Astrocyte functions

1 I NTRODUCTION

1.1 Astrocytes in general – functions and heterogeneity

Astrocytes are, as their name suggests, star-shaped glia cells, which were discovered more than 100 years ago (Kettenmann and Ransom 1995). They are the most abundant cell type in the brain (Cherniak 1990; Nedergaard et al. 2003) and fulfill diverse vital roles and contribute to normal CNS functions. Some of the well-known functions are the mainte-nance of the metabolic environment, neuronal support, and their involvement in inflam-matory processes. However, astrocytes did not receive sufficient attention over a long period, based on the assumption of a primarily passive appearance in contrast to the ex-citability of neurons. The recognition of an active contribution of astrocytes to synaptic transmission and plasticity, and the increasing awareness of their pivotal role in almost all pathological processes in the brain, revived the interest in astrocytes for basic as well as pharmacological and toxicological research (Sofroniew and Vinters 2010).

The heterogeneity of astrocytes has long been underappreciated. They have been studied as a homogeneous cell population performing ’classical’ functions, such as potassium homeostasis, regulation of the blood brain barrier, or neurotransmitter uptake. Over the last decades, not only other functions have been discovered, e.g., the stem cell capacity of astrocytes, but also the heterogeneity of different subtypes of astrocytes and their func-tional specialization in different brain regions or within one region has been realized.

Nonetheless, the understanding of their diversity lags far behind that of neurons. This heterogeneity raises, of course, new problems regarding the definition of an astrocyte. In the following, astrocyte functions as well as their heterogeneity are summarized with the aim of establishing a new basis for the characterization of astrocytes in general.

1.1.1 Astrocyte functions

The term neuroglia derives from the greek word for ‘glue’ (γλοία), which indicates a structural as well as a supplying role (nutrients, oxygen) for these non-neuronal cells.

Astrocytes are in contact with different brain cells as well as the vasculature, thereby coupling neurons and other brain cells to blood supply through astrocytic endfeet. Their numerous processes are radially distributed and one astrocyte may contact several thou-sand synapses. Moreover, astrocytes themselves are coupled through gap junctions (con-nexins), forming highly regulated networks of complex cell interactions (Sofroniew and Vinters 2010).

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The housekeeping functions, including ion homeostasis, maintenance of the extracellular matrix, the metabolic support of neurons, or the maintenance of the blood brain barrier, can be considered the ‘classical’ functions due to their early recognition (Parpura and Haydon 2009). They are well described in the literature, and were responsible for the concept of astrocytes being passive supporter cells, which are not electrically excitable like neurons, but maintain the extracellular milieu. During neurotransmission, the levels of extracellular potassium increase, which would disturb the depolarization of neurons.

Astrocytes efficiently and rapidly take up potassium by several ion channels, e.g., Kir4.1, and distribute it throughout the cytoplasm and to neighboring cells via gap junctions. This potassium buffering is facilitated by a simultaneous flux of water through water channels (e.g., Aqp4) (Song and Gunnarson 2012). Furthermore, astrocytes express many different extracellular matrix proteins and adhesion molecules, thereby generating a dynamic en-vironment, in which neurite outgrowth as well as migration is finely regulated (Thomas et al. 1996; Wiese et al. 2012). Another part of the housekeeping in the central nervous system is the formation and maintenance of the blood-brain-barrier (BBB), in which as-trocytes play a crucial role by directly interacting with brain capillary endothelial cells.

This is not only the interface for nutrient supply, but a disruption of these interactions during disease may affect the BBB integrity, which is the case e.g., in Parkinson’s disease (Cabezas et al. 2014).

Besides these homeostatic and structural functions, astrocytes primarily support neurons in many different ways. First, they support neurons metabolically by providing important nutrients. The astrocytic endfeet, which contact blood vessels, express specific glucose transporters such as GLUT-1 (Morgello et al. 1995). Moreover, they are the predominant cell type in the brain to store glucose as glycogen, which is degraded under hypoglycemic conditions to maintain axon function or increased neuronal activity via lactate exchange (Brown and Ransom 2007). Astrocytes show high glycolytic rates producing lactate, which is released and taken up by neurons to meet their energy demand during neuro-transmission (Waagepetersen et al. 1998; Westergaard et al. 1995). Second, astrocytes produce a large number of neurotrophic factors, which influence synaptogenesis and reg-ulate synaptic activity and plasticity (Christopherson et al. 2005; Pfrieger and Barres 1997; Ullian et al. 2001). And third, they regulate neurotransmission by taking up neuro-transmitters (Bak et al. 2006). The latter function has led to the concept of the tripartite synapse, in which the interactions between the pre- and the postsynaptic neuron with

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neighboring astrocytes are described (Araque et al. 1999; Perez-Alvarez and Araque 2013).

Fig. 1 describes the recycling mechanism of neurotransmitters with regard to glutamate uptake. Glutamate is released during synaptic transmission, binding to receptors at the postsynaptic membrane, and leading to a depolarization of the postsynaptic neuron.

Dur-ing synaptic transmission, the local extracellular concentration of glutamate can increase up to a millimolar range and an excessive or prolonged stimulation of postsynaptic recep-tors can lead to cell death of the postsynaptic neuron (Conti and Weinberg 1999). Thus, a rapid and efficient removal of remaining glutamate from the synaptic cleft is indispens-ible for terminating glutamate signaling and preventing excitotoxicity of glutamate

Fig. 1. The glutamate/glutamine shuttle between neurons and astrocytes. Astrocytes take up glutamate during synaptic transmission through the glutamate transporters GLT-1 and GLAST, and either release it as glutamine via the glutamine transporter SN1, store it in vesicles through vesicular glutamate transporters (vGLUT), or feed the tricarboxylic acid cycle (TCA). Conversion to glutamine is catalyzed by the gluta-mine synthetase (GS).

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(Schousboe and Waagepetersen 2005). Astrocytes express specific Na⁺-dependent trans-porters, such as the glutamate-aspartate transporter GLAST or GLT-1, which rapidly take up glutamate. In a knockout study, both transporters have been shown to be essential for preventing neurodegeneration by decreasing glutamate excitotoxicity (Rothstein et al.

1996). Astrocytes then convert the glutamate into glutamine by the glutamine synthetase, which catalyzes the ATP-dependent condensation of glutamate with ammonia, thereby also achieving ammonia detoxification (Rose et al. 2013). The newly formed glutamine is then released by the glutamine transporter SN1 and taken up by neurons, which convert it to glutamate again. Therefore, the tripartite synapse model describes a dynamic recy-cling mechanism of neurotransmitters, regulating extracellular levels and terminating synaptic transmission. Besides this recycling mechanism, astrocytes utilize glutamate as an anaplerotic substrate. It is deaminated by the glutamate dehydrogenase to form α-ke-toglutarate, which feeds the citric acid cycle (TCA) (Schousboe et al. 2013). Only re-cently, it has been proposed that astrocytes can also store glutamate in vesicles by the expression of vesicular glutamate transporters (vGLUT), and even release it in a calcium-dependent manner similar to neurons (Zorec et al. 2012). This process named gliotrans-mission is, however, still under debate (Sloan and Barres 2014). The exact mechanisms are not known and especially its relevance for and connection to neurotransmission re-mains to be clarified. A lot of current studies are focusing on calcium-signaling in astro-cytes, which represents a kind of excitability of the cells and therefore replace the as-sumption of astrocytes being ‘passive’ cells (Rusakov 2015).

Besides their role in normal brain physiology, astrocytes perform several immune func-tions under pathophysiological condifunc-tions. During inflammation or injury, a lot of differ-ent factors are released by other brain cells like microglia, which drive astrocyte activa-tion. These factors include inflammatory cytokines, such as TNFα or IL1β, which are mainly produced by activated microglia (Hanisch and Kettenmann 2007; Henn et al.

2011; Kreutzberg 1996). Growth factors such as FGF2 are mainly released by reactive astrocytes themselves or endothelial cells (Logan et al. 1992). Other factors like ATP or reactive oxygen species occur during stress responses of various cells (Fig. 2) (Sofroniew 2009).

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Fig. 2. Triggers and molecular regulators of reactive astrogliosis (Sofroniew and Vinters 2010).

All these factors trigger different responses in astrocytes, such as the activation of tran-scription factors NFκB and cJUN, or the production of the signal transducer cAMP, lead-ing to molecular and functional changes in astrocytes (Sofroniew and Vinters 2010).

These changes may have beneficial as well as detrimental effects for the surrounding tissue depending on the severity and type of injury. These different activation states of astrocytes, leading to e.g., cell proliferation, are called astrogliosis. Astrogliosis occurs in almost all diseases and injuries of the CNS and results in mild, moderate, or severe man-ifestation (Sofroniew and Vinters 2010). With increasing degree of severity, the re-entry of astrocytes into the cell cycle and following proliferation leads to the generation of a glial scar around the lesion, accompanied by a tremendous reorganization of the tissue architecture. Since glial scar formation inhibits axon regeneration, it has been considered as a fully destructive phenomenon. However, beneficial effects of glial scar formation as well as the presence of beneficial reactive astrocytes has been described, e.g., the preven-tion of oxidative stress or the inhibipreven-tion of the outspread of inflammatory and pathogenic cells and agents (Faulkner et al. 2004; Li et al. 2008; Macco et al. 2013; Sofroniew 2005).

Thus, therapeutic strategies regarding astrocyte activation have changed over the last years, from trying to prevent astrogliosis per se, to the inhibition of detrimental pathways

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and simultaneous activation of beneficial effects. However, the complexity of astrogliosis as well as the heterogeneity of reactive astrocytes aggravate the development of thera-peutic strategies (Anderson et al. 2014; Sofroniew 2009). Therefore, many studies are currently performed to elucidate the identity of reactive astrocytes e.g., on transcriptomic level, and to identify involved pathways that lead to beneficial as well as detrimental effects (Anderson et al. 2014; Falsig et al. 2006; Zamanian et al. 2012).