Glaucoma Degeneration Research Articles

Sestrin2 overexpression alleviates hydrogen peroxide-induced apoptosis and oxidative stress in retinal ganglion cells by enhancing Nrf2 activation via Keap1 downregulation

Oxidative stress-induced apoptosis of retinal ganglion cells (RGCs) contributes to the development and progression of glaucoma. Sestrin2 (Sesn2), a stress-inducible protein, has a potent antioxidant capacity that can provide cytoprotection against various noxious stimuli. However, whether Sesn2 is involved in protecting RGCs from oxidative stress remains unexplored. The purpose of this study was to evaluate the role of Sesn2 in regulating hydrogen peroxide (H2O2)-induced oxidative stress of RGCs. Here, we showed that Sesn2 expression was induced in RGCs following H2O2 exposure. Sesn2 depletion markedly exacerbated H2O2-induced apoptosis and reactive oxygen species (ROS) generation in RGCs. Notably, upregulation of Sesn2 significantly decreased H2O2-induced apoptosis and ROS generation. Moreover, Sesn2 overexpression increased the nuclear translocation of nuclear factor erythroid-derived 2-like 2 (Nrf2), elevated Nrf2/antioxidant response element (ARE)-mediated transcriptional activity and upregulated the expression of Nrf2 target genes in H2O2-stimulated RGCs. Interestingly, we found that Sesn2 promoted Nrf2/ARE activation through downregulation of kelch-like ECH-associated protein 1 (Keap1). Restoration of Keap1 or inhibition of Nrf2 significantly reversed the Sesn2-mediated protective effect in H2O2-stimulated RGCs. In conclusion, these results elucidated that Sesn2 confers a protective effect in RGCs against H2O2-induced oxidative stress by reinforcing Nrf2/ARE activation via downregulation of Keap1. Our study suggests that the Sesn2/Keap1/Nrf2 axis may play an important role in retinal degeneration in glaucoma.

Sub-region-Specific Optic Nerve Head Glial Activation in Glaucoma.

Glaucoma, a multifactorial neurodegenerative disease characterized by progressive loss of retinal ganglion cells and their axons in the optic nerve, is a leading cause of irreversible vision loss. Intraocular pressure (IOP) is a risk factor for axonal damage, which initially occurs at the optic nerve head (ONH). Complex cellular and molecular mechanisms involved in the pathogenesis of glaucomatous optic neuropathy remain unclear. Here we define early molecular events in the ONH in an inherited large animal glaucoma model in which ONH structure resembles that of humans. Gene expression profiling of ONH tissues from rigorously phenotyped feline subjects with early-stage glaucoma and precisely age-matched controls was performed by RNA-sequencing (RNA-seq) analysis and complementary bioinformatic approaches applied to identify molecular processes and pathways of interest. Immunolabeling supported RNA-seq findings while providing cell-, region-, and disease stage-specific context in the ONH in situ. Transcriptomic evidence for cell proliferation and immune/inflammatory responses is identifiable in early glaucoma, soon after IOP elevation and prior to morphologically detectable axon loss, in this large animal model. In particular, proliferation of microglia and oligodendrocyte precursor cells is a prominent feature of early-stage, but not chronic, glaucoma. ONH microgliosis is a consistent hallmark in both early and chronic stages of glaucoma. Molecular pathways and cell type-specific responses strongly implicate toll-like receptor and NF-κB signaling in early glaucoma pathophysiology. The current study provides critical insights into molecular pathways, highly dependent on cell type and sub-region in the ONH even prior to irreversible axon degeneration in glaucoma.

A broad perspective on the molecular regulation of retinal ganglion cell degeneration in glaucoma.

Glaucoma is a complex neurodegenerative disease involving RGC axons, somas, and synapses at dendrites and axon terminals. Recent research advancements in the field have revealed a bigger picture of glaucomatous neurodegeneration that encompasses multiple stressors, multiple injury sites, multiple cell types, and multiple signaling pathways for asynchronous degeneration of RGCs during a chronic disease period. Optic nerve head is commonly viewed as the critical site of injury in glaucoma, where early injurious insults initiate distal and proximal signaling for axonal and somatic degeneration. Despite compartmentalized processes for degeneration of RGC axons and somas, there are intricate interactions between the two compartments and mechanistic overlaps between the molecular pathways that mediate degeneration in axonal and somatic compartments. This review summarizes the recent progress in the molecular understanding of RGC degeneration in glaucoma and highlights various etiological paths with biomechanical, metabolic, oxidative, and inflammatory components. Through this growing body of knowledge, the glaucoma community moves closer toward causative treatment of this blinding disease.

Fixel-Based Analysis of Visual Pathway White Matter in Primary Open-Angle Glaucoma.

White matter (WM) degeneration of the visual pathways in primary open-angle glaucoma (POAG) is well documented, but its exact pathophysiology remains unclear. To date, glaucomatous WM degeneration has been exclusively studied using diffusion tensor imaging (DTI) only. However, DTI measures lack direct biological interpretation, and the approach itself suffers from multiple technical limitations. Fixel-based analysis (FBA) is a novel framework for studying WM degeneration, overcoming DTI's technical limitations and providing biologically meaningful metrics. FBA measures fiber density (FD), representing early microstructural changes, and fiber-bundle cross section (FC), representing late macrostructural changes. In this study, we use FBA to study glaucomatous degeneration of the pregeniculate optic tracts (OTs) and postgeniculate optic radiation (ORs) in POAG. This was a cross-sectional case-control study with 12 POAG patients and 16 controls. Multi-shell diffusion-weighted images were acquired. FBA was used to produce a population template, and probabilistic tractography was used to track the OTs and ORs in template space. Finally, FD and FC of the tracts of interest were compared between the two groups. Compared with the controls, the OTs of the patients exhibited a significant (familywise error corrected P < 0.05) decrease in FD and FC, whereas their ORs exhibited a significant decrease in FD but not in FC. FBA provides sensitive measures to assess WM changes in glaucoma. Our findings suggest that the OTs of glaucoma patients exhibit signs of more advanced WM degeneration compared with the ORs. This potentially implicates anterograde trans-synaptic propagation as the primary cause of glaucomatous spread along the visual pathways.

Immune Mediated Degeneration and Possible Protection in Glaucoma.

The underlying pathomechanisms for glaucoma, one of the most common causes of blindness worldwide, are still not identified. In addition to increased intraocular pressure (IOP), oxidative stress, excitotoxicity, and immunological processes seem to play a role. Several pharmacological or molecular/genetic methods are currently investigated as treatment options for this disease. Altered autoantibody levels were detected in serum, aqueous humor, and tissue sections of glaucoma patients. To further analyze the role of the immune system, an IOP-independent, experimental autoimmune glaucoma (EAG) animal model was developed. In this model, immunization with ocular antigens leads to antibody depositions, misdirected T-cells, retinal ganglion cell death and degeneration of the optic nerve, similar to glaucomatous degeneration in patients. Moreover, an activation of the complement system and microglia alterations were identified in the EAG as well as in ocular hypertension models. The inhibition of these factors can alleviate degeneration in glaucoma models with and without high IOP. Currently, several neuroprotective approaches are tested in distinct models. It is necessary to have systems that cover underlying pathomechanisms, but also allow for the screening of new drugs. In vitro models are commonly used, including single cell lines, mixed-cultures, and even organoids. In ex vivo organ cultures, pathomechanisms as well as therapeutics can be investigated in the whole retina. Furthermore, animal models reveal insights in the in vivo situation. With all these models, several possible new drugs and therapy strategies were tested in the last years. For example, hypothermia treatment, neurotrophic factors or the blockage of excitotoxity. However, further studies are required to reveal the pressure independent pathomechanisms behind glaucoma. There is still an open issue whether immune mechanisms directly or indirectly trigger cell death pathways. Hence, it might be an imbalance between protective and destructive immune mechanisms. Moreover, identified therapy options have to be evaluated in more detail, since deeper insights could lead to better treatment options for glaucoma patients.

ESNR 2019

Purpose: White matter (WM) degeneration of the visual pathways in primary open-angle glaucoma (POAG) is well documented, but its exact pathophysiology remains unclear. To date, glaucomatous WM degeneration has been exclusively studied using diffusion tensor imaging (DTI) only. However, DTI measures lack direct biological interpretation, and the approach itself suffers from multiple technical limitations. Fixel-based analysis (FBA) is a novel framework for studying WM degeneration, overcoming DTI's technical limitations and providing biologically meaningful metrics. FBA measures fiber density (FD), representing early microstructural changes, and fiber-bundle cross section (FC), representing late macrostructural changes. In this study, we use FBA to study glaucomatous degeneration of the pregeniculate optic tracts (OTs) and postgeniculate optic radiation (ORs) in POAG. Methods: This was a cross-sectional case-control study with 12 POAG patients and 16 controls. Multi-shell diffusion-weighted images were acquired. FBA was used to produce a population template, and probabilistic tractography was used to track the OTs and ORs in template space. Finally, FD and FC of the tracts of interest were compared between the two groups. Results: Compared with the controls, the OTs of the patients exhibited a significant (familywise error corrected P < 0.05) decrease in FD and FC, whereas their ORs exhibited a significant decrease in FD but not in FC. Conclusions: FBA provides sensitive measures to assess WM changes in glaucoma. Our findings suggest that the OTs of glaucoma patients exhibit signs of more advanced WM degeneration compared with the ORs. This potentially implicates anterograde trans-synaptic propagation as the primary cause of glaucomatous spread along the visual pathways.

Potential role of P2X7 receptor in neurodegenerative processes in a murine model of glaucoma

Glaucoma is a common cause of visual impairment and blindness, characterized by retinal ganglion cell (RGC) death. The mechanisms that trigger the development of glaucoma remain unknown and have gained significant relevance in the study of this neurodegenerative disease. P2X7 purinergic receptors (P2X7R) could be involved in the regulation of the synaptic transmission and neuronal death in the retina through different pathways. The aim of this study was to characterize the molecular signals underlying glaucomatous retinal injury.The time-course of functional, morphological, and molecular changes in the glaucomatous retina of the DBA/2J mice were investigated. The expression and localization of P2X7R was analysed in relation with retinal markers. Caspase-3, JNK, and p38 were evaluated in control and glaucomatous mice by immunohistochemical and western-blot analysis. Furthermore, electroretinogram recordings (ERG) were performed to assess inner retina dysfunction.Glaucomatous mice exhibited changes in P2X7R expression as long as the pathology progressed. There was P2X7R overexpression in RGCs, the primary injured neurons, which correlated with the loss of function through ERG measurements. All analyzed MAPK and caspase-3 proteins were upregulated in the DBA/2J retinas suggesting a pro-apoptotic cell death.The increase in P2X7Rs presence may contribute, together with other factors, to the changes in retinal functionality and the concomitant death of RGCs. These findings provide evidence of possible intracellular pathways responsible for apoptosis regulation during glaucomatous degeneration.

Activation of Nrf2/HO-1 pathway protects retinal ganglion cells from a rat chronic ocular hypertension model of glaucoma.

The objective of this work was to find out the effects of nuclear factor erythroid 2-related factor/heme oxygenase-1 (Nrf2/HO-1) pathway on retinal ganglion cell (RGC) injury in glaucoma. The chronic ocular hypertension (COH) rat models of glaucoma were constructed, and intraocular pressure (IOP) and RGC numbers were detected at different time points. Additionally, rats were divided into normal group (normal control rats), model group (COH model rats), and model + tBHQ group (COH model rats treated with Nrf activator, tBHQ). RGC apoptosis was detected by using TUNEL staining, and the expressions of Nrf2/HO-1 were detected by qRT-PCR and western blotting. COH model rats showed significant IOP elevation and the increased mRNA and protein expressions of Nrf2 and HO-1 from 1 to 6weeks after operation, with the evidently decreased RGC numbers at 4weeks and 6weeks after operation (all P < 0.05). Besides, rats in the model group had increased apoptosis index (AI) of RGCs and the elevated mRNA and protein expressions of Nrf2/HO-1 with remarkably reduced RGC numbers when compared with normal control rats, but the model rats treated with tBHQ exhibited an apparent decrease in AI of RGCs, as well as remarkable increases in RGC numbers and the mRNA and protein expression of Nrf2/HO-1 (all P < 0.05). Activation of Nrf2/HO-1 pathway significantly reduced the apoptosis and injury of RGCs in rats with chronic ocular hypertension (COH), thereby protecting RGCs in glaucoma, which could be a promising clinical target to prevent RGC degeneration in glaucoma.

Increased bioavailability of cyclic guanylate monophosphate prevents retinal ganglion cell degeneration

The nitric oxide – guanylyl cyclase-1 – cyclic guanylate monophosphate (NO-GC-1–cGMP) pathway has emerged as a potential pathogenic mechanism for glaucoma, a common intraocular pressure (IOP)-related optic neuropathy characterized by the degeneration of retinal ganglion cells (RGCs) and their axons in the optic nerve. NO activates GC-1 to increase cGMP levels, which are lowered by cGMP-specific phosphodiesterase (PDE) activity. This pathway appears to play a role in both the regulation of IOP, where reduced cGMP levels in mice leads to elevated IOP and subsequent RGC degeneration. Here, we investigated whether potentiation of cGMP signaling could protect RGCs from glaucomatous degeneration. We administered the PDE5 inhibitor tadalafil orally (10 mg/kg/day) in murine models of two forms of glaucoma – primary open angle glaucoma (POAG; GC-1−/− mice) and primary angle-closure glaucoma (PACG; Microbead Occlusion Model) - and measured RGC viability at both the soma and axon level. To determine the direct effect of increased cGMP on RGCs in vitro, we treated axotomized whole retina and primary RGC cultures with the cGMP analogue 8-Br-cGMP. Tadalafil treatment increased plasma cGMP levels in both models, but did not alter IOP or mean arterial pressure. Nonetheless, tadalafil treatment prevented degeneration of RGC soma and axons in both disease models. Treatment of whole, axotomized retina and primary RGC cultures with 8-Br-cGMP markedly attenuated both necrotic and apoptotic cell death pathways in RGCs. Our findings suggest that enhancement of the NO-GC-1-cGMP pathway protects the RGC body and axon in murine models of POAG and PACG, and that enhanced signaling through this pathway may serve as a novel glaucoma treatment, acting independently of IOP.

A murine glaucoma model induced by rapid in vivo photopolymerization of hyaluronic acid glycidyl methacrylate.

Glaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP) resulting in progressive loss of retinal ganglion cells (RGCs) and optic nerve degeneration, leading to blindness. New therapeutic approaches that better preserve the visual field by promoting survival and health of RGCs are highly needed since RGC death occurs despite good IOP control in glaucoma patients. We have developed a novel approach to reliably induce chronic IOP elevation in mouse using a photopolymerizable biomatrix, hyaluronic acid glycidyl methacrylate. This is achieved by rapid in vivo crosslinking of the biomatrix at the iridocorneal angle by a flash of ultraviolet A (UVA) light to impede the aqueous outflow pathway with a controllable manner. Sustained IOP elevation was induced after a single manipulation and was maintained at ~45% above baseline for >4 weeks. Significant thinning of the inner retina and ~35% reduction in RGCs and axons was noted within one month of IOP elevation. Optic nerve degeneration showed positive correlation with cumulative IOP elevation. Activation of astrocytes and microglia appeared to be an early event in response to IOP elevation preceding detectable RGC and axon loss. Attenuated glial reactivity was noted at later stage where significant RGC/axon loss had occurred suggesting astrocytes and microglia may play different roles over the course of glaucomatous degeneration. This novel murine glaucoma model is reproducible and displays cellular changes that recapitulate several pathophysiological features of glaucoma.

Increased Ap4A levels and ecto-nucleotidase activity in glaucomatous mice retina.

The pathogenesis of glaucoma involves numerous intracellular mechanisms including the purinergic system contribution. Furthermore, the presence and release of nucleotides and dinucleotides during the glaucomatous damage and the maintenance of degradation machinery through ecto-nucleotidase activity are participating in the modulation of the suitable extracellular complex balance. The aim of this study was to investigate the levels of diadenosine tetraphosphate (Ap4A) and the pattern of ecto-nucleotidase activity expression in glaucomatous retinas during the progress the pathology. Ap4A levels were analyzed by HPLC in glaucomatous retinas from the DBA/2J mice at 3, 9, 15, and 23months of age. For that, retinas were dissected as flattened whole-mounts and stimulated in Ringer buffer with or without 59mM KCl. NPP1 expression was analyzed by RT-PCR and western blot and its distribution was assessed by immunohistochemistry studies examined under confocal microscopy. Glaucomatous mice exhibited Ap4A values, which changed in stimulated retinas as long as the pathology progressed varying from 0.73 ± 0.04 (3months) to 0.170 ± 0.05pmol/mg retina (23months). Concomitantly, NPP1 expression was significantly increased (82.15%) in the DBA/2J mice at 15months. Furthermore, immunohistochemical studies showed that NPP1 labeling was stronger in OPL and IPL labeling tangentially in the vitreal part of the retina and was upregulated at 15months of age. Our findings demonstrate that Ap4A decreased levels may be related with exacerbated activity of NPP1 protein in glaucomatous degeneration and in this way contributing to elucidate different mechanisms involved in retinal impairment in glaucomatous degeneration.

Astrocyte remodeling without gliosis precedes optic nerve axonopathy

Astroyctes serve myriad functions but are especially critical in white matter tracts, where energy-demanding axons propagate action potentials great distances between neurons. Axonal dependence on astrocytes for even normal function accentuates the critical role astrocytes serve during disease. In glaucoma, the most common optic neuropathy, sensitivity to intraocular pressure (IOP) challenges RGC axons early, including degradation of anterograde transport to the superior colliculus (SC). Astrocyte remodeling presages overt axon degeneration in glaucoma and thus may present a therapeutic opportunity. Here we developed a novel metric to quantify organization of astrocyte processes in the optic nerve relative to axon degeneration in the DBA/2 J hereditary mouse model of glaucoma. In early progression, as axons expand prior to loss, astrocyte processes become more parallel with migration to the nerve’s edge without a change in overall coverage of the nerve. As axons degenerate, astrocyte parallelism diminishes with increased glial coverage and reinvasion of the nerve. In longitudinal sections through aged DBA/2 J nerve, increased astrocyte parallelism reflected elevated levels of the astrocyte gap-junction protein connexin 43 (Cx43). In the distal nerve, increased Cx43 also indicated with a higher level of intact anterograde transport from retina to SC. Our results suggest that progression of axonopathy in the optic nerve involves astrocyte remodeling in two phases. In an early phase, astrocyte processes organize in parallel, likely through gap-junction coupling, while a later phase involves deterioration of organization as glial coverage increases and axons are lost.

Annexins in Glaucoma.

Glaucoma is one of the leading causes of irreversible visual loss, which has been estimated to affect 3.5% of those over 40 years old and projected to affect a total of 112 million people by 2040. Such a dramatic increase in affected patients demonstrates the need for continual improvement in the way we diagnose and treat this condition. Annexin A5 is a 36 kDa protein that is ubiquitously expressed in humans and is studied as an indicator of apoptosis in several fields. This molecule has a high calcium-dependent affinity for phosphatidylserine, a cell membrane phospholipid externalized to the outer cell membrane in early apoptosis. The DARC (Detection of Apoptosing Retinal Cells) project uses fluorescently-labelled annexin A5 to assess glaucomatous degeneration, the inherent process of which is the apoptosis of retinal ganglion cells. Furthermore, this project has conducted investigation of the retinal apoptosis in the neurodegenerative conditions of the eye and brain. In this present study, we summarized the use of annexin A5 as a marker of apoptosis in the eye. We also relayed the progress of the DARC project, developing real-time imaging of retinal ganglion cell apoptosis in vivo from the experimental models of disease and identifying mechanisms underlying neurodegeneration and its treatments, which has been applied to the first human clinical trials. DARC has potential as a biomarker in neurodegeneration, especially in the research of novel treatments, and could be a useful tool for the diagnosis and monitoring of glaucoma.

Axogenic mechanism enhances retinal ganglion cell excitability during early progression in glaucoma

Diseases of the brain involve early axon dysfunction that often precedes outright degeneration. Pruning of dendrites and their synapses represents a potential driver of axonopathy by reducing activity. Optic nerve degeneration in glaucoma, the world's leading cause of irreversible blindness, involves early stress to retinal ganglion cell (RGC) axons from sensitivity to intraocular pressure (IOP). This sensitivity also influences survival of RGC dendrites and excitatory synapses in the retina. Here we tested in individual RGCs identified by type the relationship between dendritic organization and axon signaling to light following modest, short-term elevations in pressure. We found dendritic pruning occurred early, by 2 wk of elevation, and independent of whether the RGC responded to light onset (ON cells) or offset (OFF cells). Pruning was similarly independent of ON and OFF in the DBA/2J mouse, a chronic glaucoma model. Paradoxically, all RGCs, even those with significant pruning, demonstrated a transient increase in axon firing in response to the preferred light stimulus that occurred on a backdrop of generally enhanced excitability. The increased response was not through conventional presynaptic signaling, but rather depended on voltage-sensitive sodium channels that increased transiently in the axon. Pruning, axon dysfunction, and deficits in visual acuity did not progress between 2 and 4 wk of elevation. These results suggest neurodegeneration in glaucoma involves an early axogenic response that counters IOP-related stress to excitatory dendritic architecture to slow progression and maintain signaling to the brain. Thus, short-term exposure to elevated IOP may precondition the neural system to further insult.

The Microbead Occlusion Model of Ocular Hypertension in Mice.

Glaucoma is a common optic neuropathy that leads to vision loss through the degeneration of retinal ganglion cells (RGCs) and their axons. RGC degeneration in glaucoma is associated with sensitivity to intraocular pressure (IOP) and elevated IOP (also known as ocular hypertension) is the primary modifiable risk factor. Ocular hypertension is the primary characteristic of rodent models for glaucoma research. Intracameral injection of microbeads has evolved as a preferred method of IOP elevation in rodents, particularly in mice. Here, we outline the protocol and method for the Microbead Occlusion Model in mice. We highlight the importance of anesthesia choice and the utilization of glass micropipettes in combination with a micromanipulator and microsyringe pump for the successful execution of the model.

Retinal putative glial alterations: implication for glaucoma care.

PurposeGliosis‐like retinal alterations, presumed to be activated retinal astrocytes and Müller cells (ARAM), have been reported to occur frequently in patients with glaucoma but rarely in controls. We investigated the association between glaucomatous abnormality and the presence, the extent of retinal region, and the spatial distribution, of hyperreflective retinal alterations on optical coherence tomography (OCT) en‐face images, presumed to be ARAM.MethodsFindings of hyperreflective structures, presumed to be ARAM, in the central retinal ±24 degrees of OCT en‐face images (acquired with the SPECTRALIS® OCT) were compared between 35 younger controls, 42 older controls and 38 patients with glaucoma. Presumed ARAM was defined as reflective structures on the en‐face images other than retinal vasculature and retinal nerve fibre bundles. Chi‐square tests were used to compare the proportion of younger controls vs older controls with presumed ARAM to investigate the effect of ageing, and the proportion of patients vs age‐similar older controls with presumed ARAM to investigate the effect of disease. We also investigated the effect of glaucoma on the retinal area with presumed ARAM when it was present; we used an analysis of covariance (ancova) to compare the retinal area with hyperreflectivity in patients vs controls, adjusting for the effects of age and axial length.ResultsThe mean (S.D.) age of the younger controls, older controls, and patients with glaucoma was 26 (3), 62 (10) and 69 (8) years, respectively. The median (25th quartile, 75th quartile) of the retinal region with the hyperreflective structures, presumed to be ARAM, was zero (0,0), 1 (0,6), and 11 (0,43) degrees square in the younger controls, older controls and patients with glaucoma respectively. The chi‐square test investigating the effect of ageing found χ2(1, N = 77) = 24.8, p < 0.001, and that investigating the effect of disease found χ2(1, N = 80) = 2.3, p = 0.1. The ancova found F(1, 46) = 10.32, P = 0.02.ConclusionsThere was an effect of ageing on the presence of the hyperreflective structures, presumed to be ARAM, on OCT images. Compared to the presence of hyperreflective structures, the extent of retinal region with the hyperreflective structures has a greater potential of being an indicator of glaucomatous degeneration. Further study is needed to investigate the nature of the relation between glaucomatous abnormality and the extent of the retina with the hyperreflective structures, presumed to be ARAM.

Age-related accumulation of phosphorylated mitofusin 2 protein in retinal ganglion cells correlates with glaucoma progression

Dysregulation of axonal bioenergetics is likely a key mechanism in the initiation and progression of age-related neurodegenerative diseases. Glaucoma is a quintessential neurodegenerative disorder characterized by progressive deterioration of the optic nerve (ON) and eventual death of retinal ganglion cells (RGCs). Age and elevation of intraocular pressure are key risk factors in glaucoma, but the common early hallmarks of decreased axonal transport and increased bioenergetic vulnerability likely underlie disease initiation. We examined the correlation between bioenergetics and axonal transport with mitochondrial mutation frequency and post-translational modifications of mitofusin 2 (Mfn2) in RGCs during glaucoma progression. No increase in the frequency of mtDNA mutations was detected, but we observed significant shifts in mitochondrial protein species. Mfn2 is a fusion protein that functions in mitochondrial biogenesis, maintenance, and mitochondrial transport. We demonstrate that Mfn2 accumulates selectively in RGCs during glaucomatous degeneration, that two novel states of Mfn2 exist in retina and ON, and identify a phosphorylated form that selectively accumulates in RGCs, but is absent in ON. Phosphorylation of Mfn2 is correlated with higher ubiquitination, and failure of the protein to reach the ON. Together, these data suggest that post-translational modification of Mfn2 is associated with its dysregulation during a window of metabolic vulnerability that precedes glaucomatous degeneration. Future work to either manipulate expression of Mfn2 or to prevent its degradation could have therapeutic value in the treatment of neurodegenerative diseases where long-tract axons are vulnerable.

Interleukin-6 Deficiency Attenuates Retinal Ganglion Cell Axonopathy and Glaucoma-Related Vision Loss.

The pleotropic cytokine interleukin-6 (IL-6) is implicated in retinal ganglion cell (RGC) survival and degeneration, including that associated with glaucoma. IL-6 protects RGCs from pressure-induced apoptosis in vitro. However, it is unknown how IL-6 impacts glaucomatous degeneration in vivo. To study how IL-6 influences glaucomatous RGC axonopathy, accompanying glial reactivity, and resultant deficits in visual function, we performed neural tracing, histological, and neurobehavioral assessments in wildtype (B6;129SF2/J; WT) and IL-6 knock-out mice (B6;129S2-IL6tm1kopf/J; IL-6-/-) after 8 weeks of unilateral or bilateral microbead-induced glaucoma (microbead occlusion model). IOP increased by 20% following microbead injection in both genotypes (p < 0.05). However, deficits in wound healing at the site of corneal injection were noted. In WT mice, elevated IOP produced degenerating axon profiles and decreased axon density in the optic nerve by 15% (p < 0.01). In IL-6-/- mice, axon density in the optic nerve did not differ between microbead- and saline-injected mice (p > 0.05) and degenerating axon profiles were minimal. Preservation of RGC axons was reflected in visual function, where visual acuity decreased significantly in a time-dependent manner with microbead-induced IOP elevation in WT (p < 0.001), but not IL-6-/- mice (p > 0.05). Despite this preservation of RGC axons and visual acuity, both microbead-injected WT and IL-6-/- mice exhibited a 50% decrease in anterograde CTB transport to the superior colliculus, as compared to saline-injected controls (p < 0.01). Assessment of glial reactivity revealed no genotype- or IOP-dependent changes in retinal astrocytes. IOP elevation decreased microglia density and percent retinal area covered in WT mice (p < 0.05), while IL-6-/- mice exhibited only a decrease in density (p < 0.05). Together, our findings indicate that two defining features of RGC axonopathy—axon transport deficits and structural degeneration of axons—likely occur via independent mechanisms. Our data suggest that IL-6 is part of a mechanism that specifically leads to structural degeneration of axons. Furthermore, its absence is sufficient to prevent both structural degeneration of the optic nerve and vision loss. Overall, our work supports the proposition that functional deficits in axon transport represent a therapeutic window for RGC axonopathy and identify IL-6 signaling as a strong target for such a therapeutic.

Proteomic profiling reveals crucial retinal protein alterations in the early phase of an experimental glaucoma model.

Clinical glaucoma is difficult to assess in terms of molecular pathophysiology, prompting studies in experimental models of glaucoma. The purpose of this study was to investigate quantitative changes in retinal protein expression at the onset of experimental glaucoma in rats. Analyzing the proteome provides a suitable tool to decipher the pathophysiological processes in glaucomatous degeneration. Thermic cauterization of episcleral veins was utilized to elevate the intraocular pressure in Sprague Dawley rats. Morphological changes were surveyed on a cellular level with a staining of Brn3a-positive cells. The retinal nerve fiber layer was investigated using optical coherence tomography (OCT, Heidelberg Engineering) and the optic nerve was analyzed by an axonal grading system. Mass spectrometry-featured quantitative proteomics and immunohistochemical staining was used to identify specifically altered proteins in the course of intraocular pressure elevation and initial neurodegeneration. Proteomic data were further analyzed with Ingenuity Pathway Analysis and Cytoscape to analyze further molecular associations. The intraocular pressure rose significantly (p<0.001) for the follow-up period of 3 weeks after which animals were sacrificed. Eyes exposed to an elevated intraocular pressure showed an initial decrease of retinal ganglion cells, retinal nerve fiber layer (p<0.05) and an impairment of the optic nerve (p<0.01). Mass spectrometry led to the identification and quantification of 931 retinal proteins, whereas 32 were considerably altered. Bioinformatics-assisted clustering revealed that a majority of these proteins are functionally associated with cell differentiation, apoptosis and stress response. The creation of an interactive protein network showed that numerous altered proteins are connected regarding their cellular function. Protein kinase b, mitogen-activated protein kinase 1 and the NF-κB complex seem to be essential molecules in this context. In conclusion, these results provide further lines of evidence that substantial molecular changes occur at the onset of the disease, identifying potential key players, which might be useful as biomarkers for diagnostics and development of medical treatment in the future.

Metabolic Vulnerability in the Neurodegenerative Disease Glaucoma.

Axons can be several orders of magnitude longer than neural somas, presenting logistical difficulties in cargo trafficking and structural maintenance. Keeping the axon compartment well supplied with energy also presents a considerable challenge; even seemingly subtle modifications of metabolism can result in functional deficits and degeneration. Axons require a great deal of energy, up to 70% of all energy used by a neuron, just to maintain the resting membrane potential. Axonal energy, in the form of ATP, is generated primarily through oxidative phosphorylation in the mitochondria. In addition, glial cells contribute metabolic intermediates to axons at moments of high activity or according to need. Recent evidence suggests energy disruption is an early contributor to pathology in a wide variety of neurodegenerative disorders characterized by axonopathy. However, the degree to which the energy disruption is intrinsic to the axon vs. associated glia is not clear. This paper will review the role of energy availability and utilization in axon degeneration in glaucoma, a chronic axonopathy of the retinal projection.