Open Access

Journal of Schizophrenia Research
Research Article

The Schizophrenia Variant V1282F in SCN2A Causes
Functional Impairment of NaV1.2
Kohlnhofer B, Liu Y, Woodruff G, Lovenberg T,
Bonaventure P and Harrington AW*
Neuroscience Discovery, Janssen Pharmaceutical
Companies of Johnson & Johnson, USA*Corresponding author: Anthony W Harrington,
Neuroscience Discovery, Janssen Pharmaceutical
Companies of Johnson & Johnson, 3210 Merryfield Row,
San Diego, CA 92121, USA
Received: March 17, 2021; Accepted: April 01, 2021;
Published: April 08, 2021

Abstract
Neuropsychiatric disorders such as schizophrenia are challenging to treat
due to the biological complexity of the disease and the lack of knowledge ofthe underlying pathophysiology. Whole exome and genome sequencing studies
have identified disease-linked rare variants in patients with large effect size.
Here, we functionally characterize the schizophrenia linked variant V1282F in
SCN2A, encoding the sodium channel Nav1.2. This variant was introduced into
isogenic lines of hiPSCs using CRISPR/CAS9 genome editing tools. hiPSCs
were then differentiated into cortical neurons to understand how the variant
and gene may be contributing to disease. We observed a significant (~25%)
decrease in sodium current in the V1282F neurons compared to control neurons,
suggesting the mutation is causing a loss-of-channel function. These results
were supported by recordings in recombinant cells overexpressing either the
mutant or wildtype Nav1.2, with the mutant channel having significantly (~75%)
lower current amplitude than wildtype. We hypothesize that this phenotype may
contribute to disease either through the direct loss of neuronal activity or throughsubsequent abnormal neurodevelopment.

Introduction
Elucidating novel therapeutic targets for schizophrenia has beendifficult due to the lack of knowledge of the fundamental biology
and pathophysiology of this complex disease. Additionally, there
is poor predictive validity in current pre-clinical models. Factoring
into these problems is the lack of human model systems and ourpoor understanding of the disease genetics. Genetics are a major
contributor to schizophrenia, as the heritability of schizophrenia isestimated to be 79% from twin studies [1]. With the recent advancesin genome sequencing, we now have insight into genes that may becontributing to schizophrenia and that have the potential to be novel
pharmaceutical targets. Genome Wide Association Studies (GWAS)
and whole genome or exome sequencing efforts have identified
variants in schizophrenia patients, presumed to be disease causing
but it is not clear what the functional effects of these variants are,if any. Most of the GWAS-identified common variants are within
non-coding regions, so the field resorts to investigating rare coding
variants identified through whole genome or exome sequencing.
To transition from the bench to the bedside, it is necessary to firstunderstand how individual patient risk variants affect gene or geneproduct function and how this contributes to disease.
Many of the genes that contain variants for schizophrenia overlap
with those that contain variants for Autism Spectrum Disorder(ASD) and Intellectual Disability (ID) [2]. The gene that harbors
the greatest combined number of variants identified in ASD, ID,
and schizophrenia patient populations is the voltage-gated Sodium
Channel Alpha Subunit 2 (SCN2A). SCN2A encodes for the sodium
channel Nav1.2, which is critical for the initiation and propagationof action potentials [3]. In fact, in excess of a hundred unique denovo mutations in SCN2A have been identified and with hundredsof children born each year having mutations in SCN2A [4,5]. TheseJ Schizophr Res - Volume 7 Issue 1 - 2021
ISSN : 2471-0148 | www.austinpublishinggroup.com
Harrington et al. © All rights are reserved

variants have been identified through whole genome or exome
sequencing studies and although extremely rare, they are expected
to have high penetrance. SCN2A variants have also been identifiedin patients with Infantile Epileptic Encephalopathy (IEE) and Benign
Infantile Familial Seizures (BIFS), where the contribution of variants
to disease is better understood and those variants that have beenfunctionally analyzed have been found to be gain-of-function [6-11].
Through bioinformatic modeling, multiple SCN2A variants identified
in neuropsychiatric patients were predicted to be loss-of-function andrecently a few have been functionally evaluated using overexpression
studies in HEK293 cells. Eleven unique ASD variants and three unique
ID variants were found to either functionally inactive or inhibit
channel function [12,13]. In mice, SCN2A knockout is embryoniclethal and multiple groups have found that SCN2A+/- mice display
autistic and schizophrenia-like phenotypes [14-16].
While schizophrenia-linked mutations in SCN2A are lesscommon, mutations have been identified through whole genome
or exome sequencing, including one that leads to loss of a splice site(c.2150 A>G), one that generates a nonsense mutation (E169X), andone that creates a missense mutation (V1282F) [2,17]. However,none of them have been functionally investigated [2,17-19]. To date,there are no reports on the functionality or effect of SCN2A variants
associated with schizophrenia. In this study, we investigated one
coding variant, V1282F (Chr2: 166226804, (G/T)), located in thethird transmembrane segment of the third domain. This is a missense
variant that has been identified in two unrelated patients and is absent
in the control dataset [17]. To investigate this variant in a biologically
relevant system, we used human induced pluripotent stem cell(hiPSC) derived cortical neurons and introduced the variant using
CRISPR/CAS9 genome editing tools to analyze the variant under
endogenous expression levels. We found that the V1282F variant
causes a significant decrease in Na+ channel current density. V1282F

Citation: Kohlnhofer B, Liu Y, Woodruff G, Lovenberg T, Bonaventure P and Harrington AW. The Schizophrenia
Variant V1282F in SCN2A Causes Functional Impairment of NaV1.2. J Schizophr Res. 2021; 7(1): 1038.

Harrington AW

Austin Publishing Groupmutant channels exogenously expressed in HEK293 cells further
showed lower current amplitudes than Wildtype (WT) channels,
consistent with the findings in hiPSC-derived neurons and suggestinga loss-of-channel function associated with the V1282F mutant. To
our knowledge, this is the first report of the functional impact of a
SCN2A variant identified in a schizophrenia patient.

Methods
hiPSC maintenance and genome editing
The healthy control hiPSC line GM25430 (CVB) was obtained
from the Coriell Institute (Camden, NJ). All hiPSC lines weremaintained in mTeSR on vitronectin coated plates passaged using
ReLeSR (Stem Cell Technologies, Vancouver, Canada). To generate
SCN2AWT/V1282F and SCN2AV1282F/V1282F hiPSC lines, a guide RNA(gcatactcacATCAACAATC) was designed to target exon 19 nearchromosome position: 166226804(G/T) using the guide design toolfrom crispr.mit.edu and a SSODN donor template was designed
using 50bp homology arms incorporating the (G/T) variant (G T T
TTCAAGTGTATTTTACCAATGCCTGGTG
C T G G C T A G A C T T C C T G A T T (T) T T G A T G T
GAGTATGCTGCACTTTGCTGCTTTATTCA T T G G C A T A T A T G T). Genome editing was performed
using the Alt-R-CRISPR system (IDT, Coralville, IA). Alt-R S.p. HiFi
Cas9 Nuclease V3, Alt-R CRISPR-Cas9 crRNA, Alt-R CRISPR-Cas9
tracrRNA (ATTO 550), and SSODN were nucleofected into hiPSCs
using the P3 Primary Cell 4D-Nucleofector X kit and the Amaxa
4D-Nucleofector X Unit (Lonza, Basel, Switzerland) setting CB-150.
24 hours post-nucleofection ATTO 550+ single cells were FACS
sorted and plated. Isogenic clones were screened for the variant by
amplifying the DNA using Polymerase Chain Reaction (PCR) andprimers flanking the variant (Primer F: aggagttcctgcaaatgagttaccc andPrimer R: aatgttttgaggcatcctctcactg) followed by Sanger sequencing.
Neural precursor
differentiationcelldifferentiation

andneuronhiPSCs were differentiated into Neural Precursor Cells (NPCs)
over the course of three weeks using the STEMdiff Neural Induction
Media + SMADi Kit and protocol (STEMCELL Technologies) on
0.01% Poly-L-ornithine and Laminin (ThermoFisher, Waltham,MA) coated plates. NPCs were cultured in neural maintenancemedia: 0.5X DMEM/F12 + Glutamax, Neurobasal , 0.5X N2
supplement, 0.5X B27 Supplement , 0.5X Glutamax, 0.5X IT-S, 0.25X
2-Mercaptoethanol (Gibco-ThermoFisher, Waltham, MA), PenStrep (Corning, Corning, NY) and NEAA (Hyclone Laboratories
Inc- ThermoFisher, Waltham, MA), and 20ng/ml FGF2 (R&D
Systems, Minneapolis, MN). NPCs were differentiated into neurons
by withdrawing FGF2 once confluent. After three weeks, cells were
replated for downstream assays and 24 hours later treated with 1uM
cytosine arabinoside (ARA-C) (Sigma, St. Louis, MO) for 72 hours
to eliminate proliferating cells. After ARA-C treatment the media
was switched to neuronal maturation media containing Brain Phys
(STEMCELL Technologies), 1X PenStrep, 1X N2 supplement, 1X
B27 Supplement, 20ng/uL BDNF (Peprotech, Rocky Hill, NJ), 20ng/
uL GDNF (Peprotech), 1mM cAMP (Sigma, St. Louis, MO), 1ug/
mL Laminin, and 200nM Ascorbic Acid (Sigma). Neurons werematured for an additional month with weekly half media changes. All
assays were performed at eight weeks after FGF2 withdrawal unlessSubmit your Manuscript | www.austinpublishinggroup.com

indicated.
RNA isolation, cDNA synthesis and qRT-PCR
RNA was isolated from hiPSCs or two-month neuronalcultures using a RNeasy Mini Kit and samples were DNase treatedon column (Qiagen, Hilden, Germany). cDNA was synthesizedusing SuperScript IV First-Strand Synthesis System (Invitrogen,
Carlsbad, CA) and qRT-PCR was performed using TaqMan assays(ThermoFisher, Waltham, MA) and analyzed on a QuantStudio
Real Time PCR System (Applied Biosystems, Foster City, CA).
TaqMan assays: OCT4: Hs00999634_gH, SCN2A: Hs01109871_m1,
SCN3A: Hs00366902_m1, SCN8A: Hs00274075_m1, and GAPDH:
Hs02786624_g1. Data were normalized to GAPDH expression levels.
Data were plotted as mean ±SEM. Statistical analysis was performed
using a one-way ANOVA and results were considered significant at
p-value <0.05 (GraphPad Prism).
Immunofluorescence
NPCs were fixed in 4% Paraformaldehyde (PFA) for fiveminutes and two-month neuronal cultures were fixed in with 4%
PFA for 30 minutes. Samples were permeabilized and blocked in
0.5% PB-Triton and 5% BSA for one hour. Primary antibodies: antiNav1.2, Cat#: ASC-002 (Alomone Labs, Jerusalem, Israel); MAP2,
Cat#: AB5392 (Abcam, Cambridge, United Kingdom); SYP, Cat#:AB14692 (Abcam); SOX2, Cat#: AB97959 (Abcam); NES, Cat#:ab22035 (Abcam) were incubated at 4ºC overnight and secondary
antibodies were incubated at room temperature for one hour. Images
were captured on an Opera Phenix high-content imager at 20x
magnification (PerkinElmer, Waltham, MA) and analyzed using their
Columbus Image Data Storage and Analysis System software. Synapse
density was quantified by having the software find puncta in the areasurrounding the neurites. Images were analyzed from two lines fromeach genotype, three differentiations, with at least six wells with fiveimages from each well. Data were plotted as mean ±SEM. Statisticalanalysis was performed using a one-way ANOVA and results wereconsidered significant at p-value <0.05 (GraphPad Prism).
SCN2A pulldown and simple western blot- protein simple
wes/jess platform
Nav1.2 membrane protein was isolated from two-month neurons
by preincubation of the neurons with the 100ug/ml leupeptin (Sigma)
for 30 minutes at 37ºC, biotinylation of membrane proteins in 1.5mg/
ml sulfo-NHS-SS-biotin (Sigma) for 20 minutes at 4ºC rotating, cell
lysis, and pull-down of the biotinylated protein using neutravidinagarose beads (Pierce, Waltham, MA). Both membrane Nav1.2 andtotal Nav1.2 samples were probed using Nav1.2 ASC-002 (Alomone
Labs) at 1:500 via western using the Jess High Molecular Weight 66440 kDa Separation Module (Protein Simple, San Jose, CA). Total
Nav1.2 samples were normalized to total protein using the Protein
Simple- Total Protein Assay (Protein Simple) and membrane Nav1.2
was normalized to input Nav1.2 protein. Data were plotted as mean
±SEM. Statistical analysis was performed using a one-way ANOVA
and results were considered significant at p-value <0.05 (GraphPad
Prism).
Electrophysiology
For manual patch clamp recording, neurons were cultured at adensity of 3×105 cells on 15mm glass coverslips placed in a chamber on
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Austin Publishing Groupthe stage of an inverted microscope. Upon formation of the whole-cellconformation, the cell was constantly perfused with an extracellularsolution containing (in mM): 74.5 NaCl, 74.5 choline Cl, 2.5 BaCl2, 2KCl, 1 MgCl2, 5 glucose, 10 HEPES, 0.3 CdCl2, 3 4-aminopyridine, pH
7.4, 310 mOsm/L. The pipette electrode was filled with an intracellularsolution containing (in mM): 115 Cs methanesulfonate, 20 CsCl, 3
NaCl, 4 MgATP, 0.3 Na2GTP, 10 EGTA and 20 HEPES, pH 7.2, 290
mOsm/L. Currents were measured by whole-cell patch clamp using
an Axopatch 200B amplifier and pClamp 11 software (Molecular
Devices, San Jose, CA), digitized at 20 kHz and lowpass filtered at
5 kHz. Series resistance was 75% compensated. Currents were leaksubtracted using a P/4 protocol.
For automated Syncropatch electrophysiology (Nanion,
Germany), cDNA of the α subunit of the human Nav1.2 WT and
V1282F mutant was transfected into HEK293 cells (76 µg each per
T75 flask). Transfected cells were kept at 37ºC for the first day andmoved to 30ºC for the second day before experiment. Single-holechips with medium resistance were used. The extracellular solution
contained (in mM): 149 NaCl, 2.8 CaCl2, 4 KCl, 1 MgCl2, 5 glucose,
10 HEPES, pH 7.4, 310 mOsm/L. The intracellular solution contained(in mM): 110 CsF, 10 NaCl, 10 KCl, 10 EGTA, 10 HEPES, pH7.2, 280
mOsm/L. Currents were digitized at 20 kHz and lowpass filtered at 5kHz. Series resistance was automatically compensated. Currents wereleak subtracted.
The voltage protocol was the same for both manual andautomated patch clamp. From a holding potential of -100 mV, a
100-ms preconditioning pulse ranging from -100 mV to +40 mV(in 10 mV increments) was followed by a 10 ms test pulse to 0
mV and subsequent return to -100 mV. The time interval between
preconditioning pulses was five seconds. The peak current amplitudes
during the preconditioning pulses were used to calculate the voltage
dependence of activation (I-V/G-V). The peak current amplitudes
during the test pulses were used to calculate the current densityand voltage dependence of inactivation. All electrophysiologicalrecordings were performed at room temperature.
Electrophysiology data analysis
V1/2, the voltage at which the conductance is 50% of the
maximum value, was obtained by fitting the conductance-voltage(G-V) relationship to a Boltzmann function. Kinetic parameters wereobtained by fitting the data with a single exponential function. Datafitting and statistical analyses (two-tailed Student’s t-test or two-way
ANOVA as indicated) were performed using Origin (Northampton,
MA, USA). Results were considered statistically significant at p-value<0.05. Experimental data are reported as mean ±SEM, which result
from independent measurements on n different cells.

Results
Generation and characterization of isogenic SCN2A
V1282F hiPSCs and neurons
To investigate whether the SCN2AV1282F/V1282F variant has any effectin hiPSC-derived cortical neurons, we used CRISPR/CAS9 genome
editing tools to introduce the (G/T) variant into control hiPSCs. The(G/T) variant is in exon 19 and results in the substitution of a valine
residue for a phenylalanine residue in the third segment of the third
transmembrane domain in Nav1.2 (Figure 1A). This variant has been
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Figure 1: Generation and characterization of SCN2AV1282F/V1282F hiPSC lines.
(A) A schematic of the CRISPR/CAS9 targeting strategy introducing theV1282F variant into exon 19 of SCN2A. (B) Chromatographs of the hiPSC
lines generated for each genotype obtained by Sanger sequencing.
(C) Gene expression of the pluripotency marker OCT4 in hiPSC lines,
normalized to GAPDH. Data from iPSCs was derived from two independentlines/genotype (n= 2 WT/WT, n=2 WT/V1282F, n=2 V1282F/V1282F), one
wildtype NPC sample, and one wildtype two-month old neuron sample. All
samples were run in duplicate.

identified in two schizophrenia patients, is absent from the control
EVS population and statistics suggest this is a nonrandom associationby the Fischer’s exact test, 2-tailed p-value=0.034 [17]. However, thefunctional effects of V1282F are unknown.
We generated two hiPSC lines for each genotype: wildtype
homozygous (G/G) SCN2AWT/WT, heterozygous (G/T) SCN2AWT/V1282F, and V1282F homozygous (T/T) SCN2AV1282F/V1282F (Figure 2B).
The hiPSC lines were confirmed to express the pluripotent stem celltranscript OCT4 (Figure 1C), which confirms that the genome editing
process did not affect pluripotency of the resulting isogenic clones.
All hiPSCs were differentiated into Neural Precursor Cells (NPCs)
and probed for the NPC proteins SRY-box transcription factor 2(SOX2) and Nestin to confirm NPC identity (Figure 2A). NPCs wereJ Schizophr Res 7(1): id1038 (2021) - Page - 03

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Figure 3: Na+ current density is decreased in SCN2AV1282F/V1282F neurons.
(A) Representative Na+ current traces from SCN2AWT/WT (black) and
SCN2AV1282F/V1282F (gray) neurons.
(B) The mean time constant of Na+ channel inactivation (at 0 mV) for
SCN2AV1282F/V1282F neurons (n=42) is not significantly different (p>0.05; twotailed Student’s t-test) from that for SCN2AWT/WT neurons (n=40).
(C) The voltage dependence of channel activation and inactivation for
SCN2AWT/WT (solid and outlined squares, respectively; n=34) and SCN2AV1282F/
V1282F(solid and outlined circles, respectively; n=34) neurons is not significantly
different (p>0.05; two-way ANOVA).
(D) The mean Na+ current density of SCN2AWT/WT is significantly different fromthat of SCN2AV1282F/V1282F neurons (p=0.017; two-tailed Student’s t-test).

Figure 2: SCN2A V1282F variant does not affect synapse number or neurite
length.
(A) Immunofluorescent staining for the NPC markers NESTIN and SOX2 in
NPCs.
(B) Immunofluorescent staining for SCN2A and the neuronal marker MAP2 in
two-month cortical neurons and quantification of neurite length.
(C) Immunofluorescent staining for the neuronal marker MAP2 and thepre-synaptic marker SYP in two-month cortical neurons and quantificationof synapse number per micron dendrite. Data was derived from two
independent lines/genotype and at least three differentiations/line, at least six
wells/differentiation and five images/well were analyzed.

differentiated down the cortical neuronal lineage and cultured fortwo months. We first characterized the control and variant neurons
by staining for the Microtubule Associated Protein 2 (MAP2) andthe presynaptic protein Synaptophysin (SYP) and analyzed neurite
length and synapse density. We observed no significant differencesin neurite length or synapse density between the three genotypes(Figure 2B and 2C). Together these data suggest that the variant does
not affect neuronal differentiation, neurite elongation or synapse
formation at this early stage in vitro.
Sodium current density in SCN2AV1282F/V1282F neurons isdecreased
To determine if the missense variant alters Na+ channel
properties in these neurons, we performed whole-cell manual patch
clamp studies on SCN2AWT/WT and SCN2AV1282F/V1282F neurons. No
significant differences (p>0.05) in the kinetics of channel inactivation(Figure 3A and 3B) or the voltage dependence of channel activationand inactivation (Figure 3C) were observed between SCN2AWT/WTand SCN2AV1282F/V1282F neurons. However, the mean Na+ current
density in SCN2AV1282F/V1282F neurons (881.5±73.8, n=45) was
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Figure 4: Functional characterization of human Nav1.2 WT and V1282F
mutant channels in HEK293 cells by Syncropatch electrophysiology.
(A) Box plot of the current amplitude in cells transiently transfected with WTand V1282F. The mean amplitude of the V1282F current (119.2±14.7 pA;
n=226) is significantly smaller (p<0.0001; two-tailed Student’s t-test) than thatof the WT current (456.8±68.9 pA; n=264).
(B) Voltage dependence of WT (outlined squares) and V1282F (outlined
circles) channel inactivation. The V1/2 of V1282F channels (-54.1±0.2 mV;n=19) is significantly different (p<0.001; two-way ANOVA) from that of WT
channels (-50.5±0.1 mV; n=22).
(C) The current-voltage relationship (normalized to the current amplitude at 0mV) of V1282F channels (solid circles; n=19) is not significantly different fromthat of WT channels (solid squares; n=22) (p>0.05).

significantly (p=0.017) smaller (by ~25%) than that in SCN2AWT/WT
neurons (1162.7±88.5, n=42) (Figure 3A and 3D), suggesting that
the SCN2AV1282F/V1282F variant may alter Nav1.2 channel function (e.g.
channel open probability, single channel conductance, etc.) or the
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number of functional Nav1.2 and/or other Na+ channels at the cellsurface.
V1282F mutant channels have decreased whole-cellcurrent amplitude in HEK293 cells
Since this is a native model system in which other sodium
channels are likely contributing to the overall sodium current, wecannot isolate the sole contribution of Nav1.2 from the overall current.
To better understand the functional effect of the variant on Nav1.2
channel specifically, we recorded from HEK293 cells transiently
transfected with either Nav1.2 WT or V1282F mutant channels using
the Syncropatch automated patch clamp instrument. The V1282F
channel was functional but when compared to the WT channel, themean amplitude of the whole-cell current in cells expressing V1282F
mutant channels was significantly smaller (by ~75%; p<0.0001)(Figure 4A). In addition, V1282F produced a relatively small (~3.5mV), but statistically significant (p<0.001), hyperpolarizing shift
in the voltage dependence of channel inactivation (Figure 4B). The
voltage dependence of channel activation, on the other hand, was not
significantly (p>0.05) different between WT and V1282F channels(Figure 4C).
SCN2A V1282F variant does not alter Nav1.2 membrane
expression or localization
A decrease in sodium current could be caused by a decrease in
SCN2A expression level or a decrease in the number of Nav1.2 channels
present at the plasma membrane. To investigate these theories,
we measured SCN2A gene expression, protein and membrane
localization levels in SCN2AWT/WT, SCN2AWT/V1282F, and SCN2AV1282F/
V1282
neurons. We found that SCN2A gene expression was unaltered
between the different genotypes (Figure 5A). Interestingly, weobserved a 28% increase in total Nav1.2 protein levels in SCN2AV1282F/
V1282Fneurons compared to SCN2AWT/WT neurons (Figure 5B). Notably,
the SCN2AWT/V1282F neurons had a level of protein in between the
wildtype and homozygous variant neurons. No change in plasma
membrane Nav1.2 levels was observed (Figure 5C). Together thesedata demonstrate that the V1282F variant does not inhibit SCN2A
expression or SCN2A localization but does slightly increase SCN2A
protein levels.
SCN3A and SCN8A sodium channel gene expression
In addition to Nav1.2, other members of the voltage-gated sodium
channel family, notably Nav1.3 (SCN3A) and Nav1.6 (SCN8A), are alsosignificantly expressed in these neurons. We next asked if the V1282F
variant might alter the expression of SCN3A or SCN8A, which could
also result in changes in Na+ current density via compensatory
mechanisms. To address this question, we performed qRT-PCR toquantify the mRNA transcript levels. We identified a 20% increasein SCN3A levels in SCN2AV1282F/V1282F neurons, but no change in
SCN2AWT/V1282F neurons when compared to the SCN2AWT/WT neurons.
On the other hand, SCN8A levels were not significantly different for
the three genotypes (Figure 5A).

Discussion
Nav1.2 variants associated with IEE and BIFS are thought to resultin a gain-of-function, whereas those implicated in ASD and ID arethought to result in a loss-of-function. Several Nav1.2 variants havealso been identified as linked to schizophrenia. However, none ofSubmit your Manuscript | www.austinpublishinggroup.com

Figure 5: Expression of Na+ channels in SCN2AWT/WT, SCN2AWT/V1282F and
SCN2AV1282F/V1282F neurons.
(A) Quantification of SCN2A, SCN3A, and SCN8A gene expression in
SCN2AWT/WT, SCN2AWT/V1282F SCN2AV1282F/V1282F neurons, normalized to
GAPDH.
(B) Quantification of total Nav1.2 levels normalized to total protein.
*p-value=<0.05.
(C) Quantification of Nav1.2 membrane protein normalized to total SCN2A
input. All assays were performed in two-month old cortical neurons from atleast two independent lines for each genotype and at least two differentiations
per line were analyzed.

them have been functionally investigated. In this report, we studied
the effect of the schizophrenia associated SCN2A variant, V1282F,
in a native neuronal environment. We engineered the mutation in
isogenic hiPSC-derived cortical neurons and assessed Na+ channel
activity as well as other functional and biochemical endpoints andconsequences. To our knowledge, this is the first SCN2A variant
functionally modeled in hiPSC-derived neurons.
We found that hiPSC-derived cortical neurons containing theV1282F missense mutation in SCN2A have decreased Na+ current
density. Consistent with this, V1282F mutant channel expressed
in HEK293 cells produce smaller macroscopic currents than WT
channels. As such, our results indicate that V1282F results in lossof-function phenotypes both in hiPSC-derived neurons and in aheterologous system. Two ASD missense mutations (D82G and
T1429M) assessed in HEK293 cells have also been found to havedecreased peak currents, supporting the hypothesis that SCN2A
schizophrenia variants functionally align with ASD variants over
IEE and BIFS variants [12]. The observed increase in SCN3A gene
expression in hiPSC-derived neurons may suggest a compensatory
mechanism for a decrease in SCN2A function. While we saw a
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significant increase in SCN2A protein in these neurons, we did notobserve a significant difference in SCN2A mRNA. This increase inprotein could be rationalized by the slight but not significant increasein SCN2A mRNA and/or the possibility that the SCN2A V1282F
protein is more stable. Potentially, the increase in SCN2A protein
could also be due to a feedback mechanism to compensate for thedecrease in Na+ current, which suggest the neurons are trying toincrease channel number to compensate for the functionally impaired
protein.
Additionally, our interest lies in how a 25% decrease in current
density may affect overall network activity. With the developmentof new high-density multi-electrode arrays, it is now possible toaddress this question in future studies. Deneault et al. engineered a
SCN2A knockout in iPSC-derived neurons and found that SCN2A-/neurons had decreased sEPSC frequency but no change in amplitude[20]. Moreover, they used multi-electrode arrays to analyze network
activity and found SCN2A-/- neurons had a decreased mean firing
rate and decreased burst frequency [20]. These findings suggest that
SCN2AV1282F/V1282F neurons may also have altered network activity.
While mutations in SCN2A do have various effects on Na+ channel
function, as previously shown by others and here by us, how thesechanges in Na+ channel activity lead to a pathological mechanism of
disease is not well understood. Neuronal electrical activity is requiredfor proper development of the nervous system, specifically in neurite
outgrowth, dendritic growth, axon pathfinding and synapse formation[21,22]. Moreover, expression of voltage-gated sodium channels istightly coordinated during development. As SCN2A knockout miceare perinatal lethal, the contribution of SCN2A is vital [14]. SCN2A
is expressed along the length of the axon in early development before
it is eventually replaced by SCN8A. At this point, SCN2A becomes
localized to the axon initial segment and nodes of Ranvier [23-25]. A
decrease in the effective function of SCN2A during these early phasesof neurodevelopment could have a lasting impact, for example, laterin adolescence and early adulthood when the clinical symptoms ofschizophrenia manifest.
The hiPSC-derived cortical neuron cultures used for our studies
represent early embryonic cortical development [26]. We proposethat we may be modeling the early developmental stages of disease
with this in vitro culture system, thus the significant difference in
activity of the channel in this system likely translates to the early invivo situation. Since our neuronal cultures are more fetal-like thanadult, this could be why we did not observe decreased synapse density
in our SCN2AV1282F/V1282F neurons. However, other hiPSC diseasemodeling studies have reported decreased synapse density [27,28], so
another possibility is simply that the V1282F variant does not result
in reductions in synapse density. When SCN2A mutations linked with
ASD were computationally modeled in a mature model system, where
SCN8A protein is elevated and SCN2A protein becomes localized tothe axon initial segment, only modest phenotypes were observed[12]. These data suggest that those variants played a more active
developmental role in the maturation of the brain, which is importantfor how we design therapeutics to treat disease. Future studies should
elucidate whether SCN2A could be a potential therapeutic target, andif so, what the timing and mechanism of intervention would consistof.
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Austin Publishing GroupThe contribution of SCN2A variants to neuropsychiatric diseaseis gaining traction and our data here further reveal that a SCN2A
schizophrenia risk variant may functionally contribute to disease
by impairing Nav1.2 channel function and subsequent Na+ current
density. This agrees with evidence from a clinical study that foundthat schizophrenia patients with poorer cognitive performance
had lower levels of SCN2A expression, suggesting this gene may be
implicated more widely in schizophrenia in general [18].
hiPSC disease modeling studies of rare variants associated withschizophrenia are an important step in understanding the underlying
mechanism of disease. Moving forward, the knowledge gained fromusing these approaches will allow us to understand how certain
mutations may lead to dysfunction severe enough to cause disease as
well as to discover new therapeutics tailored to the specific dysfunctionof that gene. Here we have demonstrated that a rare variant in SCN2A
identified in two unrelated schizophrenia patients causes decreased
Na+ current. Whether this information can be leveraged into atherapeutic strategy for schizophrenia patients harboring the V1282F
mutation is a question for future studies.
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