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Citation: Alfano, V.; Romagnolo, A.;
Mills, J.D.; Cifelli, P.; Gaeta, A.;
Morano, A.; Mühlebner, A.; Aronica,
E.; Palma, E.; Ruffolo, G. Unexpected
Effect of IL-1βon the Function of
GABAAReceptors in Pediatric Focal
Cortical Dysplasia. Brain Sci. 2022,12,
807. https://doi.org/10.3390/
brainsci12060807
Academic Editors: Enrico Cherubini
and Yehezkel Ben-Ari
Received: 30 May 2022
Accepted: 17 June 2022
Published: 19 June 2022
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brain
sciences
Article
Unexpected Effect of IL-1βon the Function of GABAA
Receptors in Pediatric Focal Cortical Dysplasia
Veronica Alfano 1,2, †, Alessia Romagnolo 3, † , James D. Mills 3,4,5, Pierangelo Cifelli 6, Alessandro Gaeta 1,
Alessandra Morano 7, Angelika Mühlebner 3,8, Eleonora Aronica 3, 9, *, Eleonora Palma 1,2 ,*,‡
and Gabriele Ruffolo 1,2, ‡
1Department of Physiology and Pharmacology, Istituto Pasteur-Fondazione Cenci Bolognetti,
University of Rome Sapienza, 00185 Rome, Italy; veronica.alfano@uniroma1.it (V.A.);
alessandro.gaeta@uniroma1.it (A.G.); gabriele.ruffolo@uniroma1.it (G.R.)
2IRCCS San Raffaele Roma, 00163 Rome, Italy
3Department of (Neuro) Pathology Amsterdam Neuroscience, Amsterdam UMC Location University of
Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands; a.romagnolo@amsterdamumc.nl (A.R.);
j.d.mills@amsterdamumc.nl (J.D.M.); a.muehlebnerfahrngruber@amsterdamumc.nl (A.M.)
4Department of Clinical and Experimental Epilepsy, UCL Queen Square Institute of Neurology,
London WC1E 6BT, UK
5Chalfont Centre for Epilepsy, Chalfont St Peter SL9 0RJ, UK
6Department of Applied Clinical and Biotechnological Sciences, University of L’Aquila, 67100 L’Aquila, Italy;
pierangelo.cifelli@univaq.it
7Department of Human Neuroscience, University of Rome Sapienza, 00185 Rome, Italy;
alessandra.morano@uniroma1.it
8Department of Pathology, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands
9Stichting Epilepsie Instellingen Nederland, 2013 SW Heemstede, The Netherlands
*Correspondence: e.aronica@amsterdamumc.nl (E.A.); eleonora.palma@uniroma1.it (E.P.)
† These authors contributed equally to this work.
‡ These authors contributed equally to this work.
Abstract:
Focal cortical dysplasia (FCD) type II is an epileptogenic malformation of the neocortex, as
well as a leading cause of drug-resistant focal epilepsy in children and young adults. The synaptic
dysfunctions leading to intractable seizures in this disease appear to have a tight relationship with
the immaturity of GABAergic neurotransmission. The likely outcome would include hyperpolarizing
responses upon activation of GABA
A
Rs. In addition, it is well-established that neuroinflammation
plays a relevant role in the pathogenesis of FCD type II. Here, we investigated whether IL-1
β
,
a prototypical pro-inflammatory cytokine, can influence GABAergic neurotransmission in FCD
brain tissues. To this purpose, we carried out electrophysiological recordings on Xenopus oocytes
transplanted with human tissues and performed a transcriptomics analysis. We found that IL-1
β
decreases the GABA currents amplitude in tissue samples from adult individuals, while it potentiates
GABA responses in samples from pediatric cases. Interestingly, these cases of pediatric FCD were
characterized by a more depolarized E
GABA
and an altered transcriptomics profile, that revealed an
up-regulation of chloride cotransporter NKCC1 and IL-1
β
. Altogether, these results suggest that the
neuroinflammatory processes and altered chloride homeostasis can contribute together to increase
the brain excitability underlying the occurrence of seizures in these children.
Keywords: human GABAAreceptor; GABAAcurrent; FCD; IL-1β
1. Introduction
Focal cortical dysplasias (FCDs) are a group of malformations of cortical development
(MCD), frequently associated with drug-resistant epilepsy, and are predominant among the
pediatric population [
1
–
3
]. FCDs type II are the most common MCD in epilepsy surgery
case series [
3
]. From a histopathological point of view, FCDs type II are characterized
Brain Sci. 2022,12, 807. https://doi.org/10.3390/brainsci12060807 https://www.mdpi.com/journal/brainsci
Brain Sci. 2022,12, 807 2 of 10
by alteration of the cortical lamination and cellular abnormalities. Furthermore, these
architectural abnormalities are coupled with a “dysmature” function of the neurons found
in the dysplastic areas [
4
]. Indeed, an anomalous synaptic transmission may be one of
the key pathogenic factors leading to epilepsy in this disease. Specifically, it appears that
hallmark alterations in GABAergic neurotransmission, determining its “immature” activity,
have a pivotal role in this process, since GABA-mediated inhibitory neurotransmission
becomes unable to regulate brain excitability and eventually even drives epileptiform
activity by itself [5,6].
At present, it is a common speculation that an abnormal GABA current reversal poten-
tial (E
GABA
), mainly determined by the dysregulation of the expression of the two main
cation-chloride cotransporters (NKCC1 and KCC2) [
7
,
8
], could represent one of the key
pathophysiological events leading to this “depolarizing GABAergic transmission” [
9
,
10
].
Further support to this hypothesis comes from the observation that these pathophysiologi-
cal mechanisms are a common feature of neurodevelopmental disorders, characterized by
a high incidence of epilepsy, such as FCD [
4
], TSC [
11
], Rett syndrome [
12
,
13
] or Dravet
syndrome [14].
In particular, it is widely known that pediatric FCD shows a peculiar alteration of
GABAergic neurotransmission [
4
]. Indeed, GABA responses can behave as depolariz-
ing, especially in the most severe cases [
15
], and increased GABAergic synaptic activity
can be responsible of the network hyperexcitability, instead of acting as an inhibitory
mechanism [16].
On the other hand, it is known that the generation and recurrence of seizures have
widespread consequences on whole brain homeostasis and the onset of a “vicious cy-
cle” of neuroinflammation is likely one of the most important factors contributing to the
consolidation of the epileptogenic mechanisms [
17
]. In the specific case of FCD type II,
recent evidence supports the hypothesis that factors contributing to the maintenance of
the pro-inflammatory drive in this condition include the up-regulation of inflammatory
mediators in dysmorphic cells [
18
], imbalance of cytokines’ regulatory networks [
19
] and
the pro-inflammatory potential of the seizures themselves [20].
The prototypical pro-inflammatory interleukin-1
β
(IL-1
β
) has a prominent role in
these inflammatory processes in both epileptic patients and animal models of epilepsy [
21
].
Accordingly, we have previously shown that IL-1
β
can decrease GABA currents amplitude
in human drug-resistant temporal lobe epilepsy (TLE) by activation of IL-1
β
signaling [
22
].
Here, we investigated the effect of IL-1
β
on GABAergic neurotransmission in pediatric
brain tissues from patients affected by FCD IIb, which is the most severe form of FCD
considering the high level of neuroinflammation [
18
]. Indeed, even though several reports
supporting the role of this cytokine in neurodevelopmental epilepsies have already been
published [
23
,
24
], there is still scarce evidence regarding its potential role in the “plastic”
neurotransmission that characterizes neurodevelopmental disorders.
2. Materials and Methods
2.1. Patients
The cases included in this study were obtained from the archives of the Departments
of Neuropathology of the Amsterdam UMC (Amsterdam, The Netherlands) and the Uni-
versity Medical Center Utrecht (UMCU, Utrecht, The Netherlands). Cortical brain samples
were obtained from patients undergoing surgery for drug-resistant epilepsy and diagnosed
with FCD type IIb (with brain somatic mutations in the MTOR gene) [
2
]. After resection,
the tissue was immediately snap-frozen in liquid nitrogen and then part of the sample
was used to perform the electrophysiology experiments. All the autopsies were performed
within 16 to <48 h after death, with the acquisition of appropriate written consent for brain
autopsy and subsequent use for research purposes. Control autopsy cases had no known
history of epilepsy, a normal cortical structure for the corresponding age and no signifi-
cant brain pathology. The transcriptional profiles of post-mortem and surgical resected
tissues have previously been compared, showing minimal differences if the tissue is of
Brain Sci. 2022,12, 807 3 of 10
high quality (i.e., handled and stored as in our study) [
25
]. Tissue was obtained and used
in accordance with the Declaration of Helsinki and the Amsterdam UMC Research Code
provided by the Medical Ethics Committee. Please refer to Supplementary Table S1 for
clinical details of the patients. The electrophysiology experiments were performed with
three pediatric samples up to 5 years old (Table 1). However, for the bioinformatics analysis,
we had a larger cohort of patients and only patients below 12 years old were considered
pediatric (
18 patients
). Adult patients diagnosed with FCD IIb, including those 18 years
old, underwent transcriptomic analysis (12 patients), whilst three of these were used for the
electrophysiology experiments (Table 1). A set of electrophysiology experiments were also
performed on two FCD IIa tissues (3 years old, female; 11 years old, male). Throughout
the manuscript, patients are referred to with the symbol “#”, and their clinical features are
summarized in Table 1.
Table 1. Clinical characteristics of the patients.
Patient Age at the Time of the Surgery (y) Epilepsy Onset (y) Gender Diagnosis ASMs
#1 2 0 M FCD IIb OCZ, C, VPA,
#2 3 0 F FCD IIb LEV, OCZ, C, VPA, CL
#3 5 3 M FCD IIb C, CL
#4 18 2 M FCD IIb C, VPA, LMT, LCM
#5 44 10 F FCD IIb LEV, OCZ
#6 45 12 M FCD IIb LEV, OCZ
Three pediatric patients; three adult patients; Abbreviations: M = male, F = female, ASM = anti-seizure med-
ication, C = Carbamazepine, CL = Clobazam, LCM = Lacosamide, LEV = Levetiracetam, LMT= Lamotrigine,
OCZ = Oxcarbazepine, VPA = Valproic acid.
2.2. Membrane Preparation
All the tissues used for electrophysiology were received in dry ice and processed
immediately or stored at
−
80
◦
. Preparation of human membranes and their injection in
Xenopus laevis oocytes were performed as previously described [
26
]. Briefly, tissues were
homogenized in a membrane buffer solution (200 mM glycine, 150 mM NaCl, 50 mM EGTA,
50 mM EDTA, and 300 mM sucrose; plus 20
µ
L of protease inhibitors “P2714; Sigma”; pH 9,
adjusted with NaOH). Afterwards, the material was centrifuged for 15 min at
9500×g
.
Then, the supernatant was centrifuged for 2 h at 100,000
×
gwith an ultra-centrifuge
(Beckman-Coulter). Finally, the pellet was washed with sterile water, re-suspended in
assay buffer (glycine 5 mM) and immediately used or stored at
−
80
◦
until use. The use of
Xenopus laevis frogs and the surgical procedures for oocytes extraction and use conformed
to the Italian Ministry of Health guidelines and were approved by the same institution
(authorization no 427/2020-PR).
2.3. Xenopus Oocytes Electrophysiology
All the experiments with microtransplanted oocytes were carried out 24–48 h after
cytoplasmic injection [
26
]. GABA-evoked currents (I
GABA
) were recorded using the tech-
nique of “two-electrode voltage clamp” [
27
] after the oocytes were placed in a recording
chamber (0.1 mL volume) and continuously perfused with oocyte Ringer solution (OR:
NaCl 82.5 mM; KCl 2.5 mM; CaCl
2
2.5 mM; MgCl
2
1 mM; Hepes 5 mM, adjusted to pH 7.4
with NaOH) at room temperature (20–22
◦
C). GABA application was controlled through
a computer-operated gravity driven multi-valve perfusion system (9–10 mL/min) (Bi-
ologique RSC-200; Claix, France). With this setup, 0.5 to 1 s are enough to completely
replace the entire volume of applied solution in the recording chamber.
In all the experiments, the stability of GABA-evoked currents (I
GABA
) was evaluated
on two consecutive GABA applications, separated by a 4 min washout. Only the cells
that showed a <5% variation in current amplitude were used to test the effect of IL-1
β
.
Variation in current amplitude was calculated comparing the mean current elicited by
the two GABA applications before and after exposure to cytokines and/or inhibitors.
GABA current reversal potential (E
GABA
) was calculated by constructing current-voltage
Brain Sci. 2022,12, 807 4 of 10
(I-V) relationships that were then elaborated by a linear regression curve-fitting software
(Sigmaplot 12, Systat software Inc., Chicago, IL, USA). In a specific set of experiments, we
used IL1-Ra to block IL-1
β
‘s effect. In these experiments, we pre-incubated the cells for
30 min with the blocker alone, and then proceeded to the incubation with the cytokine
plus the inhibitor for two hours. For the incubation, IL-1
β
and IL-1Ra were diluted at the
desired concentration (specified for each experiment) in Barth’s modified saline solution
(88 mM NaCl; 1 mM KCl; 2.4 mM NaHCO
3
; 10 mM HEPES; 0.82 mM MgSO
4
; 0.33 mM
Ca(NO
3
)
2
; 20.41 mM CaCl
2
). IL-1
β
was purchased from Peprotech (London, UK) and
human IL-1Ra was purchased from Invitrogen (Waltham, MA, USA).
2.4. RNA-Seq Library Preparation and Sequencing
All library preparation and sequencing were performed at GenomeScan (Leiden,
The Netherlands
). The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina
(New England Biolabs, Ipswich, MA, USA) was used for sample processing. Sample
preparation was performed according to the protocol “NEBNext Ultra II Directional RNA
Library prep Kit for Illumina” (NEB #E7760S/L). The mRNA isolation was performed
from the total RNA using oligo-dT magnetic beads and cDNA synthesis followed. Next,
sequencing adapters were ligated to the cDNA fragments followed by PCR amplification.
Clustering and DNA-sequencing were performed using the NovaSeq6000 (Illumina, Foster
City, CA, USA) in accordance with manufacturers’ guidelines. All samples underwent
paired-end sequencing of 150 nucleotides in length and the mean read depth per sample
was 47 million reads.
2.5. Bioinformatics Analysis of RNA-Seq Data
The Bestus Bioinformaticus Decontamination Using Kmers (BBDuk) tool from the
BBTools suite was used for adapter removal, quality trimming and removal of contaminant
sequences (ribosomal/bacterial) [
28
]. A phred33 score of 20 was used to assess the quality
of the read, any read shorter than 31 nucleotides in length was excluded from the down-
stream analysis. Reads were aligned directly to the human GRCh38 reference transcriptome
(Gencode version 33) [
29
] using Salmon v0.11.3 [
30
]. Transcript counts were summarized
to the gene level and scaled using library size and average transcript length using the R
package tximport [
31
]. Genes detected in less than 20% of the samples in any diagnosis
and with counts less than 6 across all samples were filtered out. The gene counts were
than normalized using the weighted trimmed mean of M-values (TMM) method using
the R package edgeR [
32
]. The normalized counts were than log2 transformed using the
voom function from the R package limma [
33
]. The subsequent differential expression
was carried out using the R package limma. A linear model was fit for each gene and
moderated t-statistic was calculated after applying an empirical Bayes smoothing to the
standard errors. Differential expression was defined by a Benjamini–Hochberg adjusted
p-value < 0.05. The analysis compared FCD IIb pediatric tissue samples (18 patients) and
matched control cortices (6 patients).
3. Results
3.1. IL-1βAffects the GABA Current Amplitude in Oocytes Injected with FCD IIb Membranes
At first, we recorded GABA-evoked currents from oocytes microinjected with mem-
branes from adult samples of the FCD IIb brain cortex (Table 1). We recorded responses
ranging from 11.8 nA to 250.0 nA (n= 24; #4–6; 250
µ
M GABA). Subsequently, in order
to test the effect of IL-1
β
on FCD, we measured GABA current amplitude (I
GABA
) before
and after a 2 h incubation with this cytokine (25 ng/mL). We observed that this treatment
induces a decrease in I
GABA
(I
GABA
= 98.4
±
16.6 nA and 87.8
±
16.8 nA, before and af-
ter IL-1
β
incubation, n= 14; p< 0.001, Wilcoxon signed rank test; # 3–5, Table 1). This
effect was comparable to that already reported in TLE tissues [
22
] and behaved similarly,
since IL-1
β
induced GABA current decrease in 70% of the cells (14/20), while the rest
did not respond to the cytokine [
22
]. As previously shown for TLE, this I
GABA
decrease
Brain Sci. 2022,12, 807 5 of 10
was blocked by 30 min pre-treatment with 10
µ
M IL-1Ra (I
GABA
= 86.6
±
10.6 nA and
88.7 ±12.07 nA
, before and after incubation with IL-1
β
+ IL-1Ra, n= 6; p> 0.05, paired
t-test; # 5, Table 1). Interestingly, when we repeated the same experiments on pediatric FCD
IIb samples (range: 5.6 nA to 193.0 nA, n= 44; #1–3; 250
µ
M GABA), we did not obtain the
same results, observing an opposite effect of IL-1
β
, which on these tissues and in the same
experimental conditions reported above (2 h incubation, 25 ng/mL) induced a potentiation
of I
GABA
(
IGABA = 45.4 ±10.9 nA
and 52.7
±
12.9 nA, before and after IL-1
β
incubation,
n= 31
;
p< 0.001
, Wilcoxon signed rank test; # 1–3, Table 1, Figure 1). Also in this case, the
effect was blocked by 30 min pre-treatment with 10
µ
M IL-1Ra (
IGABA = 28.8 ±6.2 nA
and
27.6 ±5.0 nA
, before and after incubation with IL-1
β
+ IL-1Ra, n= 8; p> 0.05, Wilcoxon
signed rank test; # 1–3, Table 1). Subsequently, we repeated the IL-1
β
incubation ex-
periments microinjecting the oocytes with membranes from two FCD IIa tissue samples
(3-year-old female and 11-year-old male). The results showed GABA current potentiation
also in these cases (I
GABA
= 40.9
±
8.4 nA and 47.0
±
10.1 nA, before and after IL-1
β
incubation, n= 16; p< 0.01, Wilcoxon signed rank test).
Brain Sci. 2022, 12, x FOR PEER REVIEW 5 of 11
comparable to that already reported in TLE tissues [23] and behaved similarly, since IL-
1β induced GABA current decrease in 70% of the cells (14/20), while the rest did not re-
spond to the cytokine.[23]. As previously shown for TLE, this IGABA decrease was blocked
by 30 min pre-treatment with 10 μM IL-1Ra (IGABA= 86.6 ± 10.6 nA and 88.7 ± 12.07 nA,
before and after incubation with IL-1β + IL-1Ra, n = 6; p > 0.05, paired t-test; # 5, Table 1).
Interestingly, when we repeated the same experiments on pediatric FCD IIb samples
(range: 5.6 nA to 193.0 nA, n = 44; #1–3; 250 μM GABA), we did not obtain the same results,
observing an opposite effect of IL-1β, which on these tissues and in the same experimental
conditions reported above (2 h incubation, 25 ng/mL) induced a potentiation of IGABA (IGABA
= 45.4 ± 10.9 nA and 52.7 ± 12.9 nA, before and after IL-1β incubation, n = 31; p < 0.001,
Wilcoxon signed rank test; # 1–3, Table 1, Figure 1). Also in this case, the effect was blocked
by 30 min pre-treatment with 10 μM IL-1Ra (IGABA = 28.8 ± 6.2 nA and 27.6 ± 5.0 nA, before
and after incubation with IL-1β + IL-1Ra, n= 8; p > 0.05, Wilcoxon signed rank test; # 1–3,
Table 1). Subsequently, we repeated the IL-1β incubation experiments microinjecting the
oocytes with membranes from two FCD IIa tissue samples (3-year-old female and 11-year-
old male). The results showed GABA current potentiation also in these cases (IGABA = 40.9
± 8.4 nA and 47.0 ± 10.1 nA, before and after IL-1β incubation, n = 16; p < 0.01, Wilcoxon
signed rank test).
Figure 1. IL−1β potentiates GABA-evoked currents in pediatric FCD IIb tissue samples. The bar
graphs show the average GABA current percent (%) changes ± s.e.m. before and after incubation
with IL-1β (25 ng/mL; 2 h) in oocytes transplanted with adult FCD IIb tissues (blue bar; n = 14, # 4–
6, Table 1) and pediatric FCD IIb tissues (red bar; n = 31, # 1–3, Table 1). Insets represent sample
currents before (left trace) and after (right trace) incubation with IL-1β in adult (lower inset) and
pediatric (upper inset) FCD IIb. White horizontal bars represent GABA application (250 μM). There
is a significantly different effect of IL-1β between the two experimental groups. * p < 0.05 with Stu-
dent t-test.
3.2. GABA Reversal Potential in Oocytes Injected with FCD IIb Membranes
In order to better characterize the IGABA in our samples, we performed additional ex-
periments to measure EGABA in pediatric FCD IIb samples. Interestingly, we found that
EGABA was more depolarized in pediatric samples (−17.2 ± 1.1 mV, n = 13; #1–3, Table 1,
Figure 2) compared to adult ones (−22.0 ± 0.73 mV, n = 10; p < 0.05, Wilcoxon signed rank
test; #4–6, Table 1, Figure 2), suggesting that in pediatric patients, GABA currents have
unique characteristics. Indeed, these EGABA values are not comparable with those found in
neurons [8] but are consistent with those previously found using the microtransplantation
technique for the electrophysiological study of human brain tissue. [35] Furthermore, we
Figure 1.
IL
−
1
β
potentiates GABA-evoked currents in pediatric FCD IIb tissue samples. The bar
graphs show the average GABA current percent (%) changes
±
s.e.m. before and after incubation
with IL-1
β
(25 ng/mL; 2 h) in oocytes transplanted with adult FCD IIb tissues (blue bar; n= 14,
# 4–6, Table 1) and pediatric FCD IIb tissues (red bar; n= 31, # 1–3, Table 1). Insets represent sample
currents before (left trace) and after (right trace) incubation with IL-1
β
in adult (lower inset) and
pediatric (upper inset) FCD IIb. White horizontal bars represent GABA application (250
µ
M). There is
a significantly different effect of IL-1
β
between the two experimental groups. * p< 0.05 with Student
t-test.
3.2. GABA Reversal Potential in Oocytes Injected with FCD IIb Membranes
In order to better characterize the I
GABA
in our samples, we performed additional
experiments to measure E
GABA
in pediatric FCD IIb samples. Interestingly, we found that
E
GABA
was more depolarized in pediatric samples (
−
17.2
±
1.1 mV, n= 13; #1–3, Table 1,
Figure 2) compared to adult ones (
−
22.0
±
0.73 mV, n= 10; p< 0.05, Wilcoxon signed rank
test; #4–6, Table 1, Figure 2), suggesting that in pediatric patients, GABA currents have
unique characteristics. Indeed, these E
GABA
values are not comparable with those found in
neurons [
8
] but are consistent with those previously found using the microtransplantation
technique for the electrophysiological study of human brain tissue [
34
]. Furthermore,
we repeated the same measurements in two FCD IIa pediatric samples (see methods for
patients’ details) and we still found a depolarized E
GABA
, which was not statistically
different compared to that recorded in pediatric FCD IIb (−16.8 ±0.3 mV, n= 8, p> 0.05).
Brain Sci. 2022,12, 807 6 of 10
Brain Sci. 2022, 12, x FOR PEER REVIEW 6 of 11
repeated the same measurements in two FCD IIa pediatric samples (see methods for pa-
tients’ details) and we still found a depolarized EGABA, which was not statistically different
compared to that recorded in pediatric FCD IIb (−16.8 ± 0.3 mV, n = 8, p > 0.05).
Figure 2. GABA reversal potential in FCD IIb tissue samples. The two panels represent the mean
EGABA value recorded on adult (A); black circles; n = 10, # 4–6, Table 1) and pediatric (B); red squares;
n = 13, # 1–3, Table 1) FCD samples. The dots represent mean ± s.e.m. of GABA currents at corre-
spondent holding potential value (VH) normalized to the maximum currents (Imax = 73.4 for adult
and 42.4 nA for pediatric). Insets represent sample GABA−evoked currents (250 μM) at different VH
as shown in the respective panels. Note that the mean EGABA was significantly more depolarized in
pediatric FCD tissues compared to adult FCD (p < 0.05, Wilcoxon signed rank test).
3.3. Transcriptomic Analysis
In order to explain the aforementioned results, we performed transcriptomics analy-
sis on a cohort of pediatric FCD IIb tissue samples. We found that the pro-inflammatory
cytokine IL-1β and its receptor antagonist, IL-1Ra (IL-1 receptor antagonist) were signifi-
cantly upregulated in FCD IIb pediatric tissue samples (IL-1β log2FC = 4.535; IL-1Ra log2FC
of 2.042) compared to age-matched control cortices (Figure 3), suggesting IL-1Ra tries to
counteract the overexpression of IL-1β. However, their common receptor, IL-1R1 did not
show a significant upregulation in these samples (log2FC = 0.753) (Figure 3). Interestingly,
sodium-potassium-chloride cotransporter, NKCC1, was significantly up-regulated
(log2FC = 0.696) (Figure 3). Altogether, these results indicate the presence of an acute neu-
roinflammatory process in these children and altered chloride homeostasis.
Figure 2.
GABA reversal potential in FCD IIb tissue samples. The two panels represent the mean
E
GABA
value recorded on adult (
A
); black circles; n= 10, # 4–6, Table 1) and pediatric (
B
); red
squares; n= 13, # 1–3, Table 1) FCD samples. The dots represent mean
±
s.e.m. of GABA currents
at correspondent holding potential value (V
H
) normalized to the maximum currents (I
max
= 73.4
for adult and 42.4 nA for pediatric). Insets represent sample GABA
−
evoked currents (250
µ
M) at
different V
H
as shown in the respective panels. Note that the mean E
GABA
was significantly more
depolarized in pediatric FCD tissues compared to adult FCD (p< 0.05, Wilcoxon signed rank test).
3.3. Transcriptomic Analysis
In order to explain the aforementioned results, we performed transcriptomics analysis
on a cohort of pediatric FCD IIb tissue samples. We found that the pro-inflammatory cy-
tokine IL-1
β
and its receptor antagonist, IL-1Ra (IL-1 receptor antagonist) were significantly
upregulated in FCD IIb pediatric tissue samples (IL-1
β
log
2
FC = 4.535; IL-1Ra log
2
FC of
2.042) compared to age-matched control cortices (Figure 3), suggesting IL-1Ra tries to coun-
teract the overexpression of IL-1
β
. However, their common receptor, IL-1R1 did not show a
significant upregulation in these samples (log
2
FC = 0.753) (Figure 3). Interestingly, sodium-
potassium-chloride cotransporter, NKCC1, was significantly up-regulated (log
2
FC = 0.696)
(Figure 3). Altogether, these results indicate the presence of an acute neuroinflammatory
process in these children and altered chloride homeostasis.
Brain Sci. 2022, 12, x FOR PEER REVIEW 7 of 11
Figure 3. Expression levels of the genes of interest in FCD IIb pediatric tissue samples compared to
age-matched controls. IL−1β, IL−1Ra and NKCC1 are significantly upregulated in FCD IIb pediatric
tissue samples compared to controls. Expression levels are described in logarithmic scale. * p < 0.05;
**** p < 0.0001. A linear model was fit for each gene and moderated t-statistic was calculated after
applying an empirical Bayes smoothing to the standard errors. Those genes with a Benjamini–
Hochberg adjusted p-value < 0.05 were considered significant. Differential expression analysis com-
pared 18 FCD IIb patients and six age-matched control cortices.
4. Discussion
This study focused on the analysis of the effect of IL-1β on GABAergic neurotrans-
mission in FCD IIb samples. This cytokine can decrease GABA currents amplitude in adult
FCD IIb, while it increases these responses in pediatric FCD samples. Interestingly, we
found that these latter tissues were indeed characterized by a more depolarized EGABA
compared to adult FCD IIb, thus suggesting an alteration in chloride homeostasis. [8] Fi-
nally, the transcriptomic analysis revealed an up-regulation of the expression of IL-1β, IL-
1Ra and NKCC1 in a cohort of pediatric FCD IIb samples that fits well with the aforemen-
tioned electrophysiological results.
The technical approach we used to perform our functional experiments, membrane
microtransplantation in Xenopus oocytes, has the potential of allowing electrophysiologi-
cal recordings from rare human diseases using little amounts of brain tissue, thus making
it easier to test cytokines or other mediators on these pathologies. On the other hand, with
this technique, we record “whole” glial and neuronal currents without discriminating
among cellular subtypes. Nonetheless, it has been demonstrated that neurotransmitter re-
ceptors transplanted in Xenopus oocytes from transfected cells retain their functional char-
acteristics [36].
To our knowledge, this is the first time that IL-1β was reported to affect GABAergic
function in human FCD IIb tissues, even though its ability to reduce the amplitude of
GABA-evoked currents upon activation of its specific receptor was already described in
human TLE [23].
Figure 3.
Expression levels of the genes of interest in FCD IIb pediatric tissue samples compared to
age-matched controls. IL
−
1
β
,IL
−
1Ra and NKCC1 are significantly upregulated in FCD IIb pediatric
tissue samples compared to controls. Expression levels are described in logarithmic scale. * p< 0.05;
**** p< 0.0001. A linear model was fit for each gene and moderated t-statistic was calculated
after applying an empirical Bayes smoothing to the standard errors. Those genes with a Benjamini–
Hochberg adjusted p-value < 0.05 were considered significant. Differential expression analysis
compared 18 FCD IIb patients and six age-matched control cortices.
Brain Sci. 2022,12, 807 7 of 10
4. Discussion
This study focused on the analysis of the effect of IL-1
β
on GABAergic neurotransmis-
sion in FCD IIb samples. This cytokine can decrease GABA currents amplitude in adult FCD
IIb, while it increases these responses in pediatric FCD samples. Interestingly, we found
that these latter tissues were indeed characterized by a more depolarized E
GABA
compared
to adult FCD IIb, thus suggesting an alteration in chloride homeostasis [
8
]. Finally, the
transcriptomic analysis revealed an up-regulation of the expression of IL-1
β
,IL-1Ra and
NKCC1 in a cohort of pediatric FCD IIb samples that fits well with the aforementioned
electrophysiological results.
The technical approach we used to perform our functional experiments, membrane
microtransplantation in Xenopus oocytes, has the potential of allowing electrophysiological
recordings from rare human diseases using little amounts of brain tissue, thus making
it easier to test cytokines or other mediators on these pathologies. On the other hand,
with this technique, we record “whole” glial and neuronal currents without discriminating
among cellular subtypes. Nonetheless, it has been demonstrated that neurotransmitter
receptors transplanted in Xenopus oocytes from transfected cells retain their functional
characteristics [35].
To our knowledge, this is the first time that IL-1
β
was reported to affect GABAergic
function in human FCD IIb tissues, even though its ability to reduce the amplitude of
GABA-evoked currents upon activation of its specific receptor was already described in
human TLE [22].
Additionally, we also described the effect of IL-1
β
on two FCD IIa pediatric tissue
samples, where we obtained the same results as the recordings in FCD IIb tissues. Indeed,
FCD IIa and IIb represent two histopathological subtypes of FCD type II, and FCD IIb is
further distinguished from FCD IIa by the additional presence of balloon cells [2,36]. This
broadens the relevance of our findings, since we can hypothesize that the alteration in
synaptic transmission by inflammatory mediators may be a general feature of FCD, at least
of those forms frequently associated with drug-resistant epilepsy.
Here, we can infer that the mechanism underlying IL-1
β
mediated modulation of
GABAergic transmission depends upon the activation of transplanted
IL-1β
receptors
(
IL-1R1
) [
22
], since the blockade with IL-1Ra, the endogenous antagonist [
37
] which specif-
ically prevents the binding of IL-
β
to this receptor, could prevent both GABA current
decrease and increase in adult and pediatric specimens, respectively.
Alongside this mechanism, it is also possible that GABA
A
Rs modulation takes place
as a consequence of the altered expression or dysregulated function of the two main cation-
chloride cotransporters expressed in the brain: NKCC1, which mediates chloride influx, and
KCC2, responsible for chloride extrusion [
8
]. Here, we described an about 5 mV E
GABA
shift
in pediatric FCD, which correlates with the reported NKCC1 upregulation. Furthermore,
this is also supported by previous findings describing a depolarizing shift of about 7–8 mV
in membranes extracted from epileptic subiculum, where both an upregulation of NKCC1
and a downregulation of KCC2 were reported [34].
Interestingly, recent evidence supports the hypothesis that inflammatory stimuli, in
particular IL-1
β
, can alter GABAergic neurotransmission also by modulating the expression
and function of the aforementioned chloride transporters [
38
]. This is particularly relevant,
since chloride homeostasis contributes to the pathogenesis of several neurodevelopmental
disorders such as TSC, autism and epileptic syndromes [
38
]. Any kind of pathogenic factor
acting on this delicate equilibrium, such as neuroinflammation, could serve as a therapeutic
target for conditions that are often characterized by seizures resistant to known ASMs
or medically untreatable cognitive impairment. In pediatric patients, the inflammatory
processes can originate from prenatal stress, infection or traumatic injuries [
38
,
39
] that
contribute to the occurrence of seizures in the first months of life. Therefore, in these
patients, it could be relevant to therapeutically intervene as soon as possible to quench the
neuroinflammatory processes.
Brain Sci. 2022,12, 807 8 of 10
Interestingly, our data indicate that IL-1Ra is also up-regulated, suggesting an attempt
to compensate the IL-1
β
increase in our cohort of FCD IIb samples. However, the need for
epilepsy surgery in these cases suggests that the endogenous anti-inflammatory cytokines
failed to dampen inflammation, which can lead to brain hyperexcitability [40].
The role of GABAergic transmission in this scenario is indeed pivotal. An interesting
hypothesis revolves around the ability of aberrant GABAergic transmission to promote epilep-
togenesis in malformed FCD cerebral cortex, especially in pediatric
cases [4,6,41]
. In fact, the
shift in GABA reversal potential has been associated with a state of brain “dysmaturity” that is
a hallmark of several neurodevelopmental diseases such as
TSC [11,42], Dravet [14]
and Rett
syndromes [12,13].
Here, we provided novel evidence in favor of this idea, since pediatric FCD displayed
aberrant, depolarized GABA responses, which were further potentiated by incubation
with IL-1β.
Indeed, inflammatory mediators may be relevant for the pathogenesis of epilepsy
in FCD, as confirmed by the reported up-regulation of inflammatory cytokines and their
receptors and/or downstream effectors (such as IL-1
β
, IL-6, CCL3, CCL4, STAT3, C-JUN
and CCR5) in this disease [
19
,
43
]. Hence, concerning the evident implications on the
sustainment of a pro-inflammatory milieu and the detrimental consequences it possesses on
the progression of the disease, IL-1
β
would also potentiate a kind of GABAergic activity
that does not counteract seizures, but rather contributes to their generation and recurrence.
5. Conclusions
To understand the pathophysiological mechanisms underlying the dysfunction of
synaptic transmission in FCD IIb is a fundamental step towards the development of
new therapeutic strategies in this disease. Here, we highlighted a potential link between
two relevant phenomena, such as neuroinflammation and GABAergic neurotransmission,
and shed light on how the differential effect of IL-1
β
in pediatric versus adult tissues
may depend on a disturbed chloride homeostasis in pediatric FCD IIb samples. Since
endogenous anti-inflammatory response, as demonstrated by our transcriptomic analysis,
may not be enough to compensate for the excessive pro-inflammatory stimuli, we can
infer that an early pharmacological potentiation of anti-inflammatory mechanisms in FCD
IIb may favorably affect the progression of the disease and development of epilepsy in
these patients.
Supplementary Materials:
The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/brainsci12060807/s1, Table S1: Clinical information of the pediatric
study cohort used for transcriptomic analysis.
Author Contributions:
Conceptualization, E.P., E.A. and G.R.; methodology, E.P., E.A. and G.R.;
investigation, V.A., A.R., J.D.M., A.G., P.C. and G.R.; data curation, V.A., A.R., J.D.M., P.C., G.R., A.M.
(Alessandra Morano) and A.M. (Angelika Mühlebner); writing—original draft preparation, A.R., G.R.
and E.P.; writing—review and editing, E.P. and E.A.; supervision, E.P. and E.A.; funding acquisition,
E.P. and E.A. All authors have read and agreed to the published version of the manuscript.
Funding:
The work was supported by grants from Ateneo Project (Sapienza University)”, grant n
◦
RM11916B84D24429 (EP). This research was supported by intramural “DISCAB” GRANT 2022 code
07_DG_2022_05 to P.C. This project has received funding from the European Union’s Horizon 2020
Research and Innovation Program under grant agreement No. 952455; EpiEpiNet (EP, EA).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
GR was supported by Italian Ministry of Health “Ricerca corrente”. The authors
wish to thank Anwesha Ghosh for English proofreading.
Conflicts of Interest: The authors declare no conflict of interest.
Brain Sci. 2022,12, 807 9 of 10
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