Karen X. Duong-Polk

Intraocular pressure elevation induces mitochondrial fission and triggers OPA1 release in glaucomatous optic nerve.

PURPOSE To determine whether elevation of intraocular pressure (IOP) triggers mitochondrial fission and ultrastructural changes and alters optic atrophy type 1 (OPA1) expression and distribution in the optic nerve (ON) of glaucomatous DBA/2J mice. METHODS IOP in the eyes of DBA/2J mice was measured, and mitochondrial structural changes were assessed by conventional electron microscopy (EM) and EM tomography. Cytochrome c oxidase IV subunit 1 (COX), OPA1, and Dnm1, a rat homologue of dynamin-related protein-1, mRNA were measured by quantitative (q)PCR. COX and OPA1 protein distribution was assessed by immunocytochemistry and Western blot. RESULTS Excavation of the optic nerve head (ONH), axon loss, and COX reduction were evident in 10-month-old glaucomatous ONHs of eyes with >20 mm Hg IOP elevation. EM analysis showed mitochondrial fission, matrix swelling, substantially reduced cristae volume, and abnormal cristae depletion in 10-month-old glaucomatous ONH axons. The mean length of mitochondrial cross section in these axons decreased from 858.2 +/- 515.3 nm in 3-month-old mice to 583.3 +/- 298.6 nm in 10-month-old glaucomatous mice (P < 0.001). Moderate reductions of COX mRNA were observed in the 10-month-old DBA/2J mices ONHs. Larger reductions of OPA1 immunoreactivity and gene expression were coupled with larger increases of Dnm1 gene expression in 10-month-old glaucomatous ONH. Subcellular fractionation analysis indicates increased release of both OPA1 and cytochrome c from mitochondria in 10-month-old glaucomatous ONs. CONCLUSIONS IOP elevation may directly damage mitochondria in the ONH axons by promoting reduction of COX, mitochondrial fission and cristae depletion, alterations of OPA1 and Dnm1 expression, and induction of OPA1 release. Thus, interventions to preserve mitochondria may be useful for protecting against ON degeneration in glaucoma.

Memantine blocks mitochondrial OPA1 and cytochrome c release, and subsequent apoptotic cell death in glaucomatous retina

PURPOSE To determine whether intraocular pressure (IOP) elevation alters OPA1 expression and triggers OPA1 release, as well as whether the uncompetitive N-methyl-d-aspartate (NMDA) glutamate receptor antagonist memantine blocks OPA1 release and subsequent apoptotic cell death in glaucomatous DBA/2J mouse retina. METHODS Preglaucomatous DBA/2J mice received memantine (5 mg/kg, intraperitoneal injection, twice daily for 3 months) and IOP in the eyes was measured monthly. RGC loss was counted after FluoroGold labeling. OPA1, Dnm1, Bcl-2, and Bax mRNA were measured by qPCR. OPA1 protein was assessed by immunohistochemistry and Western blot. Apoptotic cell death was assessed by TUNEL staining. RESULTS Memantine treatment significantly increased RGC survival in glaucomatous DBA/2J mice and increased the 75-kDa OPA1 isoform, but did not alter the 80- and 90-kDa isoforms. The isoforms of OPA1 were significantly increased in the cytosol of the vehicle-treated glaucomatous retinas but were significantly decreased in memantine-treated glaucomatous retinas. OPA1 immunoreactivity was decreased in the photoreceptors of both vehicle- and memantine-treated glaucomatous retinas, but was increased in the outer plexiform layer of only the memantine-treated glaucomatous retinas. Memantine blocked apoptotic cell death in the GCL, increased Bcl-2 gene expression, and decreased Bax gene expression. CONCLUSIONS OPA1 release from mitochondria in glaucomatous mouse retina is inhibited by blockade of glutamate receptor activation. Because this OPA1 effect was accompanied by increased Bcl-2 expression, decreased Bax expression, and apoptosis blockade, glutamate receptor activation in the glaucomatous retina may involve a distinct mitochondria-mediated cell death pathway.

Genetic and pharmacological inhibition of JNK ameliorates hypoxia-induced retinopathy through interference with VEGF expression

Many ocular pathologies, including retinopathy of prematurity (ROP), diabetic retinopathy, and age-related macular degeneration, result in vision loss because of aberrant neoangiogenesis. A common feature of these conditions is the presence of hypoxic areas and overexpression of the proangiogenic vascular endothelial growth factor (VEGF). The prevailing current treatment, laser ablation of the retina, is destructive and only partially effective. Preventive and less destructive therapies are much more desirable. Here, we show that mice lacking c-Jun N-terminal kinase 1 (JNK1) exhibit reduced pathological angiogenesis and lower levels of retinal VEGF production in a murine model of ROP. We found that hypoxia induces JNK activation and regulates VEGF expression by enhancing the binding of phospho-c-Jun to the VEGF promoter. Intravitreal injection of a specific JNK inhibitor decreases retinal VEGF expression and reduces pathological retinal neovascularization without obvious side effects. These results strongly suggest that JNK1 plays a key role in retinal neoangiogenesis and that it represents a new pharmacological target for treatment of diseases where excessive neoangiogenesis is the underlying pathology.

Inhibition of Histone Deacetylases 1 and 3 Protects Injured Retinal Ganglion Cells

PURPOSE Thy-1 is a marker of retinal ganglion cell (RGC) differentiation. Optic nerve injury triggers reduction of Thy-1 promoter activation followed by retinal ganglion cell (RGC) death. This study determined whether MS-275, an inhibitor of the histone deacetylases 1 and 3, can inhibit these changes. METHODS Mice expressing cyan fluorescent protein (CFP) under control of the Thy-1 promoter received MS-275 (subcutaneous) or vehicle three times per week starting 1 week before optic nerve crush and continuing for 6 weeks. The same retinal area was imaged using the blue-light confocal scanning laser ophthalmoscope before and after optic nerve crush every week, and fluorescent spots were counted manually. The eyes were then processed for histopathologic analysis. RESULTS The mean proportions of fluorescent retinal neurons remaining in the vehicle group following optic nerve crush were 36 ± 8, 18 ± 6, 13 ± 10, 12 ± 4, 13 ± 5, and 13 ± 5% at weeks 1 through 6, respectively (n = 6). In contrast, the mean proportions of fluorescent retinal neurons remaining in the group treated with MS-275 were 59 ± 19, 39 ± 11, 34 ± 12, 33 ± 15, 32 ± 13, and 27 ± 15% at weeks 1 through 6, respectively (n = 7, P < 0.05 at weeks 1 through 5). Rate analysis showed that MS-275 slowed the rate of loss during the first 2 weeks by 23% (P < 0.05) and subsequently was similar. Histopathologic analysis revealed 27 ± 13% greater ganglion cell layer (GCL) neurons in the eyes from mice that received MS-275 treatment (P < 0.02). CONCLUSIONS These results indicate that treatment with MS-275 protects against the loss of RGC differentiation and promotes RGC survival following optic nerve injury.

Protection of injured retinal ganglion cell dendrites and unfolded protein response resolution after long-term dietary resveratrol.

Long-term dietary supplementation with resveratrol protects against cardiovascular disease, osteoporesis, and metabolic decline. This study determined how long-term dietary resveratrol treatment protects against retinal ganglion cell (RGC) dendrite loss after optic nerve injury and alters the resolution of the unfolded protein response. Associated changes in markers of endoplasmic reticulum stress in RGCs also were investigated. Young-adult Thy1-yellow fluorescent protein (YFP) and C57BL/6 mice received either control diet or diet containing resveratrol for approximately 1 year. Both groups then received optic nerve crush (ONC). Fluorescent RGC dendrites in the Thy1-YFP mice were imaged weekly for 4 weeks after ONC. There was progressive loss of dendrite length in all RGC types within the mice that received control diet. Resveratrol delayed loss of dendrite complexity and complete dendrite loss for most RGC types. However, there were variations in the rate of retraction among different RGC types. Three weeks after ONC, cytoplasmic binding immunoglobulin protein (BiP) suppression observed in control diet ganglion cell layer neurons was reversed in mice that received resveratrol, nuclear C/EBP homologous protein (CHOP) was near baseline in control diet eyes but was moderately increased by resveratrol; and increased nuclear X-box-binding protein-1 (XBP-1) observed in control diet eyes was reduced in eyes that received resveratrol to the same level as in control diet uncrushed eyes. These results indicate that protection of dendrites by resveratrol after ONC differs among RGC types and suggest that alterations in long-term expression of binding immunoglobulin protein, CHOP, and XBP-1 may contribute to the resveratrol-mediated protection of RGC dendrites after ONC.

Longitudinal live imaging of retinal α-synuclein::GFP deposits in a transgenic mouse model of Parkinson’s Disease/Dementia with Lewy Bodies

Abnormal α-synuclein (α-syn) accumulation in the CNS may underlie neuronal cell and synaptic dysfunction leading to motor and cognitive deficits in synucleinopathies including Parkinson’s disease (PD) and Dementia with Lewy Bodies (DLB). Multiple groups demonstrated α-syn accumulation in CNS accessory structures, including the eyes and olfactory terminals, as well as in peripheral organs of Parkinsonian patients. Retinal imaging studies of mice overexpressing fused α-syn::GFP were conducted to evaluate the presence and progression of retinal pathology in a PD/DLB transgenic mouse model. Bright-field image retinal maps and fluorescent images were acquired at 1-month intervals for 3 months. Retinal imaging revealed the accumulation of GFP-tagged α-syn in retinal ganglion cell layer and in the edges of arterial blood vessels in the transgenic mice. Double labeling studies confirmed that the α-syn::GFP-positive cells were retinal ganglion cells containing α-syn. Accumulation of α-syn persisted in the same cells and increased with age. Accumulation of α-syn::GFP was reduced by immunization with single chain antibodies against α-syn. In conclusion, longitudinal live imaging of the retina in the PDGF-α-syn::GFP mice might represent a useful, non-invasive tool to monitor the fate of α-syn accumulation in the CNS and to evaluate the therapeutic effects of compounds targeting α-syn.

Protection by an Oral Disubstituted Hydroxylamine Derivative against Loss of Retinal Ganglion Cell Differentiation following Optic Nerve Crush

Thy-1 is a cell surface protein that is expressed during the differentiation of retinal ganglion cells (RGCs). Optic nerve injury induces progressive loss in the number of RGCs expressing Thy-1. The rate of this loss is fastest during the first week after optic nerve injury and slower in subsequent weeks. This study was undertaken to determine whether oral treatment with a water-soluble N-hydroxy-2,2,6,6-tetramethylpiperidine derivative (OT-440) protects against loss of Thy-1 promoter activation following optic nerve crush and whether this effect targets the earlier quick phase or the later slow phase. The retina of mice expressing cyan fluorescent protein under control of the Thy-1 promoter (Thy1-CFP mice) was imaged using a blue-light confocal scanning laser ophthalmoscope (bCSLO). These mice then received oral OT-440 prepared in cream cheese or dissolved in water, or plain vehicle, for two weeks and were imaged again prior to unilateral optic nerve crush. Treatments and weekly imaging continued for four more weeks. Fluorescent neurons were counted in the same defined retinal areas imaged at each time point in a masked fashion. When the counts at each time point were directly compared, the numbers of fluorescent cells at each time point were greater in the animals that received OT-440 in cream cheese by 8%, 27%, 52% and 60% than in corresponding control animals at 1, 2, 3 and 4 weeks after optic nerve crush. Similar results were obtained when the vehicle was water. Rate analysis indicated the protective effect of OT-440 was greatest during the first two weeks and was maintained in the second two weeks after crush for both the cream cheese vehicle study and water vehicle study. Because most of the fluorescent cells detected by bCSLO are RGCs, these findings suggest that oral OT-440 can either protect against or delay early degenerative responses occurring in RGCs following optic nerve injury.

Differential protection of injured retinal ganglion cell dendrites by brimonidine

PURPOSE To determine whether brimonidine protects against the retraction and loss of retinal ganglion cell (RGC) dendrites after optic nerve crush (ONC). METHODS Fluorescent RGCs of mice expressing yellow fluorescent protein (YFP) under the control of the Thy-1 promoter (Thy1-YFP mice) were imaged in vivo and assigned to one of six groups according to dendrite structure. The mice then received brimonidine every other day starting 2 days before, or 2 or 6 days after, unilateral ONC. Control animals received vehicle every other day starting 2 days before ONC. Control animals received vehicle every other day starting 2 days before ONC. Total dendrite length, dendrite branching complexity, and the time until complete loss of dendrites were assessed weekly for 4 weeks. RESULTS Overall, brimonidine treatment significantly slowed the complete loss of RGC dendrites and significantly slowed the reduction of total dendrite length and branching complexity. Separate analysis of each RGC group showed brimonidine significantly delayed the time until complete loss of dendrites in four of the RGC groups. These delays generally were similar when treatment started either 2 days before or 2 days after ONC, but were smaller or absent when treatment started 6 days after ONC Protection against loss of total dendrite length and loss of branching complexity was observed in three of the RGC groups. In two of these RGC groups, protective effects persisted until the end of the study. CONCLUSIONS Brimonidine protects many RGC types against dendrite retraction, loss of branching complexity, and complete loss of dendrites following ONC. However, the pattern and magnitude of this protection differs substantially among different RGC types. These results indicate that requirements for RGC-protective therapies following optic nerve injury may differ among RGC types.

Ocular integrity following manganese labeling of the visual system for MRI.

Injection of manganese into the eye will enhance the contrast of visual system neuronal pathways imaged by MRI (MEMRI). The present study was undertaken to determine the effect of a range of MnCl2 doses upon the integrity of various ocular structures. Anesthetized mice received ocular anterior chamber injections of 50-500 nmol of MnCl2. One week later, the eyes were fixed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Additional animals received 50 nmol of MnCl2 injected into the anterior chamber and were later imaged using T1-weighted 7T MRI. Following 500 and 300nmol MnCl2, the corneal stroma and endothelium were degenerated, the anterior chamber contained a dense fibrin matrix with extensive inflammatory cell infiltration, a plaque often formed on the anterior lens, and significant retinal degeneration was observed. Following 100nmol MnCl2, retinal preservation of ocular structures was significantly better than at higher doses. In addition, there was no difference from vehicle control retina in cell counts within the ganglion cell layer, or in the width of the inner nuclear layer or outer nuclear layer. Also, there was no difference in the thickness of the inner plexiform layer. However, there was thinning of the peripheral outer plexiform layer, as well as in the outer segment layer. Visual system elements labeled in MRI of mice that received 100nmol MnCl2 included the retina, optic nerve, lateral geniculate nucleus, and superior colliculus. The preservation of ganglion cell layer cell counts and inner plexiform layer thickness following 100nmol MnCl2 suggests there was negligible injury to RGCs following this dose. These results support using 100nmol MnCl2 in mouse eyes for in vivo assessment of the integrity of RGC projections to target neurons in the brain.

Brimonidine protects against loss of Thy-1 promoter activation following optic nerve crush

BackgroundThe loss of RGCs expressing Thy-1 after optic nerve injury has an initial phase of rapid decline followed by a longer phase with slower reduction rate. This study used longitudinal retinal imaging of mice expressing cyan fluorescent protein under control of the Thy-1 promoter (Thy1-CFP mice) to determine how the α2-adrenergic agonist brimonidine influences loss of Thy1 promoter activation.MethodsBaseline images of the fluorescent retinal neurons in 30 Thy1-CFP mice were obtained using a modified confocal scanning laser ophthalmoscope. Next, brimonidine (100 ug/kg, IP) was administered either one time immediately after optic nerve crush, or immediately after optic nerve crush and then every 2 days for four weeks. A control group received a single saline injection immediately after optic nerve crush. All animals were imaged weekly for four weeks after optic nerve crush. Loss of fluorescent retinal neurons within specific retinal areas was determined by counting.ResultsAt one week after optic nerve crush, the proportion of fluorescent retinal neurons retaining fluorescence was 44±7% of baseline in control mice, 51±6% after one brimonidine treatment, and 55±6% after brimonidine treatment every other day (P<0.05 for both brimonidine treatment groups compared to the control group). Subsequently, the number of fluorescent retinal neurons in the group that received one treatment differed insignificantly from the control group. In contrast, the number of fluorescent retinal neurons in the group that received repeated brimonidine treatments was greater than the control group by 28% at two weeks after crush and by 32% at three weeks after crush (P<0.05 at both time points). Rate analysis showed that brimonidine slowed the initial rate of fluorescent cell decline in the animals that received multiple treatments (P<0.05). Differences in the rate of loss among the treatment groups were insignificant after the second week.ConclusionRepeated brimonidine treatments protect against loss of fluorescence within fluorescent retinal neurons of Thy1-CFP mice after optic nerve crush. As most of fluorescent retinal neurons in this system are RGCs, these findings indicate that repeated brimonidine treatments may protect RGC health following optic nerve crush.