Showing papers on "Retinal ganglion cell published in 2014"

Transcellular degradation of axonal mitochondria

Abstract: It is generally accepted that healthy cells degrade their own mitochondria. Here, we report that retinal ganglion cell axons of WT mice shed mitochondria at the optic nerve head (ONH), and that these mitochondria are internalized and degraded by adjacent astrocytes. EM demonstrates that mitochondria are shed through formation of large protrusions that originate from otherwise healthy axons. A virally introduced tandem fluorophore protein reporter of acidified mitochondria reveals that acidified axonal mitochondria originating from the retinal ganglion cell are associated with lysosomes within columns of astrocytes in the ONH. According to this reporter, a greater proportion of retinal ganglion cell mitochondria are degraded at the ONH than in the ganglion cell soma. Consistently, analyses of degrading DNA reveal extensive mtDNA degradation within the optic nerve astrocytes, some of which comes from retinal ganglion cell axons. Together, these results demonstrate that surprisingly large proportions of retinal ganglion cell axonal mitochondria are normally degraded by the astrocytes of the ONH. This transcellular degradation of mitochondria, or transmitophagy, likely occurs elsewhere in the CNS, because structurally similar accumulations of degrading mitochondria are also found along neurites in superficial layers of the cerebral cortex. Thus, the general assumption that neurons or other cells necessarily degrade their own mitochondria should be reconsidered.

A formula for human retinal ganglion cell receptive field density as a function of visual field location.

Abstract: In the human eye, all visual information must traverse the retinal ganglion cells. The most numerous subclass, the midget retinal ganglion cells, are believed to underlie spatial pattern vision. Thus the density of their receptive fields imposes a fundamental limit on the spatial resolution of human vision. This density varies across the retina, declining rapidly with distance from the fovea. Modeling spatial vision of extended or peripheral targets thus requires a quantitative description of midget cell density throughout the visual field. Through an analysis of published data on human retinal topography of cones and ganglion cells, as well as analysis of prior formulas, we have developed a new formula for midget retinal ganglion cell density as a function of position in the monocular or binocular visual field.

Connecting the Retina to the Brain

Abstract: The visual system is beautifully crafted to transmit information of the external world to visual processing and cognitive centers in the brain. For visual information to be relayed to the brain, a series of axon pathfinding events must take place to ensure that the axons of retinal ganglion cells, the only neuronal cell type in the retina that sends axons out of the retina, find their way out of the eye to connect with targets in the brain. In the past few decades, the power of molecular and genetic tools, including the generation of genetically manipulated mouse lines, have multiplied our knowledge about the molecular mechanisms involved in the sculpting of the visual system. Here, we review major advances in our understanding of the mechanisms controlling the differentiation of RGCs, guidance of their axons from the retina to the primary visual centers, and the refinement processes essential for the establishment of topographic maps and eye-specific axon segregation. Human disorders, such as albinism and achiasmia, that impair RGC axon growth and guidance and, thus, the establishment of a fully functioning visual system will also be discussed.

Coenzyme Q10 inhibits glutamate excitotoxicity and oxidative stress-mediated mitochondrial alteration in a mouse model of glaucoma.

Abstract: Purpose. To test whether a diet supplemented with coenzyme Q10 (CoQ10) ameliorates glutamate excitotoxicity and oxidative stress–mediated retinal ganglion cell (RGC) degeneration by preventing mitochondrial alterations in the retina of glaucomatous DBA/2J mice.

Identification of retinal ganglion cell neuroprotection conferred by platelet-derived growth factor through analysis of the mesenchymal stem cell secretome.

Abstract: The development of neuroprotective strategies to attenuate retinal ganglion cell death could lead to novel therapies for chronic optic neuropathies such as glaucoma. Intravitreal transplantation of mesenchymal stem cells slows retinal ganglion cell death in models of optic nerve injury, but the mechanism of action remains unclear. Here we characterized the neuroprotective effects of mesenchymal stem cells and mesenchymal stem cell-derived factors in organotypic retinal explant culture and an in vivo model of ocular hypertensive glaucoma. Co-culture of rat and human bone marrow-derived mesenchymal stem cells with retinal explants increased retinal ganglion cell survival, after 7 days ex vivo, by ∼2-fold and was associated with reduced apoptosis and increased nerve fibre layer and inner plexiform layer thicknesses. These effects were not demonstrated by co-culture with human or mouse fibroblasts. Conditioned media from mesenchymal stem cells conferred neuroprotection, suggesting that the neuroprotection is mediated, at least partly, by secreted factors. We compared the concentrations of 29 factors in human mesenchymal stem cell and fibroblast conditioned media, and identified 11 enriched in the mesenchymal stem cell secretome. Treatment of retinal explants with a cocktail of these factors conferred retinal ganglion cell neuroprotection, with factors from the platelet-derived growth factor family being the most potent. Blockade of platelet-derived growth factor signalling with neutralizing antibody or with small molecule inhibitors of platelet-derived growth factor receptor kinase or downstream phosphatidylinositol 3 kinase eliminated retinal ganglion cell neuroprotection conferred by mesenchymal stem cell co-culture. Intravitreal injection of platelet-derived growth factor -AA or -AB led to profound optic nerve neuroprotection in vivo following experimental induction of elevated intraocular pressure. These data demonstrate that mesenchymal stem cells secrete a number of neuroprotective proteins and suggest that platelet-derived growth factor secretion in particular may play an important role in mesenchymal stem cell-mediated retinal ganglion cell neuroprotection. Furthermore, platelet-derived growth factor may represent an independent target for achieving retinal ganglion cell neuroprotection.

The novel Rho kinase (ROCK) inhibitor K-115: a new candidate drug for neuroprotective treatment in glaucoma.

Abstract: Purpose To investigate the effect of K-115, a novel Rho kinase (ROCK) inhibitor, on retinal ganglion cell (RGC) survival in an optic nerve crush (NC) model. Additionally, to determine the details of the mechanism of K-115's neuroprotective effect in vivo and in vitro. Methods ROCK inhibitors, including K-115 and fasudil (1 mg/kg/d), or vehicle were administered orally to C57BL/6 mice. Retinal ganglion cell death was then induced with NC. Retinal ganglion cell survival was evaluated by counting surviving retrogradely labeled cells and measuring RGC marker expression with quantitative real-time polymerase chain reaction (qRT-PCR). Total oxidized lipid levels were assessed with a thiobarbituric acid-reactive substances (TBARS) assay. Reactive oxygen species (ROS) levels were assessed by co-labeling with CellROX and Fluorogold. Expression of the NADPH oxidase (Nox) family of genes was evaluated with qRT-PCR. Results The survival of RGCs after NC was increased 34 ± 3% with K-115, a significantly protective effect. Moreover, a similar effect was revealed by the qRT-PCR analysis of Thy-1.2 and Brn3a, RGC markers. Levels of oxidized lipids and ROS also increased with time after NC. NC-induced oxidative stress, including oxidation of lipids and production of ROS, was significantly attenuated by K-115. Furthermore, expression of the Nox gene family, especially Nox1, which is involved in the NC-induced ROS production pathway, was dramatically reduced by K-115. Conclusions The results indicated that oral K-115 administration delayed RGC death. Although K-115 may be mediated through Nox1 downregulation, we found that it did not suppress ROS production directly. Our findings show that K-115 has a potential use in neuroprotective treatment for glaucoma and other neurodegenerative diseases.

Jak/Stat signaling stimulates zebrafish optic nerve regeneration and overcomes the inhibitory actions of Socs3 and Sfpq.

Abstract: The regenerative failure of mammalian optic axons is partly mediated by Socs3-dependent inhibition of Jak/Stat signaling (Smith et al., 2009, 2011). Whether Jak/Stat signaling is part of the normal regenerative response observed in animals that exhibit an intrinsic capacity for optic nerve regeneration, such as zebrafish, remains unknown. Nor is it known whether the repression of regenerative inhibitors, such as Socs3, contributes to the robust regenerative response of zebrafish to optic nerve damage. Here we report that Jak/Stat signaling stimulates optic nerve regeneration in zebrafish. We found that IL-6 family cytokines, acting via Gp130-coupled receptors, stimulate Jak/Stat3 signaling in retinal ganglion cells after optic nerve injury. Among these cytokines, we found that CNTF, IL-11, and Clcf1/Crlf1a can stimulate optic axon regrowth. Surprisingly, optic nerve injury stimulated the expression of Socs3 and Sfpq (splicing factor, proline/glutamine rich) that attenuate optic nerve regeneration. These proteins were induced in a Jak/Stat-dependent manner, stimulated each other's expression and suppressed the expression of regeneration-associated genes. In vivo, the injury-dependent induction of Socs3 and Sfpq inhibits optic nerve regeneration but does not block it. We identified a robust induction of multiple cytokine genes in zebrafish retinal ganglion cells that may contribute to their ability to overcome these inhibitory factors. These studies not only identified mechanisms underlying optic nerve regeneration in fish but also suggest new molecular targets for enhancing optic nerve regeneration in mammals.

Differential responses to high-frequency electrical stimulation in ON and OFF retinal ganglion cells

Abstract: Objective. The field of retinal prosthetics for artificial vision has advanced considerably in recent years, however clinical outcomes remain inconsistent. The performance of retinal prostheses is likely limited by the inability of electrical stimuli to preferentially activate different types of retinal ganglion cell (RGC). Approach. Here we examine the response of rabbit RGCs to high-frequency stimulation, using biphasic pulses applied at 2000 pulses per second. Responses were recorded using cell-attached patch clamp methods, and stimulation was applied epiretinally via a small cone electrode. Main results. When prolonged stimulus trains were applied to OFF-brisk transient (BT) RGCs, the cells exhibited a non-monotonic relationship between response strength and stimulus amplitude; this response pattern was different from those elicited previously by other electrical stimuli. When the amplitude of the stimulus was modulated transiently from a non-zero baseline amplitude, ON-BT and OFF-BT cells exhibited different activity patterns: ON cells showed an increase in activity while OFF cells exhibited a decrease in activity. Using a different envelope to modulate the amplitude of the stimulus, we observed the opposite effect: ON cells exhibited a decrease in activity while OFF cells show an increase in activity. Significance. As ON and OFF RGCs often exhibit opposing activity patterns in response to light stimulation, this work suggests that high-frequency electrical stimulation of RGCs may be able to elicit responses that are more physiological than traditional pulsatile stimuli. Additionally, the prospect of an electrical stimulus capable of cell-type specific selective activation has broad applications throughout the fields of neural stimulation and neuroprostheses.

Axonal loss of retinal neurons in multiple sclerosis associated with optic radiation lesions

Abstract: Objective: To investigate the potential links between thinning of retinal ganglion cell axons in eyes of patients with multiple sclerosis (MS) without past optic neuritis (ON) and MS-related inflammatory damage of the posterior visual pathway. Methods: Temporal retinal nerve fiber layer (tRNFL) thickness was analyzed in eyes with no history of ON (NON) from 53 patients with relapsing-remitting MS. Fifty normal age- and sex-matched controls were examined with optical coherence tomography. Low-contrast visual acuity charts were used for functional assessment of vision. The optic tract (OT) and optic radiation (OR) were identified using probabilistic tractography, and volume of T2 fluid-attenuated inversion recovery lesions and diffusion tensor imaging (DTI) indices were measured within both structures. Cross-sectional diameter of the OT was also calculated. Results: tRNFL thickness was significantly reduced in NON eyes and was associated with reduced low-contrast visual acuity. Lesions within the OR were detected in the majority of patients. There was a significant correlation between thinning of the tRNFL and OR lesion volume (adjusted for non-OR lesion volume, age, sex, and disease duration). tRNFL thickness also correlated with OR DTI indices. No OT lesions were identified in any of the patients and no relationship between retinal nerve fiber layer loss and potential markers of OT lesions was found. Conclusion: The results demonstrate a strong tract-specific association between loss of tRNFL fibers and MS-related inflammation within OR.

Absence of transient receptor potential vanilloid-1 accelerates stress-induced axonopathy in the optic projection.

Abstract: How neurons respond to stress in degenerative disease is of fundamental importance for identifying mechanisms of progression and new therapeutic targets. Members of the transient receptor potential (TRP) family of cation-selective ion channels are candidates for mediating stress signals, since different subunits transduce a variety of stimuli relevant in both normal and pathogenic physiology. We addressed this possibility for the TRP vanilloid-1 (TRPV1) subunit by comparing how the optic projection of Trpv1−/− mice and age-matched C57 controls responds to stress from elevated ocular pressure, the critical stressor in the most common optic neuropathy, glaucoma. Over a 5 week period of elevated pressure induced by microbead occlusion of ocular fluid, Trpv1−/− accelerated both degradation of axonal transport from retinal ganglion cells to the superior colliculus and degeneration of the axons themselves in the optic nerve. Ganglion cell body loss, which is normally later in progression, occurred in nasal sectors of Trpv1−/− but not C57 retina. Pharmacological antagonism of TRPV1 in rats similarly accelerated ganglion cell axonopathy. Elevated ocular pressure resulted in differences in spontaneous firing rate and action potential threshold current in Trpv1−/− ganglion cells compared with C57. In the absence of elevated pressure, ganglion cells in the two strains had similar firing patterns. Based on these data, we propose that TRPV1 may help neurons respond to disease-relevant stressors by enhancing activity necessary for axonal signaling.

Duplication of TBK1 Stimulates Autophagy in iPSC-derived Retinal Cells from a Patient with Normal Tension Glaucoma.

Abstract: Duplication of theTBK1 gene causes normal tension glaucoma (NTG); however the mechanism by which this copy number variation leads to retinal ganglion cell death is poorly understood. The ability to use skin-derived induced pluripotent stem cells (iPSCs) to investigate the function or dysfunction of a mutant gene product in inaccessible tissues such as the retina now provides us with the ability to interrogate disease pathophysiology in vitro. iPSCs were generated from dermal fibroblasts obtained from a patient with TBK1-associated NTG, via viral transduction of the transcription factors OCT4, SOX2, KLF4, and c-MYC. Retinal progenitor cells and subsequent retinal ganglion cell-like neurons were derived using our previously developed stepwise differentiation protocol. Differentiation to retinal ganglion-like cells was demonstrated via rt-PCR targeted against TUJ1, MAP2, THY1, NF200, ATOH7 and BRN3B and immunohistochemistry targeted against NF200 and ATOH7. Western blot analysis demonstrated that both fibroblasts and retinal ganglion cell-like neurons derived from NTG patients with TBK1 gene duplication have increased levels of LC3-II protein (a key marker of autophagy). Duplication of TBK1 has been previously shown to increase expression of TBK1 and here we demonstrate that the same duplication leads to activation of LC3-II. This suggests that TBK1-associated glaucoma may be caused by dysregulation (over-activation) of this catabolic pathway.

Melanopsin ganglion cells are the most resistant retinal ganglion cell type to axonal injury in the rat retina.

Abstract: We report that the most common retinal ganglion cell type that remains after optic nerve transection is the M1 melanopsin ganglion cell. M1 ganglion cells are members of the intrinsically photosensitive retinal ganglion cell population that mediates non-image-forming vision, comprising ∼2.5% of all ganglion cells in the rat retina. In the present study, M1 ganglion cells comprised 1.7±1%, 28±14%, 55±13% and 82±8% of the surviving ganglion cells 7, 14, 21 and 60 days after optic nerve transection, respectively. Average M1 ganglion cell somal diameter and overall morphological appearance remained unchanged in non-injured and injured retinas, suggesting a lack of injury-induced degeneration. Average M1 dendritic field size increased at 7 and 60 days following optic nerve transection, while average dendritic field size remained similar in non-injured retinas and in retinas at 14 and 21 days after optic nerve transection. These findings demonstrate that M1 ganglion cells are more resistant to injury than other ganglion cell types following optic nerve injury, and provide an opportunity to develop pharmacological or genetic therapeutic approaches to mitigate ganglion cell death and save vision following optic nerve injury.

BDNF Promotes Axon Branching of Retinal Ganglion Cells via miRNA-132 and p250GAP

Abstract: A crucial step in the development of the vertebrate visual system is the branching of retinal ganglion cell (RGC) axons within their target, the superior colliculus/tectum. A major player in this process is the neurotrophin brain-derived neurotrophic factor (BDNF). However, the molecular basis for the signaling pathways mediating BDNF action is less well understood. As BDNF exerts some of its functions by controlling the expression of microRNAs (miRNAs), we investigated whether miRNAs are also involved in BDNF-mediated retinal axon branching. Here, we demonstrate that the expression pattern of miRNA-132 in the retina is consistent with its involvement in this process, and that BDNF induces the upregulation of miRNA-132 in retinal cultures. Furthermore, in vitro gain-of-function and loss-of-function approaches in retinal cultures reveal that miRNA-132 mediates axon branching downstream of BDNF. A known target of miRNA-132 is the Rho family GTPase-activating protein, p250GAP. We find that p250GAP is expressed in RGC axons and mediates the effects of miRNA-132 in BDNF-induced branching. BDNF treatment or overexpression of miRNA-132 leads to a reduction in p250GAP protein levels in retinal cultures, whereas the overexpression of p250GAP abolishes BDNF-induced branching. Finally, we used a loss-of-function approach to show that miRNA-132 affects the maturation of RGC termination zones in the mouse superior colliculus in vivo, while their topographic targeting remains intact. Together, our data indicate that BDNF promotes RGC axon branching during retinocollicular/tectal map formation via upregulation of miRNA-132, which in turn downregulates p250GAP.

Excitatory Synaptic Inputs to Mouse On-Off Direction-Selective Retinal Ganglion Cells Lack Direction Tuning

Abstract: Direction selectivity represents a fundamental visual computation. In mammalian retina, On-Off direction-selective ganglion cells (DSGCs) respond strongly to motion in a preferred direction and weakly to motion in the opposite, null direction. Electrical recordings suggested three direction-selective (DS) synaptic mechanisms: DS GABA release during null-direction motion from starburst amacrine cells (SACs) and DS acetylcholine and glutamate release during preferred direction motion from SACs and bipolar cells. However, evidence for DS acetylcholine and glutamate release has been inconsistent and at least one bipolar cell type that contacts another DSGC (On-type) lacks DS release. Here, whole-cell recordings in mouse retina showed that cholinergic input to On-Off DSGCs lacked DS, whereas the remaining (glutamatergic) input showed apparent DS. Fluorescence measurements with the glutamate biosensor intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) conditionally expressed in On-Off DSGCs showed that glutamate release in both On- and Off-layer dendrites lacked DS, whereas simultaneously recorded excitatory currents showed apparent DS. With GABA-A receptors blocked, both iGluSnFR signals and excitatory currents lacked DS. Our measurements rule out DS release from bipolar cells onto On-Off DSGCs and support a theoretical model suggesting that apparent DS excitation in voltage-clamp recordings results from inadequate voltage control of DSGC dendrites during null-direction inhibition. SAC GABA release is the apparent sole source of DS input onto On-Off DSGCs.

Repetitive mild traumatic brain injury causes optic nerve and retinal damage in a mouse model.

Abstract: There is increasing evidence that long-lasting morphologic and functional consequences can be present in the human visual system after repetitive mild traumatic brain injury (r-mTBI). The exact location and extent of the damage in this condition are not well understood. Using a recently developed mouse model of r-mTBI, we assessed the effects on the retina and optic nerve using histology and immunohistochemistry, electroretinography (ERG), and spectral-domain optical coherence tomography (SD-OCT) at 10 and 13 weeks after injury. Control mice received repetitive anesthesia alone (r-sham). We observed decreased optic nerve diameters and increased cellularity and areas of demyelination in optic nerves in r-mTBI versus r-sham mice. There were concomitant areas of decreased cellularity in the retinal ganglion cell layer and approximately 67% decrease in brain-specific homeobox/POU domain protein 3A-positive retinal ganglion cells in retinal flat mounts. Furthermore, SD-OCT demonstrated a detectable thinning of the inner retina; ERG demonstrated a decrease in the amplitude of the photopic negative response without any change in a- or b-wave amplitude or timing. Thus, the ERG and SD-OCT data correlated well with changes detected by morphometric, histologic, and immunohistochemical methods, thereby supporting the use of these noninvasive methods in the assessment of visual function and morphology in clinical cases of mTBI.

White matter consequences of retinal receptor and ganglion cell damage.

Abstract: PURPOSE Patients with Leber hereditary optic neuropathy (LHON) and cone-rod dystrophy (CRD) have central vision loss; but CRD damages the retinal photoreceptor layer, and LHON damages the retinal ganglion cell (RGC) layer. Using diffusion MRI, we measured how these two types of retinal damage affect the optic tract (ganglion cell axons) and optic radiation (geniculo-striate axons). METHODS Adult onset CRD (n = 5), LHON (n = 6), and healthy controls (n = 14) participated in the study. We used probabilistic fiber tractography to identify the optic tract and the optic radiation. We compared axial and radial diffusivity at many positions along the optic tract and the optic radiation. RESULTS In both types of patients, diffusion measures within the optic tract and the optic radiation differ from controls. The optic tract change is principally a decrease in axial diffusivity; the optic radiation change is principally an increase in radial diffusivity. CONCLUSIONS Both photoreceptor layer (CRD) and retinal ganglion cell (LHON) retinal disease causes substantial change in the visual white matter. These changes can be measured using diffusion MRI. The diffusion changes measured in the optic tract and the optic radiation differ, suggesting that they are caused by different biological mechanisms.

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

Abstract: 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.

Lesions in the posterior visual pathway promote trans-synaptic degeneration of retinal ganglion cells.

Abstract: Objective Retrograde trans-synaptic degeneration of retinal ganglion cell layer (GCL) has been proposed as one of the mechanisms contributing to permanent disability after visual pathway damage We set out to test this mechanism taking advantage of the new methods for imaging the macula with high resolution by optical coherence tomography (OCT) in patients with lesions in the posterior visual pathway Additionally, we explored the association between thinning of GCL as an imaging marker of visual impairment such as visual field defects Methods Retrospective case note review of patients with retrogeniculate lesions studied by spectral domain OCT of the macula and quadrant pattern deviation (PD) of the visual fields Results We analysed 8 patients with either hemianopia or quadrantanopia due to brain lesions (stroke = 5; surgery = 2; infection = 1) We found significant thinning of the GCL in the projecting sector of the retina mapping to the brain lesion Second, we found strong correlation between the PD of the visual field quadrant and the corresponding macular GCL sector for the right (R = 0792, p<0001) and left eyes (R = 0674, p<0001) Conclusions The mapping between lesions in the posterior visual pathway and their projection in the macula GCL sector corroborates retrograde trans-synaptic neuronal degeneration after brain injury as a mechanism of damage with functional consequences This finding supports the use of GCL thickness as an imaging marker of trans-synaptic degeneration in the visual pathway after brain lesions

RNA Sequence Reveals Mouse Retinal Transcriptome Changes Early after Axonal Injury

Abstract: Glaucoma is an ocular disease characterized by progressive retinal ganglion cell (RGC) death caused by axonal injury. However, the underlying mechanisms involved in RGC death remain unclear. In this study, we investigated changes in the transcriptome profile following axonal injury in mice (C57BL/6) with RNA sequencing (RNA-seq) technology. The experiment group underwent an optic nerve crush (ONC) procedure to induce axonal injury in the right eye, and the control group underwent a sham procedure. Two days later, we extracted the retinas and performed RNA-seq and a pathway analysis. We identified 177 differentially expressed genes with RNA-seq, notably the endoplasmic reticulum (ER) stress-related genes Atf3, Atf4, Atf5, Chac1, Chop, Egr1 and Trb3, which were significantly upregulated. The pathway analysis revealed that ATF4 was the most significant upstream regulator. The antioxidative response-related genes Hmox1 and Srxn1, as well as the immune response-related genes C1qa, C1qb and C1qc, were also significantly upregulated. To our knowledge, this is the first reported RNA-seq investigation of the retinal transcriptome and molecular pathways in the early stages after axonal injury. Our results indicated that ER stress plays a key role under these conditions. Furthermore, the antioxidative defense and immune responses occurred concurrently in the early stages after axonal injury. We believe that our study will lead to a better understanding of and insight into the molecular mechanisms underlying RGC death after axonal injury.

Tumor necrosis factor‐alpha mediates activation of NF‐κB and JNK signaling cascades in retinal ganglion cells and astrocytes in opposite ways

Abstract: Tumor necrosis factor-alpha (TNF) is an important mediator of the innate immune response in the retina. TNF can activate various signaling cascades, including NF-κB, nuclear factor kappa B (NF-κB) and c-Jun N-terminal kinase (JNK) pathways. The harmful role of these pathways, as well as of TNF, has previously been shown in several retinal neurodegenerative conditions including glaucoma and retinal ischemia. However, TNF and TNF-regulated signaling cascades are capable not only of mediating neurotoxicity, but of being protective. We performed this study to delineate the beneficial and detrimental effects of TNF signaling in the retina. To this end, we used TNF-treated primary retinal ganglion cell (RGC) and astrocyte cultures. Levels of expression of NF-κB subunits in RGCs and astrocytes were evaluated by quantitative RT-PCR (qRT-PCR) and Western blot (WB) analysis. NF-κB and JNK activity in TNF-treated cells was determined in a time-dependent manner using ELISA and WB. Gene expression in TNF-treated astrocytes was measured by qRT-PCR. We found that NF-κB family members were present in RGCs and astrocytes at the mRNA and protein levels. RGCs failed to activate NF-κB in the presence of TNF, a phenomenon that was associated with sustained JNK activation and RGC death. However, TNF initiated the activation of NF-κB and mediated transient JNK activation in astrocytes. These events were associated with glial survival and increased expression of neurotoxic pro-inflammatory factors. Our findings suggest that, in the presence of TNF, NF-κB and JNK signaling cascades are activated in opposite ways in RGCs and astrocytes. These events can directly and indirectly facilitate RGC death.

Methods of Retinal Ganglion Cell Differentiation From Pluripotent Stem Cells.

Abstract: Glaucoma, the worldwide leading cause of irreversible blindness, is characterized by progressive degeneration of the optic nerve and loss of retinal ganglion cells. Research into glaucoma pathogenesis has been hampered by difficulties in isolating and culturing retinal ganglion cells in vitro. However, recent improvements in laboratory techniques have enabled the generation of a variety of mature cell types from pluripotent stem cells, including retinal ganglion cells. Indeed, stem cell-based approaches have the potential to revolutionize the field by providing an unlimited source of cells for replacement therapies and by enabling development of in vitro disease models for drug screening and research. Consequently, research aimed at directing pluripotent stem cells to differentiate into retinal ganglion cells has expanded dramatically during the past decade, resulting in significant advances in technique and efficiency. In this paper, we review the methodology for retinal ganglion cell differentiation from pluripotent stem cells of both mouse and human origin and summarize how these techniques have opened up new avenues for modelling glaucoma. Generation of stem cell-derived retinal ganglion cells will have significant translational values, providing an in vitro platform to study the mechanisms responsible for pathogenesis and for drug screening to improve treatment options, as well as for the development of cell therapies for optic neuropathies such as glaucoma.

Histone deacetylase 3 (HDAC3) plays an important role in retinal ganglion cell death after acute optic nerve injury

Abstract: Optic nerve damage initiates a series of early atrophic events in retinal ganglion cells (RGCs) that precede the BAX-dependent committed step of the intrinsic apoptotic program. Nuclear atrophy, including global histone deacetylation, heterochromatin formation, shrinkage and collapse of nuclear structure, and the silencing of normal gene expression, comprise an important obstacle to overcome in therapeutic approaches to preserve neuronal function. Several studies have implicated histone deacetylases (HDACs) in the early stages of neuronal cell death, including RGCs. Importantly, these neurons exhibit nuclear translocation of HDAC3 shortly after optic nerve damage. Additionally, HDAC3 activity has been reported to be selectively toxic to neurons. RGC-specific conditional knockout of Hdac3 was achieved by transducing the RGCs of Hdac3 fl/fl mice with an adeno-associated virus serotype 2 carrying CRE recombinase and GFP (AAV2-Cre/GFP). Controls included similar viral transduction of Rosa26 fl/fl reporter mice. Optic nerve crush (ONC) was then performed on eyes. The ablation of Hdac3 in RGCs resulted in significant amelioration of characteristics of ONC-induced nuclear atrophy such as H4 deacetylation, heterochromatin formation, and the loss of nuclear structure. RGC death was also significantly reduced. Interestingly, loss of Hdac3 expression did not lead to protection against RGC-specific gene silencing after ONC, although this effect was achieved using the broad spectrum inhibitor, Trichostatin A. Although other HDACs may be responsible for gene expression changes in RGCs, our results indicate a critical role for HDAC3 in nuclear atrophy in RGC apoptosis following axonal injury. This study provides a framework for studying the roles of other prevalent retinal HDACs in neuronal death as a result of axonal injury.