The nature of early astroglial protection—Fast activation and signaling

Accepted Manuscript Title: The Nature of Early Astroglial Protection − Fast Activation and Signaling Authors: Julianna Kardos, L´aszl´o H´eja, Katalin Jemnitz, Rich´ard Kov´acs, Mikl´os Palkovits PII: DOI: Reference:

S0301-0082(16)30078-8 http://dx.doi.org/doi:10.1016/j.pneurobio.2017.03.005 PRONEU 1485

To appear in:

Progress in Neurobiology

Received date: Revised date: Accepted date:

12-7-2016 22-9-2016 5-3-2017

Please cite this article as: Kardos, Julianna, H´eja, L´aszl´o, Jemnitz, Katalin, Kov´acs, Rich´ard, Palkovits, Mikl´os, The Nature of Early Astroglial Protection − Fast Activation and Signaling.Progress in Neurobiology http://dx.doi.org/10.1016/j.pneurobio.2017.03.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The Nature of Early Astroglial Protection – Fast Activation and Signaling

Julianna Kardosa,*, László Héjaa, Katalin Jemnitza, Richárd Kovácsb and Miklós Palkovitsc

a

Functional Pharmacology Research Group, Institute of Organic Chemistry, Research Centre

for Natural Sciences, Hungarian Academy of Sciences; bInstitute of Neurophysiology, Charité Universitätsmedizin Berlin, Germany; cHuman Brain Tissue Bank and Laboratory, Semmelweis University Budapest, Hungary *Author to whom correspondence should be addressed: Julianna Kardos, Research Centre for Natural Sciences, Hungarian Academy of Sciences, H-1117 Budapest, Magyar tudósok körútja 2. E-mail address: [email protected], phone: +36-1-382-6612

Keywords: Astroglial signaling and metabolism Astrocytic control of excitation Astrocytic energy homeostasis Early neuroprotection Word count: 6 986 1

ABSTRACT Our present review is focusing on the uniqueness of balanced astroglial signaling. The balance of excitatory and inhibitory signaling within the CNS is mainly determined by sharp synaptic transients of excitatory glutamate (Glu) and inhibitory γ-aminobutyrate (GABA) acting on the sub-second timescale. Astroglia is involved in excitatory chemical transmission by taking up i) Glu through neurotransmitter-sodium transporters, ii) K+ released due to presynaptic action potential generation, and iii) water keeping osmotic pressure. Glu uptakecoupled Na+ influx may either ignite long-range astroglial Ca2+ transients or locally counteract over-excitation via astroglial GABA release and increased tonic inhibition. Imbalance of excitatory and inhibitory drives is associated with a number of disease conditions, including prevalent traumatic and ischaemic injuries or the emergence of epilepsy. Therefore, when addressing the potential of early therapeutic intervention, astroglial signaling functions combating progress of Glu excitotoxicity is of critical importance. We suggest, that excitotoxicity is linked primarily to over-excitation induced by the impairment of astroglial Glu uptake and/or GABA release. Within this framework, we discuss the acute alterations of Glu-cycling and metabolism and conjecture the therapeutic promise of regulation. We also confer the role played by key carrier proteins and enzymes as well as their interplay at the molecular, cellular, and organ levels. Moreover, based on our former studies, we offer potential prospect on the emerging theme of astroglial succinate sensing in course of Glu excitotoxicity.

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Contents 1. Introduction ............................................................................................................................ 5 2. Glutamate homeostasis ......................................................................................................... 9 2.1. Astroglial Glu and GABA Transporters........................................................................... 10 2.2. Transporter Trafficking, Crosstalk, Energetics ............................................................... 12 2.3. Astroglial Ca2+ signaling ................................................................................................. 13 2.4. Astroglial Glutamine Synthetase ................................................................................... 14 2.5. Oxidative metabolism of Glu by astrocytes................................................................... 15 3. Astroglial succinate sensing ................................................................................................. 16 3.1. Astroglial SUC ‘sensor’ in the brain ............................................................................... 16 3.2. Orphan G protein-coupled SUC receptor and the family of P2Y purinoreceptors ....... 17 3.3. SUC to monitor hypometabolism, oxidative stress, neuroinflammation ..................... 18 4. Ammonia homeostasis ......................................................................................................... 18 4.1. Hepatic Encephalopathy ................................................................................................ 19 5. Traumatic and ischaemic brain injuries ............................................................................... 21 6. Failing Glu homeostasis ........................................................................................................ 24 7. Glia ‘on the table’ – concluding remarks ............................................................................. 25 Author contributions ................................................................................................................ 28 Conflict of interest statement .................................................................................................. 28 Acknowledgements .................................................................................................................. 28 References ................................................................................................................................ 28 Search criteria .......................................................................................................................... 82

Abbreviations 1,4-DPCA AAT Activin ALS AMPA AQP4 AxD

1,4-Dihydrophenonthrolin-4-one-3-carboxylic acid, prolyl hydroxylase inhibitor elevating HIF-1α protein level Aspartate aminotransferase, also named Glu oxaloacetate transaminase TGF-β family member regulatory factor Amiotrophic lateral sclerosis α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid, agonist of AMPA-type Glu receptor Astroglial aquaporin channel subtype Alexander disease/fibrinoid leukodystrophy caused by GFAP missense mutation at chromosome 17 3

BBB BED Beta-catenin

Blood-brain barrier 5-Amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide hydrochloride Transcriptional co-activator of the Wnt/β-Catenin pathway regulating stem cell pluripotency and cell fate decisions during development CDD4 Plasma membrane protein and extracellular matrix receptor CNS Central nervous system CSF Cerebrospinal fluid Cx40, Cx43 Astrocite-specific gap junctional connexin protein subtypes EAAT1 (GLAST) Astroglial excitatory amino acid-Na+ symporter subtype 1 EAAT2 (GLT1, GLT-1) Astroglial excitatory amino acid-Na+ symporter subtype 2 EGF Epidermal growth factor ER Estrogen receptor Estradiol Selective estrogen receptor modulator, up-regulating EAAT1 and EAAT2 levels GABA Gamma-aminobutyric acid, the main inhibitory CNS neurotransmitter within the brain GABAAR Chloride ion channel forming GABA receptor subtype GABABR G protein coupled GABA receptor subtype B GAT2 Astroglial GABA-Na+ symporter subtype 2 GAT3 Astroglial GABA-Na+ symporter subtype 3 GDH Glu dehydrogenase GFAP Glial fibrillary acidic protein GFAP Glial fibrillary acidic protein, a type III intermediate filament protein GHB Gamma-hydroxybutyrate Gln Glutamine Glu L-glutamic acid/glutamate, the main excitatory CNS neurotransmitter within the brain GPR91 Orphan G protein coupled receptor for SUC GS Gln synthetase HA Hyperammonemia HE Hepatic encephalopathy HIF-1 Hypoxia-inducible factor-1 HSP-72 Heat shock protein-72 IBI Ischaemic brain injury IGF-I Insulin like growth factor type I IL-1β Member of the interleukin 1 family of cytokines ISF Interstitial fluid generated by water, passively following ionic gradients Ketamine (RS)-2-(2-chlorophenyl)-2-(methylamino)cyclohexanone, antagonist of NMDA type Glu receptor Lactate Astrocytic glycogen-derived hydroxycarboxylic acid MCT4 Monocarboxylate transporter subype 4 mGluRs Metabotropic Glu receptors microRNAs/miRNAs Small noncoding regulatory RNAs reducing stability and/or translation of target mRNAs miR-29a, miR-181a Astrocyte enriched microRNAs 4

Miro

MSO NCX NF-κB NH3/NH4+ NMDA Nogo-A PAPs PC PNS PPARγ Raloxifene S100β SERMs SUC Tamoxifen TBI TCA TGF-β TNF-α VTA xcα-KG

Ca2+-sensitive mitochondrial Rho GTPase adaptor protein linking the mitochondria to the motor protein kinesin-1 through Trak1/2 (Milton) proteins L-methionine-S,R-sulfoximine, inhibitor of GS Na+-Ca2+ exchanger, removing 1 Ca2+ in exchange of 3 Na+ across the plasma membrane Nuclear factor kappa-light-chain-enhancer of activated B cells Ammonia N-methyl-D-aspartate, agonist of NMDA-type Glu receptor Myelin-associated neurite outgrowth inhibitor Perisynaptic astrocytic processes Astroglial pyruvate carboxylase Peripheral nervous system Peroxisome proliferator-activated receptor γ SERM, up-regulating EAAT1 and EAAT2 levels Astroglial Ca2+ binding protein Selective ER modulators Succinic acid/succinate, the intermediary metabolite of the TCA cycle SERM, up-regulating EAAT1 and EAAT2 levels Traumatic brain injury Tricarboxylic acid Transforming growth factor-β Tumor necrosis factor-α Ventral tegmental area Cystine/Glu antiporter system Alpha-keto-glutarate

1. Introduction After the pioneering electrophysiology studies on glia cells (Kuffler and Porter, 1964), the neuro-centric perception of glia function (i.e. supplier (astroglia), shelter (oligodendroglia), cleaner (astroglia, microglia), border (astroglial interface at the blood-brain barrier, BBB)) has moved toward the understanding of glia-specific signaling functions. The relative importance of these glial functions in comparison with the neuronal ones under physiological and pathological conditions of the central nervous system (CNS) has received a broad interest in CNS research (for instance Chen et al., 2014; Christopherson et al., 2005; 5

García-Martín et al., 2015; Hagemann et al., 2013; Ishibashi et al., 2006; Jo et al., 2015; Kanemaru et al., 2007; Kékesi et al., 2015; Kim et al., 2015; Lasič et al., 2016; Lippmann et al., 2016; Oberheim et al., 2009; Pál et al., 2013, 2015; Szalay et al., 2016; as for reviews see Attwell et al., 2010; Barres, 2008; Butterworth, 2010; Cabezas et al., 2014; Capani et al., 2016; Chung et al., 2015; Héja, 2014; Hertz et al., 2015a; Hirase et al., 2014; Illes and Verkhratsky, 2016; Kettenmann et al., 2011; Kimelberg and Nedergaard, 2010; Kovács, R. et al., 2012; Kovács, Z. et al., 2015; Lim et al., 2016; MacVicar and Newman, 2015; Oliveira et al., 2015; Parpura et al., 2014; Pearson-Leary et al., 2016; Peng et al., 2014; Rial et al., 2016; Somjen, 1988; Torrente et al., 2014; Verkhratsky et al., 2014; Verkhratsky and Nedergaard, 2014; Wang and Bordey, 2008; Zorec et al., 2015). A number of physical, structural, developmental, and functional attributes of brain parenchyma, such as strength, control of neuronal proliferation and neurite extension, formation of BBB and tripartite synapse, can be ascribed to various sub-types and conditions of astroglia, the brain-specific class of glial cells (Brenner, 2014; Freeman, 2010; Oberheim et al., 2012; Sun et al., 2010; Verkhratsky et al., 2015a; Weber and Barros, 2015). The name astrocyte comes from the Latin ‘astra’ (stars) and ‘cyte’ (cell) words depicting a star-shaped cell morphology (Lenhossék, 1893). The fundamental roles of astroglia-specific glial fibrillary acidic protein (GFAP) (Mamber et al., 2012) in proper brain functioning, survival, and diseases substantiate the general and specific functions of astroglia (Bernardi et al., 2013; Boer et al., 2010; Czeiter et al., 2012; Hol and Pekny, 2015; Middeldorp and Hol, 2011). For example, GFAP affects the blood flow and performs the isolation of damaged tissue after ischemia (Hol and Pekny, 2015; Plog et al., 2015). Moreover, it is the fatal Alexander disease (AxD) that can directly be related to GFAP mutation in astrocytes (Ashrafi et al., 2013; 6

Brenner and Messing, 2015; Chen et al., 2013; Cotrina et al., 2014, Messing et al., 2012; Walker et al., 2014). These findings conclude to GFAP is being a biomarker for astroglial activation reflecting either the involvement of astrocytes in various physiological or disease conditions or predicting treatment outcome (Ojha et al., 2015; Plog et al., 2015; Sun et al., 2010; Yang and Wang, 2015). In addition to GFAP and GFAP autoantibody (Zhang et al., 2014), a variety of functional proteins can also be considered as astrocyte-specific biomarkers. These include i) gap junctional connexin 40 and 43 subtypes (Cx40 and Cx43); ii) the water channel aquaporin subtype 4 (AQP4); iii) a calcium-binding protein S100β; iv) glutamatergic markers such as the excitatory amino acid transporter subtypes 1 (EAAT1/GLAST) and 2 (EAAT2/GLT1/GLT-1), as well as glutamine (Gln) synthetase (GS); or v) pyruvate carboxylase (PC) which plays a key role for de novo biosynthesis of excitatory Lglutamic acid (glutamate, Glu) and inhibitory γ-aminobutyrate (GABA) neurotransmitters (Cesar and Hamprecht, 1995; Rajkowska and Stockmeier, 2013; Shank et al., 1985; Yu et al., 1983). Importantly, human GFAP/S100β and EAAT1/EAAT2 positive astrocytes are distinguishable (Sosunov et al., 2014). Protective and harmful roles for astroglia may be prevalent and/or co-existing. In various brain injuries including lesion, cerebral ischemia and reperfusion, stroke, perinatal or intermittent hypoxia and subsequent seizures, reactive astrogliosis they can be perceived together (Allaman et al., 2011; Ashpole et al., 2013; Binder and Steinhäuser, 2006; Cotrina et al., 2014; Gasparini et al., 2015; Jagadapillai et al., 2014; Liddelow and Barres, 2016; Liu, S. et al., 2016; Romero et al., 2014; Sajja et al., 2016; Sofroniew, 2009). Indicated by morphologic and functional adjustments, astroglial protection mechanisms can become harmful (Gleichman and Carmichael, 2014; Naegele, 2015). Apparently, such a transition depends on 7

temporal and spatial aspects such as local/penetrating, focal/global, partial/complete, chronic/acute, constant/intermittent, preconditioned/unconditioned incidence of the brain injury in question (Verkhratsky et al., 2015b). These characteristics suggest that the spatiotemporal dynamics of the cascade of events following brain injuriy may be critical when the potential of therapeutic intervention or prevention are evaluated. In considering mechanisms and targets appropriate for neuroprotection we regard the dynamic balance of excitation and inhibition (Dehghani et al., 2016) as a key prerequisite, scaling from sub-second to daily recurrence. Fast synaptic transmission enabled by Glu and GABA within the brain does imply clearance, keeping space with the demand and competing receptor desensitization. Dynamic clearance of Glu can potentially be exploited in early therapeutic intervention. Indeed, searching for “Glu transporter” in the PubMed database gives rise to more than 4000 items revealing Glu uptake-dependent functions and diseases in various brain areas (Fig. 1). Within this agenda, effects of astrocyte activation, fast signaling, and morphological responses of astroglia and associated ambient homeostasis to excitotoxicity are highlighted. We focus on fast astroglial response to neuronal excitation as a mechanism to protect neurons against un-balanced brain activity. Neuronal excitation evokes fast astroglial response by way of Glu uptake through EAAT2 and EAAT1 excitatory amino acid-Na+ symporters, and subsequent metabolism by astroglial Gln synthetase GS. A considerable portion of the transported Glu is oxidatively metabolized in the tricarboxylic acid (TCA) cycle requiring either an amino transferase or Glu dehydrogenase (GDH) to get the Glu carbon skeleton directed into the cycle. These processes actually secure energy production that is important to provide adequate energy supply to maintain the astroglial capacity for Glu uptake. In context, we discuss sub-second-to-minute timescale processes 8

such as the fast astroglial release of the inhibitory GABA (Héja et al., 2009; 2012; Kardos et al., 2015; Kersanté et al., 2013; Kirischuk et al., 2015; Lee et al., 2011; Unichenko et al., 2013), ATP, Glu and D-Serine (Halassa and Haydon, 2010; Henneberger et al., 2010; Parpura et al., 2014; for discussions of gliotransmission see Bazargani and Attwell, 2016; Sloan and Barres, 2014; Van Horn et al., 2013; Verkhratsky et al., 2016). These processes can be modulated by protein synthesis- and trafficking-related processes, including up- and downregulation of astroglial Glu transporter expression by epidermal growth factor (EGF) (Zelenaia et al., 2000), transforming growth factor-β (TGF-β) (Lee et al., 2012), insulin like growth factor type I (IGF-I) (Yin et al., 2013), and tumor necrosis factor-α (TNF-α) (Pickering et al., 2005). Likewise, the idea of succinic acid (succinate, SUC) sensing is also conferred within the framework of early astroglial protection. Holistic approximation taking into account the conditions and consequences of competing ammonia (NH3/NH4+) detoxification are also tackled.

2. Glutamate homeostasis Evidence is accumulating that astrocytes participate in excitatory neurotransmission via uptake of synaptically released Glu, and by maintaining Gln and energy provisions as well (McKenna, 2013; Schousboe et al., 2014). The energy consumer Glu uptake is maintained by the expense of Na+ gradient in astrocytes. Astroglial Na+ transients may in turn dissipate in various ways (Kirischiuk et al., 2015), for example by reverse mode operation of the Na+Ca2+ exchanger (NCX) (Matsuda et al., 1996) or the Na+-dependent GABA transporters (see below). Both astroglial functions, i.e. Glu-uptake evoked signaling and the antioxidant defense executed by the cystine/Glu antiporter system (xc-) and glutathione (Lewerenz et al al., 2013; Robinson et al., 2015), require low level of ambient Glu (0.001-0.01 mM). We place 9

astroglia in the focus of the outcome after brain injuries, because keeping on the outside-in Glu gradient is specifically related to perisynaptic astrocytic processes (PAPs) (Ghézali et al., 2016). By harboring excitatory and inhibitory amino acid transporter subtypes, synthesizing and shuttling Gln (Parpura et al., 2014; Zieliska et al., 2015), PAPs appear setting to overcome Glu excitotoxicity (Fig. 2). Unfortunately, despite the advances in health care, rapid intervention within minutes after cerebral ischemia or traumatic brain injury (TBI) is currently far from being a therapeutic reality. Within hours, however, the vital matter is whether mechanism-based medication via targets in PAPs related to Glu excitotoxicity can influence the outcome (Gleichman and Carmichael, 2014). Sub-anaesthetic ketamine dose applied for treating major depression has acted within 2 hours (Sanacora et al., 2012). It is explained by the long-lasting decrease of synaptic Glu release via indirect activation of presynaptic mGluRs offsetting an impared Glu clearance (Peng et al., 2015; Sanacora et al., 2012). 2.1. Astroglial Glu and GABA Transporters A growing and interesting topic is the study of the effect of small molecules on Glu transporter expression/trafficking and cascade of events leading to excitotoxicity after brain injury (Gleichman and Carmichael, 2014; Kong et al., 2014; Takahashi et al., 2015). There are several lines of evidence that up-regulation of Glu transporters in astrocytes compete these events, and prospect research should shortly clarify whether the expression of astroglial Glu transporters can be controlled to keep brain damage after injury as low as possible via translational activators (Colton et al., 2010; Limpert and Cosford, 2014), beta-lactam antibiotics (Jagadapillai et al., 2014, Krzyzanowska et al., 2016; Rothstein et al., 2005),

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estrogens (Karki et al., 2014; Lee et al., 2012; Pawlak et al., 2005), or glucocorticoids (Zschocke et al., 2005). In addition to matching the actual demand of Glu uptake, our focus on astroglial EAAT1, EAAT2, and GABA transporter subtypes GAT2, GAT3 along with the possible control of their expression is based on an unanticipated mechanism through which clearance of Glu by astroglial EAAT1/EAAT2 can trigger the release of GABA by enhancing neurotransmitter efflux through astroglial GAT2/GAT3 (Héja et al., 2009). Héja and co-workers first showed that removal of Glu by astroglial transporters triggers an elevation in the extracellular level of GABA and tonic inhibition (Héja et al., 2012). Intriguingly, it has been found that astroglial efflux of GABA could be blocked by inhibitors of astroglial Glu transporters, indicating that astrocytic GABA efflux depends on the inward and outward activity of astroglial EAAT1/EAAT2 and GAT2/GAT3 subtypes, respectively. It has also been demonstrated that the astroglial source for GABA is the polyamine metabolism (Héja et al., 2012; Yoon et al., 2014) (Fig. 2). Findings outlined above suggest, that backing the astroglial transporter-mediated Na+ dynamics and GABA efflux (Héja et al., 2009 and 2012; Kardos et al., 2015, Kirischuk et al., 2012, 2015; Lee et al., 2011; Rose and Chatton, 2015; Unichenko et al., 2013; Wójtowicz et al., 2013) by the controlled elevation of the expression of both EAAT1/EAAT2 (Colton et al., 2010; Jagadapillai et al., 2014; Kong et al., 2014; Karki et al., 2014; Limpert and Cosford, 2014; Takahashi et al., 2015) and GAT2/GAT3 (Doi et al., 2005; Ueda et al., 2007) may in turn prevent spreading of Glu-induced injurious cascade locally, within the tripartite synapse. Intervening at the site of over-excitation may also be specific to bypass activation of extrasynaptic Glu receptor subtypes such as N-methyl-D-aspartate (NMDA) or α-amino-311

hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) (Hardingham and Bading, 2010; Höft et al., 2014; Jaenisch et al., 2016; Lee et al., 2010; Montes de Oca Balderas and Aguilera, 2015; O’hare Doig and Fitzgerald, 2015; Verkhratsky and Kirchhoff, 2007). There are lessons to learn from vertebrate species that tolerate long-term oxygen depletion. Amongst 22 genes involved in GABAergic inhibitory response, the ones controlling extracellular GABA level (astroglial GAT2, GAT3) and tonic inhibition through extrasynaptic GABAA receptor (GABAAR) comprising the α4, α6, δ subunits dominate in the anoxic crucian carp brain, that lives on days to months without oxygen (Ellefsen et al., 2009). Furthermore, the substantial and specific decrease of GAT2 and GAT3 after oxygen deprivation indicates that the accumulation of extracellular GABA and the enhanced GABAergic tonic inhibition thereupon (Yoon et al., 2011) allow the long-term existence of crucian carp under anoxic condition. Furthermore, down-regulation of GAT2/GAT3 and increased tonic inhibition have also been demonstrated in peri-infarct cortex (penumbra) subsequent to stroke (Clarkson et al., 2010). These findings conclusively suggest that by restraining excitability and ATP spending, an overall enhancement of tonic inhibition via small molecule inhibition of astroglial GABA transporters may also be a viable approach to direct early therapeutic involvement in excitotoxicity. 2.2. Transporter Trafficking, Crosstalk, Energetics As outlined above, further than up-regulation of EAAT2 (Ghosh et al., 2016 and references cited), we claim that facilitation of Na+ symport-generated crosstalk between astroglial Glu and GABA transporters leads to the exchange of GABA for ambient Glu accompanied by an enhanced tonic inhibition via extrasynaptic GABAA receptor activation opening a new 12

possibility for a more balanced excitation-inhibition scheme (Héja et al., 2009, 2012; for reviews see Kardos et al., 2015; Kirischuk et al., 2015 and references cited) (Fig. 2). This is because the Glu-GABA exchange within PAPs can directly control neuronal excitation by negative feedback presenting enhancement of tonic inhibition with increasing excitation. Sub-second timing and sub-cellular specificity makes it ideal for an early intervention to the progress and expansion of excitotoxic conditions. To this end, mechanisms that promote Glu and GABA transporter interaction by the enhancement of their surface expression, membrane trafficking or PAP motility (Heller and Rusakov, 2015; Murphy-Royal et al., 2015), may be proposed as a strategy against various injuries related to excitotoxicity. We note that the shared feature of these mechanisms strengthening cross-talk of Glu and GABA transporters and tonic inhibition thereby may require a direct energy supply. From the point of view of teleology, such energy-coupling may be evidenced by the strikingly similar PAP localization for the Na+,K+-ATPase alpha 2 subunit and Glu transporters EAAT1 and EAAT2 in the rat somatosensory cortex (Cholet et al., 2002) (Fig. 2). Energy considerations may substantiate such a direct connection, since the uptake of a single Glu molecule in astrocytes requires one molecule of ATP (McKenna, 2013). 2.3. Astroglial Ca2+ signaling Along with the diffusion of clustered EAAT2 to the PAP membrane (Awabdh et al., 2015), astroglial mitochondria is also mobile. Their immobilization within PAPs depends on neuronal activity, Glu uptake, or Ca2+ influx through the reversed operation of NCX (Jackson and Robinson, 2015; Jackson et al., 2014). Medicinal chemistry campaigns related to the development of NCX subtype inhibitors such as 5-amino-N-butyl-2-(4-ethoxyphenoxy)benzamide hydrochloride (BED) may contribute to the development of investigational drugs 13

aiding the elucidation of the role of astroglial NCX in astroglial Ca2+ signaling (Secondo et al., 2015). Biochemical dissection of the underlying mechanisms disclosed a role for Ca2+sensitive mitochondrial Rho GTPase adaptor protein (Miro) isoforms arresting mitochondria within PAPs (Jackson and Robinson, 2015; Stephen et al., 2015). Amongst the various pathways possibly increasing Ca2+ in PAPs (for a recent review of the theme see Bazarghani and Atwell, 2016), the reverse operation of NCX exchanging Ca2+ for Na+ (Doengi et al., 2008) might be the most important (Fig. 2). Moreover, the reverse NCX operation can be increased by the robust Na+-coupled uptake of synaptically released Glu (Schummers et al., 2008; Raiteri and Raiteri, 2015). The whole scenario looks like as Ca2+ signaling through PAPs and backing energy support from mitochondria were sequential to the onset of astroglial uptake of synaptically released Glu. It is also plausible, however, that glutamatergic transmission in general, and excitotoxic condition specifically may precipitate local Ca2+ transients by activation of astroglial metabotropic mGluR2/mGluR3 (Bazarghani and Atwell, 2016) but not NMDA (Dzamba et al., 2013) receptors. 2.4. Astroglial Glutamine Synthetase Astroglia has a central role both in Glu metabolism and in detoxification through the conversion of ammonia to Gln predominantly by the astroglial GS (Brusilow et al., 2010; Kimelberg and Nedergaard, 2010). Since GS performs in both functions, Glu metabolism and ammonia detoxification, the conversion of Glu to Gln via GS competes with that of ammonia to Gln by GS (Fig. 2). Based on this scheme we can easily foresee that either excess Glu or surplus ammonia may exhaust astroglial functions such as Gln synthesis and clearance along with the volume-regulation. Astrocyte swelling brings about injured morphology and malfunction of astroglia combined with imbalance of astroglial Ca2+ homeostasis (Brusilow et 14

al., 2010; Görg et al., 2013; Haack et al., 2014; Häussinger and Görg, 2010). The selfamplifying osmotic- and oxidative stress result in altered expression and function of GS and plasmalemmal Glu transporter subtypes EAAT1 and EAAT2 leading to Glu excitotoxicity and recurrent seizures (Dhaher et al., 2015; Montana et al., 2014). Recent findings on areadependent epileptic activities associated with the application of GS inhibitor L-methionineS,R-sulfoximine (MSO) (Dhaher et al., 2015) may compromise the therapeutic use of MSO in acute liver failure (see below) (Brusilow et al., 2010). In that case, the acute treatment of hepatic encephalopathy (HE) by MSO invites clinicians to establish a seizure-circumventing therapeutic window prior its use. 2.5. Oxidative metabolism of Glu by astrocytes The mitochondrial enzyme GDH, localized mainly in astrocytes (Mastorodemos et al., 2005), catalyzes the reversible conversion of Glu to the TCA cycle metabolite α-ketoglutarate (α-KG) and ammonia (Hertz et al., 2007 and references cited): Glu + NAD+ ↔ α-KG + NH3 + NADH (Fig. 2). This scheme predicts that Glu can also be considered as an energy substrate of glucose-based oxidative metabolism in astroglia (Karaka et al., 2015 and reference cited; Lebon et al., 2002). Indeed, Karaka and co-workers (Karaka et al., 2015) provide evidence that genetic impairment of GDH and consequent Glu oxidation brings about central energydeprivation elevating hepatic glucose production. Since the astroglial Glu uptake is directly coupled to glutamatergic synaptic transmission, the scheme also identifies the homeostasis of astroglial Glu as a central provision of excitation-inhibition balance. The reversibility of the reaction catalyzed by GDH also depicts how disruption of ammonia homeostasis by the impairment of peripheral detoxification mechanisms may provoke encephalopathy (see Section 4). Also, oxygen-inducible astroglial enzyme aspartate aminotransferase (AAT) 15

catalyzes the reversible formation of α-ketoglutarate from Glu (Khanna et al., 2015 and references cited): Glu + oxaloacetate ↔ aspartate + α-KG (Fig. 2). Calculations show that about 24-27 ATP molecules are produced by the full oxidization of one molecule Glu (McKenna, 2013). Such a substantial contribution to brain energy demands may justify, in turn, its major role as an excitatory neurotransmitter within the brain.

3. Astroglial succinate sensing 3.1. Astroglial SUC ‘sensor’ in the brain Astrocytic protection of neuronal function is not limited to disease conditions. It is also of paramount importance to maintain regular neuronal activity. In addition to the signaling and metabolic functions of astrocytes outlined, recent studies also raise the possibility that glial cells play important role in detecting the local intensity of neuronal activation and controlling blood flow accordingly (Gourine et al., 2010). Accumulating evidence suggests that different energy metabolites play a role not only in neuronal but also in astroglial signaling. We have discovered that the major TCA cycle metabolite SUC and gammahydroxybutyrate (GHB) (Maitre, 1997), which enters the TCA cycle via SUC, specifically bind to recognition sites present in human and rat synaptic membrane homogenates isolated from the nucleus accumbens, the brain “reward area” (Molnár et al., 2006, 2007, 2008a, 2008b). The binding sites recognizing SUC/GHB and GABAB receptor (GABABR) ligands were clearly distinguishable (Molnár et al., 2006; 2008b). Furthermore, we have described that both SUC and GHB evoke astroglial Ca2+ transients in a subset of ATP-responsive astrocytes that are activated in a neuron-independent way in brain slices acutely isolated from the nucleus accumbens (Molnár et al., 2009; 2011a, 2011b). As the concentration of SUC in 16

plasma increases from 5 µM up to 125 µM with exercise, metabolic acidosis or in hypoglycaemic metabolic states, the apparent EC50 (50-60 µM) of the effect of SUC is within the range of physiological plasma concentration. It appears that the low-affinity SUC/GHB sensor outlined above and the high-affinity GHB receptor subtype described (Bay et al., 2014) are distinguishable proteins. Moreover, the concentration dependence of the number of ATP-responsive cells was highly identical as a function of both SUC and GHB concentration, suggesting a mutual ‘sensory’ role for SUC and GHB. These findings imply that astrocytes report the energy status of the network via Ca2+ response to the concurring input and output signals of ATP and SUC. 3.2. Orphan G protein-coupled SUC receptor and the family of P2Y purinoreceptors The SUC-evoked Ca2+ signal remaining in mice lacking GABAB receptor type 1 subunit in the presence and absence of NMDA receptor antagonist (2R)-amino-5-phosphonovaleric acid indicates action mechanisms independent of the inhibitory GABABR or the excitatory NMDA receptor subtype (Molnár et al., 2011a). It is to mention, however, that the activation of astrocytes by GHB has been claimed to be GABABR-mediated (Gould et al., 2014). In fact, roles for orphan G protein coupled receptors in neuromodulation have already been suggested (Civelli, 2012). While both SUC and GHB were successfully docked to the binding crevice of the homology model of orphan G protein coupled SUC receptor (GPR91) (He et al., 2004), GPR91 was not detectable in the brain (unpublished results). Apparently, GPR91 is present in the retina where it may have a major role in retinal angiogenesis (Hu et al., 2015; Sapieha et al., 2008). We can speculate about the physical entity being an astroglial sensor for energy metabolites SUC and GHB. Binding interaction between SUC/GHB sites and the gap-junction blocker carbenoxolone hemisuccinate may suggest some astroglial gap junction 17

protein as target (Molnár et al., 2007; 2011b). Unique blockade of the Ca2+ signal by the subtype-selective antagonist may also identify SUC ‘sensing’ by the G protein coupled purinergic P2Y1 receptor subtype (Molnár et al., 2011b). It is to mention here that SUC also reverses platelet inhibition evoked by P2Y antagonists (Spath et al., 2012). 3.3. SUC to monitor hypometabolism, oxidative stress, neuroinflammation Ariza and co-workers suggested the SUC receptor as a novel therapeutic target for oxidative and metabolic stress-related conditions (Ariza et al., 2012). Indeed, GPR91 and SUC may be key players in hypoxia-ischemia recovery and reperfusion injury (Chouchani et al., 2014; Chun et al., 2015; D’Alessandro et al., 2016; de Castro Fonseca et al., 2016; Hamel et al., 2014). Reportedly SUC is a ‘danger signal’ that induces IL-1β production via HIF-1α subtype (Tannahill et al., 2013). We conclude that the astroglial action of SUC and GHB may represent a link between low-energy sates and Ca2+ signaling in astrocytic networks, similarly to the astrocytic control of breathing by pH-dependent release of ATP (Gourine et al., 2010). Within the framework of monitoring central energy-deprivation, we may also speculate about the potential of lactate signaling, including its role played in memory formation and retrieval (Bergersen and Gjedde, 2012; DiNuzzo, 2016 and reference cited; Karus et al., 2015; Lauritzen et al., 2013; Lebedeva et al., 2015; Tsai et al., 2016) and pyruvate signaling (Zilberter et al., 2015). Besides, pyruvate facilitates the efflux of Glu from ISF by lowering blood Glu level through the reaction catalyzed by glutamate-pyruvate transaminase (Zilberter et al., and references cited).

4. Ammonia homeostasis

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As outlined before, astroglia is capable to utilize ammonia by astroglial GS that also transforms excitatory Glu to Gln. Conversely, inhibitory GABA ensues Glu via the chemical reaction catalyzed by GABA:2-oxoglutarate aminotransferase. These key metabolic processes leading to the balance of excitatory and inhibitory CNS drives may put a more holistic view of the cellular and molecular mechanisms of ammonia homeostasis in the centre1. 4.1. Hepatic Encephalopathy Reduced GS activity due to liver damage, inflammation, oxidative stress induced by drugs, ischemia/reperfusion injury, liver cirrhosis, and hepatocellular carcinoma might all be associated with HE (Romero-Gómez et al., 2015). “There is a general agreement that increasing concentration of ammonia in the brain acts as the main pathogenic factor for all types of HE” (Ferenci et al, 2002). HE can be directly linked to the breakdown of GS maintaining hepatic ammonia homeostasis due to acute/chronic liver failure, as well as portal hypertension induced porto-systemic bypass. Portal hypertension is also associated with clinical manifestations of cirrhosis, hepatitis, gastrointestinal bleeding, and HE-initiating ascites. Used to cover a broad spectrum of neuropsychiatric syndromes, HE is characterized

1

HYPERAMMONEMIA. Specifically, ammonia from the gut is detoxified by two major pathways, urea synthesis and Gln formation. Urea synthesis is responsible for the disposal of over 90 % of surplus nitrogen from dietary or endogenous nitrogen sources (van Straten et al., 2014). The crucial role of hepatic GS in the maintenance of ammonia homeostasis was demonstrated in liver-specific GS knockout mice exhibiting increased blood ammonia levels, induction of oxidative stress in brain tissue, and behavioral abnormalities (Qvartskhavaa et al., 2015). Encephalopathy induced by hyperammonemia (HA) is rare in adults in the absence of overt liver diseases (Kromas et al., 2015). Urea cycle diseases can occur due to episodes of HA initiated by metabolic stress, infections, vomiting, surgery, starvation, acute or chronic liver diseases, stress, or increased exogenous protein load (Häberle et al., 2012). Chemotherapy might also lead to severe, even fatal HA (Laemmle et al., 2015). Beta-catenin deficiency may lead to the lack of enzymes involved in hepatic Gln synthesis including GS (Clinkenbeard et al., 2012). Conversely, the constitutive activation of β-catenin pathway in several types of hepatocellular carcinomas results in strong, homogenous, and diffuse GS expression, opposing the normal distribution over a few hepatocytes around the terminal venules (Koehne de Gonzalez et al., 2015).

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by confusion, loss of consciousness and coma as farthest (Kimelberg and Nedergaard, 2010; Qvartskhavaa et al., 2015). As outlined above, the increased blood level of ammonia generates astrocyte swelling. These changes may alter NO production and interrelated HIF-1 level (Hendrickson and Poyton, 2015). Moreover, HE is associated with the release of numerous cytokines, causing both systemic and CNS inflammatory responses. In terminal HE, which is characterized by BBB opening and brain edema, microglia is also activated. Systemic infection in acute liver failure patients also results in the rupture of the BBB and the formation of vasogenic brain edema (Aldridge et al., 2015). Modeling systemic infection, lipopolysaccharide treatment of mice with acute liver failure led to hepatic coma and the BBB becoming permeable to the 25-kDa protein immunoglobulin G (Chastre et al., 2014). Hyperammonemic animals displaying edema and HE showed clear alterations of BBB function combined with the expression of BBB tight junction proteins (Rangroo Thrane et al., 2012).

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5. Traumatic and ischaemic brain injuries Increasing number of severe to mild TBIs2 due to automobile accidents, wars, terrorisms, violent behavior, falls and sporting activity together with the widespread stroke-induced secondary/original ischaemic brain injuries (IBIs) (Donnan et al., 2008; Prakash and Carmichael, 2015; Sun et al., 2016) justifies the re-evaluation and exploration of furthering possibilities in early therapeutic intervention (Arcure and Harrison, 2008; Lai et al., 2014). We note that neovascularization, the key to rehabilitation in both TBIS and IBI, is not comprehended in sufficient details enabling translation of knowledge into therapeutic intervention (Prakash and Carmichael, 2015). As a consequence, appropriate models of widely variable human conditions that would be prospective cornerstones in the development of more appropriate treatment options and therapeutics (Fluri et al., 2015) are also missing, urging for emerging new technologies and extensive study on signaling astroglia. Nevertheless, it is conceivable that early treatments of TBI and IBI may partly be related, as far as fighting the acute emergence of excitotoxicity and subsequent neuroinflammatory processes are concerned (Chen and Swanson, 2003; Jaenisch et al.,

2

NEURAL REGENERATION. We acknowledge here neurorestoratology campaigns (Qiao et al., 2015) and breakthrough findings on regeneration by trusting in early therapeutic intervention of TBI-evoked excitotoxicity as a ‘must’ for preserving the capacity of subsequent neural regeneration. The many of penetrating damage to the brain contrasts spinal cord injury, whereby functional recovery may be allowed by newly formed intraspinal detour circuits (Bareyre et al., 2004; Filli et al., 2014). Whereas limited spinal cord injury can regenerate spontaneously, the regenerative capacity of the brain through increasing progenitor proliferation and the migration of neuroblasts to the damaged brain parenchyma (Arvidsson et al., 2002; Martí-Fàbregas et al., 2010; Yang and Levison, 2006; Zhang and Chopp, 2009) is less permissive (Snyder and Park, 2002). It has been observed decades ago, that it is the glial environment of the peripheral nervous system (PNS) that enables axonal growth after CNS injury (David and Aguayo, 1981). Oligodendroglia may have a role in sustaining synaptic circuitry via down-regulation of CNS growth with the help of myelin-associated neurite outgrowth inhibitor Nogo-A (Kempf and Schwab, 2013). Conservation of central circuitry restricts growth response to brain injuries impairing regenerative therapy so far. Recent evidence on growth response built up by injured CNS neurons via astroglial TGF-β family member activins, however, does frame clues enabling regenerative therapy after stroke all over again (Omura et al., 2015). Furthermore, beneficial role for astrocyte scar formation in CNS axon regeneration has been reported recently (Anderson et al., 2016).

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2016; Lozano et al., 2015). By executing both Glu signaling and metabolism (Hadera et al., 2015), the astrocyte is probably the major cellular player specifically responsible for excitation-inhibition balance. However, the role for oligodendroglia in action potential dynamics may also be decisive (Fields, 2008 and references cited). Reportedly, anoxemia does induce impairment of neurons and oligodendrocytes, but GFAP positive astrocytes survive (d’Anglemont de Tassigny et al., 2015). It may be of interest to mention here, that astrocytes better survive neurotoxic nanomaterials such as specific Na+ channel forming polyamidoamine dendrimers than neurons (Nyitrai et al., 2013a, 2013b). Furthermore, the α subunit of HIF-1 expressed by astrocytes promotes cellular adaptation to hypoxic conditions, preserving astroglial morphology and viability under Glu toxicity (Badawi et al., 2012). The level of HIF-1α is controlled by prolyl hydroxylase. In turn, HIF-1α specifically regulates lactate release from highly glycolytic astrocytes via the enhanced expression of monocarboxylate transporter MCT4 (Rosafio and Pellerin, 2014). Moreover, it has been shown recently, that the application of prolyl hydroxylase inhibitor 1,4dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA) avoided the elimination of regenerative repair of ear injury by HIF-1α in mice (Zhang et al., 2015). These breakthrough findings on critical processes affected by proteins such as activin and HIF-1α may endow new suggestions for future studies related to regeneration after brain lesion as well as ischemia. Within the CNS, the glial environment generally form scars over the injury site that sets the axonal regeneration failing (Fawcett and Asher, 1999). Reactive astrocytes within the lesion site exhibit Glu transporter clusters with reduced Glu uptake, with elevated ambient Glu advancing cellular injury (Schreiner et al., 2013). Besides, a good deal of studies converges to the potential of astrogliosis in primary brain tumor progression (O’Brien et al., 2013). 22

Therefore, the suggested early control of reactive astrogliosis (Pekny et al., 2015) by addressing mechanisms that involve heat shock protein 72 (HSP 72), or selective estrogen receptor (ER) modulators (SERMs), such as estradiol, raloxifene, and tamoxifen can also hold therapeutic potential (Avila-Rodriguez et al., 2016; Barreto et al., 2009, 2012, 2014). In addition, on the basis of up-regulation of astroglial Glu transporter EAAT1/EAAT2 expression via activation of NF-κB and ERs (Karki et al., 2014), we may conjecture excitotoxicity as causal to reactive astrogliosis. It may have relevance in this context, that “stroke in subcortical white matter regions of the brain accounts for approximately 30% of all stroke subtypes, and white matter injury is a component of most classes of stroke damage” (Sozmen et al., 2012). It is known for a long time that ischemic tolerance can be developed by specific treatment protocols applying mild injuries (‘preconditioning’) making harms of subsequent IBI less detrimental (Nyitrai et al. 2005 and references cited). It may have relevance in the context of excitotoxicity, given that a role for astroglial EAAT2/GLT1 glutamate transporter as peroxisome proliferator-activated receptor (PPAR) γ gene has been pointed to ischemic preconditioning (Romera et al., 2007). Furthermore, up-regulation of astroglial P2X7 purinergic receptor subtype has also been described in ischemic tolerance (Hirayama et al., 2015). Increasing evidence suggests that microRNA (Friedman et al., 2009; Natera-Naranjo et al., 2010) may have the potential of being a specific biomarker of various brain conditions and pathologies (Ignacio et al., 2015; Ouayang et al., 2013; 2014; 2015 and references cited). Apparently, there may be relationship between distinguishable effects of ischemic tolerance on different hippocampal areas (Nyitrai et al., 2005) and matching microRNA miR-29a effects on neuronal vulnerability to cerebral ischemia (Ouyang et al., 2013; 2014). It is remarkable 23

that application of miR-181a antagomir in rat forebrain ischemia preserved astrocyte function such as the level of EAAT2 (Moon et al., 2013; see also Section 2.1). These findings support the view that astroglial EAAT2 may well be a major player in mechanisms leading to both IBI and ischaemic preconditioning (Krzyzanowska et al., 2016). One can speculate about the qualification necessary for the translational use of the above conclusion. Effective pretreatment outcome after microRNA application could be validated by establishing relationships between astroglial microRNA in neuronal survival and apoptosis or in response to ischemia reperfusion (Di et al., 2014; Ouyang et al., 2013; 2015 and references cited).

6. Failing Glu homeostasis Atypical composition and circulation of the interstitial fluid (ISF) are characteristics of brain physiology failing homeostasis (Marchi et al., 2016). For example, anoxic/ischaemic conditions initiating the loss of metabolic substrates such as TCA cycle anions, weaken astroglial K+ uptake. An increase of K+ in the ISF holds back water influx driven by the AQP4 channel. Furthermore, due to the loss of oxidative energy supply, Glu uptake coupled to proper Na+,K+-ATPase function is also compromised. The spillover of Glu may result in the uncontrolled activation of extrasynaptic Glu receptors and spreading excitation thereby. We are convinced that the cascade of events initiated by citotoxic restrictions (Marchi et al., 2016) pertains primarily ISF bathing PAPs. Accordingly, a “gliocentric” view of early phenomena possibly contributing to the development of edema at the brain parenchyma can be substantiated. The question is how to interfere the non-physiological (toxic) accumulation of K+ or Glu? Although arterial pulsatility would facilitate the AQP4-dependent exchange between the ISF and the cerebrospinal fluid (CSF) (Iliff et al., 2013) via the glymphatic system (Plog et al., 2015; Xie et al., 2013), it may not work properly in acute 24

anoxemia/ischaemia. Likewise, Glu export through BBB (Helms et al., 2012) may not be accounted, although hydrostatic forces could matter (Marchi et al., 2016). We note a possible role of the capillary phenomena boosting up ISF exchange with pulsatile shrinkage and swelling of nanocapillary channels within a PAP-covered (Pekny et al., 2015) synapses (Fig. 3). Remarkable findings on the decrease-increase coupling of ISF volume to the arousalsleep periods (Xie et al., 2013) corroborate this scheme. Direct path for CSF drainage through the meningeal lymphatic systems (Louveau et al., 2015; Mezey and Palkovits, 2015) may promote the egress of citotoxic ISF from PAPs. To this end, more detailed studies on meningeal Glu transporters (Berger and Hediger, 2000) and K+ channels (Kanjhan et al., 2010) are proposed.

7. Glia ‘on the table’ – concluding remarks In his biographical memoir about his colleague and lifelong friend Stephen William Kuffler (Katz, 1982), Royal Society Fellow Sir Bernard Katz points out that the principal problem concerning the roles of satellite cells in the function of the nervous system remained ‘on the table’ together with many tantalizing questions in the absence of ‘more precise information about the biochemical properties of various glial cells and about the nature of neuron-glia interaction’. Growing data and timely reviews on glia show that we have a more adequate knowledge about the biochemical properties of various glial cells and their roles in the function and disease of nervous system by now (see previous references and Araque et al., 1999; Verkhratsky et al., 2015b). Additionally, the increasing resolution of functional imaging data can in principle enable perceptible evidence on the spatio-temporal dynamics of glianeuron interaction, as well as the underlying major astroglial player channels, receptors, regulators of G receptor signaling, transporters, and enzymes (Binder and Steinhäuser, 2006; 25

Carpenter et al., 2015; Davila et al., 2013; Escartin and Murai, 2014; Eusemann et al., 2015; Gómez-Gonzalo et al., 2011; Hinton et al., 2014; Irimia and Van Horn, 2015; Jeanson et al., 2016; Khanna et al., 2015; Kim et al., 2015; Kumaria et al., 2008; Lee et al., 2012; Logothetis et al., 2001; Nolan and Scholz, 2015; Petzold and Murthy, 2011; Rouach et al., 2008). It is plausible, that major advances in understanding glia may shift prevailing neurocentric model-calculations (Markram, 2006; Naze et al., 2015) as well as neurocentric therapies (Janigro and Walker, 2014) towards issues of astroglia protection as a prerequisite to neuroprotection in various neurodegenerative conditions, including Parkinson’s and Alzheimer diseases and stroke (Garzón et al., 2016). As far as we know, pharmaceuticals thus far ignored the potential of astroglial targeting for early therapeutic interventions in a wide-continuum of excitotoxic conditions from various forms of mild IBIs to severe TBIs and subsequent seizures, presently taken as an example. It is to note that GFAP turned to be a clinically meaningful biomarker, significantly contributing to TBI outcome prediction (Czeiter et al., 2012). We could also mention here a suspected role for astrocytes in partially overlapping mechanisms of stroke and migraine aura (Dreier and Reiffurth, 2015) and seizures (Eickhoff et al., 2014), i.e. in spreading depression. Recent results on the EAAT2 preserving effect of miR-181a antagomir in rat forebrain ischemia suggest that possible control of RNA metabolism may have a therapeutic potential in the future. Up-regulation of EAAT1/EAAT2 in reactive astrogliosis features astroglial protection against Glu excitotoxicity in brain injuries. Astroglial functions, such as Glu uptake, Gln synthesis and clearance combined with volume-regulation should keep on the demand. If not, swelling elicits a cascade of events leading to inappropriate astroglial morphology and Ca2+ homeostasis and deteriorating functions. Increased blood level of ammonia resulted 26

from the impairment of hepatic conversion to urea and Gln generates astrocyte swelling as well. These findings suggest that pathogenic conditions such as HA/HE may worsen the therapeutic outcome in brain injury. Enhancement of astroglial Glu-GABA exchange through up-regulation of both EAAT1/EAAT2 and GAT2/GAT3 may get in the way of injurious Gluinduced osmotic and oxidative cascade. Involvement of astrocytes has been associated with a wide variety of acute and chronic CNS conditions3 by now. These extend over BBB dysfunction and epilepsy (Bar-Klein et al., 2014; Ji et al., 2015; Kovács et al., 2012), degenerative disorders (Cabezas et al., 2014; 2016; Capani et al., 2016; Colangelo et al., 2014; Hertz et al., 2015a; Kubik and Philbert, 2015), ischaemic tolerance (Hirayama et al., 2015), mental illnesses (Elsayed and Magistretti, 2015; Liu, X. et al., 2016), neurodevelopmental conditions such as autism spectrum disorder and schizophrenia (Durieux et al., 2015; Guillem et al., 2015), neuropathic pain (Chen et al., 2014), synaptic and cognitive impairment in disease (Chung et al., 2015), and working memory difficulties (O’Donnell et al., 2015). In order to further academic approaches already proposed ingeniously, we may also conjecture the concept of early astroglial intervention.

3

MENTAL HEALTH AND ILLNESSES. Previous and current reviews on astrocyte functions eloquently ask what steps are required to reach the physiological or pathological endpoint in mental health and illnesses (Barres, 2008; Capani et al., 2016; Elsayed and Magistretti, 2015; Hoogland and Parpura, 2015; Somjen, 1988; Verkhratsky et al., 2015b). There is no argument regarding importance of “subcellular timing” as one of the leading explanatory factor and should no doubt be the main specification of fast astroglial signaling as the most appropriate model for the study of various astroglia-neuron signaling pathways (DiNuzzo, 2016; Illes and Verkhratsky, 2016; Rial et al., 2016; Sajja et al., 2016). Within this framework, the potential significance of excitation-dependent “early” control of memory and behavior under physiological and pathological conditions is discussed. Astroglia has been implicated in both the formation (Zorec et al., 2015) - perhaps via D-serine (Henneberger et al., 2010), - and the impairment (Chung et al., 2015) of memory. It is widely accepted by now, that little stress (Pearson-Leary et al., 2016) and more exercise (Tsai et al., 2016) may counterbalance memory destruction. By conjecturing sleep and wakefulness, we may notice distinguishable, norepinephrine-regulated astroglia performance (O’Donnell et al., 2015). Involvement of astrocytes in modulation of behaviors (Durieux et al., 2015; Oliveira et al., 2015) possibly involving Glu signaling and/or metabolism (Durieux et al., 2015; Hertz et al., 2015b; Liu, X. et al., 2016) has also been proposed, particularly in depression (Peng et al., 2015; Rajkowska and Stockmeier, 2013; Sanacora et al., 2012). Indeed, antidepressants affect Cx43 channel performance (Jeanson et al., 2016).

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For that, mechanistic clues, including altered astroglial spatio-temporal Ca2+ dynamics and better disease models to study astrocyte function (Bazargani and Attwell, 2015; Hoogland and Parpura, 2015; Lecanu and Papadopoulos, 2013; Ojo et al., 2015) are yet to be addressed.

Author contributions J.K. and M.P. designed the structure, L.H. constructed figures, J.K., L.H., J.K., R.K. and M.P. wrote the manuscript.

Conflict of interest statement None.

Acknowledgements We gratefully thank Dr Orsolya Tőke for her critical comments and careful editing of the manuscript as well as professor Ferenc Hajós for the tripartite synapse photocopy shown in Fig. 3. Financial support by the KMR_12-1-2012-0112 and ERA-Chemistry OTKA 102166 grants are greatly acknowledged.

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Search criteria 

Major processes and players of excitation-inhibition balance via Glu homeostasis, including the astroglial uptake of Glu, Ca2+ signaling, Glu transporter

trafficking, Glu-GABA

echange

mechanism

and

oxidative

metabolism of Glu are highlighted. 

Discussion of a novel, astroglial “sensor” of oxidative hypometabolism, stress and neuroinflammation, such as the putative brain-derived G protein-coupled succinate receptor or family P2Y receptors is submitted.



Astrocytes are placed in the centre of Glu metabolism-reliant ammonia (NH3/NH4+) homeostasis interplaying with the progression of ammonia detoxification by the liver and hepatic encephalopathy.



Prevalent and invasive traumatic and ischaemic brain injuries are explored to validate the potential of the “gliocentric” concept of early therapeutic intervention.



Promotion of the idea of excitation-dependent pulsing of “nanocapillars” taking shape by the synapse and perisynaptic astrocytic processes offers the idea of pumping interstitial fluid off the tripartite synapse to arrive at the glymphatic and lymphatic systems. Condition of failing Glu homeostasis does compromise the mechanical clearance presenting excitotoxicity.

82

Figure legends Fig. 1. Glu transporter-related topics in the literature based on their appearance in the title and abstract of 4163 articles obtained by searching for “glutamate transporter” and synonyms in the PubMed database. Publication years range from 1964 to 2016. Size of the slices on pie charts corresponds to the number of appearances of the given terms and their synonyms. ALS: Amyotrophic lateral sclerosis; VTA: Ventral tegmental area. Fig. 2. Chart of an excitatory tripartite synapse to illustrate the time-evolution of major functions and the identification of players shaping the balance between central excitation and inhibition in perisynaptic astrocytic processes (PAPs). 1th step: Na+ influx via clearance of synaptic Glu by astrocytic glutamate-Na+ symporters EAAT1/2 triggers the reverse operation of astrocytic GABA transporters GAT2/3 resulting in GABA release. This GABA release does not require Glu receptor activation, extracellular Ca2+ or Glu decarboxylase activity, and could be blocked by inhibitors of either glial glutamate or GABA transporters. 2 nd step: Ca2+ influx prompted by the reverse operation of astroglial Na +-Ca2+ exchanger NCX induces the import of more EAAT1/2 with Na+/K+ ATPase α2, and more mitochondria enhancing oxidative energy supply. 3rd step: Progressing Glu uptake and its coupling to Gln production, ammonia detoxification, oxidative energy metabolism, and redox defence via cystine influx through the system xc-. 4th step: Running out of oxidative energy, ammonia detoxification capacity, and switching over the excitotoxic regime signaling succinate. Fig. 3. The tripartite synapse. Photocopy of an electron micrograph showing nanocapillary channels between synapses and perisynaptic astrocytic processes (PAPs). Bar scale: 500 nm. Courtesy of Ferenc Hajós.

83

Figures

Fig. 1.

84

Fig. 2.

85

Fig. 3.

86



Major processes and players of excitation-inhibition balance via Glu homeostasis, including the astroglial uptake of Glu, Ca2+ signaling, Glu transporter trafficking, Glu-GABA echange mechanism and oxidative metabolism of Glu are highlighted.



Discussion of a novel, astroglial “sensor” of oxidative hypometabolism, stress and neuroinflammation, such as the putative brain-derived G protein-coupled succinate receptor or family P2Y receptors is submitted.



Astrocytes are placed in the centre of Glu metabolism-reliant ammonia (NH3/NH4+) homeostasis interplaying with the progression of ammonia detoxification by the liver and hepatic encephalopathy.



Prevalent and invasive traumatic and ischaemic brain injuries are explored to validate the potential of the “gliocentric” concept of early therapeutic intervention.



Promotion of the idea of excitation-dependent pulsing of “nanocapillars” taking shape by the synapse and perisynaptic astrocytic processes offers the idea of pumping interstitial fluid off the tripartite synapse to arrive at the glymphatic and lymphatic systems. Condition of failing Glu homeostasis does compromise the mechanical clearance presenting excitotoxicity.