The Atoh7 remote enhancer provides transcriptional robustness during retinal ganglion cell development.

The retinal ganglion cell (RGC) competence factor ATOH7 is dynamically expressed during retinal histogenesis. ATOH7 transcription is controlled by a promoter-adjacent primary enhancer and a remote shadow enhancer (SE). Deletion of the ATOH7 human SE causes nonsyndromic congenital retinal nonattachment (NCRNA) disease, characterized by optic nerve aplasia and total blindness. We used genome editing to model NCRNA in mice. Deletion of the murine SE reduces Atoh7 messenger RNA (mRNA) fivefold but does not recapitulate optic nerve loss; however, SEdel/knockout (KO) trans heterozygotes have thin optic nerves. By analyzing Atoh7 mRNA and protein levels, RGC development and survival, and chromatin landscape effects, we show that the SE ensures robust Atoh7 transcriptional output. Combining SE deletion and KO and wild-type alleles in a genotypic series, we determined the amount of Atoh7 needed to produce a normal complement of adult RGCs, and the secondary consequences of graded reductions in Atoh7 dosage. Together, these data reveal the workings of an evolutionary fail-safe, a duplicate enhancer mechanism that is hard-wired in the machinery of vertebrate retinal ganglion cell genesis.

Neuron-astrocyte transmitophagy is altered in Alzheimer's disease.

Under physiological conditions in vivo astrocytes internalize and degrade neuronal mitochondria in a process called transmitophagy. Mitophagy is widely reported to be impaired in neurodegeneration but it is unknown whether and how transmitophagy is altered in Alzheimer's disease (AD). Here we report that the internalization of neuronal mitochondria is significantly increased in astrocytes isolated from AD mouse brains. We also demonstrate that the degradation of neuronal mitochondria by astrocytes is increased in AD mice at the age of 6 months onwards. Furthermore, we demonstrate for the first time a similar phenomenon between human neurons and AD astrocytes, and in murine hippocampi in vivo. The results suggest the involvement of S100a4 in impaired mitochondrial transfer between neurons and AD astrocytes together with significant increases in the mitophagy regulator and reactive oxygen species in aged AD astrocytes. These findings demonstrate altered neuron-supporting functions of AD astrocytes and provide a starting point for studying the molecular mechanisms of transmitophagy in AD.

Effects of intravitreal injection of human CD34+ bone marrow stem cells in a murine model of diabetic retinopathy.

Human CD34 + stem cells are mobilized from bone marrow to sites of tissue ischemia and play an important role in tissue revascularization. This study used a murine model to test the hypothesis that intravitreal injection of human CD34 + stem cells harvested from bone marrow (BMSCs) can have protective effects in eyes with diabetic retinopathy. Streptozotocin-induced diabetic mice (C57BL/6J) were used as a model for diabetic retinopathy. Subcutaneous implantation of Alzet pump, loaded with Tacrolimus and Rapamycin, 5 days prior to intravitreal injection provided continuous systemic immunosuppression for the study duration to avoid rejection of human cells. Human CD34 + BMSCs were harvested from the mononuclear cell fraction of bone marrow from a healthy donor using magnetic beads. The CD34 + cells were labeled with enhanced green fluorescent protein (EGFP) using a lentiviral vector. The right eye of each mouse received an intravitreal injection of 50,000 EGFP-labeled CD34 + BMSCs or phosphate buffered saline (PBS). Simultaneous multimodal in vivo retinal imaging system consisting of fluorescent scanning laser ophthalmoscopy (enabling fluorescein angiography), optical coherence tomography (OCT) and OCT angiography was used to confirm the development of diabetic retinopathy and study the in vivo migration of the EGFP-labeled CD34 + BMSCs in the vitreous and retina following intravitreal injection. After imaging, the mice were euthanized, and the eyes were removed for immunohistochemistry. In addition, microarray analysis of the retina and retinal flat mount analysis of retinal vasculature were performed. The development of retinal microvascular changes consistent with diabetic retinopathy was visualized using fluorescein angiography and OCT angiography between 5 and 6 months after induction of diabetes in all diabetic mice. These retinal microvascular changes include areas of capillary nonperfusion and late leakage of fluorescein dye. Multimodal in vivo imaging and immunohistochemistry identified EGFP-labeled cells in the superficial retina and along retinal vasculature at 1 and 4 weeks following intravitreal cell injection. Microarray analysis showed changes in expression of 162 murine retinal genes following intravitreal CD34 + BMSC injection when compared to PBS-injected control. The major molecular pathways affected by intravitreal CD34 + BMSC injection in the murine retina included pathways implicated in the pathogenesis of diabetic retinopathy including Toll-like receptor, MAP kinase, oxidative stress, cellular development, assembly and organization pathways. At 4 weeks following intravitreal injection, retinal flat mount analysis showed preservation of the retinal vasculature in eyes injected with CD34 + BMSCs when compared to PBS-injected control. The study findings support the hypothesis that intravitreal injection of human CD34 + BMSCs results in retinal homing and integration of these human cells with preservation of the retinal vasculature in murine eyes with diabetic retinopathy.

Astrocytes phagocytose focal dystrophies from shortening myelin segments in the optic nerve of Xenopus laevis at metamorphosis.

Oligodendrocytes can adapt to increases in axon diameter through the addition of membrane wraps to myelin segments. Here, we report that myelin segments can also decrease their length in response to optic nerve (ON) shortening during Xenopus laevis metamorphic remodeling. EM-based analyses revealed that myelin segment shortening is accomplished by focal myelin-axon detachments and protrusions from otherwise intact myelin segments. Astrocyte processes remove these focal myelin dystrophies using known phagocytic machinery, including the opsonin milk fat globule-EGF factor 8 (Mfge8) and the downstream effector ras-related C3 botulinum toxin substrate 1 (Rac1). By the end of metamorphic nerve shortening, one-quarter of all myelin in the ON is enwrapped or internalized by astrocytes. As opposed to the removal of degenerating myelin by macrophages, which is usually associated with axonal pathologies, astrocytes selectively remove large amounts of myelin without damaging axons during this developmental remodeling event.

Transcellular degradation of axonal mitochondria.

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.

Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death.

Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations.

Myelination transition zone astrocytes are constitutively phagocytic and have synuclein dependent reactivity in glaucoma.

Optic nerve head (ONH) astrocytes have been proposed to play both protective and deleterious roles in glaucoma. We now show that, within the postlaminar ONH myelination transition zone (MTZ), there are astrocytes that normally express Mac-2 (also known as Lgals3 or galectin-3), a gene typically expressed only in phagocytic cells. Surprisingly, even in healthy mice, MTZ and other ONH astrocytes constitutive internalize large axonal evulsions that contain whole organelles. In mouse glaucoma models, MTZ astrocytes further up-regulate Mac-2 expression. During glaucomatous degeneration, there are dystrophic processes in the retina and optic nerve, including the MTZ, which contain protease resistant γ-synuclein. The increased Mac-2 expression by MTZ astrocytes during glaucoma likely depends on this γ-synuclein, as mice lacking γ-synuclein fail to up-regulate Mac-2 at the MTZ after elevation of intraocular pressure. These results suggest the possibility that a newly discovered normal degradative pathway for axons might contribute to glaucomatous neurodegeneration.

Dual leucine zipper kinase is necessary for retinal ganglion cell axonal regeneration in Xenopus laevis.

Retinal ganglion cell (RGC) axons of the African clawed frog, Xenopus laevis, unlike those of mammals, are capable of regeneration and functional reinnervation of central brain targets following injury. Here, we describe a tadpole optic nerve crush (ONC) procedure and assessments of brain reinnervation based on live imaging of RGC-specific transgenes which, when paired with CRISPR/Cas9 injections at the one-cell stage, can be used to assess the function of regeneration-associated genes in vivo in F0 animals. Using this assay, we find that map3k12, also known as dual leucine zipper kinase (Dlk), is necessary for RGC axonal regeneration and acts in a dose-dependent manner. Loss of Dlk does not affect RGC innervation of the brain during development or visually driven behavior but does block both axonal regeneration and functional vision restoration after ONC. Dlk loss does not alter the acute changes in mitochondrial movement that occur within RGC axons hours after ONC but does completely block the phosphorylation and nuclear translocation of the transcription factor Jun within RGCs days after ONC; yet, Jun is dispensable for reinnervation. These results demonstrate that in a species fully capable of regenerating its RGC axons, Dlk is essential for the axonal injury signal to reach the nucleus but may affect regeneration through a different pathway than by which it signals in mammalian RGCs.

Glaucoma-associated Optineurin mutations increase transmitophagy in a vertebrate optic nerve.

We previously described a process referred to as transmitophagy where mitochondria shed by retinal ganglion cell (RGC) axons are transferred to and degraded by surrounding astrocytes in the optic nerve head of mice. Since the mitophagy receptor Optineurin (OPTN) is one of few large-effect glaucoma genes and axonal damage occurs at the optic nerve head in glaucoma, here we explored whether OPTN mutations perturb transmitophagy. Live-imaging of Xenopus laevis optic nerves revealed that diverse human mutant but not wildtype OPTN increase stationary mitochondria and mitophagy machinery and their colocalization within, and in the case of the glaucoma-associated OPTN mutations also outside of, RGC axons. These extra-axonal mitochondria are degraded by astrocytes. Our studies support the view that in RGC axons under baseline conditions there are low levels of mitophagy, but that glaucoma-associated perturbations in OPTN result in increased axonal mitophagy involving the shedding and astrocytic degradation of the mitochondria.

Evaluating the performance of OCT in assessing static and potential dynamic properties of the retinal ganglion cells and nerve fiber bundles in the living mouse eye.

Glaucoma is a group of eye diseases characterized by the thinning of the retinal nerve fiber layer (RNFL), which is primarily caused by the progressive death of retinal ganglion cells (RGCs). Precise monitoring of these changes at a cellular resolution in living eyes is significant for glaucoma research. In this study, we aimed to assess the effectiveness of temporal speckle averaging optical coherence tomography (TSA-OCT) and dynamic OCT (dOCT) in examining the static and potential dynamic properties of RGCs and RNFL in living mouse eyes. We evaluated parameters such as RNFL thickness and possible dynamics, as well as compared the ganglion cell layer (GCL) soma density obtained from in vivo OCT, fluorescence scanning laser ophthalmoscopy (SLO), and ex vivo histology.

Selective pattern of motor system damage in gamma-synuclein transgenic mice mirrors the respective pathology in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is characterised by substantial loss of both upper and lower motor neuron function, with sensory and cognitive systems less affected. Though heritable forms of the disease have been described, the vast majority of cases are sporadic with poorly defined underlying pathogenic mechanisms. Here we demonstrate that the neurological pathology induced in transgenic mice by overexpression of γ-synuclein, a protein not previously associated with ALS, recapitulates key features of the disease, namely selective damage and loss of discrete populations of upper and lower motor neurons and their axons, contrasted by limited effects upon the sensory system.

Optic Nerve Head Myelin-Related Protein, GFAP, and Iba1 Alterations in Non-Human Primates With Early to Moderate Experimental Glaucoma.

Purpose: The purpose of this study was to test if optic nerve head (ONH) myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), glial fibrillary acidic protein (GFAP), and ionized calcium binding adaptor molecule 1 (Iba1) proteins are altered in non-human primate (NHP) early/moderate experimental glaucoma (EG). Methods: Following paraformaldehyde perfusion, control and EG eye ONH tissues from four NHPs were paraffin embedded and serially (5 µm) vertically sectioned. Anti-MBP, CNPase, GFAP, Iba1, and nuclear dye-stained sections were imaged using sub-saturating light intensities. Whole-section images were segmented creating anatomically consistent laminar (L) and retrolaminar (RL) regions/sub-regions. EG versus control eye intensity/pixel-cluster density data within L and two RL regions (RL1 [1-250 µm]/RL2 [251-500 µm] from L) were compared using random effects models within the statistical program "R." Results: EG eye retinal nerve fiber loss ranged from 0% to 20%. EG eyes' MBP and CNPase intensity were decreased within the RL1 (MBP = 31.4%, P < 0.001; CNPase =62.3%, P < 0.001) and RL2 (MBP = 19.6%, P < 0.001; CNPase = 56.1%, P = 0.0004) regions. EG eye GFAP intensity was decreased in the L (41.6%, P < 0.001) and RL regions (26.7% for RL1, and 28.4% for RL2, both P < 0.001). Iba1+ and NucBlue pixel-cluster density were increased in the laminar (28.2%, P = 0.03 and 16.6%, P = 0.008) and both RL regions (RL1 = 37.3%, P = 0.01 and 23.7%, P = 0.0002; RL2 = 53.7%, P = 0.002 and 33.2%, P < 0.001). Conclusions: Retrolaminar myelin disruption occurs early in NHP EG and may be accompanied by laminar and retrolaminar decreases in astrocyte process labeling and increases in microglial/ macrophage density. The mechanistic and therapeutic implications of these findings warrant further study.

The basic science of optic nerve regeneration.

Diverse insults to the optic nerve result in partial to total vision loss as the axons of retinal ganglion cells are destroyed. In glaucoma, axons are injured at the optic nerve head; in other optic neuropathies, axons can be damaged along the entire visual pathway. In all cases, as mammals cannot regenerate injured central nervous system cells, once the axons are lost, vision loss is irreversible. However, much has been learned about how retinal ganglion cells respond to axon injuries, and many of these crucial discoveries offer hope for future regenerative therapies. Here we review the current understanding regarding the temporal progression of axonal degeneration. We summarize known survival and regenerative mechanisms in mammals, including specific signaling pathways, key transcription factors, and reprogramming genes. We cover mechanisms intrinsic to retinal ganglion cells as well as their interactions with myeloid and glial cell populations in the retina and optic nerve that affect survival and regeneration. Finally, we highlight some non-mammalian species that are able to regenerate their retinal ganglion cell axons after injury, as understanding these successful regenerative responses may be essential to the rational design of future clinical interventions to regrow the optic nerve. In the end, a combination of many different molecular and cellular interventions will likely be the only way to achieve functional recovery of vision and restore quality of life to millions of patients around the world.

Analysis of the retinal capillary plexus layers in a murine model with diabetic retinopathy: effect of intravitreal injection of human CD34+ bone marrow stem cells.

BACKGROUND: Diabetic retinopathy is a retinal vasculopathy involving all three retinal capillary plexus layers. Since human CD34+ bone marrow stem cells (BMSCs) have the potential to promote revascularization of ischemic tissue, this study tests the hypothesis that intravitreal injection of human CD34+ BMSCs can have protective effects on all layers of the retinal vasculature in eyes with diabetic retinopathy. METHODS: Streptozotocin (STZ)-induced diabetic mice were injected intravitreally with 50,000 human CD34+ BMSCs or phosphate-buffered saline (PBS) into the right eye. Systemic immunosuppression with rapamycin and tacrolimus was started 5 days before the injection and maintained for study duration to prevent rejection of human cells. All mice were euthanized 4 weeks after intravitreal injection; both eyes were enucleated for retinal flat mount immunohistochemistry. The retinal vasculature was stained with Isolectin-GS-IB4. Confocal microscopy was used to image four circular areas of interest of retina, 1-mm diameter around the optic disc. Images of superficial, intermediate, and deep retinal capillary plexus layers within the areas of interest were obtained and analyzed using ImageJ software with the Vessel Analysis plugin to quantitate the retinal vascular density and vascular length density in the three plexus layers. RESULTS: Three distinct retinal capillary plexus layers were visualized and imaged using confocal microscopy. Eyes that received intravitreal injection of CD34+ BMSCs (N=9) had significantly higher vascular density and vascular length density in the superficial retinal capillary plexus when compared to the untreated contralateral eyes (N=9) or PBS treated control eyes (N=12; P values <0.05 using ANOVA followed by post-hoc tests). For the intermediate and deep plexus layers, the difference was not statistically significant. CONCLUSIONS: The protective effect of intravitreal injection of the human CD34+ BMSCs on the superficial retinal capillary plexus layers is demonstrated using confocal microscopy in this murine model of diabetic retinopathy.

Temporal regulation of Ath5 gene expression during eye development.

During central nervous system development the timing of progenitor differentiation must be precisely controlled to generate the proper number and complement of neuronal cell types. Proneural basic helix-loop-helix (bHLH) transcription factors play a central role in regulating neurogenesis, and thus the timing of their expression must be regulated to ensure that they act at the appropriate developmental time. In the developing retina, the expression of the bHLH factor Ath5 is controlled by multiple signals in early retinal progenitors, although less is known about how these signals are coordinated to ensure correct spatial and temporal pattern of gene expression. Here we identify a key distal Xath5 enhancer and show that this enhancer regulates the early phase of Xath5 expression, while the proximal enhancer we previously identified acts later. The distal enhancer responds to Pax6, a key patterning factor in the optic vesicle, while FGF signaling regulates Xath5 expression through sequences outside of this region. In addition, we have identified an inhibitory element adjacent to the conserved distal enhancer region that is required to prevent premature initiation of expression in the retina. This temporal regulation of Xath5 gene expression is comparable to proneural gene regulation in Drosophila, whereby separate enhancers regulate different temporal phases of expression.

Glaucomatous optic nerve injury involves early astrocyte reactivity and late oligodendrocyte loss.

Glaucoma, a neurodegenerative disease affecting retinal ganglion cells (RGC), is a leading cause of blindness. Since gliosis is common in neurodegenerative disorders, it is important to describe the changes occurring in various glial populations in glaucoma animal models in relation to axon loss, as only changes that occur early are likely to be useful therapeutic targets. Here, we describe changes occurring in glia within the myelinated portion of the optic nerve (ON) in both DBA/2J mice and in a rat ocular hypertension model. In both glaucoma animal models, we found only a modest loss of oligodendrocytes that occurred after axons had already degenerated. In DBA/2J mice there was proliferation of oligodendrocyte precursor cells (OPCs) and new oligodendrocyte generation. Activation of microglia was detected only in highly degenerated DBA/2J ONs. In contrast, a large increase in astrocyte reactivity occurred early in both animal models. These results are consistent with astrocytes playing a prominent role in regulating axon loss in glaucoma.

Role of visual pigment properties in rod and cone phototransduction.

Retinal rods and cones share a phototransduction pathway involving cyclic GMP. Cones are typically 100 times less photosensitive than rods and their response kinetics are several times faster, but the underlying mechanisms remain largely unknown. Almost all proteins involved in phototransduction have distinct rod and cone variants. Differences in properties between rod and cone pigments have been described, such as a 10-fold shorter lifetime of the meta-II state (active conformation) of cone pigment and its higher rate of spontaneous isomerization, but their contributions to the functional differences between rods and cones remain speculative. We have addressed this question by expressing human or salamander red cone pigment in Xenopus rods, and human rod pigment in Xenopus cones. Here we show that rod and cone pigments when present in the same cell produce light responses with identical amplification and kinetics, thereby ruling out any difference in their signalling properties. However, red cone pigment isomerizes spontaneously 10,000 times more frequently than rod pigment. This high spontaneous activity adapts the native cones even in darkness, making them less sensitive and kinetically faster than rods. Nevertheless, additional factors are probably involved in these differences.

Programmed switch in the mitochondrial degradation pathways during human retinal ganglion cell differentiation from stem cells is critical for RGC survival.

Retinal ganglion cell (RGC) degeneration is the root cause for vision loss in glaucoma as well as in other forms of optic neuropathy. A variety of studies have implicated abnormal mitochondrial quality control (MQC) as contributing to RGC damage and degeneration in optic neuropathies. The ability to differentiate human pluripotent stem cells (hPSCs) into RGCs provides an opportunity to study RGC MQC in great detail. Degradation of damaged mitochondria is a critical step of MQC, and here we have used hPSC-derived RGCs (hRGCs) to analyze how altered mitochondrial degradation pathways in hRGCs affect their survival. Using pharmacological methods, we have investigated the role of the proteasomal and endo-lysosomal pathways in degrading damaged mitochondria in hRGCs and their precursor stem cells. We found that upon mitochondrial damage induced by the proton uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP), hRGCs more efficiently degraded mitochondria than did their precursor stem cells. We further identified that for degrading damaged mitochondria, stem cells predominantly use the ubiquitine-proteasome system (UPS) while hRGCs use the endo-lysosomal pathway. UPS inhibition causes apoptosis and cell death in stem cells, while hRGC viability is dependent on the endo-lysosomal pathway but not on the UPS pathway. These findings suggest that manipulation of the endo-lysosomal pathway could be therapeutically relevant for RGC protection in treating optic neuropathies associated with mitophagy defects. Endo-lysosome dependent cell survival is also conserved in other human neurons as we found that differentiated human cerebral cortical neurons also degenerated upon endo-lysosomal inhibition but not with proteasome inhibition.

Progressive ganglion cell degeneration precedes neuronal loss in a mouse model of glaucoma.

Glaucoma is characterized by retinal ganglion cell (RGC) pathology and a progressive loss of vision. Previous studies suggest RGC death is responsible for vision loss in glaucoma, yet evidence from other neurodegenerative diseases suggests axonal degeneration, in the absence of neuronal loss, can significantly affect neuronal function. To characterize RGC degeneration in the DBA/2 mouse model of glaucoma, we quantified RGCs in mice of various ages using neuronal-specific nuclear protein (NeuN) immunolabeling, retrograde labeling, and optic nerve axon counts. Surprisingly, the number of NeuN-labeled RGCs did not decline significantly until 18 months of age, at which time a significant decrease in RGC somal size was also observed. Axon dysfunction and degeneration occurred before loss of NeuN-positive RGCs, because significant declines in RGC number assayed by retrograde tracers and axon counts were observed at 13 months. To examine whether axonal dysfunction/degeneration affected gene expression in RGC axons or somas, NeuN and neurofilament-heavy (NF-H) immunolabeling was performed along with quantitative reverse transcription-PCR for RGC-specific genes in retinas of aged DBA/2 mice. Although these mice had similar numbers of NeuN-positive RGCs, the expression of neurofilament light, Brn-3b, and Sncg mRNA varied; this variation in RGC-specific gene expression was correlated with the appearance of NF-H immunoreactive RGC axons. Together, these data support a progression of RGC degeneration in this model of glaucoma, beginning with loss of retrograde label, where axon dysfunction and degeneration precede neuronal loss. This progression of degeneration suggests a need to examine the RGC axon as a locus of pathology in glaucoma.

Retinal ganglion cells downregulate gene expression and lose their axons within the optic nerve head in a mouse glaucoma model.

Little is known about molecular changes occurring within retinal ganglion cells (RGCs) before their death in glaucoma. Taking advantage of the fact that gamma-synuclein (Sncg) mRNA is expressed specifically and highly in adult mouse RGCs, we show in the DBA/2J mouse model of glaucoma that there is not only a loss of cells expressing this gene, but also a downregulation of gene expression of Sncg and many other genes within large numbers of RGCs. This downregulation of gene expression within RGCs occurs together with reductions in FluoroGold (FG) retrograde transport. Surprisingly, there are also large numbers of Sncg-expressing cells without any FG labeling, and among these many that have a marker previously associated with disconnected RGCs, accumulation of phosphorylated neurofilaments in their somas. These same diseased retinas also have large numbers of RGCs that maintain the intraocular portion while losing the optic nerve portion of their axons, and these disconnected axons terminate within the optic nerve head. Our data support the view that RGC degeneration in glaucoma has two separable stages: the first involves atrophy of RGCs, whereas the second involves an insult to axons, which causes the degeneration of axon portions distal to the optic nerve head but does not cause the immediate degeneration of intraretinal portions of axons or the immediate death of RGCs.