Retinal ganglion cells adapt to ionic stress in experimental glaucoma.

Introduction: Identification of early adaptive and maladaptive neuronal stress responses is an important step in developing targeted neuroprotective therapies for degenerative disease. In glaucoma, retinal ganglion cells (RGCs) and their axons undergo progressive degeneration resulting from stress driven by sensitivity to intraocular pressure (IOP). Despite therapies that can effectively manage IOP many patients progress to vision loss, necessitating development of neuronal-based therapies. Evidence from experimental models of glaucoma indicates that early in the disease RGCs experience altered excitability and are challenged with dysregulated potassium (K+) homeostasis. Previously we demonstrated that certain RGC types have distinct excitability profiles and thresholds for depolarization block, which are associated with sensitivity to extracellular K+. Methods: Here, we used our inducible mouse model of glaucoma to investigate how RGC sensitivity to K+ changes with exposure to elevated IOP. Results: In controls, conditions of increased K+ enhanced membrane depolarization, reduced action potential generation, and widened action potentials. Consistent with our previous work, 4 weeks of IOP elevation diminished RGC light-and current-evoked responses. Compared to controls, we found that IOP elevation reduced the effects of increased K+ on depolarization block threshold, with IOP-exposed cells maintaining greater excitability. Finally, IOP elevation did not alter axon initial segment dimensions, suggesting that structural plasticity alone cannot explain decreased K+ sensitivity. Discussion: Thus, in response to prolonged IOP elevation RGCs undergo an adaptive process that reduces sensitivity to changes in K+ while diminishing excitability. These experiments give insight into the RGC response to IOP stress and lay the groundwork for mechanistic investigation into targets for neuroprotective therapy.

Ocular stress enhances contralateral transfer of lenadogene nolparvovec gene therapy through astrocyte networks.

Lenadogene nolparvovec (GS010) was developed to treat a point mutation in mitochondrial ND4 that causes Leber hereditary optic neuropathy. GS010 delivers human cDNA encoding wild-type ND4 packaged into an rAAV2/2 vector that transduces retinal ganglion cells, to induce allotopic expression of hybrid mitochondrial ND4. GS010 clinical trials improved best-corrected visual acuity (BCVA) up to 5 years after treatment. Interestingly, unilateral treatment improved BCVA bilaterally. Subsequent studies revealed GS010 DNA in visual tissues contralateral to the injected eye, suggesting migration. Here we tested whether unilateral intraocular pressure (IOP) elevation could influence the transfer of viral ND4 RNA in contralateral tissues after GS010 delivery to the IOP-elevated eye and probed a potential mechanism mediating translocation in mice. We found IOP elevation enhanced viral ND4 RNA transcripts in contralateral visual tissues, including retinas. Using conditional transgenic mice, we depleted astrocytic gap junction connexin 43 (Cx43), required for distant redistribution of metabolic resources between astrocytes during stress. After unilateral IOP elevation and GS010 injection, Cx43 knockdown eradicated ND4 RNA transcript detection in contralateral retinal tissues, while transcript was still detectable in optic nerves. Overall, our study indicates long-range migration of GS010 product to contralateral visual tissues is enhanced by Cx43-linked astrocyte networks.

Microfluidic Platforms Promote Polarization of Human-Derived Retinal Ganglion Cells That Model Axonopathy.

Purpose: Axons depend on long-range transport of proteins and organelles which increases susceptibility to metabolic stress in disease. The axon initial segment (AIS) is particularly vulnerable due to the high bioenergetic demand of action potential generation. Here, we prepared retinal ganglion cells derived from human embryonic stem cells (hRGCs) to probe how axonal stress alters AIS morphology. Methods: hRGCs were cultured on coverslips or microfluidic platforms. We assayed AIS specification and morphology by immunolabeling against ankyrin G (ankG), an axon-specific protein, and postsynaptic density 95 (PSD-95), a dendrite-specific protein. Using microfluidic platforms that enable fluidic isolation, we added colchicine to the axon compartment to lesion axons. We verified axonopathy by measuring the anterograde axon transport of cholera toxin subunit B and immunolabeling against cleaved caspase 3 (CC3) and phosphorylated neurofilament H (SMI-34). We determined the influence of axon injury on AIS morphology by immunolabeling samples against ankG and measuring AIS distance from soma and length. Results: Based on measurements of ankG and PSD-95 immunolabeling, microfluidic platforms promote the formation and separation of distinct somatic-dendritic versus axonal compartments in hRGCs compared to coverslip cultures. Chemical lesioning of axons by colchicine reduced hRGC anterograde axon transport, increased varicosity density, and enhanced expression of CC3 and SMI-34. Interestingly, we found that colchicine selectively affected hRGCs with axon-carrying dendrites by reducing AIS distance from somas and increasing length, thus suggesting reduced capacity to maintain excitability. Conclusions: Thus, microfluidic platforms promote polarized hRGCs that enable modeling of axonopathy. Translational Relevance: Microfluidic platforms may be used to assay compartmentalized degeneration that occurs during glaucoma.

Intraocular Sustained Release of EPO-R76E Mitigates Glaucoma Pathogenesis by Activating the NRF2/ARE Pathway.

Erythropoietin (EPO) is neuroprotective in multiple models of neurodegenerative diseases, including glaucoma. EPO-R76E retains the neuroprotective effects of EPO but diminishes the effects on hematocrit. Treatment with EPO-R76E in a glaucoma model increases expression of antioxidant proteins and is neuroprotective. A major pathway that controls the expression of antioxidant proteins is the NRF2/ARE pathway. This pathway is activated endogenously after elevation of intraocular pressure (IOP) and contributes to the slow onset of pathology in glaucoma. In this study, we explored if sustained release of EPO-R76E in the eye would activate the NRF2/ARE pathway and if this pathway was key to its neuroprotective activity. Treatment with PLGA.EPO-E76E prevented increases in retinal superoxide levels in vivo, and caused phosphorylation of NRF2 and upregulation of antioxidants. Further, EPO-R76E activates NRF2 via phosphorylation by the MAPK pathway rather than the PI3K/Akt pathway, used by the endogenous antioxidant response to elevated IOP.

Axon hyperexcitability in the contralateral projection following unilateral optic nerve crush in mice.

Optic neuropathies are characterized by degeneration of retinal ganglion cell axonal projections to the brain, including acute conditions like optic nerve trauma and progressive conditions such as glaucoma. Despite different aetiologies, retinal ganglion cell axon degeneration in traumatic optic neuropathy and glaucoma share common pathological signatures. We compared how early pathogenesis of optic nerve trauma and glaucoma influence axon function in the mouse optic projection. We assessed pathology by measuring anterograde axonal transport from retina to superior colliculus, current-evoked optic nerve compound action potential and retinal ganglion cell density 1 week following unilateral optic nerve crush or intraocular pressure elevation. Nerve crush reduced axon transport, compound axon potential and retinal ganglion cell density, which were unaffected by intraocular pressure elevation. Surprisingly, optic nerves contralateral to crush demonstrated 5-fold enhanced excitability in compound action potential compared with naïve nerves. Enhanced excitability in contralateral sham nerves is not due to increased accumulation of voltage-gated sodium channel 1.6, or ectopic voltage-gated sodium channel 1.2 expression within nodes of Ranvier. Our results indicate hyperexcitability is driven by intrinsic responses of αON-sustained retinal ganglion cells. We found αON-sustained retinal ganglion cells in contralateral, sham and eyes demonstrated increased responses to depolarizing currents compared with those from naïve eyes, while light-driven responses remained intact. Dendritic arbours of αON-sustained retinal ganglion cells of the sham eye were like naïve, but soma area and non-phosphorylated neurofilament H increased. Current- and light-evoked responses of sham αOFF-sustained retinal ganglion cells remained stable along with somato-dendritic morphologies. In retinas directly affected by crush, light responses of αON- and αOFF-sustained retinal ganglion cells diminished compared with naïve cells along with decreased dendritic field area or branch points. Like light responses, αOFF-sustained retinal ganglion cell current-evoked responses diminished, but surprisingly, αON-sustained retinal ganglion cell responses were similar to those from naïve retinas. Optic nerve crush reduced dendritic length and area in αON-sustained retinal ganglion cells in eyes ipsilateral to injury, while crush significantly reduced dendritic branching in αOFF-sustained retinal ganglion cells. Interestingly, 1 week of intraocular pressure elevation only affected αOFF-sustained retinal ganglion cell physiology, depolarizing resting membrane potential in cells of affected eyes and blunting current-evoked responses in cells of saline-injected eyes. Collectively, our results suggest that neither saline nor sham surgery provide a true control, chronic versus acute optic neuropathies differentially affect retinal ganglion cells composing the ON and OFF pathways, and acute stress can have near-term effects on the contralateral projection.

Sensitivity to extracellular potassium underlies type-intrinsic differences in retinal ganglion cell excitability.

Neuronal type-specific physiologic heterogeneity can be driven by both extrinsic and intrinsic mechanisms. In retinal ganglion cells (RGCs), which carry visual information from the retina to central targets, evidence suggests intrinsic properties shaping action potential (AP) generation significantly impact the responses of RGCs to visual stimuli. Here, we explored how differences in intrinsic excitability further distinguish two RCG types with distinct presynaptic circuits, alpha ON-sustained (αON-S) cells and alpha OFF-sustained (αOFF-S) cells. We found that αOFF-S RGCs are more excitable to modest depolarizing currents than αON-S RGCs but excitability plateaued earlier as depolarization increased (i.e., depolarization block). In addition to differences in depolarization block sensitivity, the two cell types also produced distinct AP shapes with increasing stimulation. αOFF-S AP width and variability increased with depolarization magnitude, which correlated with the onset of depolarization block, while αON-S AP width and variability remained stable. We then tested if differences in depolarization block observed in αON-S and αOFF-S RGCs were due to sensitivity to extracellular potassium. We found αOFF-S RGCs more sensitive to increased extracellular potassium concentration, which shifted αON-S RGC excitability to that of αOFF-S cells under baseline potassium conditions. Finally, we investigated the influence of the axon initial segment (AIS) dimensions on RGC spiking. We found that the relationship between AIS length and evoked spike rate varied not only by cell type, but also by the strength of stimulation, suggesting AIS structure alone cannot fully explain the observed differences RGC excitability. Thus, sensitivity to extracellular potassium contributes to differences in intrinsic excitability, a key factor that shapes how RGCs encode visual information.

Dysfunctional cGMP Signaling Leads to Age-Related Retinal Vascular Alterations and Astrocyte Remodeling in Mice.

The nitric oxide-guanylyl cyclase-1-cyclic guanylate monophosphate (NO-GC-1-cGMP) pathway is integral to the control of vascular tone and morphology. Mice lacking the alpha catalytic domain of guanylate cyclase (GC1-/-) develop retinal ganglion cell (RGC) degeneration with age, with only modest fluctuations in intraocular pressure (IOP). Increasing the bioavailability of cGMP in GC1-/- mice prevents neurodegeneration independently of IOP, suggesting alternative mechanisms of retinal neurodegeneration. In continuation to these studies, we explored the hypothesis that dysfunctional cGMP signaling leads to changes in the neurovascular unit that may contribute to RGC degeneration. We assessed retinal vasculature and astrocyte morphology in young and aged GC1-/- and wild type mice. GC1-/- mice exhibit increased peripheral retinal vessel dilation and shorter retinal vessel branching with increasing age compared to Wt mice. Astrocyte cell morphology is aberrant, and glial fibrillary acidic protein (GFAP) density is increased in young and aged GC1-/- mice, with areas of dense astrocyte matting around blood vessels. Our results suggest that proper cGMP signaling is essential to retinal vessel morphology with increasing age. Vascular changed are preceded by alterations in astrocyte morphology which may together contribute to retinal neurodegeneration and loss of visual acuity observed in GC1-/- mice.

Astrocyte Networks as Therapeutic Targets in Glaucomatous Neurodegeneration.

Astrocytes are intimately involved in the response to neurodegenerative stress and have become an attractive target for the development of neuroprotective therapies. However, studies often focus on astrocytes as single-cell units. Astrocytes are densely interconnected by gap junctions that are composed primarily of the protein connexin-43 (Cx43) and can function as a broader network of cells. Such networks contribute to a number of important processes, including metabolite distribution and extracellular ionic buffering, and are likely to play an important role in the progression of neurodegenerative disease. This review will focus on the pro-degenerative and pro-survival influence of astrocyte Cx43 in disease progression, with a focus on the roles of gap junctions and hemichannels in the spread of degenerative stress. Finally, we will highlight the specific evidence for targeting these networks in the treatment of glaucomatous neurodegeneration and other optic neuropathies.

TRPV1 Supports Axogenic Enhanced Excitability in Response to Neurodegenerative Stress.

Early progression in neurodegenerative disease involves challenges to homeostatic processes, including those controlling axonal excitability and dendritic organization. In glaucoma, the leading cause of irreversible blindness, stress from intraocular pressure (IOP) causes degeneration of retinal ganglion cells (RGC) and their axons which comprise the optic nerve. Previously, we discovered that early progression induces axogenic, voltage-gated enhanced excitability of RGCs, even as dendritic complexity in the retina reduces. Here, we investigate a possible contribution of the transient receptor potential vanilloid type 1 (TRPV1) channel to enhanced excitability, given its role in modulating excitation in other neural systems. We find that genetic deletion of Trpv1 (Trpv1 -/-) influences excitability differently for RGCs firing continuously to light onset (αON-Sustained) vs. light offset (αOFF-Sustained). Deletion drives excitability in opposing directions so that Trpv1 -/- RGC responses with elevated IOP equalize to that of wild-type (WT) RGCs without elevated IOP. Depolarizing current injections in the absence of light-driven presynaptic excitation to directly modulate voltage-gated channels mirrored these changes, while inhibiting voltage-gated sodium channels and isolating retinal excitatory postsynaptic currents abolished both the differences in light-driven activity between WT and Trpv1 -/- RGCs and changes in response due to IOP elevation. Together, these results support a voltage-dependent, axogenic influence of Trpv1 -/- with elevated IOP. Finally, Trpv1 -/- slowed the loss of dendritic complexity with elevated IOP, opposite its effect on axon degeneration, supporting the idea that axonal and dendritic degeneration follows distinctive programs even at the level of membrane excitability.

Cortical projections to the two retinotopic maps of primate pulvinar are distinct.

Comprised of at least five distinct nuclei, the pulvinar complex of primates includes two large visually driven nuclei; one in the dorsal (lateral) pulvinar and one in the ventral (inferior) pulvinar, that contain similar retinotopic representations of the contralateral visual hemifield. Both nuclei also appear to have similar connections with areas of visual cortex. Here we determined the cortical connections of these two nuclei in galagos, members of the stepsirrhine primate radiation, to see if the nuclei differed in ways that could support differences in function. Injections of different retrograde tracers in each nucleus produced similar patterns of labeled neurons, predominately in layer 6 of V1, V2, V3, MT, regions of temporal cortex, and other visual areas. More complete labeling of neurons with a modified rabies virus identified these neurons as pyramidal cells with apical dendrites extending into superficial cortical layers. Importantly, the distributions of cortical neurons projecting to each of the two nuclei were highly overlapping, but formed separate populations. Sparse populations of double-labeled neurons were found in both V1 and V2 but were very low in number (<0.1%). Finally, the labeled cortical neurons were predominately in layer 6, and layer 5 neurons were labeled only in extrastriate areas. Terminations of pulvinar projections to area 17 was largely in superficial cortical layers, especially layer 1.