Cell Biology of the Müller Cell

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Chapter 17 Cell Biology of the Müller Cell

Introduction

In addition to microglial cells, which are the resident immune cells of the retina, the retina contains two types of macroglial cell: astrocytes and Müller cells (Fig. 17.1A). Macroglial cells support the functioning and metabolism of retinal neurons. For this reason, they constitute an anatomical and functional link between these neurons and the compartments with which they need to exchange molecules (blood vessels, vitreous chamber, and subretinal space). Most nutrients, waste products, ions, water, and other molecules are transported through macroglial cells between the retinal vessels and neurons. As a peculiarity, astrocytes play a crucial role in retinal vascularization; the localization of these cells in the mature retina is restricted to the nerve fiber and ganglion cell layers.

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Fig. 17.1 Müller cells (M) span the entire thickness of the neuroretina, and are arranged in a regular pattern. (A) Schematic drawing of the cellular constituents of a human retina. The perikarya of Müller cells are localized in the inner nuclear layer (INL). The funnel-shaped endfeet of Müller cells form the inner surface of the retina. In the outer (OPL) and inner plexiform layers (IPL), side branches which form perisynaptic membrane sheaths originate at the stem processes. Both astrocytes (AG) and Müller cells contact the superficial blood vessels (BV) and the inner surface of the retina. In the outer nuclear layer (ONL), the stem process of Müller cells forms membrane sheaths which envelop the perikarya of rods (R) and cones (C). Microvilli of Müller cells extend into the subretinal space which surrounds the photoreceptor segments (PRS). Microglial (MG) cells are located in both plexiform layers and the ganglion cell layer (GCL). A, amacrine cell; B, bipolar cell; G, ganglion cell; H, horizontal cell; P, pericyte; RPE, retinal pigment epithelium. (B–D) Confocal images of living guinea pig retina preparations. (B) Radial section, illustrating the tight package of the cellular elements shown in (A). The Müller cells are labeled with the vital dye, Mitotracker Orange (green); synapses and the outer segments of photoreceptor cells are counterlabeled with another vital dye, FM-43. (C, D) Optical horizontal sections through a flat-mounted retina, illustrating the regular pattern of Müller cell stem processes in the inner plexiform layer (D) and the almost total occupation of the ganglion cell layer by the Müller cell endfeet (green; M); only the somata of the ganglion cells (G) appear “empty” (C).

Morphology of müller cells

Heinrich Müller (1820−1864) in 1851 described the “radial fibers” of the retina which later became known as Müller cells. Müller cells are specialized radial glial cells which span the entire thickness of the neural retina, from the subretinal space to the vitreal surface (Fig. 17.1A, B). The somata of the cells are located in the inner nuclear layer, and two stem (trunk) processes radiate from the soma in opposite directions. The outer stem process draws towards the subretinal space; here, microvilli of Müller cells surround the photoreceptor inner segments. Zonulae adherentes between Müller and photoreceptor cells form the outer limiting membrane of the retina. The inner stem process projects towards the vitread surface of the retina; the end of this process is expanded into a funnel-shaped endfoot. Both the basement membrane at the inner retinal surface and the Müller cell endfoot membranes together constitute the inner limiting membrane. Lateral processes extend into the plexiform layers where they form elaborate sheaths around the neuronal synapses, as well as into the nuclear layers where they embed neuronal perikarya. Astrocytes and Müller cells ensheath the vessels of the superficial vascular plexus; the deep capillaries are ensheathed by en passant endfeet of Müller cells. Within the macula, Müller cells display a Z-shaped morphology because the outer processes accompany the centrifugally running cone axons in the Henle fiber layer (Figs 17.2 and 17.3C). The central fovea contains 20–30 atypical Müller cells. These cells are not associated with cone axons; instead, their outer process runs straight towards the inner retinal surface where the soma is located and where their large, flat endfeet contribute to the inner limiting membrane of the fovea proper.1

Müller cells constitute the cores of functional retinal columns

The human retina contains 8–10 million regularly arranged Müller cells (Figs 17.1D and 17.4C). Each Müller cell constitutes the core of a column of retinal neurons which represents the smallest functional unit for “forward information processing.”2 Such a column contains one cone per Müller cell (Figs 17.3B, C and 17.4B, C) and up to 10 rods, as well as 6 (fovea) and 4 (periphery) inner nuclear layer neurons, and 2.5 (fovea) and 0.3 (periphery) ganglion cell layer neurons, respectively.1

Müller cells interact with the neurons of their columns in a kind of symbiotic relationship. They are responsible for all functional and metabolic support of their associated neurons. Müller cells provide trophic substances to neurons, and remove metabolic waste. They play a critical role in regulation of the extracellular space volume, ion and water homeostasis, and in the maintenance of the inner blood–retinal barrier. They release gliotransmitters and other neuroactive substances, and impact synaptic activity by neurotransmitter recycling which involves the supply of neurons with precursors of neurotransmitters. All of these functions directly or indirectly modify the neuronal activity. Müller cells support the survival of photoreceptors and neurons, are responsible for the structural stabilization of the retina, and are modulators of immune and inflammatory responses. They guide the light to the photoreceptors and buffer mechanical tissue deformations. Müller cells become activated upon virtually all pathogenic stimuli. Reactive Müller cells are neuroprotective but may also stop supporting the neurons, and rather contribute to neuronal degeneration. Currently, the many roles of Müller cells in the regulation of retinal function are still not fully resolved, and are the subject of ongoing intensive research. Noteworthy, much of the current knowledge about Müller cell (dys-)function was obtained in animal models and thus awaits confirmation on human cells.

Light guidance

Because the retina is inverted, light has to pass the entire depth of the neuroretina before it arrives at the photoreceptors. In the retina as in any other organ, all cells and their processes and organelles are phase objects which scatter the incoming light. In particular, the synapses in the plexiform layers have dimensions close to 500 nm, i.e., within the wavelength range of visible light; this makes them light-scattering structures.3 The backscattering of light from the retinal cell structures is clinically used by optical coherence tomography. However, light scattering should reduce visual sensitivity and acuity, particularly outside the fovea (where the inner layers are shifted aside, and the light directly hits the cones).

To reduce light scattering, nonfoveal Müller cells act as living optical fibers that guide the light through the inner retinal layers towards the photoreceptors (Fig. 17.4A).3 The funnel-shaped endfeet of Müller cells act as light collectors at the vitread surface of the retina. The refractory index of Müller cells is slightly lower than that of photoreceptor segments (which are also light-guiding fibers) but higher than that of the surrounding retinal tissue.3 On the other hand, the refractory index of Müller cell endfeet is rather low, about halfway between that of Müller cell stem processes and that of the vitreous.3 This allows a “soft coupling” of the light path between the vitreous and the retina, and reduces light reflection at the inner retinal surface. By light guidance, Müller cells transport an image (like a fiberoptic plate) with minimal loss of light intensity from the inner retinal surface to the photoreceptors; this image is resolved in pixels corresponding to individual Müller cells (Fig. 17.4A). Because the local densities of cones and Müller cells are roughly equal (Fig. 17.4C), every cone has its “private” Müller cell which delivers its part of the image, while several (up to 10) rods are illuminated by one Müller cell (Fig. 17.4A).

Recycling of cone photopigments

Müller cells support the function of photoreceptors by other mechanisms, as well. There exist two cycles in the retina that regenerate the photopigments, the rod and the cone visual cycle.4 In the photoreceptor outer segments, all-trans retinal is reduced to all-trans retinol. Rod-derived all-trans retinol is regenerated to 11-cis retinal in the pigment epithelium while cone-derived all-trans retinol is processed by Müller cells. Müller cells convert all-trans retinol to 11-cis retinol which is subsequently oxidized to 11-cis retinal by a retinol dehydrogenase, and released into the extracellular space for uptake by the cone photoreceptors. For the transport of the retinoids, Müller cells express cellular retinol-binding protein and cellular retinal-binding protein (CRALBP).5 Furthermore, Müller cells contribute to the assembly of the photoreceptor outer segments,6 in part by the release of lactose and pigment epithelium-derived factor (PEDF),7,8 and phagocytose outer-segment discs shed from cones.9 Docosahexaenoic acid is required for outer-segment renewal and for the preservation of mitochondria.10 Müller cells incorporate docosahexaenoic acid into phospholipids, and channel it to the photoreceptors.10

Regulation of the synaptic activity by neurotransmitter uptake

Precise shaping (i.e., control in time and space) of synaptic activity depends upon the kinetics of the presynaptic neurotransmitter release and of the (re-)uptake of the transmitter molecules into the cells. In the neuroretina, photoreceptor cells, neurons, and macroglial cells express high-affinity transporters for neurotransmitters. Müller cells possess uptake and exchange systems for various neurotransmitters, particularly for glutamate and γ-aminobutyric acid (GABA) (Fig. 17.5).

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Fig. 17.5 Recycling of amino acid neurotransmitters in the outer plexiform layer. The ribbon synapse of a photoreceptor cell (PC) synthesizes glutamate which is released in the dark. The postsynaptic elements are dendrites of bipolar (BC) and horizontal cells (HC). Horizontal cells release γ-aminobutyric acid (GABA) which is formed from glutamate. The synaptic complexes are surrounded by Müller cell sheets. The right side shows neurotransmitter uptake systems and some metabolic pathways within Müller cells. Glutamate, GABA, and ammonia (NH4+) are transported into Müller cells and transformed to glutamine, alanine, and α-ketoglutarate (α-KG). These products are released from Müller cells, and taken up by neurons. Glutamine serves as a precursor for the transmitter synthesis in neurons (glutamate–glutamine cycle). Lactate, alanine, α-ketoglutarate, and glutamine are utilized by neurons as substrates for their energy metabolism. Glutamate is also used for the production of glutathione (GSH), which is an antioxidant, released from Müller cells and taken up by neurons under oxidative stress conditions. ALAT, alanine aminotransferase; AAT, aspartate aminotransferase; CGA, cystine-glutamate antiporter; CyT, cystine transporter; EAAC1, excitatory amino acid carrier 1; EAAT5, excitatory amino acid transporter 5; GABA-T, GABA transaminase; GAD, glutamic acid decarboxylase; AGC, aspartate-glutamate carrier; GAT, GABA transporter; GDH, glutamate dehydrogenase; GLAST, glutamate-aspartate transporter; GLT-1, glutamate transporter-1; GlyT, glycine transporter; GP, glutathione peroxidase; GR, glutathione reductase; GS, glutamine synthetase; GSH, glutathione; GSSG, glutathione disulfide; LDH, lactate dehydrogenase; MAS, malate-aspartate shuttle; OAA, oxaloacetate; PAG, phosphate-activated glutaminase; R•, free radicals; SSA, succinate semialdehyde; TCA, tricarboxylic acid cycle.

The major glutamate uptake carrier of Müller cells is the glutamate aspartate transporter (GLAST, or excitatory amino acid transporter-1, EAAT1).1113 In addition, EAAT2 (GLT1) and EAAT3 were found in human Müller cells.14,15 EAATs mediate the cotransport of three sodium ions and one proton, and the countertransport of one potassium ion, with each glutamate anion.16 The transport of an excess of sodium into the cell generates an inward current (the transporter is “electrogenic”; Figs 17.6A and 17.7A, B) and cellular depolarization. The amplitude of the glutamate transporter currents is voltage-dependent (Fig. 17.6A); a very negative membrane potential is essential for an efficient uptake of glutamate.17 Sodium-dependent carriers allow uphill transport of substrates into the cells against a concentration gradient. The driving force for the transport is the electrochemical sodium gradient across the plasma membrane, generated by the Na+,K+-ATPase. Other transporters, e.g., for GABA, are also electrogenic (Figs 17.6B and 17.7F). The subcellular distribution of the GABA transporter currents (Fig. 17.6B) corresponds with the observation that GABA is taken up by amacrine and Müller cells in the inner retina but exclusively by Müller cells in the outer retina.18

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Fig. 17.7 Neurotransmitters modify the membrane conductance of Müller cells. (A) Administration of glutamate (100 µM) to a rat Müller cell evoked inward currents through electrogenic glutamate transporters, while the glutamate receptor agonists, kainate (500 µM) and N-methyl-d-aspartate (NMDA) (100 µM, in the presence of 10 µM glycine and absence of magnesium) did not evoke membrane currents. This suggests that rat Müller cells express metabotropic but no functional ionotropic glutamate receptors. The current traces were recorded in whole-cell records at a membrane potential of –80 mV. (B) Time-dependent record of the whole-cell currents in a human Müller cell. The currents at +120 mV were mainly mediated by big-conductance potassium (BK) channels. The BK currents were transiently increased in response to extracellular adenosine triphosphate (ATP) (500 µM). Extracellular glutamate (500 µM) evoked a delayed transient increase in BK currents. The increase in the currents at –60 and –100 mV during the exposure of glutamate (arrow) reflects the activation of the electrogenic glutamate transporters. The arrowheads indicate transient activation of calcium-evoked cation channels. (C) Activation of metabotropic P2Y and ionotropic P2X7 receptors in a human Müller cell. Activation of P2Y receptors by ATP (100 µM) evoked repetitive transient calcium-evoked activation of BK currents (that were recorded at the potential of +120 mV; left). Activation of P2X7 receptors by 2’-/3’-O-(4-benzoylbenzoyl)-ATP (BzATP; 50 µM) evoked a sustained calcium-evoked activation of BK currents, as well as cation currents through P2X7 receptor channels (right). (D) Immunostaining of two isolated human Müller cells against the P2X7 receptor protein using two different antibodies. Arrows, perikarya; arrowheads, cell endfeet. Bars, 20 µm. (E) Extracellular glutamate (200 µM) increased the activity of a single BK channel that was recorded in a membrane patch localized at the perikaryum of a human Müller cell. Above, Time dependency of the opening probability of the channel. Below, Original records of the channel activity. The pipette potential was 0 mV (i.e., the recording was made near the resting membrane potential of the cell). C, closed-state current level; 1, open-state current level. (F) γ-aminobutyric acid (GABA) (100 µM) evoked two currents in a human Müller cell, a transient, rapidly inactivating current mediated by GABAA receptor channels (arrow), and a sustained current mediated by electrogenic GABA transporters (asterisk).

In the outer plexiform layer, the photoreceptor-derived glutamate is mainly removed by photoreceptor, horizontal, and bipolar cells.12,19 whereas Müller cells prevent the lateral spread of neurotransmitters beyond the synapse(s) where they are released, thus ensuring visual resolution (Fig. 17.5). In the inner plexiform layer, Müller cell-provided glutamate uptake takes a more active part in shaping the synaptic responses. The rapid termination of synaptic glutamate action in nonspiking inner retinal neurons and in retinal ganglion cells is predominantly mediated by the uptake of glutamate into Müller cells.20,21 Generally, Müller cells remove the bulk of extracellular glutamate in the inner retina.13,22,23

Malfunction of glial glutamate uptake contributes to glutamate toxicity

Glutamate toxicity is a major cause of neuronal loss in many retinal disorders, including glaucoma, ischemia, diabetes, and inherited photoreceptor degeneration. A malfunction and/or downregulation of GLAST may cause, or contribute to, a rise in extracellular glutamate towards excitotoxic levels.2325 Hypoxia facilitates the formation of free radicals in mitochondria; the resulting lipid peroxidation disrupts glutamate transport into Müller cells.26 This mechanism is probably implicated in the neuronal dysfunction in diabetic retinopathy27 and in ganglion cell death in Leber hereditary optic neuropathy.28 Inflammatory lipids such as arachidonic acid, a reduction in extracellular pH as occurs in ischemia, and zinc ions released from photoreceptors29 all inhibit glial glutamate uptake.30,31

Because glutamate transport is voltage-dependent (Fig. 17.6A), depolarization decreases the efficiency of glial glutamate uptake.17,32 Depolarization of Müller cells occurs in response to pathological rises in extracellular potassium (as occurs in ischemia and glaucoma), to the opening of cation channels in Müller cells (Fig. 17.7C), and to a decrease in the potassium permeability of glial membranes due to inactivation and/or downregulation of inwardly rectifying potassium (Kir) channels (Fig. 17.8A, B). An inactivation of Kir channels was observed in animal models of various retinopathies (see below). Depolarization of Müller cells can also be evoked by inflammatory lipids such as arachidonic acid and prostaglandins which potently inhibit Na+,K+-ATPase. Depolarization of Müller cells may even cause a reversal of the glutamate transport.33 A nonvesicular release of glutamate and aspartate via reversed operation of glial glutamate transporters was implicated in the excitotoxic damage of retinal ganglion cells.34,35 A release of glial glutamate might also be mediated by the cystine glutamate antiporter (which is activated under oxidative stress conditions when an elevated production of glutathione requires an increased uptake of cystine; Fig. 17.5) and by exocytosis of glutamate-containing secretory vesicles.1,36 Under various pathological conditions including ischemia, glaucoma, retinal detachment, and proliferative retinopathies, an enhanced expression or activity of GLAST was observed.11,37 This might counterbalance the depolarization-induced malfunction of glial glutamate uptake.

Production of neurotransmitter precursors

To sustain the mechanism of transmitter recycling at the synapses, the Müller cells are endowed with specific enzymes. After GABA has been taken up by Müller cells, it is readily converted to glutamate by the GABA transaminase, and glutamate is converted to glutamine by the glutamine synthetase (Fig. 17.5). Glutamine synthetase is exclusively localized to glial cells.38 Glutamine is released from Müller cells, and taken up by neurons as a precursor for the resynthesis of glutamate and GABA (glutamate–glutamine cycle; Fig. 17.5).39 In the photoreceptor cells, this cycle accounts only for a part of glutamate supply; they directly take up glutamate from the synaptic cleft, and, in addition, synthesize glutamate from α-ketoglutarate as well as from Müller cell-derived glutamine.19,40 By contrast, the production of glutamate in bipolar and ganglion cells almost entirely depends on Müller cell-derived glutamine.40 Pharmacological blockade of the glutamine synthetase in Müller cells causes a rapid loss of the glutamate content of bipolar and ganglion cells, and (within 2 minutes) the animals become functionally blind.40 Likewise, a significant amount of GABA in amacrine cells is synthesized from Müller cell-derived glutamine.40

Downregulation of the glial glutamine synthetase contributes to neuronal dysfunction and glutamate toxicity in inherited photoreceptor degeneration and retinal detachment.41,42 The downregulation is mediated (at least in part) by the basic fibroblast growth factor (bFGF).43 bFGF is rapidly released within the retina after detachment, and its expression level increases under ischemic conditions, after light injury, and in response to inherited photoreceptor degeneration and mechanical injury.1 Though bFGF is a neurotrophic factor which supports the survival of photoreceptors and neurons,4446 the bFGF-induced downregulation of glutamine synthetase might rather aggravate neuronal degeneration. Under ischemic conditions, the GABA transaminase activity is decreased, and GABA accumulates in Müller cells.47,48 This impairs the efficiency of glial GABA uptake due to the decrease in the extra- to intracellular gradient as a driving force.

Trophic support of photoreceptors and neurons

The uptake of glutamate and GABA by Müller cells links neuronal excitation with the release of metabolic substrates, as well as with the defense against oxidative stress. Müller cells produce various substrates of the oxidative neuronal metabolism such as glutamine, lactate, alanine, and α-ketoglutarate (Fig. 17.5)49; these substrates are used by photoreceptors and neurons in periods of metabolic stress in the dark. The metabolic support is regulated by neuron-derived glutamate (which stimulates the uptake of glucose and the production of lactate) and potassium (which induces hydrolyzation of glycogen in Müller cells).1,50

Antioxidative support of photoreceptors and neurons

Glutamate in Müller cells is also utilized for the production of the antioxidant, glutathione (Fig. 17.5).39 The retina has a high need of antioxidant protection; this results from light exposure together with high oxygen consumption and the presence of high polyunsaturated fatty acid levels in photoreceptors. In addition, the outer retina underlies circadian periods of hypoxia (dark) and hyperoxia (light)51 which both increase oxidative stress. Usually, retinal glutathione is confined to glial and horizontal cells.39,52 In Müller cells, glutathione constitutes about 2% of the total protein.1 In response to oxidative stress such as during ischemia, glutathione is rapidly released from Müller cells and provided to the neurons,52 where it acts as cofactor of the enzyme glutathione peroxidase. The glial release of antioxidants like glutathione, and of neuroprotective factors like bFGF and adenosine, is implicated in the protection of photoreceptors from the harmful effects of circadian light exposure.

Müller cells from aged animals contain reduced levels of glutathione; this is associated with mitochondrial damage, membrane depolarization, and reduced cell viability.53 An age-dependent decrease in retinal glutathione may accelerate the pathogenesis of retinopathies in the elderly. In diabetic retinopathy, the decreased glutamate uptake of Müller cells results in reduced glutathione synthesis54,55 and in an upregulation of glutaredoxin which catalyzes the deglutathionylation of proteins; the latter mechanism induces nuclear translocation of nuclear factor-κB and upregulation of proinflammatory factors.56 Other antioxidants provided by Müller cells include pyruvate, α-ketoglutarate, metallothionein, lysozyme, ceruloplasmin, heme oxygenase, and reduced ascorbate.1

Removal of carbon dioxide

Photoreceptor cells display the highest rate of oxidative metabolism of all cells in the body. This high metabolic activity is associated with high oxygen and glucose demands. The active glucose metabolism, in turn, generates much carbon dioxide and water. Via transcellular transport, Müller cells redistribute metabolic waste into the blood and vitreous. Carbon dioxide is cleared by a process called carbon dioxide siphoning57; it is rapidly hydrated to bicarbonate and protons by carbonic anhydrases.58 Carbonic anhydrase II is localized within Müller and amacrine cells, whereas the membrane-bound carbonic anhydrase XIV is present in glial cells and vascular endothelia.57,59,60 Bicarbonate is transported to the blood and vitreous by sodium bicarbonate cotransporters and anion exchangers, both enriched in glial endfoot membranes.57

Regulation of the extracellular pH

By the removal of carbon dioxide, Müller cells also regulate the extracellular pH. Light-evoked neuronal activity is associated with extracellular alkalinization.61 The pH shift is balanced by an efflux of protons from photoreceptors62 and by the consecutive action of carbonic anhydrases and glial sodium bicarbonate cotransporters.63 Because sodium bicarbonate transporters are electrogenic, depolarization of Müller cells by potassium released from active neurons causes an influx of bicarbonate into the cells.63 The carbonic anhydrase-generated protons remain outside the cells and cause an extracellular acidification.63 Because extracellular acidification inhibits synaptic transmission,64 Müller cell-mediated pH regulation may protect neurons against overexcitation.

Spatial potassium buffering

Neuronal activity is associated with rapid ion shifts between the intra- and extracellular spaces. Sodium, chloride, and calcium ions flow into active neurons, and potassium ions are released from neurons. Light onset causes increases in extracellular potassium in the plexiform layers, and a decrease in the subretinal space.65,66 If not corrected, increased potassium will cause neuronal depolarization and hyperexcitation. Müller cells buffer imbalances in the extracellular potassium concentration via permission of transcellular potassium currents, a process termed “spatial potassium buffering” or “potassium siphoning.”67,

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