Aldosterone: a mediator of retinal ganglion cell death and the potential role in the pathogenesis in normal-tension glaucoma

Glaucoma is conventionally defined as a chronic optic neuropathy characterized by progressive loss of retinal ganglion cells (RGCs) and optic nerve fibers. Although glaucoma is often associated with elevated intraocular pressure (IOP), significant IOP reduction does not prevent progression of the disease in some glaucoma patients. Thus, exploring IOP-independent mechanisms of RGC loss is important. We describe chronic systemic administration of aldosterone and evaluate its effect on RGCs in rat. Aldosterone was administered via an osmotic minipump that was implanted subcutaneously into the mid-scapular region. Although systemic administration of aldosterone caused RGC loss associated with thinning of the retinal nerve fiber layer without elevated IOP, the other cell layers appeared to be unaffected. After chronic administration of aldosterone, RGC loss was observed at 2 weeks in the peripheral retina and at 4 weeks in the central retina. However, administration of mineralocorticoid receptor blocker prevented RGC loss. These results demonstrate aldosterone is a critical mediator of RGC loss that is independent of IOP. We believe this rat normal-tension glaucoma (NTG) animal model not only offers a powerful system for investigating the mechanism of neurodegeneration in NTG, but can also be used to develop therapies directed at IOP-independent mechanisms of RGC loss.     

Activation of autophagy induces retinal ganglion cell death in a chronic hypertensive glaucoma model

Autophagy is reported to have important roles in relation to regulated cell death pathways and neurodegeneration. This study used chronic hypertensive glaucoma rat model to investigate whether the autophagy pathway has a role in the apoptosis of retinal ganglion cells (RGCs) after chronic intraocular pressure (IOP) elevation. Under electron microscopy, autophagosomes were markedly accumulated in the dendrites and cytoplasm of RGCs after IOP elevation. Western blot analysis showed that LC3-II/LC3-I and beclin-1 were upregulated throughout the 8-weeks period after IOP elevation. The pattern of LC3 immunostaining showed autophagy activation in the cytoplasm of RGCs to increase and peak at 4 weeks after IOP elevation. Most of these LC3B-positive RGCs underwent apoptosis by terminal deoxynucleotidyltransferase-mediated biotinylated UTP nick end labeling, and inhibition of autophagy with 3-methyladenine decreased RGC apoptosis. The activated pattern shows that autophagy is initially activated in the dendrites of the RGCs, but, thereafter autophagy is mainly activated in the cytoplasm of RGCs. This may show that autophagy is differently regulated in different compartments of the neuron. This present study showed that autophgy is activated in RGCs and has a role in autophagic cell death after chronic IOP elevation.     

Functional assessment of glutamate clearance mechanisms in a chronic rat glaucoma model using retinal ganglion cell calcium imaging

Using optical imaging of retinal ganglion cell (RGC) calcium dynamics in living intact retinal wholemount preparations, we tested whether RGCs in an experimental rat glaucoma model were more sensitive to exogenously applied glutamate as a result of deficient glutamate clearance mechanisms. In contrast to post-natal rat RGCs in purified cultures, in which the calcium influx induced by 200 microm NMDA and 10 microm glutamate was approximately equivalent, application of up to 500 microm glutamate did not affect calcium levels in RGCs in retinal wholemounts, even though the RGCs responded to 200 microm NMDA. Glutamate (500 microm) did elicit a RGC calcium response in retinal wholemounts when glutamate transporters were inhibited pharmacologically with DL-threo-beta-benzyloxyaspartate, confirming the presence of glutamate clearance mechanisms in this intact retina preparation. The effect of glutamate was then assessed on retinas from rats with chronically elevated intraocular pressure in one eye, produced by the injection of hypertonic saline into an episcleral vein. Application of up to 500 microm glutamate had no effect on RGC calcium levels, while millimolar concentrations of glutamate induced a calcium signal in RGCs that was indistinguishable from that in fellow control retinas. Therefore, there was no evidence for a global defect in glutamate uptake in this rat model of experimental glaucoma. Imaging glutamatergic calcium dynamics of RGCs in retinal wholemounts represents a novel methodology to probe glutamate transporter function and dysfunction in an intact CNS tissue system.     

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 (βB1‐CTGF) to elucidate new possible mechanisms of the disease. Therefore, IOP was measured in βB1‐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 βB1‐CTGF mice (P < 0.001). At 15 weeks, electroretinogram measurements were performed and both the a‐ and b‐wave amplitudes were significantly decreased in βB1‐CTGF retinae (both P < 0.01). Significantly fewer Brn‐3a⁺ retinal ganglion cells (RGCs) were observed in the βB1‐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 βB1‐CTGF group (P = 0.002). Furthermore, a decrease in recoverin⁺ cells was observable in the βB1‐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 βB1‐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 βB1‐CTGF mouse could serve as an excellent model for better understanding the pathomechanisms in POAG.     

Retinal ganglion cell neurodegeneration in mitochondrial inherited disorders

Since the early days of mitochondrial medicine, it has been clear that optic atrophy is a very common and sometimes the singular pathological feature in mitochondrial disorders. The first point mutation of mitochondrial DNA (mtDNA) associated with the maternally inherited blinding disorder, Leber's hereditary optic neuropathy (LHON), was recognized in 1988. In 2000, the other blinding disorder, dominant optic atrophy (DOA) Kjer type, was found associated with mutations in the nuclear gene OPA1 that encodes a mitochondrial protein. Besides these two non-syndromic optic neuropathies, optic atrophy is a prominent feature in many other neurodegenerative diseases that are now recognized as due to primary mitochondrial dysfunction.We will consider mtDNA based syndromes such as LHON/dystonia/Mitochondrial Encephalomyopahty Lactic Acidosis Stroke-like (MELAS)/Leigh overlapping syndrome, or nuclear based diseases such as Friedreich ataxia (mutations in FXN gene), deafness-dystonia-optic atrophy (Mohr-Tranebjerg) syndrome (mutations in TIMM8A), complicated hereditary spastic paraplegia (mutations in SPG7), DOA “plus” syndromes (mutations in OPA1), Charcot-Marie-Tooth type 2A (CMT2A) with optic atrophy or hereditary motor and sensory neuropathy type VI (HMSN VI) (mutations in MFN2), and Costeff syndrome and DOA with cataract (mutations in OPA3). Thus, genetic errors in both nuclear and mitochondrial genomes often lead to retinal ganglion cell death, a specific target for mitochondrial mediated neurodegeneration. Many mechanisms have been studied and proposed as the bases for the pathogenesis of mitochondrial optic neuropathies including bioenergetic failure, oxidative stress, glutamate toxicity, abnormal mitochondrial dynamics and axonal transport, and susceptibility to apoptosis.     

Retinal ganglion cell differentiation in cultured mouse retinal explants

The availability of genetically engineered mice harboring specific mutations in genes affecting one or more retinal cell types affords new opportunities for investigating the genetic regulatory mechanisms of vertebrate retina formation. When identifying critical regulatory genes involved in retina development it is often advantageous to complement in vivo analysis with in vitro characterization. In particular, by combining classical techniques of retinal explant culturing with gene transfer procedures relying on herpes simple virus (HSV) amplicon vectors, gain-of-function analysis with genes of interest can be performed quickly and efficiently. Here, details are provided for isolating and culturing explants containing retinal progenitor cells and for infecting the explants with HSV expression vectors that perturb or rescue retinal ganglion cells, the first cell type to differentiate in the retina. In addition, the availability of sensitive techniques to monitor gene expression, including detection of reporter gene expression using antibodies and detection of endogenous marker gene expression using quantitative RT-PCR, provides an effective means for comparing wild-type and mutant retinas from genetically engineered mice.     

Up‐regulation of crystallins is involved in the neuroprotective effect of wolfberry on survival of retinal ganglion cells in rat ocular hypertension model

Wolfberry (fruit of Lycium barbarum Linn) has been known for balancing ‘Yin’ and ‘Yang’ in the body, nourishing the liver and kidney, improving visual acuity for more than 2,500 years in oriental countries. The active components in wolfberry include L. barbarum polysaccharide (LBP), zeaxanthine, betaine, cerebroside and trace amounts of zinc, iron, and copper. Each of them confers distinct beneficial effects and together they help to explain widespread use of wolfberry in the eastern world. Earlier study reported the neuroprotective effects of LBP on retinal ganglion cell (RGC) in an experimental model of glaucoma and the underlying in vivo cellular mechanisms of LBP neuroprotection deserve further exploration. In this study, we adopted proteomics, functional genomics, to evaluate pharmacological effects of LBP on the neuronal survival pathways. Among the significantly changed proteins induced by LBP feeding on ocular hypertension (OH) retinas, only proteins in crystallin family were focused in this study. The proteomic results were further confirmed using the Western blotting of the retinas and immunohistochemical staining of the retinal sections. We demonstrated that neuroprotective effect of—wolfberry extract—LBP on the survival of RGCs may be mediated via direct up‐regulation of neuronal survival signal βB2‐crystallin. J. Cell. Biochem. 110: 311–320, 2010. © 2010 Wiley‐Liss, Inc.     

Osteopontin drives retinal ganglion cell resiliency in glaucomatous optic neuropathy

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Dock3 attenuates neural cell death due to NMDA neurotoxicity and oxidative stress in a mouse model of normal tension glaucoma

Dedicator of cytokinesis 3 (Dock3), a new member of the guanine nucleotide exchange factors for the small GTPase Rac1, promotes axon regeneration following optic nerve injury. In the present study, we found that Dock3 directly binds to the intracellular C-terminus domain of NR2B, an N-methyl-𝒟-aspartate (NMDA) receptor subunit. In transgenic mice overexpressing Dock3 (Dock3 Tg), NR2B expression in the retina was significantly decreased and NMDA-induced retinal degeneration was ameliorated. In addition, overexpression of Dock3 protected retinal ganglion cells (RGCs) from oxidative stress. We previously reported that glutamate/aspartate transporter (GLAST) is a major glutamate transporter in the retina, and RGC degeneration due to glutamate neurotoxicity and oxidative stress is observed in GLAST-deficient (KO) mice. In GLAST KO mice, the NR2B phosphorylation rate in the retina was significantly higher compared with Dock3 Tg:GLAST KO mice. Consistently, glaucomatous retinal degeneration was significantly improved in GLAST KO:Dock3 Tg mice compared with GLAST KO mice. These results suggest that Dock3 overexpression prevents glaucomatous retinal degeneration by suppressing both NR2B-mediated glutamate neurotoxicity and oxidative stress, and identifies Dock3 signaling as a potential therapeutic target for both neuroprotection and axonal regeneration.     

Pou4f2‐GFP knock‐in mouse line: A model for studying retinal ganglion cell development

Pou4f2 acts as a key node in the comprehensive and step‐wise gene regulatory network (GRN) and regulates the development of retinal ganglion cells (RGCs). Accordingly, deletion of Pou4f2 results in RGC axon defects and apoptosis. To investigate the GRN involved in RGC regeneration, we generated a mouse line with a POU4F2‐green fluorescent protein (GFP) fusion protein expressed in RGCs. Co‐localization of POU4F2 and GFP in the retina and brain of Pou4f2‐GFP/+ heterozygote mice was confirmed using immunofluorescence analysis. Compared with those in wild‐type mice, the expression patterns of POU4F2 and POU4F1 and the co‐expression patterns of ISL1 and POU4F2 were unaffected in Pou4f2‐GFP/GFP homozygote mice. Moreover, the quantification of RGCs showed no significant difference between Pou4f2‐GFP/GFP homozygote and wild‐type mice. These results demonstrated that the development of RGCs in Pou4f2‐GFP/GFP homozygote mice was the same as in wild‐type mice. Thus, the present Pou4f2‐GFP knock‐in mouse line is a useful tool for further studies on the differentiation and regeneration of RGCs.     

Exercise reverses age‐related vulnerability of the retina to injury by preventing complement‐mediated synapse elimination via a BDNF‐dependent pathway

Retinal ganglion cells (RGCs) become increasingly vulnerable to injury with advancing age. We recently showed that this vulnerability can be strongly modified in mice by exercise. However, the characteristics and underlying mechanisms of retinal protection with exercise remain unknown. Hence, the aim of this study was to investigate cellular changes associated with exercise‐induced protection of aging retinal cells and the role of local and peripheral trophic signalling in mediating these effects. We focussed on two molecules that are thought to play key roles in mediating beneficial effects of exercise: brain‐derived neurotrophic factor (BDNF) and AMP‐activated protein kinase (AMPK). In middle‐aged (12 months old) C57BL/6J mice, we found that exercise protected RGCs against dysfunction and cell loss after an acute injury induced by elevation of intra‐ocular pressure. This was associated with preservation of inner retinal synapses and reduced synaptic complement deposition. Retinal expression of BDNF was not upregulated in response to exercise alone. Rather, exercise maintained BDNF levels in the retina, which were decreased postinjury in nonexercised animals. Confirming a critical role for BDNF, we found that blocking BDNF signalling during exercise by pharmacological means or genetic knock‐down suppressed the functional protection of RGCs afforded by exercise. Protection of RGCs with exercise was independent of activation of AMPK in either retina or skeletal muscle. Our data support a previously unidentified mechanism in which exercise prevents loss of BDNF in the retina after injury and preserves neuronal function and survival by preventing complement‐mediated elimination of synapses.     

Activation of transient receptor potential vanilloid-1 (TRPV1) influences how retinal ganglion cell neurons respond to pressure-related stress

Our recent studies implicate the transient receptor potential vanilloid-1 (TRPV1) channel as a mediator of retinal ganglion cell (RGC) function and survival. With elevated pressure in the eye, TRPV1 increases in RGCs, supporting enhanced excitability, while Trpv1 -/- accelerates RGC degeneration in mice. Here we find TRPV1 localized in monkey and human RGCs, similar to rodents. Expression increases in RGCs exposed to acute changes in pressure. In retinal explants, contrary to our animal studies, both Trpv1 -/- and pharmacological antagonism of the channel prevented pressure-induced RGC apoptosis, as did chelation of extracellular Ca²⁺. Finally, while TRPV1 and TRPV4 co-localize in some RGC bodies and form a protein complex in the retina, expression of their mRNA is inversely related with increasing ocular pressure. We propose that TRPV1 activation by pressure-related insult in the eye initiates changes in expression that contribute to a Ca²⁺-dependent adaptive response to maintain excitatory signaling in RGCs.     

Activation of transient receptor potential vanilloid-1 (TRPV1) influences how retinal ganglion cell neurons respond to pressure-related stress

Our recent studies implicate the transient receptor potential vanilloid-1 (TRPV1) channel as a mediator of retinal ganglion cell (RGC) function and survival. With elevated pressure in the eye, TRPV1 increases in RGCs, supporting enhanced excitability, while Trpv1 -/- accelerates RGC degeneration in mice. Here we find TRPV1 localized in monkey and human RGCs, similar to rodents. Expression increases in RGCs exposed to acute changes in pressure. In retinal explants, contrary to our animal studies, both Trpv1 -/- and pharmacological antagonism of the channel prevented pressure-induced RGC apoptosis, as did chelation of extracellular Ca²⁺. Finally, while TRPV1 and TRPV4 co-localize in some RGC bodies and form a protein complex in the retina, expression of their mRNA is inversely related with increasing ocular pressure. We propose that TRPV1 activation by pressure-related insult in the eye initiates changes in expression that contribute to a Ca²⁺-dependent adaptive response to maintain excitatory signaling in RGCs.     

Fine mapping of a sedative-hypnotic drug withdrawal locus on mouse chromosome 11

We have established that there is a considerable amount of common genetic influence on physiological dependence and associated withdrawal from sedative-hypnotic drugs including alcohol, benzodiazepines, barbiturates and inhalants. We previously mapped two loci responsible for 12 and 9% of the genetic variance in acute alcohol and pentobarbital withdrawal convulsion liability in mice, respectively, to an approximately 28-cM interval of proximal chromosome 11. Here, we narrow the position of these two loci to a 3-cM interval (8.8 Mb, containing 34 known and predicted genes) using haplotype analysis. These include genes encoding four subunits of the GABA(A) receptor, which is implicated as a pivotal component in sedative-hypnotic dependence and withdrawal. We report that the DBA/2J mouse strain, which exhibits severe withdrawal from sedative-hypnotic drugs, encodes a unique GABA(A) receptor gamma2 subunit variant compared with other standard inbred strains including the genetically similar DBA/1J strain. We also demonstrate that withdrawal from zolpidem, a benzodiazepine receptor agonist selective for alpha1 subunit containing GABA(A) receptors, is influenced by a chromosome 11 locus, suggesting that the same locus (gene) influences risk of alcohol, benzodiazepine and barbiturate withdrawal. Our results, together with recent knockout studies, point to the GABA(A) receptor gamma2 subunit gene (Gabrg2) as a promising candidate gene to underlie phenotypic differences in sedative-hypnotic physiological dependence and associated withdrawal episodes.     

Delay of photoreceptor degeneration in tubby mouse by sulforaphane

In this study, the homozygous tubby (tub/tub) mutant mouse, with an early progressive hearing loss and photoreceptor degeneration, was used as a model system to examine the effects of systemic administration of a naturally occurring isothiocyanate, sulforaphane (SF), on photoreceptor degeneration. Several novel observations have been made: (i) the mRNA and protein expression of thioredoxin (Trx), thioredoxin reductase (TrxR) and NF-E2-related factor-2 (Nrf2) were significantly reduced even prior to photoreceptor cell degeneration in the retinas of tub/tub mice, suggesting that retinal expression of the Trx system is impaired and that Trx regulation is involved in the pathogenesis of retinal degeneration in this model, (ii) intraperitoneal injection with SF significantly up-regulated retinal levels of Trx, TrxR, and Nrf2, and effectively protected photoreceptor cells in tub/tub mice as evaluated functionally by electroretinography and morphologically by quantitative histology, and (iii) treatment with PD98059, an inhibitor of extracellular signal-regulated kinases (ERKs), blocked SF-mediated ERKs activation and up-regulation of Trx/TrxR/Nrf2 in the retinas of tub/tub mice. This suggests that ERKs and Nrf2 are involved in the mechanism of SF-mediated up-regulation of the Trx system to protect photoreceptor cells in this model. These novel findings are significant and could provide important information for the development of a unique strategy to prevent sensorineural deafness/retinal dystrophic syndromes and also other forms of inherited neurological disorders.