Quantitative iTRAQ analysis of retinal ganglion cell degeneration after optic nerve crush

Retinal ganglion cells (RGCs) are central nervous system (CNS) neurons that transmit visual information from the retina to the brain. Apoptotic RGC degeneration causes visual impairment that can be modeled by optic nerve crush. Neuronal apoptosis is also a salient feature of CNS trauma, ischemia (stroke), and diseases of the CNS such as Alzheimer's, Parkinson's, multiple sclerosis, and amyotrophic lateral sclerosis. Optic nerve crush induces the apoptotic cell death of ～ 70% of RGCs within the first 14 days after injury. This model is particularly attractive for studying adult neuron apoptosis because the time-course of RGC death is well established and axon regeneration within the myelinated optic nerve can be concurrently evaluated. Here, we performed a large scale iTRAQ proteomic study to identify and quantify proteins of the rat retina at 1, 3, 4, 7, 14, and 21 days after optic nerve crush. In total, 337 proteins were identified, and 110 were differentially regulated after injury. Of these, 58 proteins were upregulated (>1.3 ×), 46 were downregulated (<0.7 ×), and 6 showed both positive and negative regulation over 21 days, relative to normal retinas. Among the differentially expressed proteins, Thymosin-β4 showed an early upregulation at 3 days, the time-point that immediately precedes the induction of RGC apoptosis after injury. We examined the effect of exogenous Thymosin-β4 administration on RGC death after optic nerve injury. Intraocular injections of Thymosin-β4 significantly increased RGC survival by ～ 3-fold compared to controls and enhanced axon regeneration after crush, demonstrating therapeutic potential for CNS insults. Overall, our study identified numerous proteins that are differentially regulated at key time-points after optic nerve crush, and how the temporal profiles of their expression parallel RGC death. This data will aid in the future development of novel therapeutics to promote neuronal survival and regeneration in the adult CNS.     

Neuroprotectin D1 inhibits retinal ganglion cell death following axotomy

Neuroprotectin D1 (NPD1), a docosahexaenoic acid-derived autacoid, is an endogenous neuroprotective and anti-inflammatory mediator that is generated in the retina and brain. The effects of exogenous NPD1 on retinal ganglion cell (RGC) apoptosis and the role of 12/15-lipoxygenase (Alox15) in retina were evaluated after optic nerve transection (ONT). Treatment with NPD1 was associated with significant protection against RGC death. The percentage of RGC survival in NPD1-treated group was 30% at 2 weeks after ONT as compared with 12% of RGC survival in the ONT group without treatment. Endogenous NPD1 was a predominant lipid autocoid in uninjured and axotomized retinas. Alox15 mRNA expression was upregulated in retinas following ONT suggesting that amplification of 12/15-lipoxygenase (12/15-LOX) may represent a neuroprotective response in the rat retina. The density of RGCs was higher in the normal retina of 12/15-LOX-deficient mice as compared with congenic controls. Hence, the resident NPD1 has a potential role in the physiological and pathophysiological responses of the retina.     

Glial Cells Activation Potentially Contributes to the Upregulation of Stromal Cell-Derived Factor-1α After Optic Nerve Crush in Rats

Stromal cell-derived factor-1α (SDF-1α) plays an important role after injury. However, little is known regarding its temporal and spatial expression patterns or how it interacts with glial cells after optic nerve crush injury. We characterized the temporal and spatial expression pattern of SDF-1α in the retina and optic nerve following optic nerve crush and demonstrated that SDF-1α is localized to the glial cells that are distributed in the retina and optic nerve. CXCR4, the receptor for SDF-1α, is expressed along the ganglion cell layer (GCL). The relative expression levels of Sdf-1α mRNA and SDF-1α protein in the retina and optic nerve 1, 2, 3, 5, 7, 10 and 14 days after injury were determined using real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay, respectively, and the Cxcr4 mRNA expression was determined using real-time PCR. Immunofluorescence and immunohistochemical approaches were used to detect the localization of SDF-1α and CXCR4 after injury. The upregulation of Sdf-1α and Cxcr4 mRNA was detected as early as day one after injury in the retina and day two in the optic nerve, the expression peaks 5–7 days after injury. The expression of Sdf-1α and Cxcr4 mRNA was maintained for at least 14 days after the optic nerve crush injury. Furthermore, SDF-1α-positive zones were distributed locally in the reactive glial cells, which suggested potential autocrine stimulation. CXCR4 was mainly expressed in the GCL, which was also adjacent to the the glial cells. These findings suggest that following optic nerve crush, the levels of endogenous SDF-1α and CXCR4 increase in the retina and optic nerve, where activated glial cells may act as a source of increased SDF-1α protein.     

Early downregulation of IGF-I decides the fate of rat retinal ganglion cells after optic nerve injury

Retinal ganglion cells (RGCs) die by apoptosis after optic nerve injury. A number of reports have separately shown changes in pro-apoptotic proteins such as the Bcl-2 family members following optic nerve injury. However, induction time of these apoptotic signals has not been identified due to different treatments of the optic nerve, and insufficient time intervals for measurements. Therefore, the stream of cell death signals is not well understood. In the present study, we systematically reinvestigated a detailed time course of these cell death/survival signals in the rat retina after optic nerve crush, to determine the signal cascade leading to RGC apoptosis. The most conspicuous changes detected in the retina were the rapid inactivation of phospho-Akt and phospho-Bad proteins 2-3 days after optic nerve damage, and the subsequent gradual activation of Bax protein and caspase-3 activity accompanied by cell loss of RGCs 6 days after nerve injury. Cellular localization of these molecular changes was limited to RGCs. Furthermore, amount of insulin-like growth factor-I (IGF-I), an activator of the phosphatidyl inositol-3-kinase (PI3K)/Akt system, was initially decreased from RGCs 1-2 days just prior to the inactivation of phospho-Akt by optic nerve crush. Conversely, supplementation with IGF-I into the rat retina induced upregulation of phospho-Akt expression and cell survival of RGCs both in vitro and in vivo. Thus, injury to the optic nerve might induce early changes in cellular homeostasis with a plausible loss of trophic support for injured RGCs. Actually, IGF-I drastically enhanced neurite outgrowth from adult rat RGCs via a wortmannin-dependent mechanism in a retinal explant culture. Our data strongly indicate that IGF-I is a key molecule that induces RGC apoptosis or RGC survival and regeneration in the retina during the early stage of optic nerve injury.     

Low Intensity Repetitive Transcranial Magnetic Stimulation Does Not Induce Cell Survival or Regeneration in a Mouse Optic Nerve Crush Model

Low intensity repetitive Transcranial Magnetic Stimulation (LI-rTMS), a non-invasive form of brain stimulation, has been shown to induce structural and functional brain plasticity, including short distance axonal sprouting. However, the potential for LI-rTMS to promote axonal regeneration following neurotrauma has not been investigated. This study examined the effect of LI-rTMS on retinal ganglion cell (RGC) survival, axon regeneration and levels of BDNF in an optic nerve crush neurotrauma model. Adult C57Bl/6J mice received a unilateral intraorbital optic nerve crush. Mice received 10 minutes of sham (handling control without stimulation) (n=6) or LI-rTMS (n = 8) daily stimulation for 14 days to the operated eye. Immunohistochemistry was used to assess RGC survival (β-3 Tubulin) and axon regeneration across the injury (GAP43). Additionally, BDNF expression was quantified in a separate cohort by ELISA in the retina and optic nerve of injured (optic nerve crush) (sham n = 5, LI-rTMS n = 5) and non-injured mice (sham n = 5, LI-rTMS n = 5) that received daily stimulation as above for 7 days. Following 14 days of LI-rTMS there was no significant difference in mean RGC survival between sham and treated animals (p>0.05). Also, neither sham nor LI-rTMS animals showed GAP43 positive labelling in the optic nerve, indicating that regeneration did not occur. At 1 week, there was no significant difference in BDNF levels in the retina or optic nerves between sham and LI-rTMS in injured or non-injured mice (p>0.05). Although LI-rTMS has been shown to induce structural and molecular plasticity in the visual system and cerebellum, our results suggest LI-rTMS does not induce neuroprotection or regeneration following a complete optic nerve crush. These results help define the therapeutic capacity and limitations of LI-rTMS in the treatment of neurotrauma.     

Regulation of GDF-15, a distant TGF-β superfamily member,in a mouse model of cerebral ischemia

GDF-15 is a novel distant member of the TGF-β superfamily and is widely distributed in the brain and peripheral nervous system. We have previously reported that GDF-15 is a potent neurotrophic factor for lesioned dopaminergic neurons in the substantia nigra, and that GDF-15-deficient mice show progressive postnatal losses of motor and sensory neurons. We have now investigated the regulation of GDF-15 mRNA and immunoreactivity in the murine hippocampal formation and selected cortical areas following an ischemic lesion by occlusion of the middle cerebral artery (MCAO). MCAO prominently upregulates GDF-15 mRNA in the hippocampus and parietal cortex at 3 h and 24 h after lesion. GDF-15 immunoreactivity, which is hardly detectable in the unlesioned brain, is drastically upregulated in neurons identified by double-staining with NeuN. NeuN staining reveals that most, if not all, neurons in the granular layer of the dentate gyrus and pyramidal layers of the cornu ammonis become GDF-15-immunoreactive. Moderate induction of GDF-15 immunoreactivity has been observed in a small number of microglial cells identified by labeling with tomato lectin, whereas astroglial cells remain GDF-15-negative after MCAO. Comparative analysis of the size of the infarcted area after MCAO in GDF-15 wild-type and knockout mice has failed to reveal significant differences. Together, our data substantiate the notion that GDF-15 is prominently upregulated in the lesioned brain and might be involved in orchestrating post-lesional responses other than the trophic support of neurons.     

Intravitreal Transplantation of Human Umbilical Cord Blood Stem Cells Protects Rats from Traumatic Optic Neuropathy

Objectives

To treat traumatic optic neuropathy (TON) with transplantation of human umbilical cord blood stem cells (hUCBSC) and explore how transplanted stem cells participate in the neuron repairing process.

Methods

A total of 195 Sprague-Dawley rats were randomly assigned to three groups: sham-surgery, optic nerve injury, and stem cell transplant group. Optic nerve injury was established in rats by directly clamping the optic nerve for 30 seconds. hUCBSC was microinjected into the vitreous cavity of injured rats. Optic nerve function was evaluated by flash visual evoked potentials (F-VEP). Apoptosis in retina tissues was detected by TUNEL staining. GRP78 and CHOP gene expression was measured by RT-PCR.

Results

After injury, transplantation of hUCBSC significantly blunted a reduction in optic nerve function indicated by smaller decreases in amplitude and smaller increases in peak latency of F-VEP waveform compared to the injury alone group. Also, significant more in retinal ganglion cell (RGC) count and less in RGC apoptosis were detected after transplantation compared to injured rats. The protective effect correlated with upregulated GRP78 and downregulated CHOP mRNA expression.

Conclusion

Intravitreal transplantation of hUCBSCs significantly blunted a reduction in optic nerve function through increasing RGC survival and decreasing retinal cell apoptosis. The protective role of transplantation was associated with upregulation of GRP78 expression and downregulation of CHOP expression in retinal cells.     

Serotonin transporter-mediated molecular axis regulates regional retinal ganglion cell vulnerability and axon regeneration after nerve injury

Molecular insights into the selective vulnerability of retinal ganglion cells (RGCs) in optic neuropathies and after ocular trauma can lead to the development of novel therapeutic strategies aimed at preserving RGCs. However, little is known about what molecular contexts determine RGC susceptibility. In this study, we show the molecular mechanisms underlying the regional differential vulnerability of RGCs after optic nerve injury. We identified RGCs in the mouse peripheral ventrotemporal (VT) retina as the earliest population of RGCs susceptible to optic nerve injury. Mechanistically, the serotonin transporter (SERT) is upregulated on VT axons after injury. Utilizing SERT-deficient mice, loss of SERT attenuated VT RGC death and led to robust retinal axon regeneration. Integrin β3, a factor mediating SERT-induced functions in other systems, is also upregulated in RGCs and axons after injury, and loss of integrin β3 led to VT RGC protection and axon regeneration. Finally, RNA sequencing analyses revealed that loss of SERT significantly altered molecular signatures in the VT retina after optic nerve injury, including expression of the transmembrane protein, Gpnmb. GPNMB is rapidly downregulated in wild-type, but not SERT- or integrin β3-deficient VT RGCs after injury, and maintaining expression of GPNMB in RGCs via AAV2 viruses even after injury promoted VT RGC survival and axon regeneration. Taken together, our findings demonstrate that the SERT-integrin β3-GPNMB molecular axis mediates selective RGC vulnerability and axon regeneration after optic nerve injury.     

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.Download video file.^{(53M, mov)}     

Distribution of Mesenchymal Stem Cells and Effects on Neuronal Survival and Axon Regeneration after Optic Nerve Crush and Cell Therapy

Bone marrow-derived cells have been used in different animal models of neurological diseases. We investigated the therapeutic potential of mesenchymal stem cells (MSC) injected into the vitreous body in a model of optic nerve injury. Adult (3–5 months old) Lister Hooded rats underwent unilateral optic nerve crush followed by injection of MSC or the vehicle into the vitreous body. Before they were injected, MSC were labeled with a fluorescent dye or with superparamagnetic iron oxide nanoparticles, which allowed us to track the cells in vivo by magnetic resonance imaging. Sixteen and 28 days after injury, the survival of retinal ganglion cells was evaluated by assessing the number of Tuj1- or Brn3a-positive cells in flat-mounted retinas, and optic nerve regeneration was investigated after anterograde labeling of the optic axons with cholera toxin B conjugated to Alexa 488. Transplanted MSC remained in the vitreous body and were found in the eye for several weeks. Cell therapy significantly increased the number of Tuj1- and Brn3a-positive cells in the retina and the number of axons distal to the crush site at 16 and 28 days after optic nerve crush, although the RGC number decreased over time. MSC therapy was associated with an increase in the FGF-2 expression in the retinal ganglion cells layer, suggesting a beneficial outcome mediated by trophic factors. Interleukin-1β expression was also increased by MSC transplantation. In summary, MSC protected RGC and stimulated axon regeneration after optic nerve crush. The long period when the transplanted cells remained in the eye may account for the effect observed. However, further studies are needed to overcome eventually undesirable consequences of MSC transplantation and to potentiate the beneficial ones in order to sustain the neuroprotective effect overtime.     

A distinct effect of transient and sustained upregulation of cellular factor XIII in the goldfish retina and optic nerve on optic nerve regeneration

Unlike in mammals, fish retinal ganglion cells (RGCs) have a capacity to repair their axons even after optic nerve transection. In our previous study, we isolated a tissue type transglutaminase (TG) from axotomized goldfish retina. The levels of retinal TG (TG(R)) mRNA increased in RGCs 1-6weeks after nerve injury to promote optic nerve regeneration both in vitro and in vivo. In the present study, we screened other types of TG using specific FITC-labeled substrate peptides to elucidate the implications for optic nerve regeneration. This screening showed that the activity of only cellular coagulation factor XIII (cFXIII) was increased in goldfish optic nerves just after nerve injury. We therefore cloned a full-length cDNA clone of FXIII A subunit (FXIII-A) and studied temporal changes of FXIII-A expression in goldfish optic nerve and retina during regeneration. FXIII-A mRNA was initially detected at the crush site of the optic nerve 1h after injury; it was further observed in the optic nerve and achieved sustained long-term expression (1-40days after nerve injury). The cells producing FXIII-A were astrocytes/microglial cells in the optic nerve. By contrast, the expression of FXIII-A mRNA and protein was upregulated in RGCs for a shorter time (3-10days after nerve injury). Overexpression of FXIII-A in RGCs achieved by lipofection induced significant neurite outgrowth from unprimed retina, but not from primed retina with pretreatment of nerve injury. Addition of extracts of optic nerves with injury induced significant neurite outgrowth from primed retina, but not from unprimed retina without pretreatment of nerve injury. The transient increase of cFXIII in RGCs promotes neurite sprouting from injured RGCs, whereas the sustained increase of cFXIII in optic nerves facilitates neurite elongation from regrowing axons.     

Early remodelling of the extracellular matrix proteins tenascin‐C and phosphacan in retina and optic nerve of an experimental autoimmune glaucoma model

Glaucoma is characterized by the loss of retinal ganglion cells (RGCs) and optic nerve fibres. Previous studies noted fewer RGCs after immunization with ocular antigens at 28 days. It is known that changes in extracellular matrix (ECM) components conduct retina and optic nerve degeneration. Here, we focused on the remodelling of tenascin‐C and phosphacan/receptor protein tyrosine phosphatase β/ζ in an autoimmune glaucoma model. Rats were immunized with optic nerve homogenate (ONA) or S100B protein (S100). Controls received sodium chloride (Co). After 14 days, no changes in RGC number were noted in all groups. An increase in GFAP mRNA expression was observed in the S100 group, whereas no alterations were noted via immunohistochemistry in both groups. Extracellular matrix remodelling was analyzed after 3, 7, 14 and 28 days. Tenascin‐C and 473HD immunoreactivity in retinae and optic nerves was unaltered in both immunized groups at 3 days. At 7 days, tenascin‐C staining increased in both tissues in the ONA group. Also, in the optic nerves of the S100 group, an intense tenascin‐C staining could be shown. In the retina, an increased tenascin‐C expression was also observed in ONA animals via Western blot. 473HD immunoreactivity was elevated in the ONA group in both tissues and in the S100 optic nerves at 7 days. At 14 days, tenascin‐C and 473HD immunoreactivity was up‐regulated in the ONA retinae, whereas phosphacan expression was up‐regulated in both groups. We conclude that remodelling of tenascin‐C and phosphacan occurred shortly after immunization, already before RGC loss. We assume that both ECM molecules represent early indicators of neurodegeneration.     

Upregulation of IGF-I in the goldfish retinal ganglion cells during the early stage of optic nerve regeneration

Goldfish retinal ganglion cells (RGCs) can regrow their axons after optic nerve injury. However, the reason why goldfish RGCs can regenerate after nerve injury is largely unknown at the molecular level. To investigate regenerative properties of goldfish RGCs, we divided the RGC regeneration process into two components: (1) RGC survival, and (2) axonal elongation processes. To characterize the RGC survival signaling pathway after optic nerve injury, we investigated cell survival/death signals such as Bcl-2 family members in the goldfish retina. Amounts of phospho-Akt (p-Akt) and phospho-Bad (p-Bad) in the goldfish retina rapidly increased four- to five-fold at the protein level by 3-5 days after nerve injury. Subsequently, Bcl-2 levels increased 1.7-fold, accompanied by a slight reduction in caspase-3 activity 10-20 days after injury. Furthermore, level of insulin-like growth factor-I (IGF-I), which activates the phosphatidyl inositol-3-kinase (PI3K)/Akt system, increased 2-3 days earlier than that of p-Akt in the goldfish retina. The cellular localization of these molecular changes was limited to RGCs. IGF-I treatment significantly induced phosphorylation of Akt, and strikingly induced neurite outgrowth in the goldfish retina in vitro. On the contrary, addition of the PI3K inhibitor wortmannin, and IGF-I antibody inhibited Akt phosphorylation and neurite outgrowth in an explant culture. Thus, we demonstrated, for the first time, the signal cascade for early upregulation of IGF-I, leading to RGC survival and axonal regeneration in adult goldfish retinas through PI3K/Akt system after optic nerve injury. The present data strongly indicate that IGF-I is one of the most important molecules for controlling regeneration of RGCs after optic nerve injury.     

Comparative Evaluation of Methods for Estimating Retinal Ganglion Cell Loss in Retinal Sections and Wholemounts

To investigate the reliability of different methods of quantifying retinal ganglion cells (RGCs) in rat retinal sections and wholemounts from eyes with either intact optic nerves or those axotomised after optic nerve crush (ONC). Adult rats received a unilateral ONC and after 21 days the numbers of Brn3a⁺, βIII-tubulin⁺ and Islet-1⁺ RGCs were quantified in either retinal radial sections or wholemounts in which FluoroGold (FG) was injected 48 h before harvesting. Phenotypic antibody markers were used to distinguish RGCs from astrocytes, macrophages/microglia and amacrine cells. In wholemounted retinae, counts of FG⁺ and Brn3a⁺ RGCs were of similar magnitude in eyes with intact optic nerves and were similarly reduced after ONC. Larger differences in RGC number were detected between intact and ONC groups when images were taken closer to the optic nerve head. In radial sections, Brn3a did not stain astrocytes, macrophages/microglia or amacrine cells, whereas βIII-tubulin and Islet-1 did localize to amacrine cells as well as RGCs. The numbers of βIII-tubulin⁺ RGCs was greater than Brn3a⁺ RGCs, both in retinae from eyes with intact optic nerves and eyes 21 days after ONC. Islet-1 staining also overestimated the number of RGCs compared to Brn3a, but only after ONC. Estimates of RGC loss were similar in Brn3a-stained radial retinal sections compared to both Brn3a-stained wholemounts and retinal wholemounts in which RGCs were backfilled with FG, with sections having the added advantage of reducing experimental animal usage.     

α-Crystallin protects RGC survival and inhibits microglial activation after optic nerve crush

Aims

Activation of retinal microglial cells (RMCs) is known to contribute to retinal ganglion cell (RGC) death after optic nerve injury. The purpose of this study was to investigate the effects of intravenous injection of α-crystallin on RGC survival and RMC activation in a rat model of optic nerve crush.

Main methods

RGCs were retrogradely labeled with fluorogold. Rats were intravenously injected with normal saline or α-crystallin (0.05 g/kg, 0.5 g/kg, and 5 g/kg) at 2, 4, 6, 8, 10, and 12 days after the optic nerve crush. Activated RMCs were characterized using immunofluorescence labeling with CD11b, and TNF-α and iNOS expression was detected using immunoblot analyses. We analyzed the morphology and numbers of RGC and RMC 2 and 4 weeks after injury using fluorescence and confocal microscopy.

Key findings

The number of RGCs decreased after optic nerve injury, accompanied by significantly increased numbers of activated RMCs. Intravenous injection of α-crystallin decreased the number of RMCs, and enhanced the number of RGCs compared to saline injection. α-Crystallin administration inhibited TNF-α and iNOS protein expression induced by optic nerve injury.

Significance

Our results suggest that α-crystallin promotes RGC survival and inhibits RMC activation. Intravenous injection of α-crystallin could be a possible strategy for the treatment of optic nerve injury.     

Ocular neuroprotection by siRNA targeting caspase-2

Retinal ganglion cell (RGC) loss after optic nerve damage is a hallmark of certain human ophthalmic diseases including ischemic optic neuropathy (ION) and glaucoma. In a rat model of optic nerve transection, in which 80% of RGCs are eliminated within 14 days, caspase-2 was found to be expressed and cleaved (activated) predominantly in RGC. Inhibition of caspase-2 expression by a chemically modified synthetic short interfering ribonucleic acid (siRNA) delivered by intravitreal administration significantly enhanced RGC survival over a period of at least 30 days. This exogenously delivered siRNA could be found in RGC and other types of retinal cells, persisted inside the retina for at least 1 month and mediated sequence-specific RNA interference without inducing an interferon response. Our results indicate that RGC apoptosis induced by optic nerve injury involves activation of caspase-2, and that synthetic siRNAs designed to inhibit expression of caspase-2 represent potential neuroprotective agents for intervention in human diseases involving RGC loss.     

Neuronal Injury External to the Retina Rapidly Activates Retinal Glia,Followed by Elevation of Markers for Cell Cycle Re-Entry and Death in Retinal Ganglion Cells

Retinal ganglion cells (RGCs) are neurons that relay visual signals from the retina to the brain. The RGC cell bodies reside in the retina and their fibers form the optic nerve. Full transection (axotomy) of the optic nerve is an extra-retinal injury model of RGC degeneration. Optic nerve transection permits time-kinetic studies of neurodegenerative mechanisms in neurons and resident glia of the retina, the early events of which are reported here. One day after injury, and before atrophy of RGC cell bodies was apparent, glia had increased levels of phospho-Akt, phospho-S6, and phospho-ERK1/2; however, these signals were not detected in injured RGCs. Three days after injury there were increased levels of phospho-Rb and cyclin A proteins detected in RGCs, whereas these signals were not detected in glia. DNA hyperploidy was also detected in RGCs, indicative of cell cycle re-entry by these post-mitotic neurons. These events culminated in RGC death, which is delayed by pharmacological inhibition of the MAPK/ERK pathway. Our data show that a remote injury to RGC axons rapidly conveys a signal that activates retinal glia, followed by RGC cell cycle re-entry, DNA hyperploidy, and neuronal death that is delayed by preventing glial MAPK/ERK activation. These results demonstrate that complex and variable neuro-glia interactions regulate healthy and injured states in the adult mammalian retina.     

Dlx1 and Dlx2 function is necessary for terminal differentiation and survival of late-born retinal ganglion cells in the developing mouse retina

Dlx homeobox genes, the vertebrate homologs of Distal-less, play important roles in the development of the vertebrate forebrain, craniofacial structures and limbs. Members of the Dlx gene family are also expressed in retinal ganglion cells (RGC), amacrine and horizontal cells of the developing and postnatal retina. Expression begins at embryonic day 12.5 and is maintained until late embryogenesis for Dlx1, while Dlx2 expression extends to adulthood. We have assessed the retinal phenotype of the Dlx1/Dlx2 double knockout mouse, which dies at birth. The Dlx1/2 null retina displays a reduced ganglion cell layer (GCL), with loss of differentiated RGCs due to increased apoptosis, and corresponding thinning of the optic nerve. Ectopic expression of Crx, the cone and rod photoreceptor homeobox gene, in the GCL and neuroblastic layers of the mutants may signify altered cell fate of uncommitted RGC progenitors. However, amacrine and horizontal cell differentiation is relatively unaffected in the Dlx1/2 null retina. Herein, we propose a model whereby early-born RGCs are Dlx1 and Dlx2 independent, but Dlx function is necessary for terminal differentiation of late-born RGC progenitors.