Our study indicates that astrocytes play an important role in the
pathophysiology of FXS by affecting neuronal output and behavioral
phenotype. Along with increased excitability of CA1 pyramidal neurons,
we demonstrated activity-dependent [K+
]o accumulation in the
hippocampus of Fmr1 KO male mice, indicating disturbance of extracellular
K+ homeostasis in FXS. This is associated with reduced ability
of astrocytes to uptake excess K+ released during synaptic activity via
Kir4.1 channels, whose expression is decreased. Furthermore, we
identified Kir4.1 mRNA as a target of FMRP. Finally, viral-mediated
expression of Kir4.1 in astrocytes rescued several neuronal and behavioral
abnormalities in Fmr1 KO male mice. Together, our findings
establish defective astrocyte Kir4.1 channels as an important contributor
to neuronal hyperexcitability and behavioral impairments
in FXS.
Astrocytes are essential elements of the tripartite synapse that
dynamically regulate the extracellular environment. Specifically, they
control elevated [K+
]o following neuronal activity through K+
clearance29,34. Here we demonstrate that astrocyte K+ uptake through
Kir4.1 channels during synaptic activity is markedly reduced in Fmr1
KO male mice. The magnitude of synaptically-evoked K+ current in
Fmr1 KO astrocytes exhibits a reduction of ∼45%. Moreover, we found
that IK kinetics displays prolonged rise time and time of peak, whereas
decay time was shorter in Fmr1 KO male mice. Impaired ability of
astrocytes to remove excess K+ through Kir4.1 in FXS is consistent with
previously reported findings in glial conditional Kir4.1-/- mice. Astrocyte
K+ current evoked by single stimulation of Schaffer collaterals is
indeed almost completely abolished in the hippocampus of glial conditional
Kir4.1-/- mice35,37. Despite the reduction by ∼45% of the Kir4.1
currents in astrocytes from Fmr1 KO mice, we found that basal [K+
]o
and astroglial RMP remain intact, in contrast to the depolarized RMP
reported in astrocytes deficient for Kir4.135,44. This suggests that the
remaining Kir4.1 activity, representing ∼55% of the current, is sufficient
to maintain basal [K+
]o and normal astroglial RMP, but however fails to
ensure efficient clearance of excess K+ released during neuronal
activity.
It is important to note that we found activity-dependent [K+
]o
accumulation and in particular enlarged and prolonged [K+
]o undershoot
in the absence of FMRP, suggesting compromised activitydependent
K+ clearance. Our findings are in accordance with prominent
undershoot observed in the hippocampus of glial conditional
Kir4.1-/- mice44. Astroglial Na+
/K+ ATPases also contribute to K+ clearance
in the hippocampus45. It is noteworthy that in Kir4.1-/- mice, the
depolarization of astrocytes may impair the activity of Na+
/K+ ATPases,
therefore masking their contribution to K+ clearance. Indeed, as the
activity of Na+
/K+
-ATPases is dependent on [K+
]o and [Na+
]i
, alteration
in homeostasis of these ions is likely to alter the uptake of excess K+ by
Na+
/K+ ATPases. Thus, although we show the important role of Kir4.1
deficit in the altered extracellular K+ homeostasis from Fmr1 KO mice
using genetic tools altering Kir4.1 expression (Kir4.1-/- mice and viral
vectors to rescue Kir4.1 function), deciphering the contribution of Na+
/
K+ ATPases in activity-dependent K+ buffering in physiological conditions
is complex.
Importantly, we found that FMRP associates with Kir4.1 mRNA and
regulates Kir4.1 expression. This reduction in Kir4.1 levels did not
result from changes in the morphology of astrocytes, which we found
to be unaltered in Fmr1 KO mice, in accordance with other studies24,27.
It is noteworthy that Kir4.1 protein levels are also under the control of
Methyl-CpG-binding protein 2 (MeCP2), a transcriptional regulator
expressed in both neurons and glial cells46, and whose mutations lead
to 95% of Rett syndrome cases47. MeCP2 interacts with the promoter of
the KCNJ10 gene encoding Kir4.148 and typical upregulation of Kir4.1 at
the early postnatal development stage36,49 is absent in MeCP2 KO mice,
suggesting altered astrocyte maturation in Rett syndrome48. Our
findings suggest that dysregulation of Kir4.1 expression and function
in the absence of FMRP is responsible for impaired [K+
]o regulation
in FXS.
Previous findings show that Kir4.1 is expressed in the proximity of
neuronal soma in several brain regions including lateral habenula50,
ventral spinal cord51 and brainstem52. We observed similar pattern of
perineuronal Kir4.1 staining in the hypothalamus and somatosensory
cortex. However, in the CA1 pyramidal layer, only Kir4.1-positive fibers
are observed53 while Kir4.1 expression in the stratum radiatum is the
most intense in GFAP-positive astrocytes50,53. Processes of CA1 stratum
radiatum astrocytes contact synapse sites54 and are therefore wellsuited
for the uptake of K+ released by dendrites of pyramidal
neurons48 and maintenance of [K+
]o homeostasis. Downregulation and
dysfunction of Kir4.1 in stratum radiatum astrocytes of Fmr1 KO male
mice lead to deregulation of the K+ clearance following synaptic
activity and thus contribute to synaptopathy in FXS. Interestingly,
pronounced downregulation and reduced activity of astrocyte Kir4.1
channels have also been reported in brain tissues from epileptic
patients, and in animal models of epilepsy55. Compromised properties
of astrocyte Kir4.1 channels have been proposed to facilitate development
of seizures and promote epileptogenesis35,55,56. This is consistent
with the high incidence of seizures reported in FXS patients,
and in the Fmr1 KO mice7,17, supporting a key role for dysfunction of the
Kir4.1 channel in mechanisms associated with epilepsy.
Increased neuronal excitability in a variety of brain regions is a
prominent feature of FXS17. Multiple lines of evidence have linked
changes in neuronal excitability in FXS to changes in expression and
activity of neuronal ion channels, such as BK or CaV2.2 channels11,12,15,16.
The altered extracellular K+ homeostasis, resulting from reduced
astroglial Kir4.1-mediated K+ uptake, may also contribute to the
deregulation of these neuronal ion channels in FXS. Increased extracellular
K+ levels, by inducing neuronal depolarization, can indeed
regulate the activity of these neuronal ion channels57,58. In the present
study, we discover that loss of FMRP specifically from hippocampal
astrocytes causes hyperexcitability of CA1 pyramidal neurons. Moreover,
we uncover dysfunction of astroglial Kir4.1 channels as a major
cause of increased excitability in CA1 pyramidal neurons in FXS. Our
experiments restoring astroglial Kir4.1 to WT level by viral-mediated
delivery indeed rescued normal astrocyte K+ uptake and neuronal
excitability in Fmr1 KO hippocampus. Our data suggest that changes in
Kir4.1 expression and activity in FXS have a stronger impact on high
regimes of neuronal activity, since we did not observe significant [K+
]o
changes in basal conditions in the Fmr1 KO hippocampus. This is
supported by a modeling study showing that astrocyte Kir4.1 channels
have a pronounced effect on neuronal excitability during repetitive
stimulation and a firing regime at 3-10 Hz59.
Neuronal hyperexcitability has been associated with several ASDlinked
behavioral phenotypes. Specifically, excitatory neurons lacking
cyclin-dependent kinase-like 5 display increased excitability that is
associated with impaired hippocampal-dependent memory60. Moreover,
in a rat model of autism induced by valproic acid-exposure,
altered social behavior was observed together with intrinsic neuronal
hyperexcitability61. Here, we show that astrocyte Kir4.1 channel in the
hippocampus has an important role in the regulation of neuronal
excitability as well as cognitive and social recognition. Selective
delivery of Kir4.1 in hippocampal astrocytes rescued spatial recognition
memory, as demonstrated by NOR experiments in Fmr1 KO male
mice. Moreover, this also restored the ability of Fmr1 KO male mice to
distinguish between a familiar and a novel stranger mouse, demonstrating
an improvement of social recognition. Restoring Kir4.1 expression in ∼70% of hippocampal CA1 stratum radiatum astrocytes
was sufficient to correct neuronal hyperexcitability and behavioral
defects in Fmr1 KO mice. The expression of Kir4.1 is also decreased in
the somatosensory cortex of Fmr1 KO mice. This could be associated
with neuronal hyperexcitability in this brain region as well as sensory
hypersensitivity16, which can contribute to abnormal social
behavior62,63. Altogether, our findings support the notion that impaired
astrocyte physiology in the absence of FMRP contributes to neuronal
defects and behavioral abnormalities. These data point to astroglial
Kir4.1 channel as a potential therapeutic target to treat FXS behavioral
defects, and especially hippocampal-dependent memory
impairments.
Impaired properties of Kir4.1 channel have been reported in several
neurodegenerative diseases including Huntington’s disease64 and
amyotrophic lateral sclerosis51,65. Emerging data point to the Kir4.1
channel as an important player in the pathophysiology of various
disorders linked to ASD and intellectual disability. Mutations in the
KCNJ10 gene encoding Kir4.1 have been reported to cause SeSAME/
EAST syndrome characterized by early onset seizures, ataxia and
mental retardation66. Likewise, two mutations resulting in Kir4.1 gain of
function were identified in patients with ASD showing symptoms of
epilepsy67. Recently, a KCNJ10 gene polymorphism has been correlated
with ASD susceptibility68. Furthermore, the same study provided evidence
of reduced expression of Kir4.1 in the hippocampus of the valproic
acid-exposed rat model of ASD, suggesting the involvement of
this astrocyte channel68. The KCNJ10 gene promoter is also a binding
target of the MeCP2 protein implicated in Rett syndrome48. Mice
lacking MeCP2 display reduced astroglial Kir4.1 expression and activity
that is associated with increased extracellular K+ levels48. However, it is
unknown whether the compromised Kir4.1 channels lead to neuronal
dysfunctions in Rett syndrome. Here, we not only uncover dysfunction
of Kir4.1 channels in FXS, but also reveal that this underlies several
neuronal and behavioral deficits in FXS. Altogether, these data suggest
that alteration in astroglial Kir4.1 channels may be involved in the
pathophysiology of several neurodevelopmental disorders.
Up to now, most research has focused on neuronal cell autonomous
mechanisms to account for neuronal hyperexcitability and
behavioral defects in FXS. By focusing on the role of astrocytes in FXS,
we here provide compelling evidence for Kir4.1 dysfunction as a major
contributor to impaired extracellular K+ homeostasis, neuronal
hyperexcitability and behavioral deficits. Herein, we propose that
restoring Kir4.1 function in the hippocampus of FXS patients could be
a promising therapeutic strategy for the treatment of neuronal
hyperexcitability and cognitive defects.
Methods
Animals
All procedures on animals strictly followed the guidelines of the European
Community Council Directives of January 1st 2013 (2010/63/EU)
and French ethic committee (certificate A751901. delivered by the
French Ministry of higher education, research and innovation). All the
experiments were carried out using male mice of wild-type (WT)
C57BL/6j background, Fmr1 knockout (KO) mice5
, mice containing Creexcisable
loxP sequence in the Fmr1 gene (Fmr1fl/fl
)
69, mice containing
Cre-excisable loxP sequence in the gene encoding Kir4.1 (Kir4.1fl/fl
) and
mice with conditional deletion of Kir4.1 in glia, Kir4.1fl/fl
:hGFAP-Cre
(Kir4.1−/−
) mice (both provided by K. D. McCarthy, University of North
Carolina, USA)35,44. Both non-transgenic littermate and age-matched
WT males were used as control mice. All animals were 21- to 32-day-old
males unless otherwise stated. Adult mice were used for behavior
experiments at the age of 4–5 months. Breeding and genotyping of
mice were performed as previously described70. Mice were housed
under 12 h/12 h light/dark cycle, temperature of 22 °C, humidity 55%
and with food and water available ad libitum. All efforts were made to
minimize the number of animals used.
Preparation of hippocampal brain slices
Male WT, Fmr1 KO, Fmr1fl/fl
, Kir4.1fl/fl or Kir4.1−/− mice (P21-32) underwent
cervical dislocation and were decapitated. The brain was isolated and
the hippocampus dissected out into ice-cold artificial cerebrospinal
fluid (ACSF) containing (mM): 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1
NaH2PO4, 26.2 NaHCO3 and 11 glucose continuously bubbled with a
mixture of 95% O2 and 5% CO2. Acute transverse hippocampal slices
(350 µm) were sectioned in cold ACSF using vibratome (Leica VT
1200S) and then recovered in ACSF at room temperature for 1 h. After
recovering, slices were transferred to a recording chamber mounted
on an Olympus BX51WI microscope equipped with infrareddifferential
interference (IR-DIC) microscopy. Slices were perfused
with the oxygenated ACSF at the constant flow rate of 2 ml/min at
room temperature. All chemicals and products in this study were
purchased from Sigma Aldrich unless otherwise stated.
Measurement of extracellular K+ concentration
K+
-sensitive microelectrodes were made of thin wall borosilicate
capillaries (GC150T-7.5, Harvard Apparatus, MA) and the interior
surface was silanized during 15 min with N,N-Dimethyltrimethylsilylamine
(226289, Sigma Aldrich). The electrodes were
dried in the oven at 200 °C for 100 min. The tip of the electrode was
filled with Potassium ionophore I—Cocktail A (60031, Sigma
Aldrich) while the rest was backfilled with 200 mM KCl in ACSF. The
calibration of K+
-sensitive microelectrodes was done in the standard
solutions of the following KCl concentration (mM): 0.5, 1, 2, 4, 8, 10
and 20. The extracellular potassium concentration ([K+
]o) was
measured in acute hippocampal slices of WT and Fmr1 KO mice. The
experiments were performed in oxygenated ACSF in the presence of
100 µM picrotoxin (P1675, Sigma Aldrich). A cut between CA1 and
CA3 was made to prevent propagation of epileptiform activity. A
glass pipette filled with ACSF was used to stimulate Schaffer collaterals
(SC) and evoke synaptic activity in CA1 area. Extracellular
field recording electrode (filled with ACSF) and K+
-sensitive microelectrode
were placed in CA1 stratum radiatum in close proximity
(<10 µm). Basal [K+
]o was measured after correct positioning of the
K+
-sensitive microelectrode in the hippocampal slice and stabilization
of the baseline. Data were acquired in current-clamp mode
using Axopatch 1D and 200B amplifier, sampled at 10 kHz, filtered at
2 kHz and digitized using Digidata 1320 and 1440 (Molecular Devices).
Stimulation intensity was adjusted by applying 0.05 Hz stimulation
and inducing similar fiber volleys in WT and Fmr1 KO mice.
[K+
]o and fEPSPs were simultaneously recorded in response to 10 Hz
train stimulus over 30 s and further analysis was performed as
previously described44. Briefly, the signal obtained using the K+
-
sensitive microelectrode was corrected by subtracting fEPSP
response. The resulting trace was linearized and converted into
[K+
]o values using parameters of the log-linear fit function. The peak
amplitude and area of [K+
]o transient as well as of [K+
]o undershoot
were measured. The return time of [K+
]o after the train stimulus was
determined as the time of baseline recovery following the undershoot
using exponential fitting44. 자연스럽게 해석해줘