Ubiquinol promotes retinal ganglion cell survival and blocks the apoptotic pathway in ischemic retinal degeneration.

Coenzyme Q10 (CoQ10) protects retinal ganglion cells (RGCs) in experimental retinal ischemia and glaucoma by scavenging reactive oxygen species. We tested whether a diet supplemented with ubiquinol, the reduced form of CoQ10, promotes RGC survival and blocks the apoptotic pathway in ischemic mouse retina induced by acute high intraocular pressure (IOP) elevation. Ubiquinol (1%) treatment significantly promoted RGC survival at 2 weeks after ischemia/reperfusion. The ubiquinol treatment significantly blocked activation of astroglial and microglial cells in the ischemic retina at 2 weeks. While the ubiquinol treatment significantly decreased active Bax protein expression in the ischemic retina, phosphorylation of Bad at serine 112 and Bcl-xL protein expression were preserved in the ubiquinol-treated ischemic retina at 12 h. Consistently, the ubiquinol treatment prevented apoptotic cell death by blocking caspase-3 cleavage. These results suggest that the ubiquinol enhances RGC survival by modulating the Bax/Bad/Bcl-xL-mediated apoptotic pathway in the ischemic retina. Ubiquinol has therapeutic potential for ameliorating elevated IOP-induced ischemic retinal degeneration.

Optineurin E50K triggers BDNF deficiency-mediated mitochondrial dysfunction in retinal photoreceptor cell line.

Optineurin (OPTN) mutations are linked to glaucoma pathology and E50K mutation shows massive cell death in photoreceptor cells and retinal ganglion cells. However, little is known about E50K-mediated mitochondrial dysfunction in photoreceptor cell degeneration. We here show that overexpression of E50K expression triggered BDNF deficiency, leading to Bax activation in RGC-5 cells. BDNF deficiency induced mitochondrial dysfunction by decreasing mitochondrial maximal respiration and reducing intracellular ATP level in RGC-5 cells. However, BDNF deficiency did not alter mitochondrial dynamics. Also, BDNF deficiency resulted in LC3-mediated mitophagosome formation in RGC-5 cells. These results strongly suggest that E50K-mediated BDNF deficiency plays a critical role in compromised mitochondrial function in glaucomatous photoreceptor cell degeneration.

Increased mitochondrial fission and volume density by blocking glutamate excitotoxicity protect glaucomatous optic nerve head astrocytes.

Abnormal structure and function of astrocytes have been observed within the lamina cribrosa region of the optic nerve head (ONH) in glaucomatous neurodegeneration. Glutamate excitotoxicity-mediated mitochondrial alteration has been implicated in experimental glaucoma. However, the relationships among glutamate excitotoxicity, mitochondrial alteration and ONH astrocytes in the pathogenesis of glaucoma remain unknown. We found that functional N-methyl-d-aspartate (NMDA) receptors (NRs) are present in human ONH astrocytes and that glaucomatous human ONH astrocytes have increased expression levels of NRs and the glutamate aspartate transporter. Glaucomatous human ONH astrocytes exhibit mitochondrial fission that is linked to increased expression of dynamin-related protein 1 and its phosphorylation at Serine 616. In BAC ALDH1L1 eGFP or Thy1-CFP transgenic mice, NMDA treatment induced axon loss as well as hypertrophic morphology and mitochondrial fission in astrocytes of the glial lamina. In human ONH astrocytes, NMDA treatment in vitro triggered mitochondrial fission by decreasing mitochondrial length and number, thereby reducing mitochondrial volume density. However, blocking excitotoxicity by memantine (MEM) prevented these alterations by increasing mitochondrial length, number and volume density. In glaucomatous DBA/2J (D2) mice, blocking excitotoxicity by MEM inhibited the morphological alteration as well as increased mitochondrial number and volume density in astrocytes of the glial lamina. However, blocking excitotoxicity decreased autophagosome/autolysosome volume density in both astrocytes and axons in the glial lamina of glaucomatous D2 mice. These findings provide evidence that blocking excitotoxicity prevents ONH astrocyte dysfunction in glaucomatous neurodegeneration by increasing mitochondrial fission, increasing mitochondrial volume density and length, and decreasing autophagosome/autolysosome formation. GLIA 2015;63:736-753.

Coenzyme Q10 ameliorates oxidative stress and prevents mitochondrial alteration in ischemic retinal injury.

Coenzyme Q10 (CoQ10) acts by scavenging reactive oxygen species for protecting neuronal cells against oxidative stress in neurodegenerative diseases. We tested whether a diet supplemented with CoQ10 ameliorates oxidative stress and mitochondrial alteration, as well as promotes retinal ganglion cell (RGC) survival in ischemic retina induced by intraocular pressure elevation. A CoQ10 significantly promoted RGC survival at 2 weeks after ischemia. Superoxide dismutase 2 (SOD2) and heme oxygenase-1 (HO-1) expression were significantly increased at 12 h after ischemic injury. In contrast, the CoQ10 significantly prevented the upregulation of SOD2 and HO-1 protein expression in ischemic retina. In addition, the CoQ10 significantly blocked activation of astroglial and microglial cells in ischemic retina. Interestingly, the CoQ10 blocked apoptosis by decreasing caspase-3 protein expression in ischemic retina. Bax and phosphorylated Bad (pBad) protein expression were significantly increased in ischemic retina at 12 h. Interestingly, while CoQ10 significantly decreased Bax protein expression in ischemic retina, CoQ10 showed greater increase of pBad protein expression. Of interest, ischemic injury significantly increased mitochondrial transcription factor A (Tfam) protein expression in the retina at 12 h, however, CoQ10 significantly preserved Tfam protein expression in ischemic retina. Interestingly, there were no differences in mitochondrial DNA content among control- or CoQ10-treated groups. Our findings demonstrate that CoQ10 protects RGCs against oxidative stress by modulating the Bax/Bad-mediated mitochondrial apoptotic pathway as well as prevents mitochondrial alteration by preserving Tfam protein expression in ischemic retina. Our results suggest that CoQ10 may provide neuroprotection against oxidative stress-mediated mitochondrial alterations in ischemic retinal injury.

Activating soluble adenylyl cyclase protects mitochondria, rescues retinal ganglion cells, and ameliorates visual dysfunction caused by oxidative stress.

Oxidative stress is a key factor causing mitochondrial dysfunction and retinal ganglion cell (RGC) death in glaucomatous neurodegeneration. The cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway is involved in mitochondrial protection, promoting RGC survival. Soluble adenylyl cyclase (sAC) is one of the key regulators of the cAMP/PKA signaling pathway. However, the precise molecular mechanisms underlying the sAC-mediated signaling pathway and mitochondrial protection in RGCs that counter oxidative stress are not well characterized. Here, we demonstrate that sAC plays a critical role in protecting RGC mitochondria from oxidative stress. Using mouse models of oxidative stress, we found that activating sAC protected RGCs, blocked AMP-activated protein kinase activation, inhibited glial activation, and improved visual function. Moreover, we found that this is the result of preserving mitochondrial dynamics (fusion and fission), promoting mitochondrial bioenergetics and biogenesis, and preventing metabolic stress and apoptotic cell death in a paraquat oxidative stress model. Notably, sAC activation ameliorated mitochondrial dysfunction in RGCs by enhancing mitochondrial biogenesis, preserving mitochondrial structure, and increasing ATP production in oxidatively stressed RGCs. These findings suggest that activating sAC enhances the mitochondrial structure and function in RGCs to counter oxidative stress, consequently promoting RGC protection. We propose that modulation of the sAC-mediated signaling pathway has therapeutic potential acting on RGC mitochondria for treating glaucoma and other retinal diseases.

Administration of Bicarbonate Protects Mitochondria, Rescues Retinal Ganglion Cells, and Ameliorates Visual Dysfunction Caused by Oxidative Stress.

Oxidative stress is a key factor causing mitochondrial dysfunction and retinal ganglion cell (RGC) death in glaucomatous neurodegeneration. The cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway is involved in mitochondrial protection, promoting RGC survival. Soluble adenylyl cyclase (sAC) is a key regulator of the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway, which is known to protect mitochondria and promote RGC survival. However, the precise molecular mechanisms connecting the sAC-mediated signaling pathway with mitochondrial protection in RGCs against oxidative stress are not well characterized. Here, we demonstrate that sAC plays a critical role in protecting RGC mitochondria from oxidative stress. Using mouse models of oxidative stress induced by ischemic injury and paraquat administration, we found that administration of bicarbonate, as an activator of sAC, protected RGCs, blocked AMP-activated protein kinase activation, inhibited glial activation, and improved visual function. Moreover, we found that this is the result of preserving mitochondrial dynamics (fusion and fission), promoting mitochondrial bioenergetics and biogenesis, and preventing metabolic stress and apoptotic cell death. Notably, the administration of bicarbonate ameliorated mitochondrial dysfunction in RGCs by enhancing mitochondrial biogenesis, preserving mitochondrial structure, and increasing ATP production in oxidatively stressed RGCs. These findings suggest that activating sAC enhances the mitochondrial structure and function in RGCs to counter oxidative stress, consequently promoting RGC protection. We propose that modulation of the sAC-mediated signaling pathway has therapeutic potential acting on RGC mitochondria for treating glaucoma and other retinal diseases.

AIBP: A New Safeguard against Glaucomatous Neuroinflammation.

Glaucoma is a group of ocular diseases that cause irreversible blindness. It is characterized by multifactorial degeneration of the optic nerve axons and retinal ganglion cells (RGCs), resulting in the loss of vision. Major components of glaucoma pathogenesis include glia-driven neuroinflammation and impairment of mitochondrial dynamics and bioenergetics, leading to retinal neurodegeneration. In this review article, we summarize current evidence for the emerging role of apolipoprotein A-I binding protein (AIBP) as an important anti-inflammatory and neuroprotective factor in the retina. Due to its association with toll-like receptor 4 (TLR4), extracellular AIBP selectively removes excess cholesterol from the plasma membrane of inflammatory and activated cells. This results in the reduced expression of TLR4-associated, cholesterol-rich lipid rafts and the inhibition of downstream inflammatory signaling. Intracellular AIBP is localized to mitochondria and modulates mitophagy through the ubiquitination of mitofusins 1 and 2. Importantly, elevated intraocular pressure induces AIBP deficiency in mouse models and in human glaucomatous retina. AIBP deficiency leads to the activation of TLR4 in Müller glia, triggering mitochondrial dysfunction in both RGCs and Müller glia, and compromising visual function in a mouse model. Conversely, restoring AIBP expression in the retina reduces neuroinflammation, prevents RGCs death, and protects visual function. These results provide new insight into the mechanism of AIBP function in the retina and suggest a therapeutic potential for restoring retinal AIBP expression in the treatment of glaucoma.

AIBP controls TLR4 inflammarafts and mitochondrial dysfunction in a mouse model of Alzheimer's disease.

Microglia-driven neuroinflammation plays an important role in the development of Alzheimer's disease (AD). Microglia activation is accompanied by the formation and chronic maintenance of TLR4 inflammarafts, defined as enlarged and cholesterol-rich lipid rafts serving as an assembly platform for TLR4 dimers and complexes of other inflammatory receptors. The secreted apoA-I binding protein (APOA1BP or AIBP) binds TLR4 and selectively targets cholesterol depletion machinery to TLR4 inflammaraft expressing inflammatory, but not homeostatic microglia. Here we demonstrated that amyloid-beta (Aß) induced formation of TLR4 inflammarafts in microglia in vitro and in the brain of APP/PS1 mice. Mitochondria in Apoa1bp-/- APP/PS1 microglia were hyperbranched and cupped, which was accompanied by increased ROS and the dilated ER. The size and number of Aß plaques and neuronal cell death were significantly increased, and the animal survival was decreased in Apoa1bp-/- APP/PS1 compared to APP/PS1 female mice. These results suggest that AIBP exerts control of TLR4 inflammarafts and mitochondrial dynamics in microglia and plays a protective role in AD associated oxidative stress and neurodegeneration.

Glaucomatous optic neuropathy: Mitochondrial dynamics, dysfunction and protection in retinal ganglion cells.

Glaucoma is a leading cause of irreversible blindness worldwide and is characterized by a slow, progressive, and multifactorial degeneration of retinal ganglion cells (RGCs) and their axons, resulting in vision loss. Despite its high prevalence in individuals 60 years of age and older, the causing factors contributing to glaucoma progression are currently not well characterized. Intraocular pressure (IOP) is the only proven treatable risk factor. However, lowering IOP is insufficient for preventing disease progression. One of the significant interests in glaucoma pathogenesis is understanding the structural and functional impairment of mitochondria in RGCs and their axons and synapses. Glaucomatous risk factors such as IOP elevation, aging, genetic variation, neuroinflammation, neurotrophic factor deprivation, and vascular dysregulation, are potential inducers for mitochondrial dysfunction in glaucoma. Because oxidative phosphorylation stress-mediated mitochondrial dysfunction is associated with structural and functional impairment of mitochondria in glaucomatous RGCs, understanding the underlying mechanisms and relationship between structural and functional alterations in mitochondria would be beneficial to developing mitochondria-related neuroprotection in RGCs and their axons and synapses against glaucomatous neurodegeneration. Here, we review the current studies focusing on mitochondrial dynamics-based structural and functional alterations in the mitochondria of glaucomatous RGCs and therapeutic strategies to protect RGCs against glaucomatous neurodegeneration.

Role of A-Kinase Anchoring Protein 1 in Retinal Ganglion Cells: Neurodegeneration and Neuroprotection.

A-Kinase anchoring protein 1 (AKAP1) is a multifunctional mitochondrial scaffold protein that regulates mitochondrial dynamics, bioenergetics, and calcium homeostasis by anchoring several proteins, including protein kinase A, to the outer mitochondrial membrane. Glaucoma is a complex, multifactorial disease characterized by a slow and progressive degeneration of the optic nerve and retinal ganglion cells (RGCs), ultimately resulting in vision loss. Impairment of the mitochondrial network and function is linked to glaucomatous neurodegeneration. Loss of AKAP1 induces dynamin-related protein 1 dephosphorylation-mediated mitochondrial fragmentation and loss of RGCs. Elevated intraocular pressure triggers a significant reduction in AKAP1 protein expression in the glaucomatous retina. Amplification of AKAP1 expression protects RGCs from oxidative stress. Hence, modulation of AKAP1 could be considered a potential therapeutic target for neuroprotective intervention in glaucoma and other mitochondria-associated optic neuropathies. This review covers the current research on the role of AKAP1 in the maintenance of mitochondrial dynamics, bioenergetics, and mitophagy in RGCs and provides a scientific basis to identify and develop new therapeutic strategies that could protect RGCs and their axons in glaucoma.

Restoring AIBP expression in the retina provides neuroprotection in glaucoma.

Glaucoma is a neurodegenerative disease manifested in retinal ganglion cell (RGC) death and irreversible blindness. While lowering intraocular pressure (IOP) is the only proven therapeutic strategy in glaucoma, it is insufficient for preventing disease progression, thus justifying the recent focus on targeting retinal neuroinflammation and preserving RGCs. We have identified apolipoprotein A-I binding protein (AIBP) as the protein regulating several mechanisms of retinal neurodegeneration. AIBP controls excessive cholesterol accumulation via upregulating the cholesterol transporter ATP-binding cassette transporter 1 (ABCA1) and reduces inflammatory signaling via toll-like receptor 4 (TLR4) and mitochondrial dysfunction. ABCA1, TLR4 and oxidative phosphorylation components are genetically linked to primary open-angle glaucoma. Here we demonstrated that AIBP and ABCA1 expression was decreased, while TLR4, interleukin 1 beta (IL-1 beta), and the cholesterol content increased in the retina of patients with glaucoma and in mouse models of glaucoma. Restoring AIBP expression by a single intravitreal injection of adeno-associated virus (AAV)-AIBP protected RGCs in glaucomatous DBA/2J mice, in mice with microbead-induced chronic IOP elevation, and optic nerve crush. In addition, AIBP expression attenuated TLR4 and IL-1 beta expression, localization of TLR4 to lipid rafts, reduced cholesterol accumulation, and ameliorated visual dysfunction. These studies collectively indicate that restoring AIBP expression in the glaucomatous retina reduces neuroinflammation and protects RGCs and Muller glia, suggesting the therapeutic potential of AAV-AIBP in human glaucoma.

Stress induced aging in mouse eye.

Aging, a universal process that affects all cells in an organism, is a major risk factor for a group of neuropathies called glaucoma, where elevated intraocular pressure is one of the known stresses affecting the tissue. Our understanding of molecular impact of aging on response to stress in retina is very limited; therefore, we developed a new mouse model to approach this question experimentally. Here we show that susceptibility to response to stress increases with age and is primed on chromatin level. We demonstrate that ocular hypertension activates a stress response that is similar to natural aging and involves activation of inflammation and senescence. We show that multiple instances of pressure elevation cause aging of young retina as measured on transcriptional and DNA methylation level and are accompanied by local histone modification changes. Our data show that repeated stress accelerates appearance of aging features in tissues and suggest chromatin modifications as the key molecular components of aging. Lastly, our work further emphasizes the importance of early diagnosis and prevention as well as age-specific management of age-related diseases, including glaucoma.

Increased optic atrophy type 1 expression protects retinal ganglion cells in a mouse model of glaucoma.

PURPOSE: The goal of this study is to determine whether increased optic atrophy type 1 (OPA1) expression protects against retinal ganglion cell (RGC) death in glaucomatous DBA/2J mice. METHODS: Intraocular pressure in DBA/2J mice was measured, and pre-glaucomatous DBA/2J mice eyes were transfected with recombinant adeno-associated virus serotype 2 (AAV2) constructs including AAV2-wild type (WT) mOPA1 for two months. Increased OPA1 expression was confirmed by western blotting and RGC survival was assessed by retrograde labeling with FluoroGold. In addition, apoptotic cell death and mitochondrial structure were determined in AAV2-WT mOPA1-transfected differentiated RGC-5 cells exposed to elevated hydrostatic pressure (30 mmHg) for three days. RESULTS: WT AAV2-mOPA1 transfection significantly increased 90 kDa and 80 kDa OPA1 isoforms in the retina of glaucomatous DBA/2J mice. OPA1 immunoreactivity was increased in the inner nuclear layer, inner plexiform layer, and ganglion cell layer in nine month-old glaucomatous DBA/2J mice transfected with AAV2-WT mOPA1. Overexpression of OPA1 significantly increased RGC survival at two months after AAV2-WT mOPA1 transfection, and decreased activation of both astroglia and microglia in the retina of glaucomatous DBA/2J mice. Also, overexpression of OPA1 in differentiated RGC-5 cells resulted in less apoptotic cell death and blocked mitochondrial fission following elevated hydrostatic pressure. CONCLUSIONS: OPA1 can directly modulate RGC survival, and increasing OPA1 expression may protect against RGC death in glaucomatous optic neuropathy.

Effect of Ubiquinol on Glaucomatous Neurodegeneration and Oxidative Stress: Studies for Retinal Ganglion Cell Survival and/or Visual Function.

Oxidative stress is one of major causal factors in glaucomatous neurodegeneration. Ubiquinol promotes retinal ganglion cell (RGC) survival against glaucomatous insults such as oxidative stress. Here we investigated the effect of ubiquinol on RGC survival and/or visual function in mouse models of glaucoma and oxidative stress. DBA/2J and age-matched DBA/2J-Gpnmb+ (D2-Gpnmb+), which do not develop intraocular pressure elevation, or C57BL/6J mice were fed with ubiquinol (1%) or control diet daily for 5 or 2 months. We assessed RGC survival by Brn3a immunohistochemistry and measured expression levels of active and total BAX, peroxisome proliferator-activated receptor-gamma coactivator 1α, transcription factor A (TFAM) and oxidative phosphorylation (OXPHOS) complex protein. Following induction of oxidative stress by paraquat injection, we also assessed visual function. In glaucomatous retina, ubiquinol supplementation significantly promoted RGC survival, blocked BAX activation and increased TFAM and OXPHOS complex II protein expression. Also, ubiquinol supplementation ameliorated oxidative stress-induced visual dysfunction. These findings indicate that ubiquinol promotes RGC survival by increasing TFAM expression and OXPHOS complex II activity in glaucomatous neurodegeneration, and that ubiquinol enhances RGC survival and preserves visual function against oxidative stress. We propose that ubiquinol has a therapeutic potential for treating oxidative stress-associated glaucomatous neurodegeneration.

Corrigendum to "Inhibition of cAMP/PKA Pathway Protects Optic Nerve Head Astrocytes against Oxidative Stress by Akt/Bax Phosphorylation-Mediated Mfn1/2 Oligomerization".

[This corrects the article DOI: 10.1155/2019/8060962.].

Loss of AKAP1 triggers Drp1 dephosphorylation-mediated mitochondrial fission and loss in retinal ganglion cells.

Impairment of mitochondrial structure and function is strongly linked to glaucoma pathogenesis. Despite the widely appreciated disease relevance of mitochondrial dysfunction and loss, the molecular mechanisms underlying mitochondrial fragmentation and metabolic stress in glaucoma are poorly understood. We demonstrate here that glaucomatous retinal ganglion cells (RGCs) show loss of A-kinase anchoring protein 1 (AKAP1), activation of calcineurin (CaN) and reduction of dynamin-related protein 1 (Drp1) phosphorylation at serine 637 (Ser637). These findings suggest that AKAP1-mediated phosphorylation of Drp1 at Ser637 has a critical role in RGC survival in glaucomatous neurodegeneration. Male mice lacking AKAP1 show increases in CaN and total Drp1 levels, as well as a decrease in Drp1 phosphorylation at Ser637 in the retina. Ultrastructural analysis of mitochondria shows that loss of AKAP1 triggers mitochondrial fragmentation and loss, as well as mitophagosome formation in RGCs. Loss of AKAP1 deregulates oxidative phosphorylation (OXPHOS) complexes (Cxs) by increasing CxII and decreasing CxIII-V, leading to metabolic and oxidative stress. Also, loss of AKAP1 decreases Akt phosphorylation at Serine 473 (Ser473) and threonine 308 (Thr308) and activates the Bim/Bax signaling pathway in the retina. These results suggest that loss of AKAP1 has a critical role in RGC dysfunction by decreasing Drp1 phosphorylation at Ser637, deregulating OXPHOS, decreasing Akt phosphorylation at Ser473 and Thr308, and activating the Bim/Bax pathway in glaucomatous neurodegeneration. Thus, we propose that overexpression of AKAP1 or modulation of Drp1 phosphorylation at Ser637 are potential therapeutic strategies for neuroprotective intervention in glaucoma and other mitochondria-related optic neuropathies.

AIBP protects retinal ganglion cells against neuroinflammation and mitochondrial dysfunction in glaucomatous neurodegeneration.

Glaucoma is a leading cause of blindness worldwide in individuals 60 years of age and older. Despite its high prevalence, the factors contributing to glaucoma progression are currently not well characterized. Glia-driven neuroinflammation and mitochondrial dysfunction play critical roles in glaucomatous neurodegeneration. Here, we demonstrated that elevated intraocular pressure (IOP) significantly decreased apolipoprotein A-I binding protein (AIBP; gene name Apoa1bp) in retinal ganglion cells (RGCs), but resulted in upregulation of TLR4 and IL-1ß expression in Müller glia endfeet. Apoa1bp-/- mice had impaired visual function and Müller glia characterized by upregulated TLR4 activity, impaired mitochondrial network and function, increased oxidative stress and induced inflammatory responses. We also found that AIBP deficiency compromised mitochondrial network and function in RGCs and exacerbated RGC vulnerability to elevated IOP. Administration of recombinant AIBP prevented RGC death and inhibited inflammatory responses and cytokine production in Müller glia in vivo. These findings indicate that AIBP protects RGCs against glia-driven neuroinflammation and mitochondrial dysfunction in glaucomatous neurodegeneration and suggest that recombinant AIBP may be a potential therapeutic agent for glaucoma.

Elevated hydrostatic pressure triggers release of OPA1 and cytochrome C, and induces apoptotic cell death in differentiated RGC-5 cells.

PURPOSE: This study was conducted to determine whether elevated hydrostatic pressure alters mitochondrial structure, triggers release of the dynamin-related guanosine triphosphatase (GTPase) optic atrophy type 1 (OPA1) or cytochrome C from mitochondria, alters OPA1 gene expression, and can directly induce apoptotic cell death in cultured retinal ganglion cell (RGC)-5 cells. METHODS: Differentiated RGC-5 cells were exposed to 30 mmHg for three days in a pressurized incubator. As a control, differentiated RGC-5 cell cultures were incubated simultaneously in a conventional incubator. Live RGC-5 cells were then labeled with MitoTracker Red and mitochondrial morphology was assessed by fluorescence microscopy. Mitochondrial structural changes were also assessed by electron microscopy and three-dimensional (3D) electron microscope tomography. OPA1 mRNA was measured by Taqman quantitative PCR. The cellular distribution of OPA1 protein and cytochrome C was assessed by immunocytochemistry and western blot. Caspase-3 activation was examined by western blot. Apoptotic cell death was evaluated by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. RESULTS: Mitochondrial fission, characterized by the conversion of tubular fused mitochondria into isolated small organelles, was triggered after three days exposure to elevated hydrostatic pressure. Electron microscopy confirmed the fission and noted no changes to mitochondrial architecture, nor outer membrane rupture. Electron microscope tomography showed that elevated pressure depleted mitochondrial cristae content by fourfold. Elevated hydrostatic pressure increased OPA1 gene expression by 35+/-14% on day 2, but reduced expression by 36+/-4% on day 3. Total OPA1 protein content was not changed on day 2 or 3. However, pressure treatment induced release of OPA1 and cytochrome C from mitochondria to the cytoplasm. Elevated pressure also activated caspase-3 and induced apoptotic cell death. CONCLUSIONS: Elevated hydrostatic pressure triggered mitochondrial changes including mitochondrial fission and abnormal cristae depletion, alteration of OPA1 gene expression, and release of OPA1 and cytochrome C into the cytoplasm before the onset of apoptotic cell death in differentiated RGC-5 cells. These results suggest that sustained moderate pressure elevation may directly damage RGC integrity by injuring mitochondria.

Inhibition of cAMP/PKA Pathway Protects Optic Nerve Head Astrocytes against Oxidative Stress by Akt/Bax Phosphorylation-Mediated Mfn1/2 Oligomerization.

Glaucoma is characterized by a progressive optic nerve degeneration and retinal ganglion cell loss, but the underlying biological basis for the accompanying neurodegeneration is not known. Accumulating evidence indicates that structural and functional abnormalities of astrocytes within the optic nerve head (ONH) have a role in glaucomatous neurodegeneration. Here, we investigate the impact of activation of cyclic adenosine 3',5'-monophosphate (cAMP)/protein kinase A (PKA) pathway on mitochondrial dynamics of ONH astrocytes exposed to oxidative stress. ONH astrocytes showed a significant loss of astrocytic processes in the glial lamina of glaucomatous DBA/2J mice, accompanied by basement membrane thickening and collagen deposition in blood vessels and axonal degeneration. Serial block-face scanning electron microscopy data analysis demonstrated that numbers of total and branched mitochondria were significantly increased in ONH astrocytes, while mitochondrial length and volume density were significantly decreased. We found that hydrogen peroxide- (H2O2-) induced oxidative stress compromised not only mitochondrial bioenergetics by reducing the basal and maximal respiration but also balance of mitochondrial dynamics by decreasing dynamin-related protein 1 (Drp1) protein expression in rat ONH astrocytes. In contrast, elevated cAMP by dibutyryl-cAMP (dbcAMP) or isobutylmethylxanthine treatment significantly increased Drp1 protein expression in ONH astrocytes. Elevated cAMP exacerbated the impairment of mitochondrial dynamics and reduction of cell viability to oxidative stress in ONH astrocytes by decreasing optic atrophy type 1 (OPA1), and mitofusin (Mfn)1/2 protein expression. Following combined treatment with H2O2 and dbcAMP, PKA inhibition restored mitochondrial dynamics by increasing mitochondrial length and decreasing mitochondrial number, and this promoted cell viability in ONH astrocytes. Also, PKA inhibition significantly promoted Akt/Bax phosphorylation and Mfn1/2 oligomerization in ONH astrocytes. These results suggest that modulation of the cAMP/PKA signaling pathway may have therapeutic potential by activating Akt/Bax phosphorylation and promoting Mfn1/2 oligomerization in glaucomatous ONH astrocytes.

Glutamate receptor activation triggers OPA1 release and induces apoptotic cell death in ischemic rat retina.

PURPOSE: Glutamate receptor activation-induced excitotoxicity has been hypothesized to cause retinal ganglion cell (RGC) death in glaucoma and to link mitochondrial dysfunction in both acute and chronic neurodegenerative disorders. However, the relationships among elevated intraocular pressure (IOP), glutamate receptor-mediated excitotoxicity, and mitochondrial dysfunction in glaucoma remains unknown. The goal of this study was to determine whether the N- methyl D-aspartate (NMDA) glutamate receptor antagonist MK801 can block optic atrophy 1 (OPA1) release and subsequent apoptotic cell death, as well as whether acute IOP elevation triggers OPA1 release and alters OPA1 gene and protein expression in the rat retina after ischemia. METHODS: Sprague Dawley rats received injections of MK801 (10 mg/kg) or vehicle and then transient retinal ischemia was induced by acute IOP elevation. Following subcellular fractionation, changes in cytoplasmic and mitochondrial OPA1 were assessed by western blot analysis. Also, the expression of OPA1 mRNA was measured by Taqman qPCR, the distribution of OPA1 protein was assessed by immunohistochemistry, and apoptotic cell death was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. RESULTS: The ~65 and 90 kDa isoforms of OPA1 were increased in the cytosol in the rat retina at 6 h and at 12 h, but only the 90 kDa isoform of OPA1 was decreased at 12 h after ischemia induced by acute IOP elevation. This suggests that ischemic insult induced OPA1 release from the mitochondria in retinas. Pretreatment with MK801 blocked this effect and significantly increased OPA1 immunoreactivity in the inner retinal layers, as well as OPA1 gene expression and total protein expression in retinas at 12 h after ischemia. Further, pretreatment with MK801 prevented apoptotic cell death in retinas at 12 h after ischemia. Following acute IOP elevation, Bcl-2 mRNA expression in retinas was decreased at 3 h and 6 h but increased at 12 h and 24 h. In contrast, Bax mRNA expression in these retinas was increased in the first 12 h and then plateaued. Moreover, pretreatment with MK801 increased Bcl-2 mRNA expression, but did not alter the course of Bax mRNA expression. CONCLUSIONS: These results indicate that OPA1 release from mitochondria triggered by acute IOP elevation is inhibited by blockade of glutamate receptor activation. Because this effect was accompanied by increases of Bcl-2 expression, no changes of Bax expression, and blockade of apoptosis, these findings indicate that glutamate receptor activation following acute IOP elevation may lead to a distinct mitochondria-mediated cell death pathway in ischemic retina. These results support further studies to determine whether ischemia-induced OPA1 release may be an important component of the biochemical cascade leading to pressure-related ischemic damage in glaucomatous retina.