Modulation of GABAergic dysfunction due to SCN1A mutation linked to Hippocampal Sclerosis

Abstract

We compared GABAergic function and neuronal excitability in the hippocampal tissue of seven sporadic MTLE patients with a patient carrying a SCN1A loss‐of‐function mutation. All had excellent outcome from anterior temporal lobectomy, and neuropathological study always showed characteristic hippocampal sclerosis (Hs). Compared to MTLE patients, there was a more severe impairment of GABAergic transmission, due to the lower GABAergic activity related to the NaV1.1 loss‐of‐function, in addition to the typical GABA‐current rundown, a hallmark of sporadic MTLE. Our results give evidence that a pharmacological rescuing of the GABAergic dysfunction may represent a promising strategy for the treatment of these patients.

Full text

BRIEF COMMUNICATION
Modulation of GABAergic dysfunction due to SCN1A
mutation linked to Hippocampal Sclerosis
Gabriele Ruffolo
1,a
, Katiuscia Martinello
2,a
, Angelo Labate
3,4
, Pierangelo Cifelli
5
, Sergio Fucile
1,2
,
Giancarlo Di Gennaro
2
, Andrea Quattrone
3
, Vincenzo Esposito
2
, Cristina Limatola
1,2
,
Felice Giangaspero
2
, Eleonora Aronica
6,7
, Eleonora Palma
1,b
& Antonio Gambardella
3,4,b
1
Department of Physiology and Pharmacology, Istituto Pasteur-Fondazione Cenci Bolognetti, University of Rome Sapienza, Rome, Italy
2
IRCCS Neuromed, Pozzilli, Isernia, Italy
3
Institute of Neurology, University Magna Græcia, Catanzaro, Italy
4
Institute of Molecular Bioimaging and Physiology of the National Research Council, Catanzaro, Italy
5
Department of Biotechnological and Applied Clinical Sciences (DISCAB), Amsterdam UMC, University of L’Aquila, L’Aquila, Italy
6
Department of (Neuro)Pathology, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands
7
Stichting Epilepsie Instellingen Nederland, Heemstede, the Netherlands
Correspondence
Antonio Gambardella, Institute of Neurology,
University Magna Græcia of Catanzaro, Viale
Europa, 88100 Catanzaro, Italy. Tel: +39-
961-3647270; Fax: +39-0961-3647177;
E-mail: a.gambardella@unicz.it;
Eleonora Palma, Department of Physiology
and Pharmacology, Istituto Pasteur-
Fondazione Cenci Bolognetti, University of
Rome Sapienza, Rome, Italy. Tel: +39-
0649910059; Fax: +39-0649910851; E-mail:
eleonora.palma@uniroma1.it
Received: 11 April 2020; Revised: 11 July
2020; Accepted: 16 July 2020
Annals of Clinical and Translational
Neurology 2020; 7(9): 1726–1731
doi: 10.1002/acn3.51150
a
These authors equally contributed to this
work.
b
Shared corresponding authorship
Abstract
We compared GABAergic function and neuronal excitability in the hippocam-
pal tissue of seven sporadic MTLE patients with a patient carrying a SCN1A
loss-of-function mutation. All had excellent outcome from anterior temporal
lobectomy, and neuropathological study always showed characteristic hip-
pocampal sclerosis (Hs). Compared to MTLE patients, there was a more severe
impairment of GABAergic transmission, due to the lower GABAergic activity
related to the Na
V
1.1 loss-of-function, in addition to the typical GABA-current
rundown, a hallmark of sporadic MTLE. Our results give evidence that a phar-
macological rescuing of the GABAergic dysfunction may represent a promising
strategy for the treatment of these patients.
Introduction
Mesial temporal lobe epilepsy (MTLE) with hippocampal
sclerosis (Hs) represents the most common type of focal
drug-resistant epilepsy.
1
Although MTLE is now routinely
treated with neurosurgery, over a third of MTLE patients
do not achieve seizure freedom,
2
and surgery can have
important adverse consequences. Better treatment
options, or even prevention, of MTLE and Hs are there-
fore needed, but rational therapy remains elusive because
their causes remain unclear. A large body of evidence
indicates the biologic processes that are relevant in the
pathogenesis of Hs include glial activation, immune
response, synaptic transmission, signal transduction, ions
transport, and synaptic plasticity.
3,4
In the hippocampus,
GABAergic inhibitory dysfunction mostly contributes to
hyperexcitability in MTLE with Hs.
5–7
Of interest, recent studies illustrate SCN1A involve-
ment in the epileptogenic neuronal network underlying
MTLE associated with Hs.
8
Accordingly, we already
illustrated that MTLE with febrile seizures may be part
of the epileptic phenotype encountered in a large family
carrying a SCN1A mutation.
9,10
This M145T mutation
of SCN1A cosegregated in all affected individuals of a
large family affected by simple febrile seizures (FS),
three of whom later developed MTLE.
10
Most
1726 ª2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited.

important, the M145T mutation is located in a highly
conserved amino acid in the first transmembrane seg-
ment of domain I of the SCN1A, and functional studies
in mammalian cells demonstrated that the M145T
mutation causes a 60% reduction of current density
and a 10 mV positive shift of the activation curve.
9
More recently, one family member with refractory
MTLE and Hs has benefited from surgery, and now we
wish to report the extensive neurophysiological study
from the resected hippocampal tissue of this patient, in
comparison with seven sporadic patients with refractory
MTLE and Hs. We believe that this study may provide
important new directions to the physiopathological
comprehension of MTLE with Hs and FS, and ulti-
mately may pave the road to develop new therapeutic
approaches in MTLE.
Patients and Methods
Patients
The clinical data of seven sporadic MTLE patients with
Hs, whose hippocampal tissues were used for electrophys-
iological recordings, are described in detail in Supplemen-
tal Material and summarized in Table S1. The patient
(mutated, mNa
V
1.1) carrying a loss-of-function missense
mutation (M145T) of SCN1A is a 31-year-old man with
MTLE, whose electroclinical data were reported in detail
elsewhere.
9,10
He had simple FS every 3–6 months from
the age of 8 months until 70 months. At the age of
13 years, he began to have afebrile seizures characterized
by behavioral arrest, some lip smacking and gestural
automatisms, or nocturnal secondary generalized motor
seizures. Over the years, he also began to experience epi-
gastric auras with some experiential phenomena prior to
the loss of awareness. Since seizures became refractory to
antiepileptic drugs (AEDs) that included oxcarbazepine,
phenobarbital, topiramate, and valproate, and he under-
went an extensive presurgical evaluation. Interictal awake
and sleep EEG recording showed bilateral mesiotemporal
epileptiform spikes, which strongly predominated (>80%)
on the right side. On intensive video-polygraphic moni-
toring, several typical seizures with onset in the right
midinferomesial temporal region were recorded. Brain
MRI revealed right Hs and, at the age of 27, he under-
went surgery, in the form of right anterio-temporal lobec-
tomy. Histopathological findings disclosed type 2 Hs with
the characteristic pattern of predominant neuronal loss in
area CA1.
3,4
Since surgery, in the last 50 months, he has
only had non-disabling auras (Engel’s outcome classifica-
tion Class 1B), and he has been taking a dual therapy
with carbamazepine plus valproate.
Electrophysiology
See Supplemental Material for details. The study was
approved by the local Ethics Committees.
Results
Patch-clamp experiments in human slices
To highlight the functional implications of mNa
V
1.1
expression, patch-clamp experiments were performed on
pyramidal cells and interneurons of the resected hip-
pocampus of the mNa
V
1.1 patient, comparing the results
with those obtained from sporadic MTLE patients (pa-
tients, # 4-8, Table S1). Current steps (100–150 pA,
500 ms duration) applied to the cells were able to elicit
one or more action potentials (APs), with pyramidal neu-
rons from all patients exhibiting APs with similar charac-
teristics, in particular with no change in AP threshold
(MTLE: 53 3 mV; n =10; # 4-8, Table S1, mNa
V
1.1
patient: 53 3 mV; n =5; P=0.64; Fig. 1A,B). By
contrast, three interneurons from the mNa
V
1.1 patient
showed a significantly depolarized AP threshold
(40 3 mV) in comparison to interneurons from
MTLE patients (58 12 mV, n =5; # 4-8, Table S1;
P=0.04; Fig. 1A,B, see Table S2 for electrophysiological
parameters). These data suggest that mNa
V
1.1 expressing
interneurons may have a reduced intrinsic excitability as
expected from M145T mutation of SCN1A.
9
GABA currents rundown
To investigate how GABA currents respond in mNa
V
1.1
patient’s hippocampus, we performed microtransplanta-
tion experiments injecting oocytes with hippocampal tis-
sues from sporadic MTLE patients (without known
genetic mutations and with Hs, Fig. 2A), one control
individual (Fig. 2B) and mNa
V
1.1 patient (Fig. 2C-E).
The results confirmed that a strong, pathological GABA-
current rundown is present in drug-resistant MTLE hip-
pocampi (49.0 9.1%; n =33; patients #1,2,4,5
Table S1; Fig. 2A), whereas the same phenomenon was
absent in the control (75.8 7.0 %; n =10; # 3,
Table S1; Fig. 2B) as previously shown.
5–7
Interestingly, the sclerotic hippocampus of mNa
V
1.1
patient also showed a GABAergic rundown (43.2 3.1
%; n =19; Fig. 2C) similar to sporadic MTLE patients.
This current rundown was mitigated by pretreatment of
2 hours with endogenous factors, as BDNF, or exogenous
agents, as the cannabis derivative cannabidivarine (CBDV,
Fig. 2D; Table S3) showing again a strong similarity with
data previously published for MTLE patients.
5,11
ª2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 1727
G. Ruffolo et al.SCN1A Mutation, Hippocampal Sclerosis and GABA

Interestingly, we found that acute co-application of
cannabidiol (CBD, 5 µM, GABA 50 µM), which has been
reported to be clinically effective in Dravet syndrome,
12
potentiated both GABA-current amplitude from mNa
V
1.1 patient (I
GABA
;+29.8 4.1 %; n =13; Fig. 2E,
Table S3) and from sporadic MTLE patients (I
GABA
;
+27.5 8.8 %; n =22; #1,2, Table S3).
13
Discussion
The novelty of our study is that we performed electro-
physiological experiments from an exclusive hippocampal
tissue obtained from a patient with MTLE and Hs, who
also carried the M145T SCN1A loss-of-function mutation.
Compared to hippocampal tissue from MTLE patients,
there was a more severe impairment of GABAergic trans-
mission, due to the lower GABAergic activity related to
the Na
V
1.1 loss-of-function, in addition to the character-
istic GABA-current rundown, a hallmark of MTLE.
5–7,11
We recorded valuable APs on both pyramidal neurons
and interneurons from mNa
V
1.1 and five (of seven) spo-
radic MTLE patients. In all of them, the histopathological
study showed the features of Hs related to MTLE, with
the characteristic pattern of neuronal loss in CA1 (see
Table S1).
3,4
Nonetheless, only in hippocampal mNa
V
1.1
slices, AP threshold was more depolarized in interneurons
than in pyramidal neurons, reflecting a reduced excitabil-
ity of GABAergic interneurons related to SCN1A muta-
tion.
14,15
In spite of the limited number of recordings on
mNav 1.1 interneurons, this type of AP responses paral-
leled the functional data obtained from cells transfected
with M145T mutated cDNA.
9
Because of the limited quantity of fresh tissue from the
patient and technical difficulty of recordings from acute
hippocampal slices, part of the tissue was snap-frozen and
used for recordings in microtransplanted oocytes.
6,16
Notably, we found a GABA current rundown that was
very similar to the one recorded from hippocampi of spo-
radic MTLE patients.
5–7,11
The GABA current rundown is
considered an electrophysiological dysfunction of refrac-
tory human MTLE,
5–7
which may explain, at least in part,
the hyperexcitability underlying the ictogenesis in these
patients. Interestingly, we previously showed in epileptic
rats that GABA current rundown arises from the hip-
pocampal region (during status epilepticus) then spread-
ing to temporal cortex in the chronic phase of seizures.
7,17
We already emphasized that the pharmacological mod-
ulation of the GABAergic impairment (i.e., GABA current
rundown) underlying MTLE may represent an interesting
therapeutic approach in pharmacoresistant MTLE.
17
In
this way, it is not surprising that BDNF could ameliorate
the GABA current rundown both in the mNa
V
1.1 and
sporadic MTLE patients, as previously reported for both
refractory human MTLE
5
and preclinical animal models
of MTLE.
7
Notably, CBDV continues to show its poten-
tial in recovering the mNav 1.1 GABAergic rundown as
Figure 1. mNaV 1.1 patient exhibits depolarized action potential (AP)
threshold in hippocampal interneurons but not in pyramidal cells. (A)
Left top, superimposed AP traces recorded from one pyramidal
neuron of the mNa
V
1.1 patient (red trace) and from one MTLE
patient (black trace, #5); left bottom, superimposed phase-plane plot
obtained from APs showed on top. Right top, superimposed AP traces
recorded from one interneuron of mNa
V
1.1 patient (red trace) and
from the same MTLE patient (black trace); right bottom,
superimposed phase-plane plot obtained from APs showed on top. (B)
Bar graph representing the mean action potential threshold values
recorded from pyramidal neurons (pyr; n =10) and interneurons
(inter; n =5) in resected hippocampi of patients with MTLE (black
bars, #4-8, Table S1) and from pyramidal cells (pyr; n =5) and
interneurons (inter; n =3) of mNa
V
1.1 patient (red bars). *P=0.044;
One-way ANOVA Test, and post hoc Holm–Sidak test.
1728 ª2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association
SCN1A Mutation, Hippocampal Sclerosis and GABA G. Ruffolo et al.

Figure 2. mNaV 1.1 patient exhibits a GABA current rundown similar to MTLE hippocampal tissues. (A) Oocytes injected with hippocampal
membranes of MTLE patients (n =33; I
max
=40.5 20 nA; #1,2, 4,5 Table S1). Black dots (●) represent the amplitude of consecutive GABA
currents as % of the first response (GABA 500 µM). Data points represent means SEM. In this and following panels all the currents are normalized
to the first current (I
max
) and black horizontal bars represent the timing of GABA application. In all experiments, the holding potential was 60 mV.
Inset: Representative current traces elicited by the first and sixth GABA application (500 µM, horizontal bar); (B) Oocytes injected with hippocampal
membranes from a non-epileptic control (n =10; I
max =
49.5 17.3 nA). White dots (○) represent the amplitude of consecutive GABA currents as
% of the first response (GABA 500 µM). Inset: Representative current traces as in (A). (C) Oocytes injected with hippocampal membranes from mNa
V
1.1 patient (n =19; I
max
=30.8 5.7 nA). Red dots ( ) represent the amplitude of consecutive GABA currents as % of the first response (GABA
500 µM). (Inset) Representative current traces as in (A). Note the current rundown similar to MTLE patients. (D) Oocytes injected with hippocampal
membranes from the mNa
V
1.1 patient before and after CBDV treatment. Red dots ( ) represent the amplitude of consecutive GABA currents as %
of the first response (GABA 500 µM) while the green squares ) represent the amplitude of consecutive GABA currents on the same cells after
2 hours of incubation with CBDV 50 nM. Data points represent means SEM [ )I
max
=30.7 8.0 nA; ( )I
max
=27.8 7 nA)]. Inset:
Representative GABA current traces as in (A), before (red traces) and after 50 nM CBDV application (green traces) as indicated. P=0.002, n =19 by
Shapiro–Wilk test and Student’s t-test. (E) Representative GABA current traces evoked by 50 µM GABA in one oocyte of 13 injected with membranes
of mNa
V
1.1 before (black trace) and after CBD co-application (5 µM, green trace).
ª2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 1729
G. Ruffolo et al.SCN1A Mutation, Hippocampal Sclerosis and GABA

previously shown for MTLE.
11
Furthermore, CBD was
capable to increase GABA current amplitude in mNa
V
1.1
and MTLE patients, as reported in Dravet syndrome and
tuberous sclerosis complex.
12,13,18
Altogether our results
emphasize the role of cannabis derivatives as treatment
for refractory epilepsies.
In our patient, the overall clinical, pharmacological, and
neuropathological findings were consistent with those
observed in sporadic MTLE with Hs, including the excel-
lent outcome from anterior temporal lobectomy. In this
way, our findings reinforce the belief that known genetic
defect, which is present diffusely in the brain, do not neces-
sarily preclude a good prognosis following epilepsy surgery,
if surgery is a reasonable option based on the concordance
of other data during presurgical evaluation. It cannot be
excluded, however, that the milder loss-of-function impair-
ment of SCN1A related to the M145T mutation could also
influence the better postsurgical outcome.
Important, as previously reported in Dravet syndrome
and tuberous sclerosis complex,
13,18
CBD was capable to
increase GABA current amplitude in mNa
V
1.1 and
MTLE patients, that was recently approved as treatment
for Dravet and Lennox–Gastaut syndromes.
12
Thus, in
accordance with the “interneuron hypothesis,”
14
our find-
ings support the assumption that seizures in mNa
V
1.1
patient could arise from a minor GABA release from
interneurons together with a reduction of GABAergic
postsynaptic efficacy after repetitive stimulation (i.e., run-
down). Major therapeutic implication of these findings is
that by rescuing the GABAergic inhibitory activity it is
possible to improve the clinical outcome of this kind of
patients. In this way, this study ultimately may pave the
road to develop new antiepileptic approaches in epileptic
patients with Hs.
Acknowledgments
The work was supported by grants from AICE-FIRE;
UCB Pharma, Brussels, Belgium (IIS-2014-101617, E.P.);
“Bando di Ateneo (Sapienza University),” grant no
RM1181642D8BD87B. G.R. was supported by a fellowship
from “Istituto Pasteur Italia –Fondazione Cenci Bolog-
netti.” We are grateful to all the families and patients
who were recruited in the studies.
Conflicts of Interest
None of the authors has any conflict of interest to disclose.
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Supporting Information
Additional supporting information may be found online
in the Supporting Information section at the end of the
article.
MTLE Patients. Detailed description of patients.
Table S1. Clinical characteristics and neurophysiological
findings of MTLE patients.
Electrophysiology. Patch-clamp in human slices; Mem-
brane Preparation, Injection Procedure, and voltage-
clamp Recordings in Oocytes.
Table S2. Electrophysiological parameters in patch-
clamped human neurons.
Table S3. Effects of pharmacological agents on mNa
V
1.1
and MTLE patients.
Figure S1. Firing of hippocampal interneurons.
Statistics. Detailed description of the statistical analysis.
ª2020 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association 1731
G. Ruffolo et al.SCN1A Mutation, Hippocampal Sclerosis and GABA