Astrocytes of the eye and optic nerve: heterogeneous populations with unique functions mediate axonal resilience and vulnerability to glaucoma

The role of glia, particularly astrocytes, in mediating the central nervous system’s response to injury and neurodegenerative disease is an increasingly well studied topic. These cells perform myriad support functions under physiological conditions but undergo behavioral changes – collectively referred to as ‘reactivity’ – in response to the disruption of neuronal homeostasis from insults, including glaucoma. However, much remains unknown about how reactivity alters disease progression – both beneficially and detrimentally – and whether these changes can be therapeutically modulated to improve outcomes. Historically, the heterogeneity of astrocyte behavior has been insufficiently addressed under both physiological and pathological conditions, resulting in a fragmented and often contradictory understanding of their contributions to health and disease. Thanks to increased focus in recent years, we now know this heterogeneity encompasses both intrinsic variation in physiological function and insult-specific changes that vary between pathologies. Although previous studies demonstrate astrocytic alterations in glaucoma, both in human disease and animal models, generally these findings do not conclusively link astrocytes to causative roles in neuroprotection or degeneration, rather than a subsequent response. Efforts to bolster our understanding by drawing on knowledge of brain astrocytes has been constrained by the primacy in the literature of findings from peri-synaptic ‘gray matter’ astrocytes, whereas much early degeneration in glaucoma occurs in axonal regions populated by fibrous ‘white matter’ astrocytes. However, by focusing on findings from astrocytes of the anterior visual pathway – those of the retina, unmyelinated optic nerve head, and myelinated optic nerve regions – we aim to highlight aspects of their behavior that may contribute to axonal vulnerability and glaucoma progression, including roles in mitochondrial turnover and energy provisioning. Furthermore, we posit that astrocytes of the retina, optic nerve head and myelinated optic nerve, although sharing developmental origins and linked by a network of gap junctions, may be best understood as distinct populations residing in markedly different niches with accompanying functional specializations. A closer investigation of their behavioral repertoires may elucidate not only their role in glaucoma, but also mechanisms to induce protective behaviors that can impede the progressive axonal damage and retinal ganglion cell death that drive vision loss in this devastating condition.


Introduction
Glaucoma, a complex disease characterized by the progressive dysfunction and permanent loss of retinal ganglion cells (RGCs), is the leading cause of irreversible blindness, affecting nearly 80 million individuals worldwide (1,2).Its prevalence places it among the most common neurodegenerative diseases, and the visual impairment it causes makes it a major health and economic burden (3)(4)(5).While the broad contours of glaucomatous progression are straightforwardthe death of retinal ganglion cells and a resultant loss in visual fieldthe etiology of the disease remains incompletely understood, particularly for primary open-angle glaucoma, the most common variant (6, 7).Age and elevated intraocular pressure (IOP) are major risk factors, while genetics also play a role, but current management strategies are largely limited to the lowering of IOP (1,2).However, the relationship between IOP and progression is not absolute: many individuals with ocular hypertension do not develop glaucoma, some individuals with IOPs in the normal range do, and some percentage of treated individuals nevertheless continue losing visual acuity (2,8).Despite the demonstrated need for directly neuroprotective therapies to halt the loss of RGCs and preserve vision, either as an adjunct to IOP control or in place of it, such an approach remains out of reach, in part because of our incomplete understanding of the pathophysiology of glaucoma.Nonetheless, new mechanistic insights into the progression of glaucoma may also shed light on other age-related neurodegenerative diseases, moving the field closer to viable treatments and preventative strategies.
Astrocytes, a type of glial support cell found throughout the central nervous system (CNS), are of particular interest in the pathophysiology of glaucoma because they constitute the main cellular component of the optic nerve head (ONH), a key site of early pathological events (2,9,10).During glaucoma they transition to a reactive state, a process that drives changes in their morphology, function and gene expression that can influence their support for RGCs, alter the tissue environment and influence disease outcome (11)(12)(13)(14).Despite growing awareness of their relevance, both in glaucoma and neurodegenerative diseases more broadly, the behavior of astrocytes in glaucoma remains poorly understood.This is linked more broadly to an incomplete understanding of white matter astrocytes, the type that populates both the optic nerve and retina, in terms of heterogeneity and behavior under both physiological and pathological conditions.Although the heterogeneity of astrocytes has recently expanded as a research topic, much of this work has been done in gray matter astrocytes of the brain; the extent of differences between white matter and gray matter astrocytes is still emerging, while heterogeneity within and between white matter astrocyte populations remains largely unknown.A more in-depth understanding of the characteristics of white matter astrocytes is required if we are to better learn their unique functions in disease.

Astrocyte function and heterogeneity
The most abundant glial cell type in the central nervous system, astrocytes were once thought of as passive structural support to neurons (15).While astrocytes do play key structural roles through both their influence on the extracellular matrix (ECM) and their own intrinsic biomechanical properties, they are now recognized as performing a broad array of functions essential to supporting neurons and maintaining a healthy CNS (16-18).Individually, astrocytes contact blood vessels and neuronal structures, rapidly shuttling energy substrates to active neurons while syphoning away excess ions and neurotransmitters released during neuronal signaling (19-23).However, astrocytes also form extensive networks, linked by gap junctions, that facilitate large scale spatial buffering of factors within the neural parenchyma as well as the coordination of astrocyte behavior (24, 25).Astrocytes additionally function as secretory cells, releasing neurotrophic and synaptogenic factors, lipids with beneficial or detrimental effects on neuronal survival, and chemokines that mediate immune response by signaling microglia and peripheral immune cells (26)(27)(28)(29)(30)(31).Beyond these core functions are a wide variety of specialized behaviors that are indicative of the still-emerging extent of astrocyte heterogeneity (32)(33)(34)(35).
From the early days of modern neuroscience, a degree of astrocyte heterogeneity has been recognized, with researchers categorizing astrocytes as either protoplasmic or fibrous, based on morphology (36).These distinctions largely align with anatomical localizationprotoplasmic astrocytes generally populate synaptically dense gray matter regions, whereas fibrous astrocytes typically reside in myelinated white matter tractsand correlate to functional specialization at the broadest level (37).Gray matter astrocytes, for example, contact large numbers of synapses, where they are involved in supporting and modulating synaptic function, ranging from the uptake of excess glutamate to altering signaling sensitivity to the preservation or elimination of individual synapses; the extent of their participation has given rise to the concept of the tripartite synapse, in which astrocytes participate in synaptic regulation alongside neuronal axons and dendrites (28,38,39).Conversely, astrocytes in white matter contact neurons along their axons, modulate oligodendrocyte function to preserve unmyelinated contact points at the Nodes of Ranvier, and incorporate oligodendrocytes within their gap junction connected networks (40-43).Historically gray matter astrocytes have been more heavily investigateddespite the vulnerability of white matter regions such as the optic nerve tract in glaucoma, multiple sclerosis, and other debilitating neurological conditionsand eventually became the basis of 'normal' astrocyte function in much of the literature (33,44,45).An exception to this trend has been the investigation of energy supplementation of neurons by astrocytes, where much foundational work was done with optic nerves for practical reasons (23,46).
More recently, however, heterogeneity within these two types has emerged as well, and a number of regional populations of astrocytes have demonstrated specialized functions, many involving physiological and behavioral activities that were previously thought to be mediated by neurons.Among gray matter astrocytes, those in the cortex have been shown to participate directly in neurovascular coupling (47), while a population at the ventral surface of the medulla oblongata detects pH changes in the blood and triggers the neuronal response that drives reflex breathing (48), and those of suprachiasmatic nucleus are able to drive circadian rhythm cycles independently of neuronal time-keeping (49).Nor are region specific populations the limit of astrocyte heterogeneity, as studies within regions have identified sub-populations with morphological and transcriptional variation: within the cortex, morphological studies identified four sub-populations of protoplasmic astrocytes, a finding replicated with single cell transcriptomic analysis (50, 51).Here too is our current understanding largely dominated by findings on gray matter astrocytes, despite evidence of morphological and functional heterogeneity in white matter tracts such as the optic nerve (52)(53)(54)(55).Two exceptions documented in the optic nervethe involvement of astrocytes in axonal mitochondria turnover and non-cell autonomous provisioning of energy substrates to axonsare of particular interest in the context of glaucoma and addressed in detail later in this review (56,57).

Astrocyte reactivity
In addition to inter-and intra-regional heterogeneity of astrocytes under physiological conditions, the changes undergone by astrocytes in response to disruption of homeostasis compound the complexity of the matter still further.When the central nervous system suffers insultsuch as physical trauma, infection by pathogens, or ischemiaastrocytes respond by undergoing a transition to a reactive state (sometimes referred to as gliosis), in which their behavior is alteredacutely or chronicallyto address the altered conditions they are exposed to (58)(59)(60).In addition to their response to relatively straightforward exogenous insults, reactive astrocytes are a major area of research in chronic neurodegenerative conditions, including glaucoma (7,(61)(62)(63)(64).Astrocyte reactivity is closely linked to neuroinflammation, a broader inflammation-like state that occurs in central nervous system injury and neurodegeneration, which is characterized by the response of astrocytes, microglia, and other resident cell types, alongside contributions from the peripheral immune system (12, 65, 66).As with conventional inflammation, neuroinflammation is an essential response to insult that nonetheless can drive deleterious outcomes, particularly when it persists in a chronic state (67, 68).However, as with other elements of astrocyte behavior, the heterogeneity of these responses is only recently coming into focus (59, 60, 69).
Historically, reactivity has been associated with its most severe manifestations, including astrocyte hypertrophy, proliferation, and scar formation, and thought to be the primary barrier to regeneration in the central nervous system of mammals (70,71).Subsequent work demonstrated reactivity to be a graded phenomenon, with many of the above responses constrained to cases of particularly grievous injury (16).Still more recent research demonstrates an essential role for reactivity in preserving nervous tissue in the face of damaging stimuli, revealing a complex behavioral response that can also include neuroprotective functions (72,73).The astrocyte-specific deletion of transcription factor STAT3, for example, diminishes the reactive capacity of astrocytes and leads to a worsening of visual function in a mouse model of glaucoma (74).A high-profile effort has been made in recent years to better understand reactive astrocytes by classifying t h e m a s d e t r i m e n t a l ( N e u r o t o x i c / A 1 ) o r b e n e fi c i a l (Neuroprotective/A2) (75, 76).However, a recent consensus statement by a number of major figures in the field of astrocyte research makes it clear that this duality understates the degree of relevant heterogeneity (69).Rather, the reactive response of astrocytes depends on a multitude of factors, including the site, type, duration, and severity of insult, as well as the preexisting state of the astrocytes in question; indeed, many subsequent studies have found overlapping expression of A1 and A2 markers in reactive astrocytes from human tissue and in a variety of mouse models (77)(78)(79).Finally, further compounding this complexity is the role of aging, which appears to drive a pseudo-reactive state in astrocytes that potentiates reactive changes and may contribute to the increased vulnerability of aged individuals to neurodegenerative diseases (80)(81)(82).
This complex heterogeneity suggests that reactivity is a loose constellation of interrelated behaviors, each of which may or may not be present in a given population of reactive astrocytes.Crucially, it also suggests that although a broad understanding of astrocyte behavior may hint at their role in glaucoma, the behavior of astrocytes elsewhere in the CNSwhether under physiological or pathological conditionsmay not be predictive of those attending the axons of RGCs in the optic nerve and retina.
Therefore, the remainder of this review will focus on the astrocyte populations of the anterior visual pathwaythe retina, the optic nerve head, and myelinated optic nerveand recent notable work in the field, including a transcriptional study by our group that corroborates previous functional and morphological studies suggesting that the astrocytes of the optic nerve head form a distinct population from those of the myelinated optic nerve.

Zones of the retinal ganglion cell axon and attendant white matter astrocyte populations
Retinal ganglion cell axons, the most structurally and metabolically (45) vulnerable component of the visual pathway, traverse three distinct 'zones' (9, 14) after emerging from the somas of RGCs.These regionsthe retinal nerve fiber layer, optic nerve head, and myelinated optic nerveexpose the axons to widely divergent environments.In the retina RGC axons are unmyelinated, increasing their metabolic vulnerability, but the nerve fiber layer possesses a dense capillary bed that may offset the lack of myelin (14).Conversely, at the optic nerve head, these still unmyelinated axons undergo a sharp turn and pass through a pressure differential across the lamina, exposing them to both mechanical and metabolic stressors; perhaps as a consequence, despite extensive vascularization the ONH is a major site of early damage in glaucoma (9, 14,83,84).The myelinated portion of the optic nerve, the largest axon section by far, emerges posteriorly from the ONH; like the retina, it is not thought to be an early site of glaucomatous damage.
Throughout these three regions, RGC axons are supported by fibrous astrocytes that emerge from precursors in the optic nerve during development (85)(86)(87)(88).Despite common developmental origins, astrocytes residing in each region show evidence of specialized adaptations that may be indicative of distinct populations.The astrocytes of the optic nerve, in particular, have historically been considered as a homogeneous population, although the specialized anatomy of the optic nerve head and its role as a key site of early glaucomatous damage later gave rise to a specific interest in the functions of astrocytes there.Recent functional studies have highlighted specialized roles for these astrocytes that may be unique, and a transcriptional study of astrocytes in the optic nerve published by our group highlights that these astrocytes may be best understood as a distinct population from those of the myelinated optic nerve (55,89,90).Intriguingly, other recent work suggests that these populations are nevertheless extensively networked across anatomically large distances by gap junctions, facilitating coordination of non-cell autonomous support functions at a previously unappreciated scale (57).A greater understanding of both the heterogeneity between these populations and their capacity for coordinated behavior may shed new light on the mechanisms of glaucomatous vulnerability and new avenues of neuroprotection.

Retinal astrocytes
In the furthest anterior region of the visual pathway, a distinct population of fibrous astrocytes tiles the vitreoretinal surface in many mammalsincluding humans -and is required for retinal vascularization during development (91-94).These retinal astrocytes interweave RGC axons in the nerve fiber layer and contact the superficial retinal vasculature, but their roles in neurovascular coupling and ionic buffering remain inconclusive (21,95,96).In humans and other primates, these astrocytes fall into two morphological categories, with more 'stereotypical' stellate astrocytes contacting the vasculature in the ganglion cell layer of the retina, while astrocytes with elongated processes that run parallel to axons are found in the nerve fiber layer (97, 98).The extent of heterogeneity and specialization of these two variants is largely unknown; recent single-cell RNA sequencing experiments with human tissue have generally been able to distinguish radial Müller glia from astrocytes, but not subtypes within these categories (99,100).While at least one study found greater variation, identifying three distinct glial clusters, it could not conclusively distinguish which may have represented Müller glia and which may have been astrocytic (101).Regardless, due to the nature of the techniques involved, none of these studies were able to link transcriptional differences to morphological variation, leaving the significance of the morphological categories uncertain.
Rats and mice, which act as the basis of many rodent models of glaucoma, do not possess such obvious morphological variation among retinal astrocytes, but some studies have assessed a greater variety in morphology via intracellular tracer dye injection (102), while others have noted trends such as the association of larger astrocytes with retinal veins rather than arteries (103).Generally, the sparseness of these astrocytes, which make up approximately 0.1% of retinal cells, renders their study a particular challenge, which is exacerbated due to the transcriptional similarity of Müller glia that outnumber retinal astrocytes by at least an order of magnitude (104,105).However, retinal astrocytes are known to form gap junction coupled networks with directional properties, including unidirectional flow into Müller glia, which are not directly coupled to each other (106).Retinal astrocytes exhibit indicators of reactivitysuch as upregulation of GFAP and remodelingin both human glaucoma and animal models, but the extent of their causal role in disease progression remains unknown (107-109).Curiously, this reactivity has been repeatedly demonstrated to propagate from injured retina to uninjured contralateral eye in animal models, through a mechanism that has long been unclear (110,111).A potential mechanism for this spread has been recently elucidated through a study on resource sharing across these anatomical distances, highlighting a potential neuroprotective role behind this previously puzzling function (57).Cx43, the gap junction protein that is essential for this phenomenon, is also upregulated in retinal astrocytes in human glaucoma (112).

Optic nerve head astrocytes
Although longitudinally the shortest of these three regions, the optic nerve head is the most structurally complex and the one most directly implicated in early glaucomatous progression (9).Formed by fusion of the optic stalk and retina during development, the ONH is a transition zone between these tissues that contains elements of both, consisting of pre-laminar areas where the retina transitions to optic nerve, the lamina itself, and the posterior myelin transition zone, which is more pronounced in rodents (9, 14, 113).Although the lamina cribrosa of humans and other large mammals is more elaborate than the glial lamina of the mouse, the structures serve the same function, forming a tight seal between the positive pressure zone inside the eye and the optic nerve, isolating the retina from exposure to cerebral spinal fluid while guiding and ensheathing RGC axons as they exit the eye (14,114,115).Axons in this region are unmyelinated and characterized by elevated mitochondrial density, which may reflect increased metabolic requirements driven by both the lack of myelination and the challenge of axonal transport across a pressure gradient; the complex vascularization at this site may be similarly indicative of high metabolic needs (9, 14,116).
Optic nerve head astrocytes play a major role in the structure of the lamina: in humans, astrocytes line the collagenous beams that form the mesh-like structure and insulate axons from connective tissue and vasculature, while in mice these collagen fibers are absent and astrocytic processes are instead responsible for the structural seal (9).In keeping with their structural role, these astrocytes express high levels of the intermediate filaments GFAP, vimentin (117), and nestin (118), and are consistently oriented transversely to the path of axons, adopting in mouse a distinct morphology similar to a "catcher's mitt" in baseball (53).Uniquely among astrocytes, those at the lamina largely lack aquaporins, the specialized water channels that are so ubiquitously expressed on astrocytes elsewhere (54,119).This absence has been observed in both humans and all investigated mammalian models, indicating that this absence is a highly conserved trait; it has been speculated that this may be a requisite for maintaining additional rigidity in these astrocytes (89).
Our recent transcriptional study of astrocytes under physiological conditions in the normo-tensive mouse optic nerve highlights a number of key differences between those of the ONH and the myelinated optic nerve proper, bolstering the idea that this a distinct population (90).Unsurprisingly, given the role of ONH astrocytes in remodeling the extracellular matrix during glaucoma, under physiological conditions we found that pathways related to ECM maintenance were highly enriched in this population relative to those of the myelinated optic nerve and corpus callosum (13).Intriguingly, we also observed clear metabolic differences between myelinated and unmyelinated optic nerve, with the astrocytes of the ONH expressing elevated levels of genes related to mitochondrial protein translation and oxidative phosphorylation.This may imply that these astrocytes are able to utilize the dense vascularization of the ONH by relying on the more energetically favorable breakdown of glucose via oxidative phosphorylation.
However, this comes with potential drawbacks, which may contribute to the heightened vulnerability of this region to g l a u c o m a t o u s d a m a g e .E l e v a t e d l e v e l s of ox i d a t i v e phosphorylation result in increased production of reactive oxygen species (ROS), a source of oxidative stress and potential contributor to glaucoma (12, 120).Conversely, astrocytes provide antioxidant support in the central nervous system through glutathione production, the peroxiredoxin system, and other mechanisms, and our transcriptional results suggest that this is the case at the ONH as well, with high levels of glutathione transferase and peroxidase transcription implying that this population of astrocytes acts to ameliorate the effects of local production of ROS (90, 121, 122).Secondly, the enrichment of oxidative phosphorylation related genes was accompanied by indicators of lower levels of glycolysis, an alternative mechanism by which astrocytes generate energy in the brain (123).Although glycolysis generates less energy than oxidative phosphorylation, it is also tied to the production of lactate, which astrocytes use to provision neurons with energy during periods of elevated energy consumption (20).Our transcriptional comparison of ONH astrocytes and those of the corpus collosum suggests that less glycogen storage may be occurring at the ONH.Given that astrocytic glycogen forms the primary energy reserve in the CNS, this may exacerbate metabolic vulnerability; however, a recent study has shown long-range energy provisioning through the interconnected astrocyte network of the optic nerve that may offsetat least in the short termthis vulnerability (23,57).Nonetheless, it is reasonable to hypothesize that even a modest loss of function regarding astrocytic ROS-amelioration or energy provisioning, due to aging or other contributing factors, could precipitate axonal damage in this metabolically demanding and vulnerable region.
Astrocytes of the ONH are also thought to directly sense and respond to mechanical pressure, which may influence their contributions to remodeling during glaucoma, although many of these changes may be driven in part by damage to RGC axons, such as the 'gap-plugging' response brought on by axonal loss (124, 125).In addition to upregulating GFAP and the gap junction protein Cx43 (112), reactive ONH astrocytes in human glaucoma and animal models extensively modify the extracellular matrix at the optic nerve head (11,13,126).Finally, while neuroinflammation in glaucoma includes, and is in part mediated by, astrocyte reactivity at the optic nerve headincluding a complex milieu of behaviors such as antioxidant activity, nitric oxide production, and changing levels of reactive oxygen species formationthe topic also incorporates complex and incompletely elucidated interactions with the activities of additional cell types such as microglia (120,127,128).In order to retain the focus on astrocyte-specific functions we have largely bypassed the broader discussion of neuroinflammation in glaucoma, but the topic has been well reviewed elsewhere (7,12).

Myelinated optic nerve astrocytes
Directly posterior to the lamina, the transition to myelinated optic nerve begins.In mice a pronounced myelin transition zone can be observed, although this may be largely the function of differences in scale and lamina extent between mice and primates.Astrocytes in this liminal region play a major role in the turnover of axonal mitochondria, discussed later.The myelinated portion of the optic nerve extends posteriorly through retrobulbar space to the lateral geniculate nucleus, superior colliculus, and other regions to which RGCs synaptically project (9).Unlike in the retina and optic nerve head, this region is populated by oligodendrocytes that myelinate the axons, insulating them electrically and protecting them from noxious stimuli (41).Due to its straightforward anatomy and relative accessibility, the myelinated optic nerve is a classic model for studying the behavior of axons and glia in white matter (41,129,130).In addition to astrocytes and oligodendrocytes, this region is populated by NG2 glia, which have been previously described as oligodendrocyte precursors but may also play specific roles in neuroinflammation, and by a significantly higher density of microglia than is found at the optic nerve head (131).
Astrocytes of the myelinated optic nerve make less extensive contact with axons than those in unmyelinated regions, primarily via gaps at Nodes of Ranvier, where they take up the potassium ions released by axonal signaling and shunt them to areas of lower concentration or to the vasculature (21,(132)(133)(134).They are also responsible for providing energy substrates to axons from stored glycogen during decreased glucose availability or heightened axonal need; astrocytic energy provisioning is well studied in this portion of the optic nerve and is the focus of a more detailed section below (46, 129).Astrocytes of this region, like those of the retina and ONH, are extensively coupled by gap junctions (135), but also interact with oligodendrocytes and myelin to modulate the extent of myelination and preserve the accessibility of nodes of Ranvier (42).

Axonal transport and mitochondrial turnover
In keeping with the metabolically demanding nature of the central nervous system, axons consume large quantities of energy, both to maintain the ionic gradients required for action potentials and for pre-synaptic elements of vesicular signaling (83, 84, 136).These intense energy requirements make axons, particularly in unmyelinated regions, highly vulnerable to dysfunction in mitochondria, the organelles responsible for generating ATP to power activities within a cell (45).Mitochondrial dysfunction is thought to contribute to major neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (137-140).The optic nerve in particular is especially vulnerable to the metabolic consequences of mitochondrial dysfunction, making it the primary site of neurological damage in mitochondrial disorders such as Leber's hereditary optic neuropathy and dominant optic atrophy, known collectively as Mitochondrial Optic Neuropathies (MON) (83,84,141).Due to its length and energy demands both along the axon and at the synapse, mitochondria are located throughout the optic nerve in regions of high energy utilization, and unmyelinated axons at the optic nerve head exhibit particularly high mitochondrial density (14,83,(142)(143)(144).However, mitochondria are impermanent structures that must be routinely replaced, and deficits in mitophagythe process of eliminating aged or damaged mitochondriacan result in decreased energy utilization and the exposure of the cell to damaging ROS and other factors (83, 145).
Historically cells were thought to degrade their own mitochondria, but in the last decade a population of phagocytic astrocytes at the optic nerve head has been shown to aid in the degradation of axonal mitochondria (55,56).These astrocytes constitutively express the phagocytic marker galectin-3 (Lgals3/ Mac-2) and engage in a process termed transmitophagy, in which mitochondria are extruded by axons before being engulfed and eliminated by astrocytes.This elimination of axonal mitochondria by phagocytic ONH astrocytes is estimated to exceed mitophagy by RGCs in the ganglion cell layer, despite greater abundance of mitochondria in that region, suggesting that distal axonal mitochondria may undergo retrograde transport to this region specifically for degradation.Consistent with previous studies showing galectin-3 expression by reactive astrocytes in mouse models of ischemia (75, 146) and physical injury (147)as well as from patients with the demyelinating disease multiple sclerosis (148)expression at the ONH rises in both DBA/2J and inducible OHT mouse models of glaucoma (55), and has been detected at the ONH in human glaucomatous tissue (149).
Curiously, work by other groups in both DBA/2J and microbead-induced mouse models of glaucoma shows an increased percentage of axonal mitochondria with indicators of damage, such as depleted cristae, in this region (150, 151).The increase in a marker of phagocytic capacity (galectin-3) combined with accumulation of degradation-bound mitochondria, although superficially paradoxical, may aid in identifying the contributions of phagocytic and mitochondrial dysfunction in glaucoma.Defects in axonal transport, both anterograde and retrograde, are well established in glaucoma and emerge early in inducible OHT models (14,152), and a recently published study identifies a decrease in anterograde transport of mitochondria and an accompanying decline in axonal ATP in a mouse microbead model (153).One possibility is that axonal transport disruption impairs the replacement of damaged mitochondria, leading to their accumulation and worsening metabolic dysfunction.An alternative is suggested by early work on non-human primates implicating ONH astrocytes in axonal phagocytosis in a model of glaucoma (154,155).Astrocytes may have a more limited phagocytic capacity than professional phagocytes such as microglia and macrophages, and the need to eliminate axonal debris as glaucoma progresses may exceed this capacity and result in a 'backlog' of damaged mitochondria (156).
Regardless of the underlying cause, an accumulation of damaged mitochondria in the ONH would be expected to exacerbate energy deficits in the region and lead to the increased production of reactive oxygen species and oxidative stress (120), contributing to the indicators of neuroinflammation seen in glaucoma (12).Furthermore, the group responsible for identifying transmitophagy in the optic nerve also claims to have identified potential sites of this function in the cortex, and recent work in a mouse model of Parkinson's Disease suggests this may indeed be a specialized astrocyte function elsewhere in the CNS (56,157).Given the evidence for a link between mitochondrial dysfunction and neurodegenerative disorders, it may be that disruption of transmitophagy plays a role in neurodegeneration more broadly.Finally, upregulation of galectin-3 is also found in an activated microglia state associated with neurodegeneration (158, 159) and may act to propagate the activated state to nearby microglia (160); moreover, recent work has shown that disrupting its expression is protective in a mouse model of glaucoma (161).Thus, we should consider the possibility that astrocytic upregulation of galectin-3 potentiates the microglial (162) and/or peripheral immune (163,164) response in glaucoma, which may contribute to a neuroinflammation in the region.
A number of related questions regarding mitochondrial function and turnover in glaucoma remain unanswered.Astrocytes are known to utilize at least 2 distinct phagocytic pathways -MEGF10 and MERTKbut which, if either, is active at the ONH remains unknown; identifying the phagocytic machinery expressed by this astrocyte population may improve our understanding of the transmitophagic process under physiological and pathological conditions (146,165).While our transcriptional data confirms high expression of Lgals3 in astrocytes of this region, it unfortunately does not clarify what other factors may be involved (90).Furthermore, just as neurons have been shown to utilize astrocytes for elimination of degraded mitochondria, evidence in a mouse model of stroke has shown that astrocytes donate healthy mitochondria to neurons after injury (166).It remains unknown whether a similar behavior occurs in the optic nerve; although our data shows an increased expression of mitochondrial components by astrocytes in this region that would be consistent with such a phenomenon, it is unable to conclusively demonstrate this possibility.Finally, at least one approach for neuroprotection in glaucoma involves the supplementation of nicotinamide (vitamin B3), a precursor of the metabolic cofactor NAD + , which is vital for mitochondrial function but declines with age (167).Oral supplementation in the DBA/2J mouse model of glaucoma protected RGCs from glaucomatous progression, and human trials are ongoing to assess the viability of this approach at a clinical level (168).

Long range energy provisioning via the optic nerve astrocyte network
In times of inadequate provisioning -due to hypoglycemia or elevated metabolic demand -astrocytic glycogen stores provide a repository of usable energy within the nervous system that can preserve neuronal function until equilibrium is restored between metabolic needs and substrate availability (23).The sequestration of glycogen by astrocytes, although low by standards of other body tissues, is extensively documented, though much regarding the mechanisms of provisioning remains unresolved (169, 170).A leading hypothesis, the astrocyte-neuron lactate shuttle, states that astrocytic glycogen is converted to lactate and secreted to neurons through monocarboxylate transporters, but aspects of the hypothesis remain contentious (20,171,172).Previous studies suggest key differences in gray and white matter astrocyte provisioning, including the metabolic signals that induces substrate transfer (glutamate in the former, while K + is a primary candidate in the later) and indicate that white matter astrocytes may store lower levels of glycogen than those of gray matter (37,171,173,174).
The optic nerve has several properties that make it a favored model for studying energy provisioning from astrocytes to axons in white matter: surgical accessibility, the presence of axons and glia but no neuronal soma, and a straightforward anatomical structure (23,170).It is also a key site of disease progression in glaucoma, as well as a number of mitochondrial optic neuropathies, a group of genetic disorders that alter mitochondrial function throughout the body but primarily damage retinal ganglion cell axons, suggesting it is amongst the most metabolically vulnerable of tissues (14,83,84,141,142,175).As such, the optic nerve is a frequently utilized system for ex vivo investigation of astrocytic and axonal metabolomics (129,176).Prior work in the optic nerve has established an essential role for astrocytic glycogen in maintaining axonal function during conditions of glucose depletion or elevated signaling activity, with experimental manipulation of glycogen levels showing that pre-depletion lessens the duration of preserved function; similar results have been demonstrated with gray matter astrocytes of the hippocampus in ex vivo slices (129, 177).However, until recently direct evidence for white matter astrocytic-glycogen support in vivo has been limited and investigations have typically involved short periods of intense glucose deprivation, rather than extended periods of modest deprivation that may be more representative of disease states.
A recently published study of energy provisioning from astrocytic glycogen stores in the optic nerve shows the relevance of this phenomenon in vivo in a microbead occlusion mouse model of glaucoma (57).Unlike previous ex vivo studies of optic nerve glycogen utilization, the moderate (~35%) IOP elevation provides a stimulus more representative of glaucomatous insult, and one in which glycogen preserves axonal function over the course of days and weeks, rather than minutes and hours (129, 170).This study is particularly notable for several reasons: 1. Indication of an early energy deficit in OHT optic nervedetectable by day 4 as a decline in glycogen reserves, and an increase in phosphorylation of the cellular energy sensor AMPK, 2. in vivo demonstration of a role for astrocytic glycogen in preserving optic nerve function, with functional losses at the behavioral level and in axonal transport occurring more rapidly in glycogen depleted nerves, and 3. A clear role for the gap junction coupled astrocyte network in energy provisioning across anatomically significant distances (≥ 10mm).
The most striking of these results is the long distance, Cx43mediated provisioning of energy substrates, in which glycogen reserves in the contralateral nerve appear to metabolically buffer the optic nerve of the microbead-treated eye, helping to preserve axonal function at the expense of depleting reserves and increasing metabolic vulnerability in the donating nerve.When mice with astrocyte-specific Cx43 deletion were treated unilaterally with microbeads, they experienced more rapid declines in function and glycogen levels in the ipsilateral nerve, while the contralateral nerve retained greater glycogen reserves and remained less sensitive to future insults.These mice also demonstrated more rapid loss of axonal transport function than WT animals, which show minimal decline in either nerve one week after microbead injection, whereas KO animals show an approximately 40% reduction in the ipsilateral nerve.As with prior ex vivo studies, the greater the stores of glycogen available, the longer axons were able to continue functioning with only modest deficits, demonstrating a neuroprotective effect for astrocytic glycogen provisioning in vivo.
The implication of the Cx43 coupled astrocyte network in these results also suggests a potential mechanism for the previously observed phenomenon of astrocyte reactivity spreading from injured eye to contralateral retina (110).Cx43 is upregulated in human glaucoma, both in retinal astrocytes and at the ONH, and multiple groups have claimed that gap junctions may propagate deleterious signals in neurodegeneration and injury (112,178,179).However, the outcome of this study -Cx43-mediated preservation of function in injured tissue at the expense of increased vulnerability in distal regionssuggests a more nuanced interpretation is in order.
While axonal dysfunctionincluding disrupted axonal transport and poor mitochondrial healthare thought to be early components of glaucoma, these results suggest that the depletion of astrocytic energy stores in the optic nerve also plays an early role (9, 83, 84, 152).In keeping with this interpretation, work published in 2018 found significant depletion of glycogen in aged DBA/2J mouse optic nerves relative to age matched controls, and DBA/2J and microbead-treated mouse models showed an increase in phosphorylation of AMPKa marker of metabolic stressin optic nerve astrocytes; similar changes in AMPK phosphorylation were also observed in tissue from glaucomatous human optic nerve (180).These results suggest an energy deficit, congruent with reports of metabolic dysfunction in glaucoma, resulting from decreased energy utilization and/or elevated metabolic costs.
Intriguingly, the 2018 study showed a decrease in lactate transferring monocarboxylate transporters in optic nerve astrocytes and RGC axons in both DBA/2J mice and mouse microbead models, as well as a decrease in axonal monocarboxylate transporters in human glaucoma (180).These counterintuitive findingsa decline in components of astrocyte to neuron energy provisioning during a period of elevated axonal vulnerabilitymay point the way to avenues of research that shed light on the function of astrocytes in glaucoma and beyond.Some work suggests that reactivity may modulate 'self-preservation' pathways in astrocytes, potentially diminishing their ability to aid distressed neurons (181)(182)(183)(184).Although seemingly disadvantageous from the viewpoint of neuroprotection, acute focal injuries in the CNSwherein a penumbral zone populated by glia but depleted of neurons surrounds the infarct core and separates it from healthy neural parenchymaprovide examples of scenarios in which such an adaptation would be a net advantage for organismal survival (185,186).Alternatively, damage to mitochondria may render them unable to effectively utilize lactate from astrocytes, cutting off axons from this energy source and exacerbating metabolic dysfunction.Given that energy dysfunction and propagation of neurodegeneration are implicated in a variety of neurological injuries and diseases, developing a greater understanding of the networked behavior of astrocytes may provide future avenues for early intervention in these devastating conditions.

Conclusion
Although the heterogeneity of astrocyte functions as a research avenue is still at a formative stageparticularly with regard to white matter, fibrous astrocytesit is a topic that is likely to play an outsized role as the field moves from foundational research to the consideration of astrocytes as druggable targets in specific neurological injuries and neurodegenerative diseases such as glaucoma.Given the unique vulnerability of retinal ganglion cell axons, especially to metabolic disruption, and the specialized role localized populations of astrocytes play in supporting these axons, we expect a more complete understanding of neuron-glia coordination in the anterior visual pathway to reveal future avenues for neuroprotective treatment that can supplement or replace the current approach of IOP management.Finally, although we advise caution in inferring behaviors across astrocyte populations, the accessibility of the anterior visual pathway may make it an ideal region for future investigations of white matter astrocytes and the development of novel approaches to elucidate their behavior.