Müller cells in the healthy and diseased retina

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Abstract

Müller glial cells span the entire thickness of the tissue, and ensheath all retinal neurons, in vertebrate retinae of all species. This morphological relationship is reflected by a multitude of functional interactions between neurons and Müller cells, including a ‘metabolic symbiosis’ and the processing of visual information. Müller cells are also responsible for the maintenance of the homeostasis of the retinal extracellular milieu (ions, water, neurotransmitter molecules, and pH). In vascularized retinae, Müller cells may also be involved in the control of angiogenesis, and the regulation of retinal blood flow. Virtually every disease of the retina is associated with a reactive Müller cell gliosis which, on the one hand, supports the survival of retinal neurons but, on the other hand, may accelerate the progress of neuronal degeneration: Müller cells protect neurons via a release of neurotrophic factors, the uptake and degradation of the excitotoxin, glutamate, and the secretion of the antioxidant, glutathione. However, gliotic Müller cells display a dysregulation of various neuron-supportive functions. This contributes to a disturbance of retinal glutamate metabolism and ion homeostasis, and causes the development of retinal edema and neuronal cell death. Moreover, there are diseases evoking a primary Müller cell insufficiency, such as hepatic retinopathy and certain forms of glaucoma. Any impairment of supportive functions of Müller cells, primary or secondary, must cause and/or aggravate a dysfunction and loss of neurons, by increasing the susceptibility of neurons to stressful stimuli in the diseased retina. On the contrary, Müller cells may be used in the future for novel therapeutic strategies to protect neurons against apoptosis (somatic gene therapy), or to differentiate retinal neurons from Müller/stem cells. Meanwhile, a proper understanding of the gliotic responses of Müller cells in the diseased retina, and of their protective vs. detrimental effects, is essential for the development of efficient therapeutic strategies that use and stimulate the neuron-supportive/protective—and prevent the destructive—mechanisms of gliosis.

Introduction

Generally, the mammalian retina contains three types of glial cells. In addition to microglial cells, there are two forms of neuron-supporting macroglial cells, astrocytes and Müller (radial glial) cells. As an exception, oligodendrocytes can be found in the myelinated nerve fiber bundles (‘medullary rays’) of rabbits and hares, as a fourth type of glia. Microglial cells are the blood-derived resident immune cells within the retina that have an important role in host defense against invading microorganisms, initiation of inflammatory processes, and tissue repair. They are normally located in the innermost retinal layers (nerve fiber, ganglion cell, and inner plexiform layers). In species with completely or locally vascularized retinae, astrocytes are also located in these innermost retinal layers (in avascular retinae/retinal areas, they are absent). Retinal astrocytes are in contact with the superficial vascular plexus via processes which wrap around the vessels, and with the vitreo–retinal border (inner limiting membrane=ILM) to which they also extend processes. These latter processes form endfeet at the ILM, intermingled with the many endfeet of the Müller cells.

The Müller cell is the principal glial cell of the vetebrate retina; in the avascular retinae of many vertebrates (including mammals) it constitutes the only type of macroglial cells. Müller cells are specialized radial glial cells which span the entire thickness of the retina (Fig. 1A, B) and contact/ensheath all retinal neuronal somata and processes (Fig. 1D). The Müller cell population forms a dense, regular pattern (Fig. 1C, bottom); each of these cells can be considered as the core of a columnar ‘micro-unit’ of retinal neurons (Fig. 1D; Reichenbach and Robinson, 1995a, Reichenbach and Robinson, 1995b). Thus, Müller cells constitute an anatomical link between the retinal neurons and the compartments with which these need to exchange molecules, i. e., the retinal blood vessels, the vitreous body, and the subretinal space (which, together with the retinal pigment epithelium=RPE, constitutes the pathway to the choroidal blood vessels).

This link is not merely anatomical but also functional. For this purpose, Müller cells are endowed with a wealth of different ion channels, ligand receptors, transmembraneous transporter molecules, and enzymes (Newman and Reichenbach, 1996; Sarthy and Ripps, 2001). Many of these molecules are specifically expressed by Müller cells (i.e., they are not found in retinal neurons or other retinal cell types) or, at least, are most abundant in Müller cells (→2.2.–2.6.). A key feature of normal mature Müller cells is the high K+ conductance of their plasma membrane. It is provided by a high density of specialized K+ channels (Fig. 2); several types of these channels have been characterized in mammalian Müller cells. They comprise

  • inwardly rectifying channels of the Kir family, mainly Kir4.1 (weakly rectifying at resting membrane potential; mainly located at the vitread endfoot and perivascular membrane areas, and in the microvilli) and Kir2.1 (strongly rectifying, rather evenly distributed in the membrane between endfoot and soma) (Kofuji et al., 2002);

  • tandem-pore (TASK) channels which allow for outward currents at depolarized membrane potentials and show a subcellular distribution similar to that of the Kir2.1 channels, plus a prominent expression in the microvilli (Skatchkov et al., 2006), and

  • Ca2+-dependent K+ channels of big conductance (BK channels), requiring intracellular Ca2+ rises and/or membrane depolarizations to enter the open state (Bringmann et al., 1997).

A block or down-regulation of the K+ channels depolarizes the cell membrane (Newman, 1989; Pannicke et al., 2000b). The high K+ conductance of the Müller cell membrane, accompanied by a very negative resting membrane potential of about -80 mV, is characteristic for normal mature Müller cells (Fig. 3; →2.1.), and is the essential precondition for virtually all neuron-supportive functions of the cells (→2.3.–2.6.).

As the Müller cells are the dominant (or even the only) type of macroglial cells in the retina, they play a wealth of crucial roles in supporting the neurons and their functions that are carried out by the concerted action of astrocytes, oligodendrocytes, and ependymal cells in other regions of the central nervous system (Fig. 5, Table 1). From early stages of retinal development, they are essential in creating and maintaining the neuroretinal architecture (Willbold et al., 1997), and support neuronal survival and regular information processing (Reichenbach et al., 1993; Newman and Reichenbach, 1996). The importance of Müller cells for the maintenance of retinal structure and function is elucidated by the observation that selective Müller cell destruction causes retinal dysplasia, photoreceptor apoptosis and, at a final state, retinal degeneration and proliferation of the RPE (Dubois-Dauphin et al., 2000). Specifically, in the healthy retina Müller cells

  • are involved in retinal glucose metabolism, providing retinal neurons with nutrients such as lactate/pyruvate for their oxidative metabolism (Poitry-Yamate et al., 1995; Tsacopoulos and Magistretti, 1996) and removing metabolic waste products;

  • regulate the retinal blood flow (Paulson and Newman, 1987) and contribute to the formation and maintenance of the blood-retinal barrier (Tout et al., 1993) (→2.2.);

  • contribute to the neuronal signaling processes, particularly by rapid uptake and recycling of neurotransmitters (Matsui et al., 1999) and by providing precursors of neurotransmitters to neurons (→2.3.);

  • maintain the ion and water homeostasis of the retinal tissue including the pH (Newman, 1996; Newman and Reichenbach, 1996; Bringmann et al., 2004) (→2.4., 2.5.); and

  • release factors (e.g., D-serine and glutamate) which control the excitability of neurons (Newman and Zahs, 1998; Stevens et al., 2003), and are likely to be involved in the recycling of photopigments, as they express cellular retinaldehyde-binding protein (CRALBP) (Bunt-Milam and Saari, 1983), bind all-trans-retinal, convert it into 11-cis-retinol, and release it into the extracellular space for uptake by cone photoreceptors (Das et al., 1992) (→2.6.).

It has been known for many years that retinal neurons (particularly, photoreceptor cells) are highly susceptible to various forms of injury including insufficient blood supply. By contrast, Müller glial cells are strikingly resistant to ischemia, anoxia, or hypoglycemia (Silver et al., 1997; Stone et al., 1999); this can be attributed to their peculiar energy metabolism (→2.2.). Thus, Müller cells survive most retinal injuries, and remain available as players in the pathogenic events.

For instance, under pathological conditions Müller cells may act as modulators of immune and inflammatory responses, by producing proinflammatory cytokines in response to infection, for example (Caspi and Roberge, 1989; Roberge et al., 1991; Drescher and Whittum-Hudson, 1996). Furthermore, Müller cells are capable of phagocytosing fragments of retinal cells and foreign substances (Mano and Puro, 1990; Stolzenburg et al., 1992; Francke et al., 2001a) (Fig. 7D–F). Most noteworthy, Müller cells become ‘activated’ or ‘reactive’ in response to virtually every pathological alteration of the retina. This reaction is called Müller cell gliosis; it is one component of a complex retinal response to pathogenic stimuli which also includes microglial activation, alterations of the vasculature, and immigration of blood-derived leukocytes into the retinal tissue. Very probably, the initiation of (at least, certain steps or forms of) Müller cell gliosis requires an interaction between microglia and Müller cells, such as shown in the case of retinal light damage (Harada et al., 2002). In particular, micoglial cells modulate the production of a variety of trophic factors by Müller cells, including some that promote survival and some that promote death of photoreceptor cells (Harada et al., 2002).

Müller cell gliosis is characterized by both non-specific responses, i. e., stereotypic alterations independent of the causal stimulus, and specific responses which depend on the given pathogenic factor or mechanism. The most sensitive non-specific response to retinal diseases and injuries, which can be used as a universal early cellular marker for retinal injury, is the upregulation of the intermediate filament protein, glial fibrillary acidic protein (GFAP) (Bignami and Dahl, 1979; Eisenfeld et al., 1984; Bringmann and Reichenbach, 2001). Another non-specific Müller cell response is an activation of the extracellular signal-regulated kinases (ERKs) which was observed early during experimental retinal detachment, retinal ischemia-reperfusion, and endotoxin-induced uveitis, respectively (Geller et al., 2001; Akiyama et al., 2002; Takeda et al., 2002), as well as in the glaucomatous eye (Tezel et al., 2003) (Table 2).

A prominent example of the specific gliotic responses of Müller cells is an altered expression of glutamine synthetase (GS), a Müller cell-specific enzyme normally involved in neurotransmitter recycling (→2.3.) and ammonia detoxification (→2.2.). After a loss of major glutamate-releasing neurons such as occurs after photoreceptor degeneration evoked by light or retinal detachment (Grosche et al., 1995; Lewis et al., 1989), the expression of GS in Müller cells is reduced whereas an enhanced expression is observed during hepatic retinopathy when GS activity is necessary to detoxify the tissue from elevated levels of ammonia (Reichenbach et al., 1995b). On the other hand, no alteration of the GS expression by Müller cells is observed in diabetic retinopathy and after optic nerve crush (Mizutani et al., 1998; Chen and Weber, 2002). However, after optic nerve crush a translocation of the GS protein within the Müller cells is observed towards their endfeet in the ganglion cell layer, where injury of ganglion cells might result in the release of excess glutamate (Chen and Weber, 2002).

To understand the clinical consequences of Müller cell gliosis it is essential to note that it may include a de-differentiation of the cells. As a most important step of this de-differentiation, the cells reduce the K+-conductance of their membrane (particularly, the Kir4.1-mediated currents; this is generally associated with a mislocation of the Kir4.1 channels in the Müller cell membrane) (Fig. 2, Fig. 3; Bringmann et al., 2000). This will cause a severe loss of the functions involved in normal neuron-glia interaction, most of which require a hyperpolarized membrane potential. A similar mislocation of the Kir4.1 protein has been described in retinas of mice carrying a genetic inactivation of the dystrophin gene product, Dp71, which is proposed to be involved in the clustering of Kir4.1 channels in the plasma membrane (Connors and Kofuji, 2002). This mislocation of Kir4.1 protein was associated with an enhanced vulnerability of retinal ganglion cells to ischemic stress (Dalloz et al., 2003). Thus, as in the brain, gliosis in the retina is Janus-faced, contributing to both damage and protection of neurons (Bringmann and Reichenbach, 2001).

Early after injury, gliosis is neuroprotective, and is thought to represent a cellular attempt to protect the tissue from further damage, e.g., by release of neurotrophic factors and antioxidants (Schütte and Werner, 1998; Frasson et al., 1999; Honjo et al., 2000; Oku et al., 2002). Some of the factors released by activated Müller cells such as the vascular endothelial growth factor (VEGF), however, may have both neuroprotective (Yasuhara et al., 2004) and detrimental effects, as VEGF may exacerbate disease progression by inducing vascular leakage and neovascularization (→3.2., 3.4., 3.5.). Likewise, in response to ischemia and early in diabetic retinopathy Müller cells increase the expression of the inducible form of nitric oxide synthase (Goureau et al., 1994; Abu-El-Asrar et al., 2001). Enzymatically formed nitric oxide exerts beneficial effects by counteracting ischemia through dilating retinal vessels, and low nitric oxide protects neurons from glutamate toxicity via closing of N-methyl-D-aspartate (NMDA) receptor channels (Kashii et al., 1996). However, higher concentrations of nitric oxide and subsequent formation of free nitrogen radicals are cytotoxic for neurons, and are involved in the development of diabetic retinopathy, for example (Roth, 1997; Goureau et al., 1999; Koeberle and Ball, 1999) (→3.2.).

At later stages and/or in more severe cases of gliosis the de-differentiation of the cells contributes to neuronal cell death, e.g., via impairment of neurotransmitter removal (i.e., excitotoxicity) and dysregulation of the ion and water homeostasis after down-regulation of K+ channels (e.g., formation of edema) (→3.1., 3.5.). Generally, an impairment of supportive functions of Müller cells may have an additive effect on dysfunction and loss of neurons, by increasing the susceptibility of neurons to stressful stimuli in the diseased retina.

Finally, Müller cells may re-enter the proliferation cycle (→3.4., 3.5.) to establish a glial scar (Burke and Smith, 1981). Glial scars are one reason for the failure of the central nervous system to regenerate. During retinal detachment, for example, Müller cell processes grow through the outer limiting membrane and fill the spaces left by dying photoreceptors (Lewis and Fisher, 2000). Within the subretinal space, the Müller cell processes then form a fibrotic layer that completely inhibits the regeneration of outer photoreceptor segments (Anderson et al., 1986). Glial scars involve the expression of inhibitory molecules on the surface of reactive glial cells which additionally inhibit regular tissue repair and neuroregeneration (Fawcett and Asher, 1999).

In summary, a proper understanding of the gliotic responses of Müller cells in the diseased retina, and of their protective and detrimental effects, is essential for the development of efficient therapeutic strategies that increase the supportive/protective and/or decrease the destructive roles of gliosis. Therefore, this article summarizes the present knowledge about some of the key functions of Müller cells in the healthy retina, and of the involvement of Müller cells in various retinopathies.

Section snippets

Retinal development

From early embryonic stages, the immature Müller cells are important for the histotypic organization of the developing retina, and for the proper wiring of its neuronal circuits. They provide an orientation scaffold and migration substrate for postmitotic young neurons (Willbold et al., 1997) as well as for their growing neurites (Stier and Schlosshauer, 1998). It is noteworthy that after de-differentiation, reactive Müller cells appear to recover this capability; in degenerated retinas of aged

Retinal detachment

The neural retina may become separated from the pigment epithelium during trauma or inflammatory eye diseases, or in the presence of retinal holes and tears. Detachment increases the distance between the choriocapillaris and the neural retina, and results in a decreased oxygen and nutrient supply of the photoreceptor cells (Stone et al., 1999; Linsenmeier and Padnick-Silver, 2000). The decreased energy supply was suggested to cause photoreceptor cell death and subsequent retinal degeneration (

Future directions

Since Müller cells contact all retinal neurons and show a high resistance against various pathogenic stimuli, they are well-situated as targets for therapeutic interventions to inhibit neuronal degeneration. Somatic gene therapy may be carried out via Müller cells. A gene transfer, e.g., of neurotrophic factors, to Müller cells may help to support their protective role in the survival of retinal neurons. Müller cells were found to be primarily transfected when genes, e.g., the gene for BDNF,

Acknowledgements

Some of the work presented in this article was conducted with grants provided by the Deutsche Forschungsgemeinschaft (BR 1249/2-1; RE 849/8-3; RE 849/10-1; WI 880/13-2; GRK 1097/1) and the Interdisziplinäres Zentrum für Klinische Forschung (IZKF) at the Faculty of Medicine of the University of Leipzig (Projects C5 and C21).

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