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Amutation in sigma-1 receptor causes juvenile amyotrophic lateral sclerosis.

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A Mutation in Sigma-1 Receptor Causes
Juvenile Amyotrophic Lateral Sclerosis
Amr Al-Saif, MD,1 Futwan Al-Mohanna, PhD,2 and Saeed Bohlega, MD3
Objective: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by loss of motor
neurons in the brain and spinal cord, leading to muscle weakness and eventually death from respiratory failure. ALS
is familial in about 10% of cases, with SOD1 mutations accounting for 20% of familial cases. Here we describe a
consanguineous family segregating juvenile ALS in an autosomal recessive pattern and describe the genetic variant
responsible for the disorder.
Methods: We performed homozygosity mapping and direct sequencing to detect the genetic variant and tested the
effect of this variant on a motor neuron-like cell line model (NSC34) expressing the wild-type or mutant gene.
Results: We identified a shared homozygosity region in affected individuals that spans 120kbp on chromosome
9p13.3 containing 9 RefSeq genes. Sequencing the SIGMAR1 gene revealed a mutation affecting a highly conserved
amino acid located in the transmembrane domain of the encoded protein, sigma-1 receptor. The mutated protein
showed an aberrant subcellular distribution in NSC34 cells. Furthermore, cells expressing the mutant protein were
less resistant to apoptosis induced by endoplasmic reticulum stress.
Interpretation: Sigma-1 receptors are known to have neuroprotective properties, and recently Sigmar1 knockout
mice have been described to have motor deficiency. Our findings emphasize the role of sigma-1 receptors in motor
neuron function and disease.
ANN NEUROL 2011;70:913–919
myotrophic lateral sclerosis (ALS, Online Mendelian
Inheritance in Man #105400) is a progressive neurodegenerative disorder that affects both upper and lower
motor neurons and leads to death from respiratory failure. It has an annual incidence of 1–3:100,000. Whereas
90% of cases do not have a family history of the disease
(sporadic ALS [SALS]), 10% have >1 affected family
member (familial ALS [FALS]). The mean age of onset
for SALS and FALS is 56 and 46 years, respectively,
whereas the juvenile form of ALS, with a milder presentation, has an age of onset <25 years. Several loci have
been linked to FALS in both the dominant and recessive
forms.1 Mutations of the gene coding Cu/Zn superoxide
dismutase SOD1 (ALS1) account for 20% of familial
and 5% of sporadic cases.2 Mutations in 2 genes, ALS2
and SETX, have been reported in juvenile ALS families.3,4 In addition, about 5% of ALS patients have frontotemporal dementia (FTD). Two genetic loci in families
segregating both phenotypes have been identified, ALSFTD1 at chromosome 9q21-225 and ALS-FTD2 at
chromosome 9p13.3-21.3.6,7 Recently, Luty et al
described variants in the 30 untranslated region (UTR) of
SIGMAR1 in 3 ALS-FTD/FTD families with autosomal
dominant mode of inheritance. They suggested that these
variants affected the stability of SIGMAR1 transcripts.8
Sigma-1 receptor (Sig-1R) is an endoplasmic reticulum (ER) chaperone that binds a wide range of ligands,
including neurosteroids, psychostimulants, and dextrobenzomorphans. It was first described by Martin et al in
1976 as sigma-opioid receptor but was later found to be
a distinct nonopioid receptor.9,10 Sig-1R was first cloned
in 1996 and found to have 223 amino acids that are
similar to yeast sterol C8-C7 isomerase.11 Recently,
dimethyltryptamine has been described as an endogenous
Sig-1R ligand.12 Physiologically, Sig-1R is involved in
ion channel modulation through interaction with Kþ
channels and inositol 1,3,5-triphosphate receptors
(IP3Rs). They have also been shown to be involved in
lipid transport and neuronal cell differentiation.13–16 Sig1R is ubiquitously expressed, and in the nervous system,
it is enriched in motor neurons of the brainstem and spinal cord, with subcellular enrichment in postsynaptic
View this article online at DOI: 10.1002/ana.22534
Received May 18, 2011, and in revised form Jun 8, 2011. Accepted for publication Jun 24, 2011.
Address correspondence to Dr Al-Saif, P.O. Box 3354, MBC #3, Riyadh, 11211, Saudi Arabia. E-mail:
From the 1Department of Genetics, 2Department of Biological and Medical Research, 3Department of Neurosciences, King Faisal Specialist Hospital and
Research Center, Riyadh, Saudi Arabia.
C 2011 American Neurological Association
of Neurology
sites.17 It has been implicated in several disorders, such
as Alzheimer disease, schizophrenia, stroke, amnesia, and
depression.18 Agonists of Sig-1R have been shown to
have neuroprotective effects in several in vivo and in
vitro models of ischemia.19,20 In the present study, we
describe a mutation in SIGMAR1 that is associated with
juvenile amyotrophic sclerosis and show the effect of this
mutation on the normal function of Sig-1R.
Subjects and Methods
Affected individuals from the ALS002 family were diagnosed
with juvenile ALS at King Faisal Specialist Hospital and
Research Center, Riyadh, Saudi Arabia. Four of the 6 affected
patients participated in the genetic study. The study was
approved by the Research Advisory Counsel at King Faisal Specialist Hospital and Research Center. Members of the family
were recruited for the study, 5 to 10ml of peripheral blood was
collected, and informed consent was obtained. DNA was
extracted from lymphocytes using Puregene DNA extraction kit
(Qiagen, Valencia, CA).
Homozygosity Mapping and Mutation
Genome-wide single nucleotide polymorphism (SNP) genotyping was performed on genomic DNA from participating individuals using GeneChip human mapping 250K Sty arrays
(Affymetrix, Santa Clara, CA) according to the manufacturer’s
protocol. Raw data were analyzed using Genotyping Console
4.0 software (Affymetrix). For mutation screening, primers were
designed to amplify the 4 exons of SIGMAR1 (GenBank reference sequence: NM_005866.2). Polymerase chain reaction
(PCR) was performed using 30ng of genomic DNA, 1 HotStarTaq PCR buffer (Qiagen), 200lM deoxynucleotide triphosphates, 200pM of each primer, and 1U of HotStarTaq DNA
Polymerase (Qiagen) in a total volume of 25ll reaction. PCR
amplicons were then cleaned and bidirectionally sequenced
using the BigDye Terminator sequencing kit (Applied Biosystems, Foster City, CA). Samples were run on an ABI3730 automated sequencer (Applied Biosystems), and generated sequence
data were analyzed by Sequencher software (Gene Codes Corporation, Ann Arbor, MI).
Sig-1R Cloning
SIGMAR1 ORF was PCR amplified using cDNA obtained
from a human B cell lymphoma cell line (SU-DHL-8). The
primers used contained restriction sites. In addition, the 30
primer contained the sequence for FLAG polypeptide tag NDYKDDDDK-C. After enzymatic digestion of the PCR product, it was ligated into pcDNA3 mammalian expression vector
(Invitrogen, Carlsbad, CA). Following transformation in One
Shot TOP10 Chemically Competent Cells (Invitrogen), PCR
was used to screen for positive colonies. Plasmids from positive
colonies were then extracted and sequenced to confirm the
incorporation of SIGMAR1 ORF.
Site-Directed Mutagenesis
The E102Q mutation was introduced to SIGMAR1-pcDNA3
vector according to the mutagenesis protocol described by
Zheng et al.21 SIGMAR1 ORF from produced colonies was
sequenced to confirm incorporation of the mutation and to
exclude the introduction of new mutations during the mutagenesis process.
NSC34 Cell Line Maintenance and Transient
A mouse motor neuron like cell line (NSC34) was obtained
from Cellutions Biosystems (Toronto, ON, Canada). Cells were
grown at 37 C with 5% CO2 in Dulbecco modified Eagle medium (D5796; Sigma, St Louis, MO) supplemented with 10%
fetal bovine serum (FBS), 100U/ml penicillin, 100lg/ml streptomycin, and 4mM L-glutamine. For transient transfection, plasmids extracted by PureLink HiPure Plasmid Maxiprep Kit (Invitrogen) were used for NSC34 transfection using Lipofectamine
2000 (Invitrogen) according to the manufacturer’s protocol.
Stable Knockdown of Sig-1R in NSC34 Cells
pSUPER.neo RNA interference vector was used for stable transfection of NSC34 cells. Briefly, NSC34 cells grown on 10cm
culture plates were transfected using 60ll Lipofectamine 2000
(Invitrogen) and 24lg of the pSUPER.neo vector containing
short hairpin against mouse Sigmar1 30 UTR sequence
GAGAGGACCTGGAGAAGTA to establish the NSC34-r1Rsh cell line. Twenty-four hours after transfection, medium was
exchanged for medium containing 250lg/ml G418. Eleven
days after growing them under selective pressure, cells were serially diluted in 96-well culture plates and continued to grow
under selection. Single colonies were then isolated and
Sucrose Gradient Fractionation
NSC34-r1R-sh cells transiently transfected with vectors expressing the wild-type or E102Q mutated SIGMAR1 were lysed the
next day using TME buffer (10mM Tris, 5mM MgCl2, and
0.5mM ethylenediaminetetraacetic acid) and homogenized by a
glass homogenizer using 25 strokes. Homogenates were centrifuged at 900g for 10 minutes at 4 C, then supernatants (4ml)
were layered on top of a sucrose gradient composed of 10 layers
(15–60%), 2ml each, and centrifuged at 27,000rpm in a Beckman L8-80M ultracentrifuge for 18 hours at 4 C. Samples of
2ml were then collected from the top of the gradient for a total
of 12 fractions. Proteins in the sucrose fractions were precipitated using the trichloroacetic acid protein precipitation method
and resuspended in an equal volume of Laemmli buffer containing 5% b-mercaptoethanol (BME), 75 mM dithiothreitol
(DTT), and 1% Triton-X.
Immunoblot Analysis
Protein samples were incubated at 55 C for 10 minutes, then
run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes.
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Al-Saif et al: Sig-1R and Juvenile ALS
FIGURE 1: Family pedigree and homozygosity mapping. (A) Pedigree of the juvenile amyotrophic lateral sclerosis family
(ALS002) shows disease status in multiple generations and genotypes for the c.304G>C mutation in participating individuals
(red asterisks indicate second cousin relationship). (B) Homozygosity tracks for affected individuals from Affymetrix genotyping
console software show shared homozygosity for 120kbp between 4 affected individuals (upper panel), and RefSeq genes
track from the UCSC genome browser shows genes and their isoforms in the mapped locus (lower panel). Note that other
small regions of homozygosity are shared by affected individuals (red arrowheads); however, they were also detected in 2
unaffected siblings and may reflect common haplotypes in the population.
Membranes were blocked in Tris-buffered saline (TBS) buffer
containing 5% nonfat milk and 0.1% Tween-20 for 1 hour,
then incubated with primary antibodies overnight (monoclonal
anti-FLAG antibodies, Sigma, F1804). Membranes were washed
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3, 10 minutes each, in TBS/0.1% Tween-20, then incubated
with antimouse antibody conjugated to horseradish peroxidase
for 1 hour. After 3 washes, signals were detected using ECL
Plus chemiluminescence reagent (Amersham, Piscataway, NJ).
of Neurology
FIGURE 2: The mutation affects a conserved amino acid in the transmembrane (TM) domain of sigma-1 receptor (Sig-1R). (A)
Sequence chromatograms for a normal (IV:6), a carrier (III:3), and an affected (IV:7) individual showing the G>C mutation at
position 304 (arrowhead). (B) Amino acid alignment for 22 species from the University of California at Santa Cruz genome
browser showing the conservation of the mutated amino acid (glutamic acid at position 102) across vertebrates (arrowhead).
(C) The mutated amino acid is located in a TM domain of Sig-1R. ER 5 endoplasmic reticulum. Source: Human Protein Reference Database (
Terminal Deoxynucleotide Transferase–Mediated Deoxyuridine Triphosphate Nick-End
Labeling (TUNEL) Assay
NSC34-r1R-sh cells grown in 6-well plates were transiently transfected with vectors expressing the wild-type or mutant forms of
SIGMAR1. At the same time, cells were cotransfected with a vector expressing red fluorescent protein (RFP) as a control for transfection. The next day, medium was replaced with serum-free medium containing 3lM thapsigargin and incubated for 24 hours.
Cells were then washed and fixed with paraformaldehyde (PFA).
Terminal deoxynucleotide transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay was performed using
MEBSTAIN Apoptosis Kit Direct (MBL International, Woburn,
MA) according to the manufacturers’ protocol. Cells were then
dried on microscope slides and visualized under a fluorescence
microscope. The percentage of TUNEL-positive cells of the total
number of cells (DAPI stained) was calculated from 3 separate
experiments. More than 150 cells were counted from each experiment. One-tail Student t test was used to determine the p value.
Description of Phenotype
In an extended consanguineous family from the eastern
region of Saudi Arabia (ALS002), we diagnosed 6 individuals with juvenile ALS according to El Escorial criteria
for the diagnosis of ALS.22 Lower limb spasticity and
weakness were noted at the age of 1 to 2 years. This was
accompanied by exaggerated tendon reflexes. Weakness of
hand and forearm muscles was noted at the age of 9 to
10 years and progressed slowly and proximally, leading to
paralysis of forearm extensors and triceps. By the age of
20 years, 2 patients used wheelchairs (individuals III:7
and IV:8, Fig 1A). Patients did not show respiratory or
bulbar muscle weakness. Sphincteric, sensory, and cerebellar functions were normal. Neurophysiological tests
showed evidence of denervation and reinnervation in
limb muscles; motor unit potentials were enlarged, polyphasic, and fast firing, with normal sensory nerve action
potentials and somatosensory evoked potentials. Motor
conduction velocities were also normal. Brain magnetic
resonance imaging did not reveal any abnormalities in
the subcortical regions or in the internal capsules. Cognitive function was preserved; full scale intelligence quotient testing (Wechsler Adult Intelligence Scale III) was
performed and showed normal results.
Homozygosity Mapping and Identification of a
Mutation in the SIGMAR1 Gene
Given the consanguinity of the family and the autosomal
recessive mode of inheritance (see Fig 1A), we assumed
that a founder mutation is segregating. Therefore, we
carried out a homozygosity mapping using GeneChip
human mapping 250K Sty arrays (Affymetrix) for 4
patients who participated in this study and their parents
and siblings. We identified a solitary shared homozygosity region in the affected individuals that was not shared
by their unaffected siblings (see Fig 1B). This relatively
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Al-Saif et al: Sig-1R and Juvenile ALS
FIGURE 3: Translocation of E102Q mutant sigma-1 receptor (Sig-1R) to lower density membranes. Sucrose gradient fractions
show the difference in membrane distribution between wild-type (WT) and E102Q mutant Sig-1R–transfected NSC34-r1R-sh
cells. Fractions 1 to 12 are from top to bottom of the gradient (15–60% sucrose). E102Q mutant Sig-1Rs are enriched in lowdensity fractions (F3–F5), where they form detergent-resistant complexes around 50kDa. In comparison, the majority of wildtype Sig-1Rs are located in high-density fractions (F8–F10). The band of about 30kDa is nonspecific, as it was detected in nontransfected cell lysate (data not shown). Monoclonal anti-FLAG antibody (Sigma, F1804) was used to detect wild-type and mutant Sig-1R.
small region spans approximately 120kbp on chromosome 9p13.3 and overlaps the ALS-FTD2 locus. It is
flanked by SNPs rs10972203 and rs6476455, and contains 9 RefSeq genes. We sequenced the coding region of
all genes in the mapped locus. Analysis of sequences
revealed a missense mutation in exon 2 of the SIGMAR1
gene encoding Sig-1R (c.304G>C) that leads to the substitution of glutamine for glutamic acid at amino acid
position 102 (E102Q) (Fig 2A). This mutation segregated perfectly with the phenotype (see Fig 1A). We
screened 271 population-matched controls for the presence of this variant, and none was found to carry it. No
variants were detected in the coding region of the
remaining 8 genes. In silico analysis showed that the glutamic acid at position 102 of SIGMAR1 is highly conserved across vertebrates (see Fig 2B). In addition, SIFT
algorithm predicted this amino acid substitution not to
be tolerated.23
Aberrant Membrane Distribution of Sig-1RE102Q
The E102Q mutation is located in a predicted transmembrane domain of Sig-1R (see Fig 2C). Therefore, we
looked for the subcellular distribution of the mutated
protein in the motor neuron-like cell line NSC34. We
first established an NSC34 cell line stably expressing an
RNA short hairpin targeting SIGMAR1 mRNA (NSC34r1R-sh cell line) to specifically knock down endogenous
Sig-1R. We then transfected these cells with a mammalian expression vector (pcDNA3, Invitrogen) expressing
wild-type or E102Q mutant Sig-1R fused to FLAG NDYKDDDDK-C polypeptide tag (Sig-1RWT-FLAG and
Sig-1RE102Q-FLAG, respectively).21 We then carried
out a sucrose gradient fractionation of transfected cell
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homogenates. We found that Sig-1RE102Q was enriched
in lower density fractions (F3–F5) (Fig 3) compared to
Sig-1RWT, which was mainly localized to higher density
fractions (F8–F10), indicating a shift of the mutant form
to lower density membranes. In addition, the majority of
the mutant protein formed detergent-resistant complexes
around 50kDa that may represent Sig-1R dimers (the
molecular weight of Sig-1R is 25kDa). They were also
present in Sig-1RWT low-density fractions but at lower
levels. These complexes did not dissociate even after loading in a buffer containing 150mM DTT and 2% TritonX, which may indicate a strong hydrophobic interaction.
NSC34 Cells Expressing Sig-1RE102Q Show
Enhanced Apoptosis
Sig-1R has been shown to suppress apoptosis induced by
ER stress24; therefore, we examined the effect of the
E102Q mutation on the antiapoptotic function of Sig1R in ER-stressed cells. We transiently transfected
NSC34-r1R-sh cells with Sig-1RWT-FLAG or Sig1RE102Q-FLAG and compared their responses to the
Caþ2 adenosine triphosphatase inhibitor and ER stressor
thapsigargin. Cells were incubated in serum-free medium
with 3lM thapsigargin for 24 hours. Next, TUNEL
assay was performed. Cells transfected with Sig-1RE102QFLAG showed a statistically significant higher percentage
of TUNEL-positive cells compared to Sig-1RWT-FLAG–
transfected cells, p value ¼ 0.01 (1-tail Student t test)
(Fig 4). This difference of about 5% represents a 1.7fold increase of apoptotic cells, which is close to what
Hayashi and Su reported on the effect of Sig-1R knockdown on cell viability (about 2-fold increase of apoptotic
cells).24 This result supports a deleterious effect of the
of Neurology
FIGURE 4: NSC34 cells expressing the E102Q mutant
sigma-1 receptor (Sig-1R) show reduced viability. TUNEL
assay shows higher percentage of apoptotic cells in Sig1RE102Q-FLAG–transfected NSC34-r1R-sh cells compared to
Sig-1RWT-FLAG–transfected cells after treatment with 3lm
thapsigargin in serum-free medium for 24 hours. Experiments were performed in triplicates, with >150 cells
counted from each slide. Error bars 5 6standard error of
the mean; p 5 0.01. WT 5 wild type.
E102Q mutation on the viability of motor neurons in
juvenile ALS patients.
Using homozygosity mapping in a juvenile ALS family,
we identified SIGMAR1 as a causative gene for juvenile
ALS. The missense mutation discovered is located in a
predicted transmembrane domain; it is also part of a
stretch of amino acids that has been shown to be important for ligand binding.25 Hayashi and Su found that
Sig-1Rs are localized to cholesterol-enriched loci in the
ER (ER lipid rafts), and in a sucrose gradient fractionation, they showed that agonist-stimulated Sig-1Rs translocate to higher density membranes.26 Our sucrose fractionation experiment shows that the majority of Sig1RE102Q is located in low-density fractions compared to
Sig-1RWT. The significance of this distribution on the
function of the mutant Sig-1R and its response to agonists requires further experiments. Although it is not
clear at this time how this mutation affects the function
of Sig-1R, the autosomal recessive mode of inheritance
suggests a loss of function mechanism. Furthermore, the
enhanced apoptosis observed in Sig-1RE102Q expressing
NSC34-r1R-sh cells, which have minimal levels of endogenous wild-type Sig-1R, can be explained by loss of
antiapoptotic function of Sig-1R.
Genes mutated in ALS have a wide variety of functions, such as detoxification (SOD1), DNA and RNA
processing (SETX, FUS, and TARDBP), vesicular traffick918
ing (ALS2 and VAPB), and axonal transport (DCTN1).1
Although the exact function of Sig-1R is not clear at present, studies have shown that it is involved in the regulation
of Kþ channels and IP3R-mediated Caþ2 signaling.13,27
Sig-1Rs also have chaperone activity at the ER, and in
vitro experiments showed that Sig-1Rs suppress aggregation
of misfolded proteins.24 Intriguingly, motor neurons in
ALS patients show evidence of activation of the unfolded
protein response and elevation of GRP78 chaperone (a
Sig-1R–binding protein).28,29 In addition, Caþ2 concentration in the ER is important for the proper folding of nascent proteins. This indicates that loss of Sig-1R function
may result in easily stressed motor neurons that accumulate
unfolded proteins and eventually leads to their degeneration. This hypothesis is supported by the reported upregulation of Sig-1R in ER stress.24 Furthermore, a recent
study showed that Sigmar1 knockout mice have a shorter
latency period in rotorod experiments, suggesting motor
deficiency in these mice.17 Our findings together with the
recent report of variants in the 30 UTR of SIGMAR1 in
ALS-FTD/FTD families suggests that the genetic variant
in the chromosome 9p13.3-21.3 locus—reported in multiple studies to be associated with ALS-FTD6,7—might be
related to SIGMAR1.
Here we presented a mutation in the SIGMAR1
gene associated with juvenile ALS. This gene is involved
in ER stress response, Caþ2 metabolism, and chaperone
activity, all of which are implicated in neurodegeneration.30 We showed that the discovered mutation alters
membrane distribution of Sig-1R and reduces cell viability. The role of Sig-1R in the pathogenesis of ALS needs
further investigation. Furthermore, the neuroprotective
properties of Sig-1R agonists19,20 together with the
described effect of agonists/antagonists on TDP-43 protein localization8 make Sig-1R a potential target for ALS
therapeutic trials.
We thank patients and their families for participating in
this study. We also thank all colleagues who helped us by
providing reagents and for their valuable advice in experimental procedures.
Potential Conflicts of Interest
Nothing to report.
Dion PA, Daoud H, Rouleau GA. Genetics of motor neuron disorders: new insights into pathogenic mechanisms. Nat Rev Genet
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