Defining the molecular identity and morphology of glia limitans superficialis astrocytes in mouse and human.

Astrocytes are a highly abundant glial cell type that perform critical homeostatic functions in the central nervous system. Like neurons, astrocytes have many discrete heterogenous subtypes. The subtype identity and functions are, at least in part, associated with their anatomical location and can be highly restricted to strategically important anatomical domains. Here, we report that astrocytes forming the glia limitans superficialis, the outermost border of brain and spinal cord, are a highly specialized astrocyte subtype and can be identified by a single marker: Myocilin (Myoc). We show that Myoc+ astrocytes cover the entire brain and spinal cord surface, exhibit an atypical morphology, and are evolutionarily conserved from rodents to humans. Identification of this highly specialized astrocyte subtype will advance our understanding of CNS homeostasis and potentially be targeted for therapeutic intervention to combat peripheral inflammatory effects on the CNS.

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.

Neuroprotection by WldS depends on retinal ganglion cell type and age in glaucoma.

BACKGROUND: Early challenges to axonal physiology, active transport, and ultrastructure are endemic to age-related neurodegenerative disorders, including those affecting the optic nerve. Chief among these, glaucoma causes irreversible vision loss through sensitivity to intraocular pressure (IOP) that challenges retinal ganglion cell (RGC) axons, which comprise the optic nerve. Early RGC axonopathy includes distal to proximal progression that implicates a slow form of Wallerian degeneration. In multiple disease models, including inducible glaucoma, expression of the slow Wallerian degeneration (WldS) allele slows axon degeneration and confers protection to cell bodies. METHODS: Using an inducible model of glaucoma along with whole-cell patch clamp electrophysiology and morphological analysis, we tested if WldS also protects RGC light responses and dendrites and, if so, whether this protection depends upon RGC type. We induced glaucoma in young and aged mice to determine if neuroprotection by WldS on anterograde axonal transport and spatial contrast acuity depends on age. RESULTS: We found WldS protects dendritic morphology and light-evoked responses of RGCs that signal light onset (αON-Sustained) during IOP elevation. However, IOP elevation significantly reduces dendritic complexity and light responses of RGCs that respond to light offset (αOFF-Sustained) regardless of WldS. As expected, WldS preserves anterograde axon transport and spatial acuity in young adult mice, but its protection is significantly limited in aged mice. CONCLUSION: The efficacy of WldS in conferring protection to neurons and their axons varies by cell type and diminishes with age.

Redistribution of metabolic resources through astrocyte networks mitigates neurodegenerative stress.

In the central nervous system, glycogen-derived bioenergetic resources in astrocytes help promote tissue survival in response to focal neuronal stress. However, our understanding of the extent to which these resources are mobilized and utilized during neurodegeneration, especially in nearby regions that are not actively degenerating, remains incomplete. Here we modeled neurodegeneration in glaucoma, the world's leading cause of irreversible blindness, and measured how metabolites mobilize through astrocyte gap junctions composed of connexin 43 (Cx43). We elevated intraocular pressure in one eye and determined how astrocyte-derived metabolites in the contralateral optic projection responded. Remarkably, astrocyte networks expand and redistribute metabolites along distances even 10 mm in length, donating resources from the unstressed to the stressed projection in response to intraocular pressure elevation. While resource donation improves axon function and visual acuity in the directly stressed region, it renders the donating tissue susceptible to bioenergetic, structural, and physiological degradation. Intriguingly, when both projections are stressed in a WT animal, axon function and visual acuity equilibrate between the two projections even when each projection is stressed for a different length of time. This equilibration does not occur when Cx43 is not present. Thus, Cx43-mediated astrocyte metabolic networks serve as an endogenous mechanism used to mitigate bioenergetic stress and distribute the impact of neurodegenerative disease processes. Redistribution ultimately renders the donating optic nerve vulnerable to further metabolic stress, which could explain why local neurodegeneration does not remain confined, but eventually impacts healthy regions of the brain more broadly.

Progression and Pathology of Traumatic Optic Neuropathy From Repeated Primary Blast Exposure.

Indirect traumatic optic neuropathy (ITON) is a condition that is often associated with traumatic brain injury and can result in significant vision loss due to degeneration of retinal ganglion cell (RGC) axons at the time of injury or within the ensuing weeks. We used a mouse model of eye-directed air-blast exposure to characterize the histopathology of blast-induced ITON. This injury caused a transient elevation of intraocular pressure with subsequent RGC death and axon degeneration that was similar throughout the length of the optic nerve (ON). Deficits in active anterograde axon transport to the superior colliculus accompanied axon degeneration and first appeared in peripheral representations of the retina. Glial area in the ON increased early after injury and involved a later period of additional expansion. The increase in area involved a transient change in astrocyte organization independent of axon degeneration. While levels of many cytokines and chemokines did not change, IL-1α and IL-1ß increased in both the ON and retina. In contrast, glaucoma shows distal to proximal axon degeneration with astrocyte remodeling and increases in many cytokines and chemokines. Further, direct traumatic optic neuropathies have a clear site of injury with rapid, progressive axon degeneration and cell death. These data show that blast-induced ITON is a distinct neuropathology from other optic neuropathies.

Loss of odor-induced c-Fos expression of juxtaglomerular activity following maintenance of mice on fatty diets.

Diet-induced obesity (DIO) decreases the number of OMP+ olfactory sensory neurons (OSN) in the olfactory epithelium by 25% and reduces correlate axonal projections to the olfactory bulb (OB). Whether surviving OSNs have equivalent odor responsivity is largely unknown. Herein, we utilized c-fos immediate-early gene expression to map neuronal activity and determine whether mice weaned to control (CF), moderately-high fat (MHF), or high-fat (HF) diet for a period of 6 months had changes in odor activation. Diet-challenged M72-IRES-tau-GFP mice were exposed to either a preferred M72 (Olfr160) ligand, isopropyl tiglate, or clean air in a custom-made Bell-jar infusion chamber using an alternating odor exposure pattern generated by a picosprizer™. Mice maintained on fatty diets weighed significantly more and cleared glucose less efficiently as determined by an intraperitoneal glucose tolerance test (IPGTT). The number of juxtaglomerular cells (JGs) decreased following maintenance of the mice on the MHF diet for cells surrounding the medial but not lateral M72 glomerulus within a 4 cell-column distance. The percentage of c-fos + JGs surrounding the lateral M72 glomerulus decreased in fat-challenged mice whereas those surrounding the medial glomerulus were not affected by diet. Altogether, these results show an asymmetry in the responsiveness of the 'mirror image' glomerular map for the M72 receptor that shows greater sensitivity of the lateral vs. medial glomerulus upon exposure to fatty diet.

Antioxidants prevent inflammation and preserve the optic projection and visual function in experimental neurotrauma.

We investigated the role of oxidative stress and the inflammasome in trauma-induced axon degeneration and vision loss using a mouse model. The left eyes of male mice were exposed to over-pressure air waves. Wild-type C57Bl/6 mice were fed normal, high-vitamin-E (VitE), ketogenic or ketogenic-control diets. Mice lacking the ability to produce vitamin C (VitC) were maintained on a low-VitC diet. Visual evoked potentials (VEPs) and retinal superoxide levels were measured in vivo. Tissue was collected for biochemical and histological analysis. Injury increased retinal superoxide, decreased SOD2, and increased cleaved caspase-1, IL-1α, IL-1ß, and IL-18 levels. Low-VitC exacerbated the changes and the high-VitE diet mitigated them, suggesting that oxidative stress led to the increase in IL-1α and activation of the inflammasome. The injury caused loss of nearly 50% of optic nerve axons at 2 weeks and astrocyte hypertrophy in mice on normal diet, both of which were prevented by the high-VitE diet. The VEP amplitude was decreased after injury in both control-diet and low-VitC mice, but not in the high-VitE-diet mice. The ketogenic diet also prevented the increase in superoxide levels and IL-1α, but had no effect on IL-1ß. Despite this, the ketogenic diet preserved optic nerve axons, prevented astrocyte hypertrophy, and preserved the VEP amplitude. These data suggest that oxidative stress induces priming and activation of the inflammasome pathway after neurotrauma of the visual system. Further, blocking the activation of the inflammasome pathway may be an effective post-injury intervention.

Astrocyte remodeling without gliosis precedes optic nerve Axonopathy.

Astroyctes serve myriad functions but are especially critical in white matter tracts, where energy-demanding axons propagate action potentials great distances between neurons. Axonal dependence on astrocytes for even normal function accentuates the critical role astrocytes serve during disease. In glaucoma, the most common optic neuropathy, sensitivity to intraocular pressure (IOP) challenges RGC axons early, including degradation of anterograde transport to the superior colliculus (SC). Astrocyte remodeling presages overt axon degeneration in glaucoma and thus may present a therapeutic opportunity. Here we developed a novel metric to quantify organization of astrocyte processes in the optic nerve relative to axon degeneration in the DBA/2 J hereditary mouse model of glaucoma. In early progression, as axons expand prior to loss, astrocyte processes become more parallel with migration to the nerve's edge without a change in overall coverage of the nerve. As axons degenerate, astrocyte parallelism diminishes with increased glial coverage and reinvasion of the nerve. In longitudinal sections through aged DBA/2 J nerve, increased astrocyte parallelism reflected elevated levels of the astrocyte gap-junction protein connexin 43 (Cx43). In the distal nerve, increased Cx43 also indicated with a higher level of intact anterograde transport from retina to SC. Our results suggest that progression of axonopathy in the optic nerve involves astrocyte remodeling in two phases. In an early phase, astrocyte processes organize in parallel, likely through gap-junction coupling, while a later phase involves deterioration of organization as glial coverage increases and axons are lost.

Axogenic mechanism enhances retinal ganglion cell excitability during early progression in glaucoma.

Diseases of the brain involve early axon dysfunction that often precedes outright degeneration. Pruning of dendrites and their synapses represents a potential driver of axonopathy by reducing activity. Optic nerve degeneration in glaucoma, the world's leading cause of irreversible blindness, involves early stress to retinal ganglion cell (RGC) axons from sensitivity to intraocular pressure (IOP). This sensitivity also influences survival of RGC dendrites and excitatory synapses in the retina. Here we tested in individual RGCs identified by type the relationship between dendritic organization and axon signaling to light following modest, short-term elevations in pressure. We found dendritic pruning occurred early, by 2 wk of elevation, and independent of whether the RGC responded to light onset (ON cells) or offset (OFF cells). Pruning was similarly independent of ON and OFF in the DBA/2J mouse, a chronic glaucoma model. Paradoxically, all RGCs, even those with significant pruning, demonstrated a transient increase in axon firing in response to the preferred light stimulus that occurred on a backdrop of generally enhanced excitability. The increased response was not through conventional presynaptic signaling, but rather depended on voltage-sensitive sodium channels that increased transiently in the axon. Pruning, axon dysfunction, and deficits in visual acuity did not progress between 2 and 4 wk of elevation. These results suggest neurodegeneration in glaucoma involves an early axogenic response that counters IOP-related stress to excitatory dendritic architecture to slow progression and maintain signaling to the brain. Thus, short-term exposure to elevated IOP may precondition the neural system to further insult.

The challenge of regenerative therapies for the optic nerve in glaucoma.

This review arose from a discussion of regenerative therapies to treat optic nerve degeneration in glaucoma at the 2015 Lasker/IRRF Initiative on Astrocytes and Glaucomatous Neurodegeneration. In addition to the authors, participants included Jonathan Crowston, Andrew Huberman, Elaine Johnson, Richard Lu, Hemai Phatnami, Rebecca Sappington, and Don Zack. Glaucoma is a neurodegenerative disease of the optic nerve, and is the leading cause of irreversible blindness worldwide. The disease progresses as sensitivity to intraocular pressure (IOP) is conveyed through the optic nerve head to distal retinal ganglion cell (RGC) projections. Because the nerve and retina are components of the central nervous system (CNS), their intrinsic regenerative capacity is limited. However, recent research in regenerative therapies has resulted in multiple breakthroughs that may unlock the optic nerve's regenerative potential. Increasing levels of Schwann-cell derived trophic factors and reducing potent cell-intrinsic suppressors of regeneration have resulted in axonal regeneration even beyond the optic chiasm. Despite this success, many challenges remain. RGC axons must be able to form new connections with their appropriate targets in central brain regions and these connections must be retinotopically correct. Furthermore, for new axons penetrating the optic projection, oligodendrocyte glia must provide myelination. Additionally, reactive gliosis and inflammation that increase the regenerative capacity must be outweigh pro-apoptotic processes to create an environment within which maximal regeneration can occur.

Early astrocyte redistribution in the optic nerve precedes axonopathy in the DBA/2J mouse model of glaucoma.

Glaucoma challenges the survival of retinal ganglion cell axons in the optic nerve through processes dependent on both aging and ocular pressure. Relevant stressors likely include complex interplay between axons and astrocytes, both in the retina and optic nerve. In the DBA/2J mouse model of pigmentary glaucoma, early progression involves axonopathy characterized by loss of functional transport prior to outright degeneration. Here we describe novel features of early pathogenesis in the DBA/2J nerve. With age the cross-sectional area of the nerve increases; this is associated generally with diminished axon packing density and survival and increased glial coverage of the nerve. However, for nerves with the highest axon density, as the nerve expands mean cross-sectional axon area enlarges as well. This early expansion was marked by disorganized axoplasm and accumulation of hyperphosphorylated neurofilamants indicative of axonopathy. Axon expansion occurs without loss up to a critical threshold for size (about 0.45-0.50 µm(2)), above which additional expansion tightly correlates with frank loss of axons. As well, early axon expansion prior to degeneration is concurrent with decreased astrocyte ramification with redistribution of processes towards the nerve edge. As axons expand beyond the critical threshold for loss, glial area resumes an even distribution from the center to edge of the nerve. We also found that early axon expansion is accompanied by reduced numbers of mitochondria per unit area in the nerve. Finally, our data indicate that both IOP and nerve expansion are associated with axon enlargement and reduced axon density for aged nerves. Collectively, our data support the hypothesis that diminished bioenergetic resources in conjunction with early nerve and glial remodeling could be a primary inducer of progression of axon pathology in glaucoma.