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Why are Astrocytes Important?

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Neurochem Res (2015) 40:389401
DOI 10.1007/s11064-014-1403-2
OVERVIEW
Why are Astrocytes Important?
Alexei Verkhratsky Maiken Nedergaard
Leif Hertz
Received: 28 May 2014 / Revised: 22 July 2014 / Accepted: 26 July 2014 / Published online: 12 August 2014
Springer Science+Business Media New York 2014
Abstract Astrocytes, which populate the grey and white
mater of the brain and the spinal cord are highly hetero-
geneous in their morphology and function. These cells are
primarily responsible for homeostasis of the central ner-
vous system (CNS). Most central synapses are surrounded
by exceedingly thin astroglial perisynaptic processes,
which act as astroglial cradle critical for genesis, mat-
uration and maintenance of synaptic connectivity. The
perisynaptic glial processes are densely packed with
numerous transporters, which provide for homeostasis of
ions and neurotransmitters in the synaptic cleft, for local
metabolic support and for release of astroglial derived
scavengers of reactive oxygen species. Through
Special Issue: In honor of Michael Norenberg.
A. Verkhratsky (&)
Faculty of Life Sciences, The University of Manchester, Oxford
Road, Manchester M13 9PT, UK
e-mail: Alexej.Verkhratsky@manchester.ac.uk
A. Verkhratsky
Achucarro Center for Neuroscience, IKERBASQUE, Basque
Foundation for Science, 48011 Bilbao, Spain
A. Verkhratsky
University of Nizhny Novgorod, Nizhny Novgorod 603022,
Russia
M. Nedergaard
Division of Glia Disease and Therapeutics, Center for
Translational Neuromedicine, University of Rochester Medical
School, Rochester, NY 14580, USA
L. Hertz
Laboratory of Brain Metabolic Diseases, Institute of Metabolic
Disease Research and Drug Development, China Medical
University, Shenyang, China
perivascular processes astrocytes contribute to bloodbrain
barrier and form glymphatic drainage system of the
CNS. Furthermore astrocytes are indispensible for gluta-
matergic and c-aminobutyrate-ergic synaptic transmission
being the supplier of neurotransmitters precursor glutamine
via an astrocytic/neuronal cycle. Pathogenesis of many
neurological disorders, including neuropsychiatric and
neurodegenerative diseases is defined by loss of homeo-
static function (astroglial asthenia) or remodelling of
astroglial homoeostatic capabilities. Astroglial cells further
contribute to neuropathologies through mounting complex
defensive programme generally known as reactive
astrogliosis.
Keywords Astrocytes
Glutamate
Astroglial cradle

Glymphatic system
Astrogliopathology
Neurological
disorders
Introduction
The 50 years since Michael D. Norenberg graduated in
medicine from University of Rochester in 1965 coincide
with the most prolific increase in our knowledge of astro-
cyte function and importance. The same year witnessed the
first publication from Nicholls and Kuffler [1] on Na? and
K? in neurones and glial cells in the leech nervous system;
in the same year Holger Hyden published his observations
on rates of oxygen consumption in neurones and glial cells
microdissected from Deiters nucleus and on rhythmic
enzyme changes in neurones and glia during sleep [2, 3],
and van Harrevelds reported first studies showing an
extracellular space in brain [4]. Several years later van den
Berg and Garfinkel [5] suggested that glutamine flows from
a small to a large metabolic compartment in brain, while
123
390
c-aminobutyrate (GABA) does so in the reverse direction,
and Benjamin and Quastel [6] proposed that glutamate
released from neurones is partly taken up by glia, con-
verted into glutamine and returned to the neurones where it
is metabolized to glutamate and GABA. The same year,
Mike published his first paperon astrocyte division in
hepatic encephalopathy [7], the first established disease
with a major glial contribution. 5 years later a seminal
study by Martinez-Hernandez et al. [8] provided the first
demonstration that glutamine synthetase (GS) is localized
in glial cells, but not in neurones. It was followed by Fine
structural localization of GS in astrocytes of rat brain [9],
pinpointing GS expression selectively to astrocytes. This
not only confirmed the suggestions by van den Berg and
Garfinkel and Benjamin and Quastel but solidly associated
them with neuronalglial interactions.
In this essay we shall overview some major aspect of
astroglial metabolism; and its connection to synthesis and
metabolism of neurotransmitters. We shall continue with
describing a general role of astroglial cells in synaptic
connectivity and synaptic transmission and we shall end
with brief overview of astroglial contribution to
neuropathology.
The GlutamineGlutamate/GABA Cycle
Formation de novo of the transmitters glutamate and
GABA (which is synthesized in GABAergic neurones from
glutamate) requires specific interactions between neurones
and astrocytes via the glutamineglutamate (GABA) cycle
with a flux equal to rate of glucose utilization in neurones
[10, 11]. These neurotransmitters are amino acids which
are produced from glucose (GABA via glutamate) in
astrocytes in the CNS and in the astrocyte-like Muller cells
in retina. However, they cannot be synthesized from glu-
cose in neurones, which lack the enzyme pyruvate car-
boxylase (PC) [12, 13]. After glycolytic degradation of
glucose to pyruvate, two molecules of pyruvate are needed
to form a new molecule of the tricarboxylic acid (TCA)
cycle constituent citric acid (Fig. 1). One pyruvate mole-
cule is degraded by the pyruvate dehydrogenase complex
to acetylcoenzyme A, i.e., using the same pathways as
when glucose is metabolized for energy production. In both
cases the acetate moiety condenses with pre-existing oxa-
loacetate in the TCA cycle to form citrate. For energy
production, citrate during one turn of the TCA cycle loses 2
of its carbon atoms and regenerates oxaloacetate, which
again condenses with acetyl CoA for further TCA cycle
activity and energy production. No new, extra TCA cycle
constituent is formed, which could be expendable for
production of glutamate. For this de novo synthesis a sec-
ond pyruvate molecule is converted by carboxylation,
123
Neurochem Res (2015) 40:389401
catalyzed by PC, to a new molecule of oxaloacetate,
providing an extra oxaloacetate molecule, which also can
condense with acetyl CoA. In this manner the the pool of
TCA intermediates is expanded, permitting use for gluta-
mate synthesis of the TCA cycle constituent a-ketoglutar-
ate (a-KG), which is formed after partial TCA cycling. An
important, debated question is whether this process is
catalyzed by glutamate dehydrogenase (GDH), as often
concluded from experiments in isolated cells, or by
aspartate aminotransferase, suggested by a large stimula-
tion of glutamate/glutamine formation in astrocytes in
intact brain in the presence of aspartate [14, 15]. Moreover,
both cytosolic and mitochondrial aspartate aminotransfer-
ase activity are very high in brain [16], allowing rapid
nitrogen exchange between glutamate and aspartate.
It has been suggested that glutamate synthesis might be
directly coupled to astrocytic glutamate (and GABA)
degradation [17] in intact tissue with its maintained inter-
actions between neurons and astrocytes, which are of key
importance for the glutamine/glutamate/(GABA) cycle.
These interactions, which do not occur in cultured astro-
cytes, may facilitate preferential use of aspartate amino-
transferase rather than of GDH for the inter-conversions
between a-KG and glutamate, whereas GDH consistently
has been found to play a major role in the cultured astro-
cytes [18, 19]. Models of relatively simple pathways for
aspartate aminotransferase-mediated exchange between a-
KG and glutamate are shown in Fig. 2a, b. Figure 2a pre-
sents the pathway suggested by Pardo et al. [14] on the
basis of their key finding that aspartate increases glutamate
formation in intact brain. A comprehensive pathway for
interconnected glutamate synthesis and degradation, which
is capable of explaining the source of aspartate is shown in
Fig. 2b. As discussed in more detail below *15 % of
glutamate released from neurones is oxidatively degraded
[20], and recent research suggest that this oxidation mainly
or perhaps almost exclusively occurs in astrocytes [2126].
These findings are consistent with, but do not prove, that
glutamate synthesis and degradation depend upon each
other, let alone follow the pattern indicated in Fig. 2b. The
corresponding metabolic pathway for GABA synthesis is
slightly more complex [13], but a similar interconnection
between glutamate production and GABA oxidation in
astrocytes (Fig. 2c) may exist.
Glutamate is converted to glutamine by the GS localised
in the cytosol. Inhibition of this enzyme not only almost
immediately interrupts glutamatergic activity [27], but may
have many other severe consequences, perhaps as a result
of the interruption of a large part of astrocytic glucose
metabolism. Thus, in retina a hormonally-induced increase
in Muller cell GS protects against neuronal injury [28],
whereas GS inhibition increases cell death [12, 29]. Glu-
tamine exit from astrocytes and entry intro neurones are
Neurochem Res (2015) 40:389401
Fig. 1 The astrocytic part of the synapse provides net synthesis of
glutamine (GLN), via the concerted action of PC and pyruvate
dehydrogenase (PDH), generating oxaloacetate (OAA) and acetyl-
CoA, the combination of which leads to synthesis of citrate (CIT).
This subsequently leads to a net synthesis of a-ketoglutarate (a-KG)
allowing synthesis of glutamate (GLU), catalyzed by either glutamate
dehydrogenase (GDH) or an amino acid aminotransferase (AA). GLU
is used for synthesis of GLN catalyzed by glutamine synthetase (GS).
GLN is transferred to the glutamatergic neurone to be used for
synthesis of GLU catalyzed by phosphate-activated glutaminase
(PAG). Released GLU is taken up into the astrocyte and transformed
into GLN completing the GLU-GLN cycle. Alternatively, the GLU
equally important as glutamine synthesis for regulation of
de novo synthesis of glutamate and GABA. Moreover,
whereas astrocytic glutamine synthesis is only involved in
the de novo synthesis of glutamate, glutamine transport in
the glutamine/glutamate (GABA) cycle is also essential
during the return of at least most released transmitter glu-
tamate to neurones after an initial uptake in astrocytes, a
process that will be described in more detail later. Gluta-
mine exits from astrocytes via the transporter SN1, which
mediates electroneutral and bidirectional glutamine trans-
port [30]. Its activity is regulated at many levels, for
example by extracellular pH, because protons compete
with Na? required for its transport activity. There are also
consistent observations that SN1 is down-regulated by
protein kinase C phosphorylation, probably by internali-
zation [30]. Released glutamine is accumulated into
391
taken up may be oxidatively metabolized which subsequently requires
de novo synthesis of GLN via the anaplerotic processes indicated in
bold arrows. Reproduced from [13], which together with other
contributions discusses metabolic interactions in detail, even in the
brain in vivo. However, the figure also shows that NH4? is required in
astrocytes and released in neurones, and mechanisms transferring
NH4?/NH3 between the two cell types are discussed in other articles
[143]. So are the transporters releasing glutamine from astrocytes and
accumulating it in neurones and the powerful transporters accumu-
lating glutamate in astrocytes, as well as associations between
glutamate uptake and metabolism. AT aspartate aminotransferase;
MAL malate; ME malic enzyme; PYR pyruvate
glutamatergic neurones by a different transporter, SAT1 or
at some places SAT2 and is subsequently converted to
glutamate by phosphate-activated glutaminase (PAG).
Another debated question regards ammonia (NH3 and
NH4?), which is needed for glutamate formation in astro-
cytes and released during the glutaminase action in neu-
rones. Diverse and complex amino acid cycles have been
suggested to be involved in ammonium transport from
neurones to astrocytes, although they probably do not
operate to any significant degree in the intact brain [16, 17,
3133]. Rather, ammonium may be transported as such,
especially since NH4? is rapidly accumulated in astrocytes
by Na?, K?-ATPase activity [34].
Accumulated glutamine is converted to glutamate in
glutamatergic neurones within the mitochondrial mem-
brane, enters the mitochondrial matrix, and is returned to
123
392
Fig. 2 a Cartoon describing metabolic pathway from pyruvate to
glutamate/glutamine in astrocytes, as suggested by Pardo et al. [14].
Joint PC and pyruvate dehydrogenase activation generates a new
molecule of citrate as described in Fig. 1. Citrate-derived a-ketoglu-
tarate (a-KG) exits the mitochondrial membrane and is transaminated
with aspartate (Asp) to form glutamate (Glu), with concomitant
oxaloacetate (OAA) formation from aspartate. OAA is carried back to
the mitochondria via malate (Mal). OGC, malate/a-ketoglutarate
exchanger b Proposed expansion by Hertz [17] of the model shown in
a. The expanded model shows astrocytic production of glutamine
(pathway 1), its transfer to glutamatergic neurones (without indication
of any extracellular space, because there is no other function for
extracellular glutamine than astrocyte-to-neurone transfer) and extra-
cellular release as the transmitter glutamate (pathway 2), and
subsequent reuptake of glutamate and oxidative metabolism in
astrocytes (pathway 3), with connections between pathways 1 and 3
shown as pathway 4. Biosynthesis of glutamine is shown in brown
and metabolic degradation of glutamate in blue. Redox shuttling and
astrocytic release of glutamine and uptake of glutamate are shown in
the cytoplasm in a pseudomalateaspartate shuttle [13, aminooxyactic acid (AOAA) and by the ketodicarboxylate
3537] (Fig. 3). The initial evidence for utilization of this
pathway was complete inhibition of glutamate release in
cerebellar granule neurons incubated in the presence of
glutamine by the aspartate aminotransferase inhibitor
123
Neurochem Res (2015) 40:389401
black, and neuronal uptake of glutamine, hydrolysis to glutamate, and
its release is shown in red. Reactions involving or resulting from
transamination between aspartate and oxaloacetate (OAA) are shown
in green. Small blue oval is pyruvate carrier into mitochondria and
small purple oval malate carrier out from mitochondria. AGC1,
aspartate/glutamate exchanger, aralar, which is required both for
initial glutamate synthesis (not indicated), and for glutamate entry
into mitochondria during its oxidative degradation; a-KG, a-ketoglu-
tarate; Glc, glucose; Pyr, pyruvate; OGC, malate/a-ketoglutarate
exchanger. All reactions are stoichiometrically accounted for. c Pro-
posed pathway for connection between astrocytic glutamate produc-
tion and GABA oxidation [17]. Color coding and most abbreviations
are as in b. Most differences from b are in the lower right corner of
the figure. The suggested glutamateGABA exchange at the mito-
chondrial membrane has not been described in mammalian brain, but
otherwise the reactions are conventional and in accordance with the
known metabolism of GABA via succinic semialdehyde (SSA) to
succinate (Succ.), a TCA cycle constituent
carrier inhibitor phenylsuccinate [35]. Later, experiments
in rat brain mitochondria indicated that this was not a
phenomenon restricted to cultured cells [36]. This pseu-
domalateaspartate shuttle plays also a role in the more
Neurochem Res (2015) 40:389401
Fig. 3 Metabolic pathway for conversion of glutamine to glutamate in
cultured cerebellar granule neurones by a pseudomalateaspartate
shuttle. Glutamine enters the intermembranaceus space from the
cytosol (red arrow at bottom of figure). Here glutamate (GLU) is
formed by the glutaminase (phosphate-activated glutminase: PAG)
and enters the mitochondrial lumen, where it is transaminated to
aspartate (Asp), coupled with transamination of oxaloacetate (OAA) to
a-ketoglutarate (a-KG). As in the malateaspartate shuttle a-KG exits
the mitochondrial membrane in exchange with malate (Mal), and
intramitochondrial malate is oxidized to oxaloacetate. The mitoc-
hondrially generated aspartate is the source of the cytosolic malate
exchanging with a-KG after it has been transaminated to oxaoacetate
and reduced to malate. Two transmitochondrial carriers are involved,
1: the glutamate/aspartate exchanger AGC1 (red), which requires
aralar (not included in Fig. 2), and 2: the a-ketoglutarate/malate
carrier OGC (blue). This process is similar to that operating in the
malateaspartate shuttle, with the exception that glutamate in the latter
originates in the cytosol, not in the mitochondrial intermembranaceous
space. MEM: space between outer and inner mitochondrial mem-
branes. From [17] (Color figure online)
complex GABA formation in intact cells [13]. Different
results have been, however, obtained in cortical synapto-
somes [38]. The malateaspartate shuttle is best known in
connection with pyruvate formation where it is the major
shuttle carrying NADH formed during conversion of gly-
ceraldehyde-3-phosphate to 1-3-biphosphoglycerate (one
of the reactions occurring during glycolysis) into mito-
chondria for oxidation. Participation of a pseudoaspartate
malate shuttle during exit of glutamate from mitochondria
to cytosol implies that an oxidative reaction must simul-
taneously occur in the cytosol, i.e., that glucose must be
metabolized. One glucose molecule is converted to two
molecules of glyceraldehyde-3-phosphate, and it is
unknown if the second molecule is used for a different
purpose or if metabolism of one molecule of glucose is
capable of supporting exit of two molecules of glutamate
from mitochondria to cytosol. Anyhow, this process is
likely to contribute to the known stochiometric relationship
between glucose metabolism and glutamate flux in vivo
393
[10, 11] and to the requirement for oxidation of at least
some glucose during brain metabolism, even when lactate
or ketone bodies are freely available. Ketone bodies enter
the TCA cycle after generation of acetyl CoA but are
unable to form oxaloacetate, since they are not metabolized
via pyruvate and thus are not substrates for pyruvate car-
boxylation. The glucose/glutamate ratio in vivo is much
lower when a large amount of ketone bodies is metabolized
together with glucose [39]. This can be explained by the
ability of ketone bodies to contribute to glutamate forma-
tion in astrocytes, as will be described together with oxi-
dative degradation of glutamate. However, their
metabolism is not associated with malateaspartate shuttle
activity and can therefore not contribute to the pseudoas-
partatemalate shuttle described above. This may explain
why metabolism of some glucose is a requirement for
metabolism in the adult brain cells. When glucose is the
sole substrate it, and malateaspartate shuttle activity, will
be responsible for all glutamate formation in astrocytes.
After glutamate and GABA have been released from
neurones as transmitters, some transmitter is partly re-
accumulated into neurones. This applies especially to
GABA [13], but some glutamate my also be taken up by
axonal terminals in glutamatergic neurones [40]. Never-
theless, the majority of released glutamate is probably as
previously mentioned accumulated by powerful virtually
exclusively astrocytic transporters [41] and thus returned to
astrocytes. Here a part (*15 %) is oxidized, whereas the
remaining *85 % is again converted to glutamine and
returned to neurones through the glutamineglutamate/
GABA cycle for reuse as transmitter [20]. Although 15 %
of total cycle flux may appear to be a very small astrocytic
involvement, the precise amount oxidized varies somewhat
between different laboratories, and with the extremely high
flux rate glutamate oxidation combined with de novo
synthesis could be of considerable, but yet unknown,
importance. Moreover, glutamine can move between indi-
vidual astrocytes in the astrocytic functional syncytium and
reaches further away during brain activation than at rest
[42]. Accordingly, even the mere cycling of released
transmitter between neurons and astrocytes might provide
opportunities for an astrocytic role in determining which
neurons should receive renewed supply of glutamate. As
already mentioned, SN1-mediated release of glutamine
from astrocytes and thus replenishment of the neurotrans-
mitter pools of glutamate and GABA is also a strictly
regulated process [30].
Moreover, the total flux in the glutamineglutamate/
(GABA) cycle is so large and the association between
glutamate uptake and metabolism so close that glutamate
oxidation in astrocytes is more than high enough to support
its own energy-consuming uptake [24]. The first step is
formation of a-KG from glutamate, by GDH as generally
123
394
assumed and repeatedly demonstrated in isolated astro-
cytes, or by aspartate aminotransferase [17, 43]. It is in
support of the latter concept that most functions remain
unchanged in GDH knockout mice, except for a reduced
glutamate oxidation in cultured, and thus isolated astro-
cytes [44]. The newly generated a-KG may either after
circling to malate exit into the cytosol and be converted by
the astrocyte-enriched [45] cytosolic malic enzyme to
pyruvate (Fig. 1) which then can re-enter the TCA cycle
for complete oxidative degradation catalyzed by the
pyruvate dehydrogenase complex. Formation of glutamate
from glucose and complete oxidation of generated gluta-
mate in astrocytes produces almost as much energy (ATP)
as direct oxidative degradation of glucose would have done
[46]. Another possibility would be that malate does not
leave the TCA cycle but after incorporation of acetyl CoA
is converted to citrate and from there to a-KG (Fig. 1) from
which another molecule of glutamate is formed. Such a
coupling between glutamate oxidation and glutamate syn-
thesis would allow renewed glutamate synthesis without
involvement of pyruvate carboxylation. Which of these
possible metabolic routes is followed may vary under dif-
ferent conditions. However, renewed synthesis of gluta-
mate after incorporation of only acetyl coenzyme A would
allow astrocytic glutamate synthesis from ketone bodies
without further glucose metabolism, and the large differ-
ence between glucose/glutamate ratio in the presence and
absence of a high concentration of ketone bodies [39]
suggests that this pathway may be followed to a major
extent, at least in the presence of a high ketone body
concentration.
Although a non-negligible amount of GABA is re-
accumulated into GABA-ergic neurones [13], there is also
a considerable GABA transport in the glutamineglutamate
(GABA) cycle [47] and degradation in astrocytes by a
well-established pathway [48].The first step of this path-
way is conversion by GABA transaminase (GABA-T) to
succinic semialdehyde [49], which in turn is converted to
succinate, a TCA cycle intermediate, by succinic aldehyde
dehydrogenase. However GABA-T is a mitochondrial
enzyme [50], and GABA uptake obviously is cytosolic. In
a study of metabolism in cultured astrocytes McKenna and
Sonnewald [51] noted increased formation of [U-13-
C]aspartate from added [U-13C]glutamate as well as of
aspartate content, when the cells were also incubated with
GABA, and tentatively interpreted this as a result of
increased entry of [U-13C]glutamate into the TCA cycle to
allow for the transamination of GABA. However, in intact
tissue, where GABA production and degradation may be
linked in a similar manner as discussed above for gluta-
mate, extracellular glutamate may not be needed for this
purpose, since glutamate could be derived from mito-
chondrial glutamate, the synthesis of which would
123
Neurochem Res (2015) 40:389401
ultimately depend upon OAA generated during glutamate
production as illustrated in Fig. 2c. Such a process would
also give rise to increased aspartate formation, again con-
sistent with the observations by McKenna and Sonnewald
[51]. Thus the experimental results by these authors may
provide support for the model suggested in Fig. 2c.
In conclusion, the glutamineglutamate/(GABA) cycle
is an example of an intense neuronalastrocytic interaction
which is essential for normal brain function. Energetically
it is much more costly than neuronal production, reuptake
and oxidation of glutamate (and GABA) would have been.
Such a process seems to occur in the very immature brain
[17], and the development of the glutamineglutamate/
GABA cycle may be one of the factors explaining the
difference between the functional capability of the imma-
ture and mature brain. It may also contribute to illustrate
the importance of astrocytes which in rodents [52] and man
[53, 54] mainly develop and mature peri- and postnatally.
In adults the quantitative characteristics of the glutamine
glutamate(GABA) cycle are today routinely studied both in
rodent and human brain in vivo, pioneered by the Shul-
manRothmanBehar group in Yale [55] and Rolf Grutter
[56], now in Lausanne. Ongoing attempts to make such
examinations virtually non-invasive and of short duration
[57] may make the technique available for clinical tests.
Fostering and Maintaining Synaptic Connectivity: The
Astroglial Cradle
Glutamate and GABA are tightly connected to other fun-
damental functions of astroglia, which constitute major
elements in controlling synaptic connectivity in the CNS.
First and foremost, astrocytes are necessary for synapse
formation, the perinatal astrogliogenesis, which occurs
shortly before glutamatergic synaptogenesis and is indis-
pensable for its major wave [58]. Astroglial role in syna-
ptogenesis is mediated through secretion of numerous
molecules that include cholesterol, thrombospondins,
estradiol, c-protocadherins, integrins, &c. [59, 60].
Astrocytes maintain synaptic connectivity throughout
life by establishing intimate structural contacts with syn-
aptic structures. Modern view on a synapse highlights its
multi-component nature, as indeed most of the synaptic
contacts in the CNS are composed of pre- and postsynaptic
terminals, astroglial perisynaptic sheath, microglial pro-
cesses (that frequently contact the synapse, arguably to
detect its functional state [61]) and parts of extracellular
matrix [6265]. Astroglial membranes cover *60 % of all
synapses in the hippocampus, with large perforated syn-
apses being covered more frequently than small macular
synapses [66]. Perisynaptic astroglial membranes are sin-
gular structures in being exceedingly thin (with diameter
Neurochem Res (2015) 40:389401
between 100 nm and 200 nm and sometimes as thin as
50 nm [67] ). The perisynaptic processes are generally
devoid of organelles; there are neither mitochondria nor
endoplasmic reticulum, similarly there is no evidence for
the presence of neurotransmitter-containing vesicles in
these tiny perisynaptic processes [6769]. At the same time
perisynaptic astroglial processes are packed with numerous
molecules responsible for transmembrane transport of ions,
neurotransmitters, glutamine and metabolic substrates as
well as with neurotransmitter receptors of both ionotropic
and metabotropic varieties. This synaptic structure that
combines neuronal and glial membranes is a remarkable
result of evolution: molecules required for the act of syn-
aptic transmission are specifically concentrated in neuronal
membranes (e.g. molecules for vesicular transmitter release
in the presynapse and receptors in the postsynapse),
whereas molecules responsible for synaptic support are
moved to a much larger surface area of the perisynaptic
process. Incidentally, the perisynaptic astroglial membrane
accounts for *80 % of the total astroglial plasmalemma,
while contributing only a minor fraction (*410 %) to the
cellular volume, which reflected by a very high surface to
volume ratio of *25 lm-1 [67, 70] As a result even rel-
atively small ion fluxes may rapidly generate large con-
centration gradients, which, for example in the form of
localised Na? signals may substantially change the efficacy
and direction of multiple ion transporters of the SLC family
[71, 72]. Finally astroglial perisynaptic processes demon-
strate remarkable and rapid morphological plasticity, and
are able to dynamically regulate the synaptic coverage and
hence affect extracellular concentration of neurotransmit-
ters, thus regulating synaptic connectivity [73, 74].
The astroglial cradle [65, 75], formed by perisynaptic
processes, regulates all aspects of synaptic function. As has
been mentioned before, astrocytes are indispensable for
synaptogenesis and synaptic maturation through secreting
various factors [60]. Another function of the astroglial
cradle is isolation of synapses lying within the territory of a
single astrocyte and prevention of neurotransmitter spill-in
and spill-over [65]. This isolation is achieved by limiting
the extracellular space though tight association of the as-
troglial membrane with the synapse as well as though
numerous neurotransmitter transporters densely populating
perisynaptic astroglial membranes. Astroglial transporters
and channels are also critical for regulating K? concen-
tration in the synaptic cleft, which maintains neuronal
excitability. This is achieved by several systems that
include Kir4.1 inward rectifier potassium channels, the
Na?/K? ATase, the K?/Cl- co-transporter KCC1 and Na?/
K?/Cl- co-transporter NKCC1 [7679]. Similarly astro-
cytes regulate extracellular concentration of Cl- anions
through either Cl- efflux via anion channels or Cl- accu-
mulation by NKCC1. Astrocytes also may be involved in
395
regulation of Ca2? concentrations in the synaptic cleft;
because Ca2? levels may drop substantially during periods
of high neuronal activity; decrease in extracellular Ca2?
concentration was reported to activate intracellular Ca2?
release in astrocytes; with some of this release Ca2? being
excreted into the extracellular milieu via reversed mode of
the Na?/Ca2? exchanger [80]. Finally, astroglial cells
regulate extracellular pH by transporting H? with (1) Na?/
H? exchanger; (2) with lactate transporter MCT-1 that co-
transports 1 H? with 1 lactate and (3) with glutamate
transporters that remove 1 H? from the extracellular space
together with each glutamate molecule. Astroglial regula-
tion of extracellular pH is also linked to Na?/bicarbonate
exchanger [81] or co-transporter [82]. The astroglial cradle
is fundamental for water homeostasis, and hence for the
extracellular volume; with water being transported
through aquaporin AQP4 [83, 84] and by neurotransmitter
and ion transporters that incorporate a water transport
pathway, such as for example NKCC1 [76, 85]. Astrocytes
are central elements for neurotransmitter homeostasis, with
their role in regulation of glutamatergic and GABAergic
transmission being described in detail above. At the same
time astrocytes are solely responsible for regulation of
adenosine in the CNS by virtue of astroglia-specific
adenosine kinase [86] and astrocytic nucleoside transport-
ers [87]. Finally the astroglial cradle may contribute to
metabolic glial neuronal exchange, by supplying active
synapses with lactate through the astrocyteneurone lac-
tate shuttle (ANLS, [88]), although this concept remains a
matter for much debate [89, 90].
Astrocytes and Glymphatic System of the CNS
An additional, and somewhat unexpected role of astrocytes
is the support of metabolic homeostasis via facilitating
complex interactions between the blood vasculature, the
interposing cerebrospinal fluid (CSF) and surrounding
interstitial fluid (ISF). In addition to their perisynaptic
processes, astrocytes extend dense, laminal processes that
comprise the glial limiting membrane, or glia limitans, a
selectively permeable structure that forms a boundary
between CNS tissue, and the CSF in the subarachnoid
spaces and throughout the ventricular system [91]. An
additional barrier is formed by astrocytic endfoot pro-
cesses juxtaposed to the cerebrovasculature, permitting bi-
directional communication between neurons and astro-
cytes, in addition to enabling astrocytes to communicate
with the supplying circulations of the blood and CSF [91,
92].
Immunogold electron microscopy and mathematical
modelling have been used to demonstrate that paravascular
astrocytic processes ensheath *97 % of the entire
123
396
Fig. 4 Schematic outline of the
glymphatic system. Convective
glymphatic fluxes of
cerebrospinal fluid (CSF) and
interstitial fluid (ISF) propel the
waste products of neuron
metabo-lism into the para-
venous space, from which they
are directed into lymphatic
vessels and ultimately return to
the general circulation for
clearance by the kidney and
liver. From [96] with permission
cerebrovasculature, leaving only 20 nm clefts between
endfeet and blood vessels while forming small channels
that allow the CSF in the subarachnoid space to flow into
the brain along the exterior of the entire blood circulatory
pathway [93]. Dynamic two-photon and contrast enhanced
MRI imaging have revealed that CSF-tracers introduced
into the subarachnoid space flow into paravascular spaces
rapidly relevant to their movement in subarachnoid spaces
[92, 94, 95]. CSF flows into the brain preferentially along
large surface arteries, such as the basal artery or the
communicating arteries of the circle of Willis, before being
cleared from the brain along paravascular spaces sur-
rounding the deep draining veins, e.g. the caudal rhinal
vein and the great vein of Galen. CSF also flows into the
VirchowRobin spaces at the surface of the cerebral cortex,
where they then distribute rapidly by moving into the brain
along smaller leptomeningeal arteries and extending their
distribution throughout the basement membrane of pro-
gressively smaller penetrating arterioles, ultimately reach-
ing the basal lamina of capillaries [92, 94, 95].
Once CSF enters paravascular spaces, it may exchange
its soluble contents, selectively, with the ISF of the brain
parenchyma. The directional influx of CSF into the brain
along para-arterial spaces and efflux along para-venous
spaces facilitates the bulk flow clearance of interstitial
solutes, including dextrans of various molecular size, inulin
and soluble b-amyloid [96]. In peripheral tissues the bulk
flow of interstitial solutes deposits wastes from cellular
metabolism in the secondary circulation provided by the
lymphatic system. Paravascular CSF channels, aided by
astrocytes, facilitate this critical process along the glym-
phatic system in the CNS, which facilitates a tertiary
circulation important for waste removal (Fig. 4). Exchange
of CSF and ISF throughout the glymphatic system is in part
dependent on the pulsatility of cerebral arteries [97], and is
most pronounced during sleep [98]. Astrocytes support
123
Neurochem Res (2015) 40:389401
CSFISF exchange by expressing aquaporin-4 water
channels in a highly polarized expression distribution that
is localized predominantly to vessel-facing astrocytic
endfeet. The genetic deletion of these channels in rodents
results in a *70 % impairment to the efficiency of CSF
ISF exchange [92]. Lipids may also be secreted by astro-
cytes in an autocrine fashion, into paravascular spaces,
potentially to signal to other astrocytes, ultimately effect-
ing ion fluxes and downstream phosphorylation pathways
that themselves contribute to modulating the release or
uptake of many other signalling agents [99]. In this way, it
may be possible for astrocytes to regulate neural networks
on a macroscopic scale.
Astrocytes as Central Elements for Neuropathology
Neurological diseases can be conceptually defined as a
failures of CNS homeostasis. As a logical evolution of this
statement we may assume that astroglial cells, being cen-
tral elements of homeostatic system, contribute to patho-
genesis of all neurological diseases. This glio-centric view
on neuropathology is growing [100105], albeit remaining
in an opposition to the prevailing view of preponderance of
neuronal mechanisms in defining the progression and out-
come of neurological disorders.
Pathological changes in astroglia are documented in
numerous neurological diseases. Conceptually these can be
classified into astrocytopathology, that is manifested in loss
or pathological modification of astroglial function, and
astroglial reactivity. Reactive astrogliosis is a multi-stage,
complex and context-specific defensive response of
astroglia to pathological insults, which produces multiple
reactive phenotypes with both neuroprotective and neuro-
toxic capabilities [105109]. Astrocytopathology is often
expressed as astroglial asthenia or atrophy either
Neurochem Res (2015) 40:389401
morphological or functional. In many types of toxic
encephalopathies (for example poisoning with lead, alu-
minium or mercury) or neurodegenerative diseases (such as
Huntington disease, amyotrophic lateral sclerosis or Wer-
nicke-Korsakoff encephalopathy) the common denomina-
tor of astroglial asthenia is represented by substantial
decrease in the efficacy of glutamate uptake, which
underlies neuronal extinction through excitotoxicity [104,
110114]. In hepatic encephalopathy, the deficient gluta-
mate uptake is combined with an enhanced glutamine
production and reduced K? buffering due to competition
between NH4? and K? [115118]. In Alzheimers disease
as well as in some other types of dementia, astrocytes show
early signs of morphological atrophy, which may result in
reduction of astroglial synaptic coverage and insufficiency
of the astroglial cradle that may impair synaptic connec-
tivity and subsequently neuronal survival [103, 119].
Incidentally, astroglial morphological atrophy has been
discovered to appear very early in the entorhinal and pre-
frontal cortices which represent brain regions with highest
vulnerability to AD type pathology [120, 121]. That
astrocytic protein changes may precede neuronal changes
in Parkinsons disease was shown more than 50 years ago
[122]. Moreover, astrocytes and glycogenolysis, an astro-
cytic function, are crucial for memory formation [123,
124].
Astroglial atrophy and degeneration has been also
implicated in pathogenesis of neuropsychiatric diseases.
The signs of reduction in astroglial numbers and in GFAP
positive profiles have been identified in schizophrenia and
major depression [102, 125]; the astroglial asthenia may
result in abnormal glutamate/glutamine catabolism and
hence in deregulated neurotransmission which is currently
considered to be a major pathogenic factor of psychiatric
disorders [126, 127]. Incidentally, 2 weeks of the in vivo
treatment with the antidepressant fluoxetine or anti-bipolar
drug carbamazepine alters expression and/or editing of
many genes in freshly isolated astroglial cells. Astrocytes
also may contribute to evolution of schizophrenia through
kynurenic acid of which astroglia are the main producers
[128]. Kynurenic acid acts as an endogenous inhibitor of
NMDA receptors, and this endogenous inhibition has been
linked to pathogenesis of schizophrenia, in which elevated
levels of kynurenic acid has been detected in both brain
tissue and in CSF [129]. Increased production of kynurenic
acid can be the result of astroglia infection with Toxo-
plasma gondii, an infection which is a defined risk factor
for schizophrenia [130].
Astrocytopathology has also been considered recently in
the context of pathophysiology of autistic spectrum disor-
ders, or ASDs [131]. The ASD covers multiple neurode-
velopmental pathologies many of which are connected with
abnormal formation of neuronal networks and disbalanced
397
neurotransmission. Development of ASD may be associ-
ated with astroglial atrophy and impaired astroglia-depen-
dent synaptogenesis and abnormal cholesterol metabolism,
which have been detected in ASD [132]. Environmental
factors that affect astroglia (for example exposure to lead
or aluminium), as well as oxidative stress (which is also
contained by astrocytes and their crucial importance for
glutathione synthesis [133] ) are the major risk factors for
ASD.
Astroglial reactivity is similarly involved in numerous
neuropathologies. Epilepsy, for example, is associated with
prominent reactive astrogliosis observed in human post-
mortem tissues and in various animal models. Astrogliosis
develops at the early stages of the disease and progresses
through the disruption of astroglial territorial domains
[134]. This morphological metamorphosis is associated
with functional changes which involve aberrant calcium
signalling, increased expression of glutamate receptors,
decreased expression of K? channels and aquaporins, and
decrease in glutamate transport and glutamate enzymatic
processing by GS [100, 135137]. These functional re-
modellings underlie compromised K? buffering and glu-
tamate homeostasis, which may contribute to an increased
neuronal excitability and generation of seizures. In Batten
disease (neuronal ceroid lipofuscinosis) reactive astroglio-
sis is one of the earliest histopathological features; inhibi-
tion of astroglial reactivity substantially accelerates
progression of the disease [138, 139]. In neurodegenerative
diseases, in AD and in ALS reactive gliosis develops in
response to formation of senile plaques or appearance of
dying neurones [140, 141], whereas in thalamic dementia
astrogliosis occurs at the early stages and may be a primary
pathogenic step underlying neuronal death [142].
Conclusion: The All Important Astroglia
Astrocytes are highly heterogeneous cells, which steadily
increase in complexity along evolution of mammals. The
brain of high primates and humans contains several types
of astroglial cells that are absent in other vertebrates. These
highly diversified astroglial cells represent the homeostatic
scaffold of the CNS responsible for every conceivable task
associated with maintaining the functional environment.
Through many cascades responsible for homeostasis
astrocytes regulate emergence, maturation and function of
neuronal networks, and are thus indispensable elements for
information processing, learning and memory. Astrocytes
are also central elements of neuropathology being central
for protective astrogliosis; in many forms of neuropathol-
ogies astroglial cells are affected by degenerative changes
that suppress their functional and neuroprotective capabil-
ities, thus facilitating pathological progression.
123
398
Acknowledgments MN research is supported by the National
Institutes of Health (NIH); AV was supported by the Alzheimers
Research Trust (UK), by European Commission, by IKERBASQUE
and by a research grant of Nizhny Novgorod State University.
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