Retinal Ganglion Cell Axons Research Articles

Nanotubules and Cellular Communication in Trabecular Meshwork Cells.

Glaucoma causes dysfunction to tissues located in the anterior and posterior eye. In the anterior eye, the trabecular meshwork (TM) is the site of pathogenesis, where decreased TM cell numbers and alterations to the amount and composition of extracellular matrix hinder outflow of aqueous humor fluid from the anterior chamber. This causes intraocular pressure (IOP) elevation. Elevated IOP, a main risk factor for primary open-angle glaucoma, damages the axons of retinal ganglion cells in the posterior eye, which ultimately leads to blindness. Thus, clinical treatment paradigms for glaucoma are focused on reducing IOP. Normotensive IOPs are established by balancing the production of aqueous fluid from the ciliary body with drainage through the TM to Schlemm's canal. When IOP becomes elevated, TM cells coordinate a homeostatic response to lower IOP, which requires effective and efficient cellular communication. Tunneling nanotubes (TNTs) are transient specialized structures that allow cells to communicate with one another. Actin-rich tubes allow direct transmission of signals and cargoes between cells. This is important to overcome limitations of diffusion-based signaling in aqueous environments such as the anterior eye. Here, we describe a live-cell imaging method for monitoring TNTs in primary TM cells.

Mouse Model of Glucocorticoid-Induced Glaucoma.

Extended glucocorticoid (GC) treatment can lead to ocular hypertension and induce iatrogenic open-angle glaucoma. GC-induced glaucoma mimics many clinical and pathological features of primary open-angle glaucoma (POAG), and therefore mouse models of GC-induced glaucoma are utilized to study pathophysiology of glaucoma. We have recently demonstrated that weekly periocular injections of dexamethasone-21-acetate (Dex-Ac) lead to robust and significant intraocular pressure (IOP) elevation, retinal ganglion cell (RGC) loss, and optic nerve degeneration in mice. Our mouse model exhibits signature features of POAG including significant IOP elevation due to reduced outflow facility, progressive optic nerve degeneration, and structural and functional loss of RGCs. Dex-induced IOP elevation is associated with increased aqueous outflow resistance due to trabecular meshwork (TM) dysfunction including excessive extracellular matrix deposition and induction of endoplasmic reticulum stress. Mouse model of Dex-induced glaucoma represents an ideal animal model to investigate both glaucomatous damage to the TM and RGC axons and to develop novel therapies. Here, we present a detailed protocol for developing this model in laboratory settings.

Corticosteroids and glaucoma: How do treatments trigger ocular nerve damage? - A systematic literature review

Corticosteroids are steroid hormone derivatives produced by the adrenal glands. Corticosteroids in the health sector have been widely utilized as anti-inflammatory agents because of their strong and rapid effects. This study aimed to identify the impact of long-term corticosteroid use on eye damage. This study uses a systematic literature review, which is guided by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) to the article screening process. The data of this study, in the form of demographic variables, route of administration, steroid type, comorbidities, and patient risk factors, were descriptively analyzed. As a result, 15 of the 22 articles were selected for analysis. Chronic corticosteroid administration may precipitate optic neuropathy, manifesting as glaucoma, which is characterized by progressive structural degeneration of retinal ganglion cells (RGCs) and concomitant visual function deterioration. The locus of primary pathological insult in glaucomatous conditions is the optic nerve head, specifically at the optic disc. This anatomical site is notable for the abrupt 90-degree angular deviation of RGC axons as they transition from the retinal nerve fiber layer to their trajectory within the optic nerve proper. The use of corticosteroids can damage ocular nerve tissue through an increase in intraocular pressure (IOP) beyond 21 mmHg. This increase in IOP is due to changes in the microstructure of the trabeculum webbing, resulting in increased obstruction to the outflow of aqueous humor.

Bioengineering Strategy to Promote CNS Nerve Growth and Regeneration via Chronic Glutamate Signaling

Being part of the mature mammalian central nervous system, impairments of the retina and optic nerves caused by trauma or diseases often cannot be restored. Progressive degeneration of retinal ganglion cells (RGCs) in glaucoma and other optic neuropathies gradually leads to permanent vision loss, which currently has no cure. The purpose of this study is to develop a biocompatible scaffold to support RGC survival and guide axon growth, facilitating optic nerve repair and regeneration. We here report that electrical stimulation (ES) significantly promoted neurite outgrowth and elongation from primary RGCs, mediated through glutamate receptor signaling. To mimic prolonged glutamate stimulation and facilitate sustained nerve growth, we fabricated biocompatible poly-γ-benzyl-L-glutamate (PBG) scaffolds for controlled glutamate release. These PBG scaffolds supported RGC survival and robust long-distance nerve growth in both retinal explants and isolated RGC cultures. In contrast, control polycaprolactone (PCL) scaffolds with similar physical structures showed little benefits on RGC survival or nerve growth. Moreover, PBG scaffolds promoted the differentiation and neurite outgrowth from embryonic stem cell-derived RGC progenitors. The aligned PBG scaffold drove directed nerve elongation along the fiber alignment. Transplantation of PBG-coated biocompatible conduits induced robust optic nerve regeneration in adult mice following nerve transection. Together, the findings present the exciting possibility of driving optic nerve regeneration and RGC progenitor cell differentiation by imitating ES or glutamate signaling. PBG presents a permissive biomaterial in supporting robust and directed axon growth with promising clinical applications in the future. Statement of SignificanceWe here reported compelling findings that demonstrate the potent regenerative effects of a bioengineered scaffold incorporating poly-γ-benzyl-L-glutamate (PBG) on the optic nerve. Retinal ganglion cell (RGC) axons, which form the optic nerve, are incapable of regenerating in adulthood, posing a significant hurdle in restoring vision for patients with optic nerve diseases or injuries. Built upon the finding that electrical stimulation promotes RGC axonal growth through glutamate signaling, we developed PBG scaffolds to provide sustained glutamate stimulation and showed their exceptional effects on driving directed axonal elongation in cultured RGCs and neural progenitors, as well as supporting robust optic nerve regeneration after transection in vivo. The findings hold great promise for reversing vision loss in patients with optic nerve conditions.

Axon guidance during mouse central nervous system regeneration is required for specific brain innervation.

Reconstructing functional neuronal circuits is one major challenge of central nervous system repair. Through activation of pro-growth signaling pathways, some neurons achieve long-distance axon regrowth. Yet, functional reconnection has hardly been obtained, as these regenerating axons fail to resume their initial trajectory and reinnervate their proper target. Axon guidance is considered to be active only during development. Here, using the mouse visual system, we show that axon guidance is still active in the adult brain in regenerative conditions. We highlight that regenerating retinal ganglion cell axons avoid one of their primary targets, the suprachiasmatic nucleus (SCN), due to Slit/Robo repulsive signaling. Together with promoting regeneration, silencing Slit/Robo invivo enables regenerating axons to enter the SCN and form active synapses. The newly formed circuit is associated with neuronal activation and functional recovery. Our results provide evidence that axon guidance mechanisms are required to reconnect regenerating axons to specific brain nuclei.

Rotenone-Induced Optic Nerve Damage and Retinal Ganglion Cell Loss in Rats.

Rotenone is a mitochondrial complex I inhibitor that causes retinal degeneration. A study of a rat model of rotenone-induced retinal degeneration suggested that this model is caused by indirect postsynaptic N-methyl-D-aspartate (NMDA) stimulation triggered by oxidative stress-mediated presynaptic intracellular calcium signaling. To elucidate the mechanisms by which rotenone causes axonal degeneration, we investigated morphological changes in optic nerves and the change in retinal ganglion cell (RGC) number in rats. Optic nerves and retinas were collected 3 and 7 days after the intravitreal injection of rotenone. The cross-sections of the optic nerves were subjected to a morphological analysis with axon quantification. The axons and somas of RGCs were analyzed immunohistochemically in retinal flatmounts. In the optic nerve, rotenone induced axonal swelling and degeneration with the incidence of reactive gliosis. Rotenone also significantly reduced axon numbers in the optic nerve. Furthermore, rotenone caused axonal thinning, fragmentation, and beading in RGCs on flatmounts and decreased the number of RGC soma. In conclusion, the intravitreal injection of rotenone in rats induced morphological abnormities with a reduced number of optic nerve axons and RGC axons when the RGC somas were degenerated. These findings help elucidate the pathogenesis of optic neuropathy induced by mitochondrial dysfunction.

Retinal Input Is Required for the Maintenance of Neuronal Laminae in the Ventrolateral Geniculate Nucleus.

Retinal ganglion cell (RGC) axons provide direct input into several brain regions, including the dorsal lateral geniculate nucleus (dLGN), which is important for image-forming vision, and the ventrolateral geniculate nucleus (vLGN), which is associated with nonimage-forming vision. Through both activity- and morphogen-dependent mechanisms, retinal inputs play important roles in the development of dLGN, including the refinement of retinal projections, morphological development of thalamocortical relay cells (TRCs), timing of corticogeniculate innervation, and recruitment and distribution of inhibitory interneurons. In contrast, little is known about the role of retinal inputs in the development of vLGN. Grossly, vLGN is divided into two domains, the retinorecipient external vLGN (vLGNe) and nonretinorecipient internal vLGN (vLGNi). Studies previously found that vLGNe consists of transcriptionally distinct GABAergic subtypes distributed into at least four adjacent laminae. At present, it remains unclear whether retinal inputs influence the development of these cell-type-specific neuronal laminae in vLGNe. Here, we elucidated the developmental timeline for these laminae in the mouse vLGNe, and results indicate that these laminae are specified at or before birth. We observed that mutant mice without retinal inputs have a normal laminar distribution of GABAergic cells at birth; however, after the first week of postnatal development, these mutants exhibited a dramatic disruption in the laminar organization of inhibitory neurons and clear boundaries between vLGNe and vLGNi. Overall, our results show that while the formation of cell-type-specific layers in mouse vLGNe does not depend on RGC inputs, retinal signals are critical for their maintenance.

Development of the Binocular Circuit.

Seeing in three dimensions is a major property of the visual system in mammals. The circuit underlying this property begins in the retina, from which retinal ganglion cells (RGCs) extend to the same or opposite side of the brain. RGC axons decussate to form the optic chiasm, then grow to targets in the thalamus and midbrain, where they synapse with neurons that project to the visual cortex. Here we review the cellular and molecular mechanisms of RGC axonal growth cone guidance across or away from the midline via receptors to cues in the midline environment. We present new views on the specification of ipsi- and contralateral RGC subpopulations and factors implementing their organization in the optic tract and termination in subregions of their targets. Lastly, we describe the functional and behavioral aspects of binocular vision, focusing on the mouse, and discuss recent discoveries in the evolution of the binocular circuit.

Modulating amacrine cell-derived dopamine signaling promotes optic nerve regeneration and preserves visual function.

As part of the central nervous system, the optic nerve, composed of axons from retinal ganglion cells (RGCs), generally fails to regenerate on its own when injured in adult mammals. An innovative approach to promoting optic nerve regeneration involves manipulating the interactions between amacrine cells (ACs) and RGCs. Here, we identified a unique AC subtype, dopaminergic ACs (DACs), that responded early after optic nerve crush by down-regulating neuronal activity and reducing retinal dopamine (DA) release. Activating DACs or augmenting DA release with levodopa demonstrated neuroprotective effects and modestly enhanced axon regeneration. Within this context, we pinpointed the DA receptor D1 (DRD1) as a critical mediator of DAC-derived DA and showed that RGC-specific Drd1 overexpression effectively overcame subtype-specific barriers to regeneration. This strategy markedly boosted RGC survival and axon regeneration after crush and preserved vision in a glaucoma model. This study unveils the crucial role of DAC-derived DA signaling in optic nerve regeneration, holding promise for therapeutic insights into neural repair.

Mistargeted retinal axons form synaptically segregated subcircuits in the visual thalamus of albino mice.

A Hebbian model of circuit remodeling predicts that two sets of inputs with sufficiently distinct activity patterns will synaptically capture separate sets of target cells. Mice in which a subset of retinal ganglion cells (RGCs) target the wrong region of the dorsal lateral geniculate nucleus (dLGN) provide the conditions for testing this prediction. In albino mice, mistargeted RGC axons form an island of terminals that is distinct from the surrounding neuropil. Blocking retinal activity during development prevents the formation of this island. However, the synaptic connectivity of the island was unknown. Here, we combine light and electron microscopy to determine if this activity-dependent island of axon terminals represent a synaptically segregated subcircuit. We reconstructed the microcircuitry of the boundary between the island and non-island RGCs and found a remarkably strong segregation within retinogeniculate connectivity. We conclude that, when sets of retinal input are established in the wrong part of the dLGN, the developing circuitry responds by forming a synaptically isolated subcircuit from the otherwise fully connected network. The fact that there is a developmental starting condition that can induce a synaptically segregated microcircuit has important implications for our understanding of the organization of visual circuits and for our understanding of the implementation of activity dependent development.

Problems and prospects for restoration of the optic nerve

Restoring visual function after damage or complete destruction of the optic nerve in adult patients has many natural barriers to neuroregeneration. Research to restore vision has focused on maintaining retinal ganglion cells (RGCs), stimulating axonal growth toward the brain, and restoring their proper synaptic connections. Unfortunately, mammalian RGC axons under normal conditions do not regenerate after injury and ultimately die. In this review, we summarize the currently known mechanisms of RGC survival and axonal regeneration in mammals, including specific intrinsic signaling pathways, key transcription factors, reprogramming genes, inflammation-related regeneration factors, and stem cell therapy. We also review the current understanding of the phenomena impeding optic nerve regeneration and possible ways to overcome these obstacles. The most important research results obtained in recent decades may be informative for the development of methods for treating the damaged visual system.

Assessment of Brain-Derived Neurotrophic Factor on Retinal Structure and Visual Function in Rodent Models of Optic Nerve Crush.

The effects of brain-derived neurotrophic factor (BDNF) on retinal ganglion cell (RGC) survival and visual function were assessed in rat and mouse models of optic nerve (ON) crush. ONs were crushed on Day 1, followed by intravitreal injections of a vehicle or BDNF on Days 1 and 8. The spatial frequency threshold was measured using optokinetic tracking on Days 7 and 14. On Day 15, ganglion cell complex (GCC) thickness was quantified using optical coherence tomography. Furthermore, all eyes were enucleated for immunohistochemical analysis of the surviving RGC somas and axons. BDNF significantly reduced the RGC soma in mice and increased GCC thickness in intact eyes, with apparent axonal swelling in both species. It displayed significantly greater RGC soma survival in eyes with ON injury, with moderately thicker axonal bundles in both species and a thicker GCC in rats. Visual function was significantly reduced in all ON-crushed animals, regardless of BDNF treatment. Thus, we obtained a comprehensive analysis of the structural and functional impact of BDNF in intact and ON-crushed eyes in two rodent models. Our results provide a foundation for further BDNF evaluation and the design of preclinical studies on neuroprotectants using BDNF as a reference positive control.

Transcriptional profiling of retinal astrocytes identifies a specific marker and points to functional specialization.

Astrocyte heterogeneity is an increasingly prominent research topic, and studies in the brain have demonstrated substantial variation in astrocyte form and function, both between and within regions. In contrast, retinal astrocytes are not well understood and remain incompletely characterized. Along with optic nerve astrocytes, they are responsible for supporting retinal ganglion cell axons and an improved understanding of their role is required. We have used a combination of microdissection and Ribotag immunoprecipitation to isolate ribosome-associated mRNA from retinal astrocytes and investigate their transcriptome, which we also compared to astrocyte populations in the optic nerve. Astrocytes from these regions are transcriptionally distinct, and we identified retina-specific astrocyte genes and pathways. Moreover, although they share much of the "classical" gene expression patterns of astrocytes, we uncovered unexpected variation, including in genes related to core astrocyte functions. We additionally identified the transcription factor Pax8 as a highly specific marker of retinal astrocytes and demonstrated that these astrocytes populate not only the retinal surface, but also the prelaminar region at the optic nerve head. These findings are likely to contribute to a revised understanding of the role of astrocytes in the retina.

Noninvasive Light Flicker Stimulation Promotes Optic Nerve Regeneration by Activating Microglia and Enhancing Neural Plasticity in Zebrafish.

Forty-hertz light flicker stimulation has been proven to reduce neurodegeneration, but its effect on optic nerve regeneration is unclear. This study explores the effect of 40-Hz light flicker in promoting optic nerve regeneration in zebrafish and investigates the underlying mechanisms. Wild-type and mpeg1:EGFP zebrafish were used to establish a model of optic nerve crush. Biocytin tracing and hematoxylin and eosin staining were employed to observe whether 40-Hz light flicker promotes regeneration of retinal ganglion cell axons and dendrites. Optomotor and optokinetic responses were evaluated to assess recovery of visual function. Immunofluorescence staining of mpeg1:EGFP zebrafish was performed to observe changes in microglia. Differentially expressed genes that promote optic nerve regeneration following 40-Hz light flicker stimulation were identified and validated through RNA-sequencing analysis and quantitative real-time PCR (qRT-PCR). Zebrafish exhibited spontaneous optic nerve regeneration after optic nerve injury and restored visual function. We observed that 40-Hz light flicker significantly activated microglia following optic nerve injury and promoted regeneration of retinal ganglion cell axons and dendrites, as well as recovery of visual function. Transcriptomics and qRT-PCR analyses revealed that 40-Hz light flicker increased the expression of genes associated with neuronal plasticity, including bdnf, npas4a, fosab, fosb, egr4, and ier2a. To our knowledge, this study is the first to demonstrate that 40-Hz light flicker stimulation promotes regeneration of retinal ganglion cell axons and dendrites and recovery of visual function in zebrafish, which is associated with microglial activation and enhancement of neural plasticity.

Transcriptional control of visual neural circuit development by GS homeobox 1.

As essential components of gene expression networks, transcription factors regulate neural circuit assembly. The homeobox transcription factor encoding gene, gs homeobox 1 (gsx1), is expressed in the developing visual system; however, no studies have examined its role in visual system formation. In zebrafish, retinal ganglion cell (RGC) axons that transmit visual information to the brain terminate in ten arborization fields (AFs) in the optic tectum (TeO), pretectum (Pr), and thalamus. Pretectal AFs (AF1-AF9) mediate distinct visual behaviors, yet we understand less about their development compared to AF10 in the TeO. Using gsx1 zebrafish mutants, immunohistochemistry, and transgenic lines, we observed that gsx1 is required for vesicular glutamate transporter, Tg(slc17a6b:DsRed), expression in the Pr, but not overall neuron number. gsx1 mutants have normal eye morphology, yet they exhibit impaired visual ability during prey capture. RGC axon volume in the gsx1 mutant Pr and TeO is reduced, and AF7 that is active during feeding is missing which is consistent with reduced hunting performance. Timed laser ablation of Tg(slc17a6b:DsRed)-positive cells reveals that they are necessary for AF7 formation. This work is the first to implicate gsx1 in establishing cell identity and functional neural circuits in the visual system.

Interleukin-4 protects retinal ganglion cells and promotes axon regeneration

BackgroundThe preservation of retinal ganglion cells (RGCs) and the facilitation of axon regeneration are crucial considerations in the management of various vision-threatening disorders. Therefore, we investigate the efficacy of interleukin-4 (IL-4), a potential therapeutic agent, in promoting neuroprotection and axon regeneration of retinal ganglion cells (RGCs) as identified through whole transcriptome sequencing in an in vitro axon growth model.MethodsA low concentration of staurosporine (STS) was employed to induce in vitro axon growth. Whole transcriptome sequencing was utilized to identify key target factors involved in the molecular mechanism underlying axon growth. The efficacy of recombinant IL-4 protein on promoting RGC axon growth was validated through in vitro experiments. The protective effect of recombinant IL-4 protein on somas of RGCs was assessed using RBPMS-specific immunofluorescent staining in mouse models with optic nerve crush (ONC) and N-methyl-D-aspartic acid (NMDA) injury. The protective effect on RGC axons was evaluated by anterograde labeling of cholera toxin subunit B (CTB), while the promotion of RGC axon regeneration was assessed through both anterograde labeling of CTB and immunofluorescent staining for growth associated protein-43 (GAP43).ResultsWhole-transcriptome sequencing of staurosporine-treated 661 W cells revealed a significant upregulation in intracellular IL-4 transcription levels during the process of axon regeneration. In vitro experiments demonstrated that recombinant IL-4 protein effectively stimulated axon outgrowth. Subsequent immunostaining with RBPMS revealed a significantly higher survival rate of RGCs in the rIL-4 group compared to the vehicle group in both NMDA and ONC injury models. Axonal tracing with CTB confirmed that recombinant IL-4 protein preserved long-distance projection of RGC axons, and there was a notably higher number of surviving axons in the rIL-4 group compared to the vehicle group following NMDA-induced injury. Moreover, intravitreal delivery of recombinant IL-4 protein substantially facilitated RGC axon regeneration after ONC injury.ConclusionThe recombinant IL-4 protein exhibits the potential to enhance the survival rate of RGCs, protect RGC axons against NMDA-induced injury, and facilitate axon regeneration following ONC. This study provides an experimental foundation for further investigation and development of therapeutic agents aimed at protecting the optic nerve and promoting axon regeneration.

Avoidance of axonal stimulation with sinusoidal epiretinal stimulation

Objective. Neuromodulation, particularly electrical stimulation, necessitates high spatial resolution to achieve artificial vision with high acuity. In epiretinal implants, this is hindered by the undesired activation of distal axons. Here, we investigate focal and axonal activation of retinal ganglion cells (RGCs) in epiretinal configuration for different sinusoidal stimulation frequencies. Approach. RGC responses to epiretinal sinusoidal stimulation at frequencies between 40 and 100 Hz were tested in ex-vivo photoreceptor degenerated (rd10) isolated retinae. Experiments were conducted using a high-density CMOS-based microelectrode array, which allows to localize RGC cell bodies and axons at high spatial resolution. Main results. We report current and charge density thresholds for focal and distal axon activation at stimulation frequencies of 40, 60, 80, and 100 Hz for an electrode size with an effective area of 0.01 mm2. Activation of distal axons is avoided up to a stimulation amplitude of 0.23 µA (corresponding to 17.3 µC cm−2) at 40 Hz and up to a stimulation amplitude of 0.28 µA (14.8 µC cm−2) at 60 Hz. The threshold ratio between focal and axonal activation increases from 1.1 for 100 Hz up to 1.6 for 60 Hz, while at 40 Hz stimulation frequency, almost no axonal responses were detected in the tested intensity range. With the use of synaptic blockers, we demonstrate the underlying direct activation mechanism of the ganglion cells. Finally, using high-resolution electrical imaging and label-free electrophysiological axon tracking, we demonstrate the extent of activation in axon bundles. Significance. Our results can be exploited to define a spatially selective stimulation strategy avoiding axonal activation in future retinal implants, thereby solving one of the major limitations of artificial vision. The results may be extended to other fields of neuroprosthetics to achieve selective focal electrical stimulation.

Investigation of the Effects of a Novel NOX2 Inhibitor, GLX7013170, against Glutamate Excitotoxicity and Diabetes Insults in the Retina.

Glutamate excitotoxicity and oxidative stress represent two major pathological mechanisms implicated in retinal disorders. In Diabetic Retinopathy (DR), oxidative stress is correlated to NADPH oxidase (NOX), a major source of Reactive Oxygen Species (ROS), and glutamate metabolism impairments. This study investigated the role of NOX2 and the novel NOX2 inhibitor, GLX7013170, in two models of a) retinal AMPA excitotoxicity [AMPA+GLX7013170 (10-4 M, intravitreally)] and b) early-stage DR paradigm (ESDR), GLX7013170: 14-day therapeutic treatment (topically, 20 μL/eye, 10 mg/mL (300 × 10-4 M), once daily) post-streptozotocin (STZ)-induced DR. Immunohistochemical studies for neuronal markers, nitrotyrosine, micro/macroglia, and real-time PCR, Western blot, and glutamate colorimetric assays were conducted. Diabetes increased NOX2 expression in the retina. NOX2 inhibition limited the loss of NOS-positive amacrine cells and the overactivation of micro/macroglia in both models. In the diabetic retina, GLX7013170 had no effect on retinal ganglion cell axons, but reduced oxidative damage, increased Bcl-2, reduced glutamate levels, and partially restored excitatory amino acid transporter (EAAT1) expression. These results suggest that NOX2 in diabetes is part of the triad, oxidative stress, NOX, and glutamate excitotoxicity, key players in the induction of DR. GLX7013170 is efficacious as a neuroprotective/anti-inflammatory agent and a potential therapeutic in retinal diseases, including ESDR.

G protein-coupled estrogen receptor expression in postnatal developing mouse retina.

Estrogen has emerged as a multifaceted signaling molecule in the retina, playing an important role in neural development and providing neuroprotection in adults. It interacts with two receptor types: classical estrogen receptors (ERs) alpha and beta, and G protein-coupled estrogen receptor (Gper). Gper differs from classical ERs in structure, localization, and signaling. Here we provide the first report of the temporal and spatial properties of Gper transcript and protein expression in the developing and mature mouse retina. We applied qRT-PCR to determine Gper transcript expression in wild type mouse retina from P0-P21. Immunohistochemistry and Western blot were used to determine Gper protein expression and localization at the same time points. Gper expression showed a 6-fold increase during postnatal development, peaking at P14. Relative total Gper expression exhibited a significant decrease during retinal development, although variations emerged in the timing of changes among different forms of the protein. Gper immunoreactivity was seen in retinal ganglion cells (RGCs) throughout development and also in somas in the position of horizontal cells at early time points. Immunoreactivity was observed in the cytoplasm and Golgi at all time points, in the nucleus at early time points, and in RGC axons as the retina matured. In conclusion, our study illuminates the spatial and temporal expression patterns of Gper in the developing mouse retina and provides a vital foundation for further investigations into the role of Gper in retinal development and degeneration.

Ca2+/Calmodulin-Dependent Protein Kinase II Enhances Retinal Ganglion Cell Survival But Suppresses Axon Regeneration after Optic Nerve Injury.

Neuroprotection after injury or in neurodegenerative disease remains a major goal for basic and translational neuroscience. Retinal ganglion cells (RGCs), the projection neurons of the eye, degenerate in optic neuropathies after axon injury, and there are no clinical therapies to prevent their loss or restore their connectivity to targets in the brain. Here we demonstrate a profound neuroprotective effect of the exogenous expression of various Ca2+/calmodulin-dependent protein kinase II (CaMKII) isoforms in mice. A dramatic increase in RGC survival following the optic nerve trauma was elicited by the expression of constitutively active variants of multiple CaMKII isoforms in RGCs using adeno-associated viral (AAV) vectors across a 100-fold range of AAV dosing in vivo. Despite this neuroprotection, however, short-distance RGC axon sprouting was suppressed by CaMKII, and long-distance axon regeneration elicited by several pro-axon growth treatments was likewise inhibited even as CaMKII further enhanced RGC survival. Notably, in a dose-escalation study, AAV-expressed CaMKII was more potent for axon growth suppression than the promotion of survival. That diffuse overexpression of constitutively active CaMKII strongly promotes RGC survival after axon injury may be clinically valuable for neuroprotection per se. However, the associated strong suppression of the optic nerve axon regeneration demonstrates the need for understanding the intracellular domain- and target-specific CaMKII activities to the development of CaMKII signaling pathway-directed strategies for the treatment of optic neuropathies.