elevated intraocular pressure (IOP). Using ELISA, immunoblotting, immunolabeling and quantitative microscopy, we measured and compared whole retina and RGC-related expression of IL-6 and IL-6Rα in normal retina (young C57), retina susceptible to glaucomatous neurodegeneration (young DBA/2), aging retina (aged C57) and aging retina challenged by elevated IOP (aged DBA/2). We found that: 1) neurodegenerative stressors induce alterations in whole retina expression of IL-6 and IL-6Rα, 2) these whole retina changes do not reflect the immediate milieu of RGCs, where IL-6 and IL-6Rα expression is spatially variable and 3) the extent and magnitude of this spatial variability is stressor-dependent. Our data provide the first evidence that neurodegenerative stressors produce microenvironments of IL-6 signaling in retina and that the nature and magnitude of spatial regulation is dependent on the identity of the stressor.

Interferon-gamma, macrophages, and virus spread after HSV-1 injection.

PURPOSE: After uniocular anterior chamber (AC) injection of HSV-1, the anterior segment of BALB/c mice becomes inflamed and infected; however, virus does not spread from the anterior segment to cause retinitis in the injected eye. The purpose of these studies was to determine whether interferon (IFN-)-γ and Mac-1(+) cells play a role in preventing direct anterior-to-posterior spread of HSV-1 in the injected eye. METHODS: One AC of adult female BALB/c mice was injected with HSV-1 (KOS). The location of IFN-α, IFN-ß, and IFN-γ in the injected eye was determined by immunofluorescence, and mRNA expression was quantified by qPCR. Injected eyes of IFN-γ knockout or clodronate-treated macrophage-depleted mice were examined to determine whether the absence of IFN-γ or Mac-1(+) macrophages affected the sites or timing of virus spread. RESULTS: IFN-α, IFN-ß, and IFN-γ were observed in the anterior segment of injected eyes through 72 hours and mRNA levels of IFN-ß and IFN-γ were increased in virus-infected eyes 48 to 120 hours after infection. However, the absence of IFN-γ or macrophages did not affect either the sites or the timing of HSV-1 infection in injected eyes. CONCLUSIONS: Protection of the retina of the injected eye does not depend on a single cell type or cytokine. In addition, in the eye, as in other sites of the body, there are redundancies in the innate response to virus infection.

Infiltrating cells and IFNgamma production in the injected eye after uniocular anterior chamber inoculation of HSV-1.

PURPOSE: After uniocular anterior chamber (AC) inoculation with HSV-1, the anterior segment of the injected eye becomes inflamed and infected; however, virus does not spread from the anterior segment and infect the retina of the injected eye. The purpose of this study was to identify early infiltrating cells and to determine whether infiltrating cells produce interferon (IFN)gamma. METHODS: Euthymic, female, BALB/c mice were injected in one AC with 3 x 10(4) PFU of HSV-1 (KOS) in a volume of 2 microL. Mice from each group were killed at 12, 24, 36, 48, and 72 hours post injection (pi), the eyes were enucleated, and frozen sections were stained with antibodies specific for IFNgamma, Mac-1 (CD11b), CD49b, F4/80, CD4, CD8, and CD11c. The same antibodies were also used to stain single-cell suspensions of ocular cells for flow cytometry. RESULTS: In the anterior segment of the injected eye, the ciliary body, and iris were virus infected and inflamed, and infiltrating cells increased throughout the period of observation. Mac-1(+), CD49b(+), and F4/80(+) cells colocalized with IFNgamma in the anterior segment as early as 12 hours pi, and the percentage of Mac-1(+) cells increased in the injected eye beginning at 24 hours pi and continued to 72 hours pi. CONCLUSIONS: Taken together, these results demonstrate that Mac-1(+) cells are important IFNgamma-producing cells in the injected eye before day 3 and suggest that the IFNgamma produced by these cells is involved in inhibition of anterior to posterior spread of virus in the injected eye.

Membership in genetic groups predicts Alzheimer disease.

The multiple polymorphisms contributing to Alzheimer disease (AD) have been difficult to identify. Three essentially sufficient risk sets were found using a fuzzy latent classification statistical model; that is, grade-of-membership analysis, and genotypes for APOE, APOCI, LDLr, cystatin C, and cathepsin D (180 cases, 120 controls). These were: (a) CST3:GA and CTSD:CT; (b) APOE44 and LDLr8:GG and LDLr13:TT; and (c) APOE34 and LDLr13:TC. Consonance with one of the groups and high aggregate membership carried >800-fold elevated risk for AD. The absence of these combinations defined low risk. APOE3/- with heterozygous promoter and receptor genotypes predicted long life without dementia.

Apolipoprotein gene E4 allele promoter polymorphisms as risk factors for Alzheimer's disease.

Alzheimer's disease is a complex neurodegenerative disorder, characterized by progressive cognitive decline and distinct neuropathology. The apolipoprotein gene E4 allele (APOE 4) is a major risk factor for the disease. Promoter polymorphisms at -491 and -427 may also contribute to the risk. We examined the two polymorphisms in 178 Alzheimer's patients and 141 controls. The -491AA genotype was overrepresented among the patients (68 versus 54%, P=0.01). However, in patients who were APOE4 carriers, the -491AA genotype more than doubled the risk [odds ratio (OR)=2.5; 95% confidence interval (CI)=1.2-5.4], especially in combination with -427TT [odds ratio (OR)=3.3; 95% confidence interval (CI)=1.5-7.7]. Moreover, the -491A/-427T/APOE4/APOC1A haplotype was threefold higher for patients. These results contribute to the evidence that regulation of APOE4 expression modulates risk for Alzheimer's disease.