Received: 2 June 2017
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Revised: 4 July 2017
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Accepted: 5 July 2017
DOI: 10.1002/glia.23196
RESEARCH ARTICLE
Subcellular reorganization and altered phosphorylation of the astrocytic gap junction protein connexin43 in human and experimental temporal lobe epilepsy Tushar Deshpande1 | Tingsong Li1,2 | Michel K. Herde1 | Albert Becker3 | Hartmut Vatter4 | Martin K. Schwarz5 | Christian Henneberger1,6,7 | Christian Steinhäuser1
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Peter Bedner1
1 Institute of Cellular Neurosciences, Medical Faculty, University of Bonn, Germany
Abstract
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Dysfunctional astrocytes are increasingly recognized as key players in the development and pro-
Department of Neurology, Children’s Hospital, Chongqing Medical University, Chongqing, China
3
Department of Neuropathology, Medical Faculty, University of Bonn, Bonn, Germany 4
Department of Neurosurgery, Medical Faculty, University of Bonn, Bonn, Germany 5
Department of Epileptology, Medical Faculty, University of Bonn, Bonn, Germany 6
Institute of Neurology, University College London, London, UK 7
German Center for Degenerative Diseases (DZNE), Bonn, Germany Correspondence Christian Steinhäuser, Institute of Cellular Neurosciences, University of Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany. Email
[email protected]. de Funding information €ckkehrerprogramm (C.H.), German NRW-Ru Research Foundation, Grant/Award Number: SFB1089 B03 to C.H., SPP1757 HE6949/1 and HE6949/3 to C.H., STE552/3 to C.S., a SPP1757 young investigator grant to M.K.H.; European Union (ERA-NET NEURON project “BrIE” to C.S.)
gression of mesial temporal lobe epilepsy (MTLE). One of the dramatic changes astrocytes undergo in MTLE with hippocampal sclerosis (HS) is loss of gap junction coupling. To further elucidate molecular mechanism(s) underlying this alteration, we assessed expression, cellular localization and phosphorylation status of astrocytic gap junction proteins in human and experimental MTLE-HS. In addition to conventional confocal analysis of immunohistochemical staining we employed expansion microscopy, which allowed visualization of blood-brain-barrier (BBB) associated cellular elements at a sub-mm scale. Western Blot analysis showed that plasma membrane expression of connexin43 (Cx43) and Cx30 were not significantly different in hippocampal specimens with and without sclerosis. However, we observed a pronounced subcellular redistribution of Cx43 toward perivascular endfeet in HS, an effect that was accompanied by increased plaque size. Furthermore, in HS Cx43 was characterized by enhanced C-terminal phosphorylation of sites affecting channel permeability. Prominent albumin immunoreactivity was found in the perivascular space of HS tissue, indicating that BBB damage and consequential albumin extravasation was involved in Cx43 dysregulation. Together, our results suggest that subcellular reorganization and/or abnormal posttranslational processing rather than transcriptional downregulation of astrocytic gap junction proteins account for the loss of coupling reported in human and experimental TLE. The observations of the present study provide new insights into pathological alterations of astrocytes in HS, which may aid in the identification of novel therapeutic targets and development of alternative anti-epileptogenic strategies.
KEYWORDS
albumin extravasation, astrocyte, astrocytic endfeet, hippocampal sclerosis, temporal lobe epilepsy
1 | INTRODUCTION
focal epilepsy is mesial temporal lobe epilepsy (MTLE), where epileptic activity is mostly originating from the hippocampus, amygdala, and
Epilepsy is a chronic brain disorder characterized by recurrent unpro-
entorhinal cortex. Seizures arising from the temporal lobe are particu-
voked seizures that affect 1–2% of the population worldwide
larly difficult to control with antiepileptic therapies, and about 75% of
(Hesdorffer et al., 2011). The most frequent and severe form of adult
€ scher, 2005). MTLE these patients are pharmacoresistant (Schmidt & Lo is frequently associated with hippocampal sclerosis (HS), histopatholog-
Tushar Deshpande and Tingsong Li contributed equally to this work.
Glia. 2017;1–12.
ically characterized by prominent neuronal loss in the hippocampal
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C 2017 Wiley Periodicals, Inc. V
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CA1 and CA4 regions, reactive astrogliosis, granule cell dispersion, and
Comparative analyses were performed in MTLE patients and in the uni-
€mcke, Beck, Lie, & Wiestler, 1999). Traditionaxonal reorganization (Blu
lateral intracortical kainate mouse model of MTLE.
ally, epilepsy has been considered to be caused by neuronal dysfunctions. However, this neurocentric view of epilepsy has recently been
2 | MATERIALS AND METHODS
challenged by a number of studies demonstrating a crucial role of glial cells in influencing neuronal activity, brain homeostasis and neuroprotection. Astrocytes, the most abundant glial population, fulfill various important functions in the brain including direct modulation of synaptic transmission by release, uptake, degradation and recycling of transmitters, control of the extracellular ion and water homeostasis, regulation of local blood flow, maintenance of BBB integrity, and supply of nutrients to neurons (Verkhratsky & Parpura, 2015). Importantly, astrocytes are electrically and metabolically connected to each other by gap junctions composed mainly of Cx43 and Cx30 to form functional networks (Giaume, Koulakoff, Roux, Holcman, & Rouach, 2010; Nagy & Rash, 2000). These coupled networks are crucially involved in the modulation of neuronal activity and synaptic transmission through clearance and spatial buffering of excess extracellular K1 and neurotransmitters. Accordingly, reduction or loss of interastrocytic coupling cause accumulation of K1 and glutamate in the extracellular space which in turn promote neuronal hyperactivity and seizures (Bedner et al., 2015; Pannasch et al., 2011; Steinhäuser, Seifert, & Bedner, 2012; Wallraff et al., 2006).
2.1 | Patient information A total of 38 hippocampal neurosurgical specimens were obtained from patients with pharmacoresistant MTLE. Based on histopathological diagnosis, 19 patients suffered from HS characterized by severe neuronal loss, atrophy and intense GFAP immunoreactivity in the Ammon’s horn. Specimens from another 19 patients, constituting the “non-HS” group, did not show significant hippocampal atrophy or neuronal death. Instead, mild to moderate astrogliosis and/or microglial activation was often seen in the non-HS. Clinical details of the patients are given in Supporting Information Table S1. In all patients, generation of temporal lobe seizures had been traced to the hippocampus with noninvasive and invasive diagnostics as described elsewhere (Behrens et al., 1994; Elger, Hufnagel, & Schramm, 1993). Informed consents were obtained from all patients for additional morphological studies. All procedures were approved by the ethics committee of Bonn University Medical center and conform to standards set by the Declaration of Helsinki (1989).
Epilepsy-associated changes in connexin expression have been examined in a number of human and experimental studies, with inconsistent results (for review see Giaume, et al. 2010; Steinhäuser
2.2 | Mouse model of MTLE
et al., 2012). This discrepancy may be partly due to the fact that Cx43
Maintenance and handling of animals were according to the local
expression levels do not necessarily reflect the extent of functional
government regulations. Experiments have been approved by the state
coupling, since connexin proteins are tightly regulated. For instance,
North Rhine Westphalia (LANUV approval number 84–02.04.2015.
Cx43, the gap junction protein responsible for about 80% of interastro-
A393). The unilateral intracortical kainate mouse model of MTLE has
cytic coupling in the hippocampus (Gosejacob et al., 2011), possesses
been described recently (Bedner et al., 2015). Briefly, male FVB mice
at least 21 phosphorylation sites within the cytoplasmic C-terminus,
aged 3–4 months were anesthetized (medetomidine, 0.3 mg/kg, i.p.
which are targeted by a number of kinases, including protein kinase C
and ketamine, 40 mg/kg, i.p.) and placed in a stereotaxic frame
(PKC), protein kinase a (PKA), and mitogen-activated protein kinase
equipped with a manual microinjection unit (TSE Systems GmbH, Bad
(MAPK). Phosphorylation of Cx43 affects the intercellular communica-
Homburg, Germany). Seventy nl of a 20 mM solution of kainic acid
tion between astrocytes by altering gating properties of the channels,
(Tocris, Bristol, UK) in 0.9% sterile NaCl were stereotactically injected
trafficking and degradation of the protein as well as assembly/ disassembly of gap junctions (Axelsen, Calloe, Holstein-Rathlou, & Nielsen, 2013; Solan & Lampe, 2009). Although several studies found increased levels of connexin mRNA and/or protein levels in human epileptic specimens, our recent functional analysis revealed complete loss of coupling in hippocampal tissue resected from patients with MTLE-HS, while coupled astrocytes were abundantly present in the hippocampus of patients with nonsclerotic MTLE. Importantly, in a mouse model closely recapitulating key alterations of chronic human MTLE-HS, we could show that astrocyte uncoupling and the consequential impairment of K1 buffering temporally precede neurodegeneration and spontaneous seizure generation
into the neocortex just above the right dorsal hippocampus. The stereotactic coordinates were 1.9 mm posterior to bregma, 1.5 mm from midline and 1.7 mm from the skull surface. Control mice were given injections of 70 nl saline under the same conditions. After injection, the scalp incision was sutured and anesthesia stopped with atipamezol (300 mg/kg, i.p.). Brains of these mice were perfusion fixed with 4% PFA followed by overnight fixation in 4% PFA, 3 months post injection. For Western blotting, entire hippocampi from the dorsal region of the temporal lobe were snap frozen in liquid nitrogen followed by storage at 2808C till further use.
2.3 | Western blotting
(Bedner et al., 2015). Though these data strongly suggest that astrocyte
Plasma membrane-associated proteins were isolated from mouse or
uncoupling represents a causal event in epileptogenesis, the underlying
human hippocampi using a Plasma Membrane Protein Extraction Kit
mechanism(s) are unclear. To address this issue, we quantitatively
(Abcam, catalogue number: ab65400) as per manufacturer’s instructions.
assessed expression, subcellular localization and phosphorylation status
Briefly, tissues were homogenized in the homogenization buffer supplied
of Cx43 and Cx30 in sclerotic and nonsclerotic epileptic hippocampi.
with the kit. The resultant lysate was centrifuged at 11,000g for 30 min.
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The supernatant constituted the cytoplasmic while the pellet constituted
For histochemical detection of the vasculature in the hippocampus,
the total membrane fraction. The pellet was extracted with the two par-
biotinylated lycopersicon esculentum (tomato) lectin (Vector laborato-
tially miscible polymer solutions provided with the kit. The final pellet
ries, catalogue number: B1175) was used. In brief, after overnight incu-
obtained after extraction was dissolved in 4% didodecyl maltoside in
bation with primary antibodies, lectin (1:100) was added and incubated
50 mM bis-Tris buffer containing HaltTM Protease and Phosphatase
for 30 min at room temperature. Then, a mix of streptavidin coupled
Inhibitor Single-Use Cocktail (Thermoscientific, catalogue number:
with Alexa Fluor ®647 (1:500, Invitrogen) and other appropriate sec-
78442). Five mg of protein of each sample were subjected to SDS PAGE
ondary antibodies was added. After each incubation, sections were
followed by Western blotting on PVDF membranes. Alternatively, total
washed three times.
(whole-cell) tissue lysates were prepared by homogenizing human or
Images with optical thickness of 1 mm (CA1 subregion) were
mouse hippocampi in a modified Radio Immunoprecipitation Assay
acquired using a confocal laser scanning microscope (Leica TCS SP8,
(RIPA) buffer (20 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.5%
Germany). For colocalization studies, 63x lenses were used.
sodium deoxycholate, 0.5% Nonidet P-40; pH 7.4) supplanted with HaltTM Protease and Phosphatase Inhibitor Single-Use Cocktail. Thirty mg of these protein lysates were subjected to SDS PAGE followed by Western blotting. All blotted membranes were blocked with 5% nonfat milk in TBST for 1 hr followed by overnight incubation with primary antibodies (custom-made rabbit anti-Cx43 antibody, dilution 1:2500 (Khan et al., 2016); rabbit anti-Cx30 antibody, Invitrogen/Thermoscientific, catalogue number 71–2200, dilution 1:250; rabbit anti-E-cadherin anti-
2.5 | Expansion microscopy The expansion microscopy (ExM) protocol with conventional primary and secondary antibodies was adopted from Chen, Tillberg, and Boyden (2015) and Chozinski et al. (2016). Hippocampal slices (60 mm) were stained as described above. For Cx43 labelling, biotinylated antirabbit secondary antibody (1:200, Jackson ImmunoResearch, catalogue
body, Abcam, ab15148, catalogue number: 71–2200, dilution 1:1000) in
number: 111–066-144) was used. The sections were incubated with
2.5% milk. The next day, after 3 times washings in TBST, the membranes
1 mM methylacrylic acid-NHS (Sigma Aldrich) linker for 1 hr. After
were probed with ECLTM Rabbit IgG, HRP-linked F(ab0 )2 fragment
washing in PBS 3 times, sections were incubated with monomer
(Amersham, catalogue number: NA 9340, dilution 1:5000) in 2.5% milk
solution containing 8.6% sodium acrylate, 2.5% acrylamide, 2% N, N0 -
for 1 hr. For phospho-specific antibodies (p-Cx43 S255 from Santa Cruz
methylenebisacrylamide, and 29.2% NaCl in PBS for 45 min. A gelling
Biotechnology, catalogue number: sc-12899-R; p-Cx43 S368 from Invi-
solution was prepared by adding ammonium persulfate (0.2%), TEMED
trogen, catalogue number 48–3000) nonfat milk was replaced by bovine
(0.2%) and 4-hydroxy-TEMPO (0.01%) to the monomer solution. Slices
serum albumin. WesternBright Sirius Chemiluminescent substrate
were placed in the gelling solution on a glass slide and the preparation
(Advansta, catalogue number: K-12043-D20) was used to visualize the
was covered with a coverslip. After 2 hr of incubation at 378C, cover-
TM
blots by GeneGnomeXRQ
imager (Syngene, UK). Densitometry was
slips and excess gel around the slices were removed. The gel pieces containing the tissue slices were incubated overnight at 378C in a
performed using GeneTools (Syngene).
digestion buffer containing 50 mM Tris, 0.5% Triton-X100, 1 mM EDTA, 0.8 M guanidine hydrochloride and 16 U/ml of proteinase K
2.4 | Immunohistochemistry
(pH 8). The next day, the digestion buffer was removed and the gel Prefixed hippocampal specimens were cut with a vibratome into 20 or 60 mm thick sections. After permeabilization and blocking (2 hr, room temperature) with 0.5% Triton
TM
X-100 and 10% normal goat serum
(NGS) or normal donkey serum (NDS) in PBS, the sections were incubated overnight (48C) in 5% NGS/NDS in PBS containing 0.1% Triton TM X-100 and primary antibodies. After washing three times with PBS, the sections were incubated with secondary antibodies conjugated with Alexa Fluor® 488, Alexa Fluor® 594 or Alexa Fluor
®
647 (Invitrogen,
dilution 1:500 each) in PBS with 2% NGS/NDS and 0.1% Triton
TM
X-100 for 1.5 hr at room temperature. After several washing steps in
pieces were incubated with streptavidin coupled with Alexa 647 (Jackson ImmunoResearch catalogue number: 016–600-084) for 1.5 hr. They were then washed with deionized water. After five washes (2.5 hr), slices containing the expanded tissue samples were transferred to a custom mounting chamber filled with distilled water, mounted by supergluing its edges to the chamber’s bottom and sealed with a coverslip on the top. Imaging was performed on a Leica SP8 confocal microscope using a 40x/1.1NA objective and hybrid detectors. The expansion factor was determined by measuring gel sizes before and after expansion.
PBS, nuclei were counterstained with Hoechst (1:100 in water). The following primary antibodies were used: rabbit anti-Cx43 (1:500, SigmaAldrich, catalogue number: C6219), mouse anti-S-100B (1:500, Abcam,
2.6 | Stereology
catalogue number: ab66028), mouse anti-NG2 (1:200, Millipore, cata-
Confocal images acquired at constant settings were used to extract the
logue number: MAB2029), goat anti-CD31 (1:200, RD Sys, catalogue
following parameters using image analysis software Fiji (Schindelin
number: AF3628), goat anti-Albumin (1:200, Abcam, catalogue number:
et al., 2012): average fluorescent intensity, area occupied by Cx43
ab19194). For immunostaining of PDGFRb and CD31, NGS in the block-
signal, average plaque size, plaque number and distribution ratio.
ing solutions was replaced by NDS, and the secondary antibodies were
Images were converted into 8-bit format and were thresholded at
also changed accordingly. For immunostaining of albumin, no serum was
three different grey values (pixel intensity measured in an 8-bit format)
used to the blocking solutions.
55, 75, and 90. All of these thresholding conditions maintained the
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statistical inference between the experimental groups. Therefore, the
Remarkably, plasma membrane levels of Cx43 and Cx30 did not differ in
intermediate condition threshold of 75 was used for further analysis.
non-HS and HS (Cx43, 4.43 6 2.22 in non-HS vs. 3.35 6 0.90 in HS,
The values were recorded for each optical plane and then averaged
n 5 5 and 6, respectively, p 5 .3; Cx30, 0.16 6 0.14 in non-HS vs. 0.14 6
across all the planes in a slice from mouse or a patient. Mean pixel
0.04 in HS, n 5 5 and 6, respectively, p 5 .74; Figure 1b). The three
intensity and the distribution ratio were measured in nonthresholded,
phospho-bands of Cx43 were similar in both conditions (not shown),
12-bit images. Pixel intensities or grey values were measured in arbi-
suggesting an unchanged phosphorylation status of the C-terminus in
trary units (a.u.). The distribution ratio was calculated as the ratio of
sclerosis.
average Cx43 intensities around the blood vessels to the intensities in
In conclusion, despite enhanced Cx43 levels there was no indica-
the rest of the tissue. The region around the blood vessel was defined
tion for increased insertion of Cx43 channels into the plasma membrane
as a 5 lm wide rim from the innermost margin of Cx43 around a blood
in tissue obtained from MTLE-HS patients.
vessel into the brain parenchyma (Supporting Information Figure S2). For each mouse or patient, these values were obtained from three slices and then were averaged.
3.2 | Accumulation of Cx43 around blood vessels in the sclerotic human CA1 region To confirm the Western blot data and investigate the cellular and sub-
2.7 | Statistical analysis
cellular distribution of connexins in human MTLE specimens, immuno-
Data are given as mean 6 standard deviation (SD). Error bars represent
histochemistry was performed. In accordance with total protein
SD. Differences between data were tested for significance using Student’s
expression found with Western blot, antibody staining revealed higher
t-test. To test for normal distribution, Kolmogorov-Smirnov and Shapiro-
Cx43 immunoreactivity in the sclerotic CA1 region than in non-HS
Wilk tests were applied. The level of significance was set at p < .05.
tissue (non-HS, 99.75 6 40.26; HS, 183.03 6 64.15, n 5 18 slices from six specimens per group, p 5 .02; Figure 2). The enhanced expression was also reflected in the total area occupied by Cx43 immunolabeled
3 | RESULTS
structures, which were much larger in the sclerotic CA1 region
3.1 | Inefficient transport of connexins to the plasma membrane in human HS
(468.01 6 426.55 mm2) as compared with non-HS (37.88 6 30.31 mm2) (n 5 18 slices from 6 specimens per group. p 5 .03). We noted that in HS, Cx43 expression was nonuniformly distributed and immunoreactiv-
To compare connexin expression between HS and non-HS human speci-
ity was preferentially seen around blood vessels (Figure 2a) while a seg-
mens, total protein lysates were immunoblotted and the membranes
regated expression was not observed for Cx30 (Supporting Information
probed with antibodies directed against Cx43, Cx30, E-cadherin and
Figure S1). To get a quantitative measure of the spatial pattern of
GAPDH (Figure 1a). GAPDH was used as a loading control because its
Cx43 immunoreactivity, we calculated a distribution ratio by dividing
expression was previously shown to be unaffected in epilepsy (Becker
the mean Cx43 staining intensity around blood vessels by the mean
et al., 2002). E-cadherin was included to test its suitability as a loading
intensity of the remaining CA1 area (Supporting Information Figure
control in Western blots of plasma membrane protein fractions (see
S2). In non-HS specimens, this ratio was 1.07 6 0.24 (n 5 6), suggesting
below). Antibodies directed against Cx43 detected three bands at
almost uniform distribution of Cx43. In contrast, a ratio of 1.83 6 0.47
around 40 kDa (P0, P1, P2), reflecting different phosphorylation states
(n 5 6) was determined in the sclerotic CA1 region (Figure 2b). To fur-
of the protein (Figure 1a) (Solan & Lampe, 2009). Normalized total Cx43
ther characterize the staining pattern, number and size of Cx43-
protein levels in HS specimens significantly (178.12 6 40.6%) exceeded
positive plaques were determined. In HS, both parameters significantly
those of non-HS (7.89 6 1.79 vs. 4.43 6 0.94; n 5 4 per group,
exceeded those in the nonsclerotic CA1 region (plaque numbers,
p 5 .004). There was, however, no statistically significant difference
5494 6 3992 vs. 885 6 708, p 5 .02; plaque size, 0.06 6 0.018 mm2 vs.
between the phospho-bands (not shown). Cx30 and E-cadherin levels
0.04 6 0.004 mm2, p 5 .02, n 5 18 slices from 6 specimens per group;
did not differ between the two groups (Cx30, 0.55 6 0.20 in non-HS vs.
Figure 2b).
0.46 6 0.22 in HS; E-cadherin, 0.10 6 0.01 in non-HS vs. 0.12 6 0.03 in
In the CNS, Cx43 expression is confined to astrocytes and ependy-
HS). Thus, total Cx43 expression was enhanced in the sclerotic hippo-
mal cells (Rash, Yasumura, Dudek, & Nagy, 2001), although its presence
campus, while Cx30 and E-cadherin expression remained unchanged.
in pericytes and endothelial cells has also been proposed in some stud-
Loss of plasma membrane-associated connexin has been hypothe-
ies (Li, Sato, Haimovici, Okamoto, & Roy, 2003; Orellana et al., 2009).
sized to be one of the mechanisms underlying disruption of gap junction
In human MTLE specimens we could not detect co-localization of
coupling (Malone, Miao, Parker, Juarez, & Hernandez, 2007). Therefore,
Cx43 with the endothelial markers lectin (Figure 2a, Supporting Infor-
we next isolated plasma membrane-associated gap junction proteins and
mation Figure S3B) and CD31 (data not shown) or with the pericyte
assessed their expression by Western blotting (Figure 1b). Here,
markers NG2 (Supporting Information Figure S3A) and PDGFRb (data
E-cadherin was used as a loading control, with neither its normalized
not shown). Since Cx43 was also closely associated with S100b (Sup-
total protein levels (cf. above) nor its absolute plasma membrane fraction
porting Information Figure S3B) we conclude that perivascular Cx43
differing between non-HS and HS samples (non-HS, 1.41 3 107 6 1.3 3
was located in astrocytic endfeet. We used S100b instead of GFAP as
10 a.u., n 5 5; HS, 1.30 3 10 6 2.36 3 10 a.u., n5 6, p 5 .42).
an astrocytic marker because the diffuse staining of GFAP in HS makes
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Western blot analysis revealed unaltered plasma membrane Cx43 levels in human HS despite of increased total Cx43. (a) Total (whole-cell) protein lysates from human specimens were probed for E-cadherin, Cx43, GAPDH, and Cx30 expression. Antibodies against Cx43 detected 3 bands (P0, P1, and P2) representing different phosphorylation states of the protein. GAPDH was used as a loading control. The bar graph shows quantification of the Western blots. Total amount of Cx43 in HS specimens was significantly higher compared to nonHS. E-cadherin and Cx30 protein levels did not differ in the two groups, respectively. *p < .05, t-test. (b) Plasma membrane-associated proteins were isolated from human MTLE specimens. E-cadherin was used as a loading control. Cx43 and Cx30 levels were not different in specimens from patients with HS vs. non-HS
FIGURE 1
it impossible to uncover subcellular structures (Supporting Information
diffraction limited microscopy was unable to resolve the fine details of
Figure S4; see also Bedner et al., 2015).
blood vessel-gap junction interface. In order to circumvent this limita-
Together, these data indicate an upregulation and extensive
tion, ExM was employed (Chen et al., 2015; Chozinski et al., 2016).
structural reorganization of Cx43 in human MTLE-HS. In the sclerotic
Briefly, this technique involves embedding the immunostained tissue
CA1 region, the gap junction protein accumulated in large plaques at
slices in a polyacrylamide gel followed by enzymatic digestion and
presumed endfeet around blood vessels, while more uniform, punctate
expansion in deionized water. An expansion factor defined as the ratio
immunolabeling characterized nonsclerotic MTLE tissue.
of gel dimensions after and before expansion was determined for each specimen (average expansion factor 4.57 6 0.67, n 5 16). There was no
3.3 | Expansion microscopy reveals details of perivascular Cx43 expression
difference in the average expansion factors of sclerotic and nonsclerotic specimens (4.36 6 0.75, n 58 for sclerotic vs. 4.74 6 0.53, n 58 for nonsclerotic). ExM allowed assignment of the location of Cx43 with
As described above, the sclerotic CA1 region was characterized by a
respect to vessels and endfeet in much more detail than standard con-
mesh-like appearance of diffuse GFAP staining and enhanced accumu-
focal microscopy (Figure 3, Supporting Information Figure S5). Most of
lation of Cx43 around blood vessels. Confocal microscopy images
the Cx43 in the perivascular space was found to be present on the
shown in Supporting Information Figure S3B indicated that most of
parenchymal side of astrocytic endfeet (arrows, Supporting Information
the Cx43 immunoreactivity was astrocytic. However, conventional
Figure S5) as GFAP or S100b separated the Cx43 signal from the
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Increased immunoreactivity and altered subcellular distribution of Cx43 in the sclerotic human hippocampus. (a) Cx43 (red), lectin (green) and Hoechst (blue) triple immunostaining. CA1 regions of sclerotic and nonsclerotic hippocampi were analyzed with a confocal microscope (1 mm optical sections). Note the upregulation and redistribution of Cx43 around lectin-positive blood vessels. Cx43 did not colocalize with lectin indicating its absence in endothelial cells. Scale bar: left panel, 25 mm; right panel, 10 mm. (b) Quantification of Cx43 immunoreactivity. The fluorescence distribution ratio was defined as the ratio of mean fluorescence intensity of Cx43 around blood vessels over mean fluorescence intensity in the rest of the section. Distribution ratio, mean fluorescence intensity, number of Cx43 plaques, total area occupied by Cx43 signal and average plaque size were significantly increased in HS. *p < .05, t-test FIGURE 2
Expansion microscopy reveals details of perivascular Cx43 expression in the human CA1 region. Immunostained tissue slices (60 mm) of the CA1 region were embedded in polyacrylamide gel followed by enzymatic digestion and expansion in deionized water. (a) Antibody staining against GFAP (green), CD31 (cyan) and Cx43 (red) displaying CD31-positive endothelial cells surrounded by GFAPpositive endfoot-like structures. GFAP and Cx43 immunoreactivity were increased in human HS (bottom). Some Cx43-positive puncta were found between astrocytic processes (solid arrows). Although Cx43 immunoreactivity was mostly associated with GFAP, some small Cx43positive puncta were seen in the perivascular space in close association with CD31 (dotted arrows). Scale bars: left panel, 10 mm; right panel, 1 mm. (b) Antibody staining against NG2 (yellow), CD31 (cyan), and Cx43 (red) identified NG2-positive pericytes surrounding CD31positive endothelial cells. In general, Cx43 did not colocalize with NG2 or CD31 but some of the Cx43-positive plaques and puncta on the parenchymal side of the vessel were associated with pericytes (solid arrows) or endothelial cells (dotted arrows). Boxed areas are shown enlarged in the right panels. Scale bars: left panels, 10 mm; right panels, 0.5 mm FIGURE 3
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Connexin expression in the unilateral intracortical kainate MTLE model 3 months after kainate injection. The noninjected (contralateral) side served as a control. (a) Lysates from dorsal hippocampal specimens were probed for total E-cadherin, Cx43, GAPDH, and Cx30 protein by Western blotting. Antibodies against Cx43 detected 3 bands (P0, P1, and P2) representing different phosphorylation states of the protein. GAPDH was used as a loading control. Bar graphs show densitometric analysis of the Western blots. Ipsilaterally, Cx43 was upregulated while no differences between ipsi- and contralateral sides were found for E-cadherin and Cx30. There was a significant increase in the ipsilateral Cx43 P2 band. (b) Plasma membrane-associated proteins were isolated from dorsal hippocampi and E-cadherin used as a loading control. Cx43 and Cx30 levels were not different in contra- vs. ipsilateral sides although P2 isoform of Cx43 was increased ipsilaterally. *p < .05, t-test
FIGURE 4
vessel wall. Cx43 formed large plaques around blood vessels in both,
recapitulates key features of the human condition, including loss of
sclerotic and nonsclerotic CA1 regions. However, in the sclerotic CA1,
astrocyte gap junction coupling (Bedner et al., 2015). Dorsal hippo-
accumulation of Cx43 was more pronounced. Importantly, many Cx43
campi were harvested from mice 3 months post kainate injection.
puncta were found to be hemmed between GFAP positive processes
Whole-cell protein lysates were immunoblotted and the resultant
(solid arrows, Figure 3a) even in sclerotic specimens. A minor proportion
membranes probed with antibodies directed against Cx43, Cx30, E-
of Cx43 immunoreactivity of relatively smaller puncta was in close asso-
cadherin and GAPDH (Figure 4a). GAPDH-normalized Cx43 protein
ciation with endothelial cells (dotted-arrows, Figure 3a,b). Pericytes, on
levels were significantly higher (n 5 4 per group, p 5 .02) in the ipsilat-
the other hand, faced large Cx43 plaques mainly on the parenchymal
eral (3.19 6 0.5) vs. contralateral hippocampus (2.18 6 0.38). This
side (solid arrows, Figure 3b). It can be concluded that, although the
increase was primarily due to an enhanced P2 band (contralateral,
majority of perivascular Cx43 immunoreactivity was astrocytic, a minor
0.21 6 0.07; ipsilateral, 0.48 6 0.16, n 5 4, p 5 .03). In contrast, Cx30
endothelial or pericytic expression of Cx43 cannot be ruled out.
(contralateral, 0.22 6 0.04; ipsilateral, 0.31 6 0.13; n 5 4, p 5 .23) and E-cadherin (contralateral, 1.52 6 0.73; ipsilateral, 1.44 6 0.30; n 54,
3.4 | The unilateral intracortical Kainate model of MTLE recapitulates the Cx43 expression patterns seen in human MTLE-HS
p 5 .85) levels were not different. Thus, whole-cell Cx43 expression was enhanced ipsilaterally, while Cx30 and E-cadherin expression remained unchanged. Plasma membrane-associated protein was isolated from the hippocampi of kainate-injected mice and subjected to
To study potential functional implications of the altered Cx43 expres-
Western blot analysis. Similar to human HS and non-HS specimens, in
sion pattern in human MTLE, we employed a mouse model that closely
the MTLE model E-cadherin-normalized plasma membrane Cx43 levels
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Increased expression and altered subcellular distribution of Cx43 in the sclerotic hippocampus in the MTLE model. (a) Triple antibody staining against Cx43 (red), lectin (green) and Hoechst (blue) revealed upregulation and redistribution toward lectin-positive blood vessels, of Cx43 in the sclerotic CA1 region. Cx43 did not colocalize with lectin, indicating its absence in endothelial cells. Scale bars: left panel, 25 mm; right panels, 10 mm. (b) Quantification of Cx43 expression. Distribution ratio, mean fluorescence intensity, number of Cx43 plaques, total area occupied by Cx43 signal and average plaque size were significantly increased in the ipsilateral hippocampus. *p < .05, ttest FIGURE 5
did not differ between ipsi- (2.55 6 0.43) and contralateral hippocam-
experimental sclerosis, albeit at the same time this tissue is character-
pus (2.35 6 0.45, n 5 4 in each group, p 5 .54) (Figure 4b). However,
ized by complete lack of astroglial coupling (Bedner et al., 2015). To fur-
ipsilaterally a significant shift toward the P2 band was observed (con-
ther investigate this apparent discrepancy, the phosphorylation status
tralateral, 0.12 6 0.02; ipsilateral, 0.27 6 0.05, p 5 .002, n 5 4 per
of Cx43 was investigated. Phosphorylation at positions S255 and S368
group), indicating altered Cx43 phosphorylation. Next, we assessed
of the Cx43 C-terminal is known to reduce the junctional conductance
with immunostaining expression and spatial distribution of Cx43 in the
(Cottrell, Lin, Warn-Cramer, Lau, & Burt, 2003; Lampe et al., 2000). To
mouse model. Similar to human MTLE, the mean fluorescence intensity
evaluate these two sites, human and mouse hippocampal samples were
(contralateral, 112.4 6 19.5 a.u, ipsilateral, 203.9 6 47.1 a.u., p 5 .04,
subjected to SDS-PAGE followed by Western blotting and then probed
n 5 3) as well as the area occupied by Cx43 immunoreactivity (contra-
with phosphospecific antibodies recognizing phosphorylation at S255
lateral, 132.8 6 49.7; ipsilateral, 1,131 6 456, p 5 .02, n 5 3) were sig-
and S368. In human samples, these antibodies identified 3 bands
nificantly higher in the ipsilateral CA1 region. Moreover, in the sclerotic
between 40 to 55 kDa, with the middle band being the most prominent
mouse hippocampus, Cx43 immunoreactivity accumulated around
(Figure 6a). Mouse samples showed only one band between 40 and
blood vessels with the distribution ratio being significantly increased
55 kDa (Figure 6b). Phosphoband intensities were summed up and
(contralateral, 1.49 6 0.13; ipsilateral, 1.09 6 0.17, p 5 .04, n 5 3)
normalized to total Cx43 intensity. In human HS, phosphorylation was
(Figure 5). Cx43 plaque number (contralateral, 2,697 6 928; ipsilateral,
increased at S255 (0.15 6 0.04; n 5 6 vs. 0.09 6 0.04, n 5 5; p 5 .038)
9287 6 3489, p 5 .03, n 5 3) and size (contralateral, 0.045 6 0.002;
but not at S368 (Figure 6c). In experimental MTLE, phosphorylation was
ipsilateral, 0.075 6 0.02, p 5 .049, n 5 3) were also higher in the
increased at both sites ipsilaterally (S255: 0.07 6 0.02 vs. 0.03 6 0.004,
ipsilateral CA1 region (Figure 5b). Thus, with regard to Cx43 protein
n 5 4 mice, p 5 .03; S368: 0.06 6 0.02 vs. 0.01 6 0.005, n 5 4 mice,
expression,
contralateral
p < .01) (Figure 6d). Accordingly, enhanced phosphorylation at sites that
hippocampus in the mouse model closely recapitulated those between
entail reduced open probability (S255) and a smaller unitary conduct-
human HS and non-HS specimens.
ance (S368) of the Cx43 channels might have contributed to loss of
the
alterations
between
ipsi-
and
astrocyte coupling in human and experimental HS.
3.5 | Phosphorylation of Cx43 at serine 255 and/or 368 is augmented in sclerosis
3.6 | Albumin extravasation in hippocampal sclerosis
The above data indicated unchanged amounts of Cx43 protein in the
BBB dysfunction accompanied by albumin extravasation has been
plasma membrane and rather enhanced plaque numbers in human and
implicated in epileptogenesis (Ivens et al., 2007; Van Vliet et al., 2007).
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9
Western blot analysis with phosphospecific antibodies revealed enhanced phosphorylation of Cx43 in HS. Plasma membrane lysates form human (a) and mouse (b) MTLE specimens were probed for Cx43 phosphorylated at S255 and S368. P-Cx43 S255 and PCx43 S368 antibodies identified 3 bands between 40 to 55 kDa in human samples, with the middle band being the most prominent. Mouse samples showed only one band between 40 and 55 kDa. Quantitative evaluation of Western blots from human (c) and mouse (d) MTLE samples. Phospho-band intensities were averaged and normalized by total Cx43 band intensities. P-Cx43 S255 levels were significantly increased in both, human and experimental MTLE-HS while P-Cx43 S368 was only elevated in experimental MTLE-HS. *p < 0.05, t-test
FIGURE 6
We have compared albumin immunoreactivity in the CA1 region of HS
4 | DISCUSSION
vs. non-HS tissue, both in human and experimental MTLE. In both species, albumin extravasation was found in sclerotic hippocampi indicat-
The present study aimed at investigating molecular mechanism(s)
ing a compromised BBB (Figure 7a,b, bottom panels). In contrast,
underlying the recently described loss of gap junction-mediated cou-
extravascular albumin immunoreactivity was absent in non-HS (human)
pling between astrocytes in the sclerotic human and mouse hippocam-
and contralateral (experimental MTLE) hippocampal specimens (Figure
pus (Bedner et al., 2015). Our results show that total (whole-cell) Cx43,
7a,b, top panels). Maximum albumin immunoreactivity was found in
but not Cx30, protein levels are upregulated in the sclerotic hippocam-
the vicinity of blood vessels. In the mouse model, extravasation of albu-
pus, both in human and experimental MTLE. The observed increase in
min was already seen 5 days after status epilepticus (data not shown),
Cx43 immunoreactivity in the human HS is in accordance with earlier
i.e. before the onset of spontaneous seizures (Bedner et al., 2015), and
studies using postmortem tissue as control (Collignon et al., 2006; Fon-
we have previously shown that extracellular albumin administration
seca, Green, & Nicholson, 2002). Data on the expression of connexins
entails a reduction in astrocytic coupling (Braganza et al., 2012). These
in experimental epilepsy are less consistent (for review see Giaume
findings are in line with the hypothesis that leakage of albumin through
et al., 2010; Steinhäuser et al., 2012), probably due to differences
a compromised BBB occurs early during epileptogenesis and might
between used animal models, seizure duration, and investigated brain
contribute to the morphological and functional alterations astrocytes
areas. In our recently established MTLE model (Bedner et al., 2015)
undergo in the sclerotic hippocampus.
the relative expression levels of Cx30 and Cx43 in the ipsi- and
10
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ET AL.
Albumin extravasation in human and experimental MTLE-HS. Triple immunostaining for albumin (red), CD31 (green) and Hoechst (blue) in the CA1 region of human and mouse hippocampus. (a) In the nonsclerotic human hippocampus (top), albumin immunoreactivity was largely confined to blood vessels, while marked albumin staining was detected in hippocampal sclerosis indicating BBB leakage. (b) In the mouse model, 3 months after kainate-induced status epilepticus pronounced albumin extravasation was observed in the ipsi- (bottom), but not in the contralateral (top) hippocampus. Scale bar: left panels, 25 mm; enlarged (boxed) images to the right, 10 mm
FIGURE 7
contralateral hippocampus closely recapitulated those observed in the
perivascular endfeet. Recently, analysis of Na1 signaling between
sclerotic and nonsclerotic human hippocampus, respectively. Intrigu-
astrocytes came to a similar conclusion (Langer et al., 2017). Therefore,
ingly, the amount of plasma membrane-associated Cx43 was not differ-
the enrichment of Cx43 protein around blood vessels should, if any-
ent between sclerotic and nonsclerotic hippocampi, neither in
thing, increase functional coupling. However, we previously observed
experimental nor human MTLE, indicating that in HS translocation of
the opposite, i.e. loss of functional coupling in sclerosis (Bedner et al.,
Cx43 to the plasma membrane is impaired. Our findings rule out down-
2015). As shown in Figure 3, Cx43 was also hemmed between GFAP
regulation of connexins as a cause for the reported loss of functional
branches, possibly indicating gap junctions between astrocytic endfeet.
coupling in HS. Alternatively, impaired assembly (docking) of gap junc-
However, these images did not allow deciding whether the labelled
tions, e.g. because of altered morphology and loss of physical contact
branches belonged to the same or different cells. We cannot rule out
between astrocyte processes, or closure of gap junctions, e.g. due to
that in HS, endfeet of adjacent astrocytes no longer contact each other
post-translational modification, might have caused uncoupling. To
and that the enriched Cx43 immunoreactivity along vessel represents
investigate the first possibility, we assessed with immunohistochemis-
hemichannels or monomers. Electron microscopy could help answering
try the expression pattern of Cx43. ExM was employed to study details
these questions. However, the expanded images clearly unraveled that
of Cx43 distribution at a resolution higher than obtainable by
a vast majority of Cx43 was associated with astrocytic processes while
diffraction-limited confocal microscopy (Chozinski et al., 2016). We
colocalization of Cx43 puncta with endothelial or pericytic markers
found that in the sclerotic hippocampus, Cx43 expression is strongly
was only sporadically observed, if at all. Intriguingly, most of the Cx43
shifted toward astrocytic endfeet ensheathing blood vessels. The small
signals in the perivascular space were found on the parenchymal side
Cx43-positive puncta with apparently random placement and no asso-
of the astrocytic processes, as GFAP or S100b separated them from
ciation with distinct structures that we have found in nonsclerotic tis-
the vessel wall. The physiological significance of this expression pattern
sue is apparently in conflict with an earlier study demonstrating
is unclear and needs further investigation.
enriched Cx43 protein levels in perivascular astrocytic endfeet in the
We next checked whether post-translational modification of Cx43 is
hippocampus of control mice (Rouach, Koulakoff, Abudara, Willecke, &
involved in astrocyte uncoupling in HS. Total conductance of coupled
Giaume, 2008). However, no quantitative measure of enrichment was
cells is influenced by the open probability and the unitary conductance of
provided in the latter study. The authors of this study also showed that
the channel (Moreno, 2005) and both parameters depend on the phos-
gap junction-mediated intercellular diffusion of fluorescent tracers
phorylation status of Cx43 (Moreno & Lau, 2007). Phosphorylation by
preferentially occurs along the vessel walls, implying that a substantial
MAPK at S255 decreases the open probability of Cx43 channels (Theve-
part of interastrocytic coupling is mediated by junctional channels in
nin et al., 2013) while S368 is targeted by PKC, leading to an increased
DESHPANDE
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ET AL.
11
incidence of lower single of channel conductance (Ek-Vitorin, King, Hey-
young investigator grant to M.K.H.) and European Union (ERA-NET
man, Lampe, & Burt, 2006). In our study, the fraction of plasma
NEURON project “BrIE” to C.S.).
membrane-associated Cx43 phosphorylated at S255 was significantly increased in human and experimental sclerosis, which would be in line with the observed loss of coupling. In contrast, elevated phosphorylation
CONFLICT OF INTERE ST
at S368 was only observed on the ipsilateral side in the animal model, but
The authors have no conflict of interest to declare.
not in human HS. It has to be noted that human specimens are only available from the late chronic state of the disorder and that the preceding pharmacological treatment may have affected cellular properties. Hence, it is possible that the animal model reflects an earlier epilepsy stage and/ or that phosphorylation of Cx43 in human astrocytes was affected by the antiepileptic therapy. Nevertheless, since complete uncoupling of astrocytes was found not only in experimental but also in human epilepsy, phosphorylation at S368 appears not to be critical in uncoupling. It has also to be considered that there are several other residues in the Cx43 Cterminal tail whose phosphorylation might be altered in HS and add to the inhibition of interastrocytic coupling. Mass spectrometric analysis might help unraveling the status of all putative phosphorylation sites within the Cx43 C-terminal. Previous work has shown that proinflammatory cytokines induce uncoupling of astrocytes (Bedner et al., 2015; Meme et al., 2006). Since cytokines like IL-1b and TNFa are upregulated and released in epilepsy (Avignone, Ulmann, Levavasseur, Rassendren, & Audinat, 2008; Vezzani & Granata, 2005) and binding to their receptors activates kinases, including MAPKs, it is reasonable to assume that the observed changes in Cx43 phosphorylation are induced by these mediators. However, seizures are known to open the BBB and cause albumin leakage into the brain parenchyma (Van Vliet et al., 2007), which in turn decreases gap junction coupling of astrocytes (Braganza et al., 2012). Therefore, albumin extravasation was assessed in human and experimental MTLE. Perivascular albumin immunoreactivity was seen only in HS specimens, a finding that in in accordance with an earlier report (Ravizza et al., 2008). Extravasation of albumin leads to activation of transforming growth factor beta (TGFb) receptor-mediated signaling in astrocytes (Heinemann, Kaufer, & Friedman, 2012; Ivens et al., 2007). TGFb signaling involves activation of MAPKs, which may initiate transcription of several genes, including proinflammatory cytokines, or directly affect phosphorylation of Cx43 as discussed above. In conclusion, the present study demonstrates that the recently reported loss of astrocyte gap junction coupling in human and experimental TLE is not a result of reduced connexin expression. Rather, redistribution of Cx43 protein and/or altered phosphorylation of its Cterminal might contribute to uncoupling in MTLE-HS. Importantly, our
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ET AL.
Additional Supporting Information may be found online in the sup-
How to cite this article: Deshpande T, Li T, Herde MK, et al. Subcellular reorganization and altered phosphorylation of the astrocytic gap junction protein connexin43 in human and experimental temporal lobe epilepsy. Glia. 2017;00:000–000. https:// doi.org/10.1002/glia.23196
