Elucidation of Relevant Neuroinflammation Mechanisms Using Gene Expression Profiling in Patients with Amyotrophic Lateral Sclerosis
Elucidation of Relevant Neuroinflammation Mechanisms Using Gene Expression Profiling in Patients with Amyotrophic Lateral Sclerosis
Yu Hui Won 1 2 3
Min-Young Lee 0 1 3
Young-Chul Choi 1 3
Yoon Ha 1 3
Hyongbum Kim 1 3
Do- Young Kim 1 3
Myung-Sun Kim 0 1 3
Ji Hea Yu 0 1 3
Jung Hwa Seo 0 1 3
MinGi Kim 0 1 3
Sung- Rae Cho 0 1 3
Seong-Woong Kang 1 3
0 Department and Research Institute of Rehabilitation Medicine, Yonsei University College of Medicine , Seoul , Korea , 3 Department of Neurology, Gangnam Severance Hospital, Yonsei University College of Medicine , Seoul , Korea , 4 Department of Neurosurgery, Spine & Spinal Cord Institute, College of Medicine, Yonsei University , Seoul , Korea , 5 Department of Pharmacology, Yonsei University College of Medicine , Seoul , Korea , 6 Department of Dermatology, Cutaneous Biology Research Institute, Yonsei University College of Medicine , Seoul , Korea , 7 Brain Korea 21 PLUS Project for Medical Science, Yonsei University , Seoul , Korea , 8 Department of Rehabilitation Medicine, Gangnam Severance Hospital, Rehabilitation Institute of Neuromuscular Disease, Yonsei University College of Medicine , Seoul , Korea , 9 Department of Medicine, the Graduate School of Yonsei University , Seoul , Korea
1 Funding: This study was supported by grants from the National Research Foundation (SRC, NRF- 2014R1A2A1A11052042; 2015M3A9B4067068), the Ministry of Science and Technology, Republic of Korea; the Korean Health Technology R&D Project (YHW, HI15C1529), Ministry of Health & Welfare, Republic of Korea; the aDongwhao Faculty Research Assistance Program of Yonsei University College of Medicine , SRC, 6-2016-0126
2 Department of Physical Medicine and Rehabilitation, Research Institute of Clinical Medicine of Chonbuk National University-Biomedical Research Institute of Chonbuk National University Hospital , Jeonju , Korea
3 Editor: Kwang-Hyun Baek, CHA University , REPUBLIC OF KOREA
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by damage of motor neurons. Recent reports indicate that inflammatory responses occurring within the central nervous system contribute to the pathogenesis of ALS. We aimed to investigate disease-specific gene expression associated with neuroinflammation by conducting transcriptome analysis on fibroblasts from three patients with sporadic ALS and three normal controls. Several pathways were found to be upregulated in patients with ALS, among which the toll-like receptor (TLR) and NOD-like receptor (NLR) signaling pathways are related to the immune response. GenesÐtoll-interacting protein (TOLLIP), mitogen-activated protein kinase 9 (MAPK9), interleukin-1β (IL-1β), interleukin-8 (IL-8), and chemokine (C-X-C motif) ligand 1 (CXCL1)Ðrelated to these two pathways were validated using western blotting. This study validated the genes that are associated with TLR and NLR signaling pathways from different types of patient-derived cells. Not only fibroblasts but also induced pluripotent stem cells (iPSCs) and neural rosettes from the same origins showed similar expression patterns. Furthermore, expression of TOLLIP, a regulator of TLR signaling pathway, decreased with cellular aging as judged by changes in its expres-
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
sion through multiple passages. TOLLIP expression was downregulated in ALS cells under
conditions of inflammation induced by lipopolysaccharide. Our data suggest that the TLR
and NLR signaling pathways are involved in pathological innate immunity and
Competing Interests: The authors have declared
that no competing interests exist.
neuroinflammation associated with ALS and that TOLLIP, MAPK9, IL-1β, IL-8, and CXCL1
play a role in ALS-specific immune responses. Moreover, changes of TOLLIP expression
might be associated with progression of ALS.
Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by damage of
motor neurons, microglial activation, and wide astrogliosis in the motor cortex and spinal cord
]. Several genes associated with the disease have been identified; however, the pathogenic
mechanisms are still elusive, and so far no curative therapeutic treatment has been developed.
Suggested pathogenic mechanisms of ALS contain genetic factors, glutamate excitotoxicity,
oxidative stress, weakened axonal transport, changed protein turnover, apoptosis,
mitochondrial dysfunction, neurotrophic deficiency, and neuroinflammation . Evidence accumulated
over the past decade indicates that immune activation and inflammation might be implicated
in ALS pathogenesis. Astrocytes, microglia, and immune-related cells have all been shown to
be actively involved in ALS pathogenesis [
]. The immune responses associated with
neurodegenerative changes have been termed neuroinflammation. In pathologically affected
areas of the central nervous system (CNS) in both human patients with ALS and mouse models
of the disease, noticeable neuroinflammation can be observed .
With these lines of evidence indicating that neuroinflammation is related to the
pathogenesis of ALS, we hypothesized that mRNA expression of components of immune-related
signaling pathways would be different in patients with sporadic ALS and that such differences might
reveal the pathogenic factors related to neuroinflammation. To identify differentially expressed
genes involved in the pathogenesis of ALS, we carried out gene expression profiling of
fibroblast cells from patients with ALS and normal controls using RNA sequencing transcriptome
analysis. In addition, we studied the certain inflammation-related genes in three different types
of cells—fibroblasts, iPSCs, and neural lineage cells, and confirmed the genes that were
considered to be representing certain inflammatory pathways. The aim of this study was to explore
disease-specific gene expressions associated with neuroinflammation in ALS using gene
expression profiling in order to better comprehend the pathogenesis of ALS.
Materials and Methods
The study was approved by the Ethics Committee and the participants signed informed
consent prior to the study. The human fibroblast samples were obtained with approval of
participants with their written informed consent to participate in this study. The Institutional Review
Board of Severance Hospital, Yonsei University Health System approved this consent
procedure and the entire study (no. 4-2012-0028).
Patients with sporadic ALS who were diagnosed using El Escorial criteria [
] and normal
healthy individuals free from any pharmacological treatment were included in this study.
These three healthy individuals have been represented as normal controls in the following
content. The patients were recruited at the Rehabilitation Institute of Neuromuscular Disease. All
patients with ALS had been previously screened for SOD1 gene mutation and showed no
mutation. Regarded on personal health histories obtained by interviews, control donors were all
unrelated and the normal phenotype was identified. To assess total functional condition of
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patients, the Revised ALS Functional Rating Scale (ALSFRS-R), scored 0–48, was used [
scores were recorded within a week of dermal punch biopsy. None of the patients with ALS or
normal subjects included in the study displayed signs of infection before biopsy.
Preparation of fibroblast cells
Punch biopsy was conducted by a dermatology specialist and performed in the upper lateral
quadrant of the buttock in patients with confirmed ALS. Dermal fibroblasts were also obtained
from normal subjects. The biopsy sample was transferred to a culture dish in Dulbecco’s
modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and
penicillin/streptomycin and incubated in a humidified 5% CO2 atmosphere at 37°C.
Cell proliferation and senescence analysis
For analysis of cell proliferation fibroblasts were seeded in 6-well plates (10,000 cells/plate) in
DMEM containing 10% FBS and penicillin/streptomycin. The number of cells per plate was
determined from counts obtained with an ADAM automatic cell counter 2, 4, 6, and 8 days
after plating as described in the manufacturer’s protocol (NanoEnTek Inc, South Korea).
Flow cytometric analysis of cellular senescence was carried out using a Quantitative Cellular
Senescence Assay Kit (Cell Biolabs, San Diego, CA, USA). Briefly, fibroblasts were treated with
pretreatment solution at 37°C for 2 h. Next, senescence-associated β-galactosidase (SA-β-gal)
substrate solution was added to the cells for 4 h. The stained cells were washed with
phosphatebuffered saline (PBS), harvested by trypsinization, and flow cytometric analysis was performed
in PBS containing 1% FBS on a FACSLSRII flow cytometer (BD Bioscience, San Jose, CA,
USA). For microscopy studies, fibroblasts were washed with PBS, fixed for 15 min with the
fixing solution at room temperature, briefly washed in PBS, and incubated with SA-β-Gal
substrate solution at 37°C without CO2 and with protection from light for 16 h. The blue stained
cells were analyzed under light microscopy.
Generation of iPSCs
The following protocol was previously described [
]. Following to the protocol of
manufacturer’s, episomal vector mixtures (total 3 μg) encoding defined reprogramming factors were
electroporated by using a microporator system (Neon; Invitrogen, Carlsbad, CA, USA). After
being pulsed three times with a voltage of 1,650 for 10 ms, the cells were grown further in
DMEM (containing 10% FBS). Otherwise, CytoTuneTM Sendai virus solution (Thermo Fisher
Scientific, Waltham, MA) including defined reprogramming four factors is mixed, and added
onto ALS and normal fibroblasts (MOI = 3). Seven days after transfection or transduction, cells
were transferred onto a feeder layer. iPSC colonies similar to human embryonic stem cells
(hESCs) were picked up mechanically and further cultured for characterization.
Cell cultures for iPSCs
Human iPSCs (normal and ALS) were cultured on mouse SIM Thioguanine/Ouabain-resistant
mouse fibroblast cell line (STO) under previously described growth conditions [
iPSCs (normal and ALS) generated in this study were maintained in hESC medium composed
of DMEM/F12 medium supplemented with 20% (vol/vol) knockout serum replacement
(Invitrogen, Carlsbad, CA), 4.5 g/L L-glutamine, 1% nonessential amino acids, 0.1 mM
2-mercaptoethanol, and 10 ng/mL basic fibroblast growth factor (bFGF) (Invitrogen, Carlsbad, CA)
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Induction of neural rosettes
For induction of neural rosettes, Embryonic bodies (EBs) were cultured in suspension for 4
days in hESC media excluding bFGF but supplemented with 5mM dorsomorphin (DM)
(Calbiochem, Darmstadt, Germany) and 5 to 10 μM SB431542 (SB) (Sigma, St. Louis, MO, USA).
On day 4, EBs were attached in Matrigel-coated culture dishes (BD Biosciences, San Jose, CA,
USA) in DMEM/F12 N2 supplemented media (N2 media) with 20 ng/ml bFGF (R&D Systems,
Minneapolis, USA) and 19 to 21 μg/ml human insulin solution (Sigma, St. Louis, MO) for
another 5 days. The emerged rosette structures were mechanically isolated using pulled glass
pipettes within the EB colonies, and isolated neural rosette clumps were replaced in
Matrigelcoated dishes. Replated neural rosettes were then expanded for an additional 6 to 7 days at 90%
According to the manufacturer’s instructions, total RNA was isolated from cultured fibroblasts
obtained from patients with ALS and normal subjects using Trizol (Invitrogen Life
Technologies, Carlsbad, CA, USA) [
]. RNA purity was evaluated with an Agilent 2100 Bioanalyzer
(Agilent Technologies, Palo Alto, CA, USA) and measured by the A260/A280 ratio for quality
control analysis. RNA integrity was evaluated by visual valuation of the 28S:18S rRNA ratio
using gel electrophoresis.
RNA sequencing and transcriptome data analysis
RNA sequencing was performed by Macrogen Inc (Seoul, Korea). The mRNA was changed
into a library of templates suitable for successive cluster generation using the reagents provided
in the Illumina1 TruSeq™ RNA Sample Preparation Kit [
]. The transcriptome analysis is
composed of RNA-seq experiment and data handling. RNA-seq experiment is TruSeq mRNA
library construction which is organized by 8 steps: purify and fragment mRNA, synthesize first
strand cDNA, synthesize second strand cDNA, perform end repair, adenylate 3' ends A single,
ligate adapters, enrich DNA fragments, and enriched library validation. First, using magnetic
beads which poly-T oligo-attached, purifying the poly-A containing mRNA molecules. After
purification, using divalent cations under raised temperature, the mRNA is split into small
fragments. The cleaved RNA fragments primed with random hexamers into first strand cDNA
using random primers and reverse transcriptase. These cDNA fragments are then subjected to
an end repair process using an end repair (ERP) mix involving the addition of a single “A”
nucleotide, followed by ligation of the adapters. On the 3’ end of the adapter, there is a
corresponding single ‘T’ nucleotide, and it provides a paired overhang for binding the adapter to the
fragment. To create the final cDNA library, the products are purified and enriched by PCR
]. Illumina utilizes a unique reaction. The reaction is called “bridged” amplification reaction
which reacts on the surface of the flow cell. A flow cell, which contains millions of unique
clusters, is loaded into the HiSeq 2000 for automated cycles of imaging and extension. Solexa’s
Sequencing-by-Synthesis utilizes four proprietary nucleotides with reversible fluorophore and
termination properties. Every individual sequencing cycle occurs in the presence of all four
nucleotides, leading to better accuracy than methods in which only one nucleotide is present in
the reaction mix [
]. To evaluate expression levels, RNA-seq reads were mapped to the
human genome. Transcript counts at the gene level were calculated. Data analysis performed
by TopHat and Cufflinks. For RNA-seq reads, TopHat is a fast splice junction mapper. Using
the ultra-high-throughput short read aligner Bowtie, TopHat aligns RNA-seq reads to
mammalian-sized genomes. After then, for identifying the splice junctions between exons, it
analyzes the mapping results. Cufflinks tests for differential expression and regulation in RNA-seq
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samples and assembles transcripts, estimates their abundances. Transcripts with a fold
induction 2 and Benjamin-Hochberg adjusted p value 0.05 were considered significant and were
included in downstream analysis.
We used the database for annotation, visualization, and integrated discovery (DAVID)
software (http://david.abcc.ncifcrf.gov/) [
] and Kyoto Encyclopedia of Genes and Genomes
(KEGG) pathways [
] for analysis. Using DAVID software, we searched for pathways that
were upregulated in subjects with ALS. Within these identified pathways, genes that are also
involved in neuroinflammation were selected for further validation.
Reverse transcription polymerase chain reaction (RT-PCR)
To validate the results of transcriptome analysis, we performed RT-PCR of genes that were
components of significantly upregulated KEGG pathways and known to be involved in
neuroinflammation. For RT-PCR, the following reaction-specific primers were used: cluster of differentiation 14
(CD14), forward 50-cagcctagacctcagccaca-30 and reverse 50-tcccgtccagtgtcaggtta-30; interferon-α/β
receptor-1 (IFNAR-1), forward 50-cgatgagtctgtcgggaatg-30 and reverse 50-gaccaatctgagctttgcga-30;
toll-interacting protein (TOLLIP), forward 50-aagaatccccgctggaataa-30 and reverse 50-gaggttgatc
atgccctcct-30; phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA),
forward 50-attccagacgcatttccaca-30 and reverse 50-gagcagcacgaggaagatca-30; interleukin-1
receptorassociated kinase 4 (IRAK4), forward 50-agcttgcagcaatggttgac-30 and reverse 50-tagctgcaccctgag
caatc-30; mitogen-activated protein kinase 1 (MAPK1), forward 50-accaaccatcgagcaaatga-30 and
reverse 50-acggtgcagaacgttagctg-30; mitogen-activated protein kinase 9 (MAPK9), forward 50-cggac
agcgtgcactaactt-30 and reverse 50-tttcaccagctctcccatga-30; interleukin-1β (IL-1β), forward 50-gtacctg
agctcgccagtga-30 and reverse 50-tgaagcccttgctgtagtgg-30; interleukin-8 (IL-8), forward 50-caaacctttc
caccccaaat-30 and reverse 50-accctctgcacccagttttc-30; chemokine (C-X-C motif) ligand 1 (CXCL1),
forward 50-tcaccccaagaacatccaaa-30 and reverse 50-actatgggggatgcaggatt-30; caspase recruitment
domain-containing protein 9 (CARD9), forward 50-gatgtacaaggaccgcatcg-30 and reverse 50-gcctcac
actggaacacctg-30; glyceraldehyde-3-phosphate dehydrogenase (GADPH), forward 50-caaggtcatccat
gacaactttg-30 and reverse 50-gtccaccaccctgttgctgtag-30. The GAPDH gene was used as the internal
control. Using RT-PCR, this study confirmed the induction of inflammation with the following
targets. cyclooxygenase-2 (COX-2) forward 50-cttcacgcatcagtttttcaag-30 and reverse 50-tcaccgtaaatatg
atttaagtccac-30. To confirm the iPSCs by RT-PCR, OCT-3/4, forward 5’-atcctgggggttctatttgg-3’ and
reverse 5’-ctccaggttgcctctcactc-3’; NANOG, forward 5’-ttccttcctccatggatctg-3’ and reverse 5’-tctg
ctggaggctgaggtat-3’; SRY-box 2 (SOX2), forward 5’-aaccccaagatgcacaactc-3’ and reverse 5’-cgg
ggccggtatttataatc-3’. To confirm the neural rosettes, NESTIN, forward 5’-gaaacagccatagagggcaaa-3’
and reverse 5’-tggttttccagagtcttcagtga-3’; paired box 6 protein (PAX6), forward 5’-gtgtccaacggatg
tgtgag-3’ and reverse 5’-ctagccaggttgcgaagaac-3’; forkhead box G1 (FOXG1), forward 5’-aggagggcg
agaagaagaac-3’ and reverse 5’-tcacgaagcacttgttgagg-3.
Real time quantitative polymerase chain reaction (RT-qPCR)
As described in detail in the previous study [
], for additional validation, real time
quantitative PCR (RT-qPCR) was performed in triplicate on a LightCycler 480 (Roche Applied Science,
Mannheim, Germany) using the LightCycler 480 SYBR Green master mix (Roche Applied
Science), and thermocycler conditions of 10 min template preincubation step at 95°C followed by
40 cycles at 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s. The melting curve analysis began at
95°C for 5 s, followed by 1 min at 60°C. The specificity of the produced amplification product
was confirmed by the melting curve analysis and showed a distinct single sharp peak with the
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expected Tm for all samples. A distinct single peak indicates that a single DNA sequence was
amplified during RT-qPCR. The GAPDH gene was used as the internal control. The expression
level of each gene of interest was obtained using the 2−ΔΔCt method.
As mentioned in the previous study [
], to confirm the expression of genes that were
validated by RT-PCR and RT-qPCR, 30 μg of extracted proteins were dissolved in sample buffer,
boiled for 5 min, and loaded onto a 10% sodium dodecyl sulfate (SDS) reducing gel. The
separated proteins were then blotted onto polyvinylidene difluoride membranes (Amersham
Pharmacia Biotech, Little Chalfont, UK) using a transblot system (Bio-Rad, Hercules, CA,
USA) at 100V for 1 h. The membranes were blocked for 1 h in Tris-buffered saline (TBS)
containing 5% nonfat dry milk (Bio-Rad), washed three times with TBS containing 0.01%
Tween 20 (TBST) for 15 min, and incubated overnight at 4°C with first antibodies specific to
the target proteins. The first antibodies are TOLLIP (1:1000 Abcam, Cambrige, MA),
MAPK9 (1:1,000 Abcam, Cambrige, MA), IL-1β (1:1,000 Abcam, Cambrige, MA), IL-8
(1:1,000 Abcam, Cambrige, MA), CXCL1 (1:1,000 Abcam, Cambrige, MA), and GAPDH
(1:1,000 Santa Cruz Biotechnology, CA, USA). The next day, the blots were washed three
times with TBST and incubated for 1 h with horseradish peroxidase (HRP)-conjugated
secondary antibodies (1:3,000 Santa Cruz Biotechnology, CA). After washing the blots three
times with TBST, the blots were visualized with an enhanced chemiluminescence detection
system (Amersham Pharmacia Biotech) [
Alkaline phosphatase staining and immunostaining
Alkaline phosphatase (AP) activity was measured with the leukocyte AP staining kit (System
Biosciences, CA, USA) according to the manufacturer’s instructions. For detecting AP activity,
the 4% paraformaldehyde fixed cells were stained AP solution for 20 min in room temperature
(RT). Samples were observed with inverted microscope (NIKON, Japan). For immunostaining
of pluripotent stem cell markers such as OCT4 and SSEA4, cells were fixed in 4%
paraformaldehyde solution (20min, RT), permeabilized with 0.1% Triton X-100 (10min, RT), and blocked
in 10% goat serum (1 h, RT). The iPSCs were incubated with primary antibodies such as
OCT3/4 (Santa Cruz, Texas, USA) and SSEA4 (Stemgent, MA, USA). The cells were observed
under fluorescent microscope (Cal Zeiss, Germany).
Lipopolysaccharide (LPS) and IL-1β treatment
Normal and ALS fibroblast cells were seeded in 6-well plates and maintained until they reached
80% confluence before incubation in DMEM serum-free media (SFM). Fibroblast cells were
washed five times with PBS and then treated with or without stimulation with 10 ng/mL LPS
(Sigma, St. Louis, MO) for 3 h. To confirm the induction of inflammation, expression of
human IL-1β was validated using RT-PCR. Expression of TOLLIP was validated by RT-PCR
of the cultured cells after LPS treatment at passages 4, 8, and 12. Normal- and patient-derived
iPSCs were washed five times with PBS and then treated with 1 μg/mL or 5 μg/mL LPS for 24 h,
or remained without any treatment. Normal- and patient-derived iPSCs and neural rosettes were
washed five times with PBS and then treated with 10 ng/mL or 100 ng/mL hIL-1β (R&D Systems,
Minneapolis) for 3 h, or remained without any treatment. To confirm the induction of
inflammation, expression of COX-2 was validated using RT-PCR. Expression of TOLLIP was validated by
RT-PCR of the cultured cells after hIL-1β treatment.
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Genomic DNA extracted from this individual’s sample was used for library preparation.
Massively parallel sequencing was done on the MiSeq System (Illumina). Additionally the primers
C9ORF72 forward 5'-ccagcttcggtcagagaagaaat-3’ and reverse 5'-gggtctagcaagagcaggtg-3’ were
used for the PCR. The PCR reaction was performed with 20 ng of genomic DNA as the
template in a 30 μℓ reaction mixture by using a EF-Taq (SolGent, Korea) as follows: activation of
Taq polymerase at 95°C for 5 min, 35 cycles of 95°C for 1 min, 58°C, and 72°C for 1 min each
were performed, finishing with 10 min step at 72°C. The amplification products were purified
with a multiscreen filter plate (Millipore Corp., Bedford, MA, USA). Sequencing reaction was
performed using a PRISM BigDye Terminator v3.1 Cycle sequencing Kit. The DNA samples
containing the extension products were added to Hi-Di formamide (Applied Biosystems,
Foster City, CA, USA). The mixture was incubated at 95°C for 5 min, followed by 5 min on ice and
then analyzed by ABI Prism 3730XL DNA analyzer (Applied Biosystems, Foster City, CA).
Data were expressed as mean ± standard deviation. Using Statistical Package for Social Sciences
(SPSS) version 20.0, statistical analyses were performed. Non-parametric statistical analysis
such as Mann-Whitney U test was used for the comparison of two groups. Student t-test was
also used to confirm the statistical results. A P-value <0.05 was considered statistically
Characteristics of subjects
Dermal punch biopsies were taken from three patients with ALS and three normal controls to
obtain fibroblast cells for this study. Basic demographic and clinical information of the study
subjects are described in Table 1. Mean disease duration of ALS was 27.7 months. Two patients
with ALS were managed with percutaneous endoscopic gastrostomy (PEG) and overnight
noninvasive ventilator, whereas subject 3 showed relatively mild symptomatic manifestations of
dysarthria and dysphagia. In the patient number 3 case, as the disease progression had been
rapidly worse, six months after the skin biopsy, the patient experienced respiratory dysfunction
and dysphagia. Thus, since then, the patient number 3 also had used ventilator and PEG tube
like the same as patient number 1 and 2 (Table 1).
Normal controls were all males, aged 32, 63, and 76 years respectively. Mean ages of patients
with ALS and normal controls were 54.3 years and 57.0 years, respectively. Performing genetic
analysis on three ALS cases with no shared genetic similarities (at least we are not told whether
they are from a specific mutation or family) is irrelevant. To verify the above, we selected
certain genes that have been reported as ALS-related genes, and confirmed the sequence using
genomic DNA (gDNA) from patient-derived fibroblasts. When massively parallel sequencing
was done on the MiSeq System (Illumina) to verify mutant genes related with ALS, there were
no ALS-related mutant genes including SOD1, TDP-43, FUS, UBQLN2 and VCP that cause
Furthermore, we confirmed the GGGGCC (G4C2) hexanucleotide repeat expansion in
C9orf72 which affects ALS pathogenic phenomenon. The diagnosis of C9orf72-related ALS/
FTD is established by detection of a heterozygous pathogenic GGGGCC (G4C2)
hexanucleotide repeat expansion in C9orf72 on molecular genetic testing [
]. When this study
confirmed the sequence of gDNA from the fibroblasts to count the exact repeat numbers of
GGGGCC (G4C2) hexanucleotide in C9ORF72, there were two patients with double repeat of
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GGGGCC (G4C2), and another did not have any GGGCC (G4C2) hexanucleotide repeat (S1
Fig). Because this GGGCC (G4C2) hexanucleotide repeat number is not the significant repeat
to show pathogenic phenomena, we concluded that these ALS patients were not genetic but
Cell proliferation and cellular senescence
To evaluate cell proliferation, fibroblasts from 6-well plates were harvested and counted with
an automatic cell counter on days 2, 4, 6, and 8. In addition, the growth curves of cultured
fibroblasts at passages 4, 8, and 12 were generated. When cell proliferation was observed until
passages 12, fibroblasts from normal subjects in passages 4 exhibited a significantly higher
proliferation rate than fibroblasts from patients with ALS, which showed a slow growth rate
(p<0.05). The proliferation of fibroblasts from both normal subjects and patients with ALS
decreased after passages 8 (Fig 1A).
To evaluate cellular senescence, the senescence status of fibroblasts from normal subjects
and patients with ALS was investigated by performing morphometric analysis for SA-β-Gal
activity. When fibroblasts from patients with ALS were cultured in passages 4, the cells had
already entered a senescent state compared to fibroblasts from normal subjects. Fibroblasts
from patients with ALS displayed more positive SA-β-Gal staining by light microscopy (Fig
1B). When flow cytometric analysis was concurrently performed to quantify the SA-β-Gal
activity, fibroblasts from both normal subjects and patients with ALS showed an increase in
SA-β-Gal activity with progression of passages (Fig 1C). Fibroblasts from patients with ALS
showed a higher level of senescence than fibroblasts from normal subjects, as indicated by
relative ratio for SA-β-Gal activity (Fig 1D). Even though there was no statistical significance,
senescence showed an increasing pattern while the cell passages were increasing.
Differentially expressed genes and pathway analysis
RNA was prepared from cultured fibroblasts of patients with ALS and normal subjects at
passages 4. We performed transcriptome analysis by RNA sequencing to identify genes that were
differentially expressed in patients with ALS compared to normal controls; Total 17,025 genes
were differentially expressed, and listed (S1 Table). Among total genes at the ALS patients, 626
transcripts were 2.0-fold lower, and 589 transcripts were higher than normal control samples
Using DAVID software, several pathways were identified to be significantly upregulated in
subjects with ALS (Table 2). Among these pathways, the Toll-like receptor (TLR) signaling
pathway and NOD-like receptor (NLR) signaling pathway are related to innate immunity, and
these two pathways are statistically significant (p<0.05). Significantly upregulated genes related
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Fig 1. Cellular proliferation and senescence. (A) Growth curves of fibroblasts from patients with ALS and normal subjects at passages 4, 8, and 12
during culture for 8 days (*P < 0.05). (B) β-galactosidase (SA-β-Gal) activity of fibroblasts from patients 1, 2, and 3 with ALS and normal subjects 1, 2, and
3 at passages 4, 8, and 12. This image magnified by a factor of 100 (Scale bar, 100 μm). (C) Flow cytometric analysis of fibroblasts from patients with ALS
and normal subjects at passages 4, 8, and 12. (D) A graph showing the relative ratio of SA-β-Gal activity on normal subjects and patients with ALS at
passages 4, 8, and 12.
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to TLR signaling pathway were IRAK4, MAPK1, IL-8, TOLLIP, IL-1β, MAPK9, PIK3CA,
CD14, and IFNAR-1. In addition, genes related to the NLR signaling pathway such as CXCL1,
MAPK1, CARD9, IL-8, IL-1β, and MAPK9 were significantly upregulated.
Validation of expression results by RT-PCR, RT-qPCR and western blotting
We next performed RT-PCR of the above 11 genes for validation. Expression levels of these
genes in ALS patient-derived fibroblasts were relative in fibroblasts from normal subjects. This
result was determined by RT-PCR as follows: CD14 (5.25-fold, p = 0.024), IFNAR-1 (1.53-fold,
p = 0.094), TOLLIP (2.59-fold, p<0.001), PIK3CA (0.84-fold, p = 0.436), IRAK4 (0.87-fold,
p = 0.730), MAPK1 (0.96-fold, p = 0.863), MAPK9 (1.76-fold, p<0.001), IL-1β (2.89-fold,
p = 0.077), IL-8 (3.20-fold, p = 0.004), CXCL1 (3.92-fold, p = 0.063), and CARD9 (1.14-fold,
p = 0.387) (Fig 2A and 2B).
We performed RT-qPCR for more accurate quantitative analysis. As a result, we confirmed
the similar expression patterns like the result of RT-PCR (Fig 3A). This result was determined
by RT-qPCR as follows: TOLLIP (1.67-fold, p = 0.002), MAPK9 (1.57-fold, p = 0.004), IL-1β
(5.20-fold, p = 0.069), IL-8 (3.86-fold, p = 0.099), and CXCL1 (4.8-fold, p = 0.041).
Western blotting was performed for further validation for the significantly upregulated
genes that were confirmed by RT-PCR. The amount of protein encoded by these genes in
fibroblasts from patients with ALS relative to that in fibroblasts from normal subjects as determined
by western blotting was as follows: TOLLIP (2.55-fold, p<0.001), MAPK9 (1.45-fold,
p<0.001), IL-1β (1.57-fold, p = 0.004), IL-8 (2.40-fold, p<0.001), and CXCL1 (2.33-fold,
p<0.001) (Fig 3B and 3C).
Comparison of differential gene expression between normal and ALS patient-derived iPSCs and neural rosettes
Since ALS is a CNS disease, there is a limitation on gene screening between fibroblasts cultured
from normal controls and patients. To overcome the limitation, we established iPSCs derived
Fig 2. RT-PCR verification of genes identified in transcriptome analysis. (A) RT-PCR analysis of 11 genes.
(B) Comparison of relative gene expression in normal subjects and patients with ALS. Eleven genes from the
Tolllike receptor and NOD-like receptor signaling pathways were verified using RT-PCR and genesÐCD14,
interferon-α/β receptor-1 (IFNAR-1), toll-interacting protein (TOLLIP), mitogen-activated protein kinase 9
(MAPK9), interleukin 1 beta (IL-1β), interleukin 8 (IL-8), and chemokine (C-X-C motif) ligand 1 (CXCL1)Ðwere
upregulated in the ALS group. *P < 0.05, ²P < 0.1
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Fig 3. Validation of identified genes by RT-qPCR and western blotting. (A) Quantitative comparison of relative gene expressions between normal
subjects and ALS patients by RT-qPCR. (B) TOLLIP, IL-1β, IL-8, MAPK9, and CXCL1 proteins were overexpressed in the subjects with ALS by western
blotting. (C) Comparison of relative expression from normal subjects and patients with ALS for the five proteins verified by western blotting. *P < 0.05,
²P < 0.1
from fibroblasts and generated neural rosettes differentiated from the iPSCs. When the
expression patterns of inflammation-related genes in three different types of cells were compared
between normal controls and ALS patients, these different types of cells showed similar gene
The iPSCs from patient-derived fibroblasts were verified their pluripotency through AP
staining (Fig 4A and 4B). Additionally, expression of OCT4 and SSEA4 through
immunocytochemistry, and the expressions of OCT4, SOX2 and NANOG were confirmed in RNA level
(Fig 4C and 4D). Neural rosettes from iPSCs were also confirmed in RNA level to verify their
success of differentiation through the expressions of NESTIN, PAX6, and FOXG1 (Fig 4E).
With these iPSCs and neural rosettes, we confirmed the patterns of gene expressions that are
related to inflammation. This result in iPSCs was determined by RT-qPCR as follows: CD14
(2.13-fold, p = 0.180), IFNAR-1 (0.91-fold, p = 0.485), TOLLIP (1.56-fold, p = 0.002), PIK3CA
(1.50-fold, p = 0.009), IRAK4 (6.49-fold, p = 0.002), MAPK1 (0.95-fold, p = 0.699), MAPK9
(0.89-fold, p = 0.589), IL-1β (1.97-fold, p = 0.132), IL-8 (8.38-fold, p = 0.002), CXCL1 (3.5-fold,
p = 0.002), and CARD9 (1.27-fold, p = 0.394) (Fig 4F).
This result in neural rosettes was determined by RT-qPCR as follows: CD14 (1.33-fold, p =
0.065), IFNAR-1 (0.97-fold, p = 0.818), TOLLIP (1.50-fold, p = 0.065), PIK3CA (1.04-fold, p =
0.589), IRAK4 (1.71-fold, p = 0.132), MAPK1 (1.21-fold, p = 1.000), MAPK9 (1.15-fold, p =
0.818), IL-1β (1.98-fold, p = 0.026), IL-8 (1.14-fold, p = 0.589), CXCL1 (1.79-fold, p = 0.026),
and CARD9 (1.30-fold, p = 0.065) (Fig 4G).
Changes in TOLLIP expression according to progression of passages
We did not expect to see overexpression of TOLLIP in fibroblasts of patients with ALS because
it is known to have a modulatory role whereas the other five genes function as inducers of the
inflammatory immune response. We hypothesized that expression of TOLLIP would change
with the cellular aging process. To investigate whether expression of TOLLIP changes
throughout the progression of disease, we performed western blot analysis of TOLLIP expression in
fibroblasts of patients with ALS collected at passages 4, 8, and 12, respectively. At passages 4
and 8, TOLLIP was overexpressed in patients with ALS relative to controls (2.52-fold, p<0.001;
1.17-fold, p = 0.011); however, its expression decreased with increasing passages and the
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Fig 4. Verification of genes identified in transcriptome analysis in normal- and patient-derived iPSCs and
neural rosettes. (A) Morphology of the expanded human iPSCs (Scale bar, 100 μm). (B) Alkaline phosphatase
staining of iPSCs (Scale bar, 100 μm). (C) The expression of OCT4 and SSEA4, which are human ESC-specific
markers, was detected by immunocytochemistry. DAPI signals indicate the total cell presence in the image (scale
bars, 100 μm). (D) The expression of OCT4, SOX2 and NANOG, which are human ESC-specific markers, was
detected by RT-PCR (lane1, ALS patient-derived iPSCs; lane 2, normal-derived iPSCs; and lane 3, human
fibroblast as a negative control). (E) The expression of NESTIN, PAX6, and FOXG1 which are the specific markers
for the human neural rosette, was detected by RT-PCR (lane 1, ALS patient-derived neural rosettes; lane 2,
normal-derived neural rosettes; and lane 3, human fibroblast as a negative control). (F) Quantitative comparison of
relative gene expressions between normal subjects and ALS patients by RT-qPCR in iPSCs. (G) Quantitative
comparison of relative gene expressions between normal subjects and ALS patients by RT-qPCR in neural
rosettes. NR, neural rosette, *P < 0.05, ²P < 0.1
expression of TOLLIP in patients with ALS was lower than that in controls at passages 12
(0.85-fold; Fig 5).
The expression of inflammation-related genes according to induced inflammation
During the study, we established iPSCs derived from normal controls and patients with ALS.
After then, we differentiated established iPSCs into neural rosettes. When we confirmed
expression of proinflammatory genes under the induction of inflammation with LPS or IL-1β,
expressions of IL-1β or COX-2 by RT-PCR were upregulated in ALS patients compared with
normal controls in iPSCs (Fig 6A and 6B) and neural rosettes (Fig 6C). First of all, we
confirmed on iPSCs that COX-2 was expressed in a LPS dose-dependent manner. Moreover, while
COX-2 was more expressed in the patient than normal, we could verify that the inflammation
was more induced in ALS patient group than normal group (Fig 6A).
When another inflammatory inducing factor, IL-1β, was administrated, COX-2 was
increased in a dose-dependent manner (Fig 6B). Additionally, on neural rosettes, when IL-1β
was administrated, COX-2 increased its expression more in ALS patient group (Fig 6C). This
result was similar with expression patterns in fibroblasts. Since the patterns of the
proinflammatory gene expression according to induced inflammation in the three different types of cells
—iPSCs, neural rosettes, and fibroblasts—were similar, these results showed that the
phenomena were disease related. Consequently, the above results might give the proof that the gene
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Fig 5. Validation of TOLLIP expression with progression of passages by western blotting. (A) Fibroblasts
from normal subjects and patients with ALS were subcultured to the 12th passage. Western blot analysis of protein
extracted from the 4th, 8th, and 12th passages (P4, P8, and P12, respectively) was performed using antibodies
against TOLLIP and GAPDH (as a control). (B) With advancing passages, the expression levels of TOLLIP in
patients with ALS were reduced compared to those in normal subjects. The numbers correspond to the ratio of the
expression level of TOLLIP in patients with ALS compared to that in normal subjects. *P < 0.05
expressions related to inflammation were upregulated in ALS via transcript screening on
We then tested whether the inflammatory response induced by LPS stimulation leads to a
change in TOLLIP expression in ALS according to progression of passages. The induction of
an inflammatory response by LPS stimulation was confirmed by expression of the
proinflammatory cytokine IL-1β using RT-PCR, and IL-1β was found to be overexpressed in fibroblasts
from ALS patients compared with controls (Fig 6D). Expression of TOLLIP after LPS
treatment was increased in fibroblasts from ALS patients at passages 4 but decreased with the
progression of passages (Fig 6E).
From the confirmation of differentially expressed genes in normal- and patient-derived
fibroblasts, this study demonstrated that the TLR and NLR signaling pathways are involved in ALS
pathogenesis and identified TOLLIP, MAPK9, IL-1β, IL-8, and CXCL1. The iPSCs and neural
rosettes were generated from the patient-derived fibroblasts, and these established cells showed
the similar pattern with the fibroblast in the gene screening of TLR and NLR signaling
pathways. In addition, we found that TOLLIP, a negative regulator of the TLR signaling pathway,
was initially overexpressed, but its expression decreased with cellular aging.
TLRs recognize the specific patterns of pathogens and play a vital role in activating innate
immunity. Recognition of pathogens by TLRs initiates signal transduction pathways,
generating the expression of several genes such as those encoding proinflammatory cytokines (tumor
necrosis factor-α [TNF-α], IL-1β, IL-6, and IL-12) and chemokines (IL-8, regulated on
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Fig 6. Expression of COX-2, IL-1β, and TOLLIP after treatment with lipopolysaccharide or IL-1β as an
inflammatory stimulus. (A) Validation of COX-2 by induced inflammation with LPS in fibroblasts from ALS
patients (lane 1, untreated ALS patient-derived iPSCs; lane 2, 1 μg/mL LPS-treated ALS patient-derived iPSCs;
lane 3, 5 μg/mL LPS-treated ALS patient-derived iPSCs; lane 4, untreated normal-derived iPSCs; lane 5, 1 μg/mL
LPS-treated normal-derived iPSCs; lane 6, 5 μg/mL LPS-treated normal-derived iPSCs). (B) Validation of COX-2
by induced inflammation with IL-1β in fibroblasts from ALS patients. Relative index graphs for comparison of
COX2 to GAPDH between ALS patients and normal controls (lane1, untreated ALS patient-derived iPSCs; lane 2, 10
ng /mL IL-1β-treated ALS patient-derived iPSCs; lane 3, 100 ng/mL IL-1β-treated ALS patient-derived iPSCs; lane
4, untreated normal-derived iPSCs; lane 5, 10 ng/mL IL-1β-treated normal-derived iPSCs; lane 6, 100 ng/mL
IL1β-treated normal-derived iPSCs). (C) Validation of COX-2 to compare in ALS patient- and normal control-derived
neural rosettes (lane 1, untreated ALS patient-derived neural rosettes; lane 2, 100 ng/mL IL-1β-treated ALS
patient-derived neural rosettes; lane 3, untreated normal-derived neural rosettes; lane 4, 100 ng/mL IL-1β-treated
normal-derived iPSCs). (D) Validation of IL-1β by induced inflammation with LPS in fibroblasts from ALS patients
(lane 1, untreated ALS fibroblasts; lane 2, 10 ng/mL LPS-treated ALS fibroblasts; lane 3, untreated normal
fibroblasts; lane 4, 10 ng/mL LPS-treated normal fibroblasts) (E) Changes in TOLLIP expression according to
progression of culture passages. TOLLIP expression in LPS-stimulated fibroblasts from patients with ALS
decreased with increasing passage. *P < 0.05
activation, normal T-cell expressed and secreted [RANTES], and macrophage inflammatory
protein-1 [MIP-1], via the nuclear factor-kappaB [NF-κB] pathway), in addition to members
of the MAPK pathway. Both innate immune responses and further development of
antigenspecific acquired immunity are regulated by these gene products [
]. The previous study
reported that CD14 and TLR2 were upregulated in animal models of various
neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and ALS [
These findings led researchers to focus on the TLR signaling pathway associated with innate
immunity in ALS pathogenesis. Expression of mutant SOD1 in the mouse model of ALS
facilitated the microglial neurotoxic inflammatory response through TLR2 [
]. Mutant SOD1
binds CD14, which is a co-receptor of TLR2 and TLR4. Microglial activation induced by
mutant SOD1 was attenuated using CD14 blocking antibody or when the microglia lacked
CD14 expression, suggesting that microglial activation via the CD14 and TLR pathways is a
neuropathological hallmark of ALS [
]. In addition, a previous study showed that CD14
and TLR2, potential indicators of the innate immune response, were upregulated in the spinal
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cords of mice and patients in ALS . The results of this study supported that the TLR
signaling pathway is associated with immune-related pathogenesis in ALS.
In addition to verifying the role of the TLR signaling pathway, this study yielded new
observation that the NLR signaling pathway is involved in neuroinflammation in ALS. The NLR
signaling pathway is thought to be a TLR-independent system for intracellular recognition of
certain pathogens. NOD proteins recognize the core structures of bacterial peptidoglycans in
the cytoplasm and have been implicated in the induction of NF-κB activity and the activation
of caspases. These NOD proteins seem to function as cytosolic sensors for the induction of
apoptosis and the induction and regulation of inflammatory responses [
]. NOD1 and NOD2
proteins undergo oligomerization upon recognizing the peptidoglycan motif, resulting in
activation of the MAPK pathway and the transcription factor NF-κB [
]. Thus, the NLR
signaling pathway induces transcription of proinflammatory cytokines -IL-1β, IL-18, TNF-α,
IL6- and chemokines such as CXCL1.
The proinflammatory cytokines IL-1β and IL-8 are induced by the TLR and NLR signaling
pathways. IL-1β is a vital mediator of the inflammatory response and is implied in a variety of
cellular phenomenon, including cell proliferation, differentiation, and apoptosis. Pathologic
activation of glia in ALS has been widely characterized and is marked by enhanced production
of potentially cytotoxic molecules such as reactive oxygen species, inflammatory mediators
such as COX-2, and proinflammatory cytokines such as IL-1β, TNF-α, and IL-6 [
]. In ALS,
mutant SOD1 activates caspase-1 and IL-1β in microglia. Mutant SOD1-induced IL-1β
correlates with amyloid-like misfolding and is independent of dismutase activity, suggesting that
IL1β affects significantly to disease progression in the mouse model of ALS by promoting
]. However, this result is controversial. Because mice with lacking IL-1β
show unchanged disease onset, the result suggests that inflammation is not a starting factor in
the disease [
]. IL-8, a chemokine produced by macrophages and other cells, is an essential
mediator of the immune response in the innate immune system. IL-8 can be secreted by any
cell type possessing the TLR that is involved in the innate immune response. This cytokine
primarily targets the neutrophil granulocytes and is often associated with inflammation. In
addition, IL-8 levels are known to increase under oxidative stress leading to recruitment of
inflammatory cells and a further increase in oxidative stress mediators, making IL-8 a key
factor in localized inflammation [
]. A previous investigation reported that an increased level of
IL-8 in the cerebrospinal fluid (CSF) of patients with ALS suggests stimulation of a
proinflammatory cytokine cascade after microglial activation [
]. Elevated levels of IL-8 in CSF from
patients with ALS showed a negative correlation with ALSFRS-R score and alterations in
chemokines were presumed to correlate with the clinical course of ALS [
]. Our data also showed
upregulation of IL-1β in fibroblasts from ALS patients; however, it is not clear whether
upregulation of IL-1β is the result or the cause of activation of TLR and NLR signaling. IL-8 has been
reported to be a potent inducer of the CXCR1, CXCR2, and the chemokine CXCL1 [
CXCL1 has been reported to be overexpressed in gastric, colon, and skin cancers [
] and is
known to recruit oligodendrocytes in multiple sclerosis when coupled with CXCR2 .
CXCL1 was overexpressed in ALS fibroblasts in our study; however, its role in ALS
pathogenesis is not clear.
MAPK9 was reported to be associated with mitochondrial dysfunction and glucocorticoid
receptor signaling in neurodegenerative disorders using pathway analysis [
analysis in ALS using a genome-wide association study identified the MAPK signaling pathway as
the one of the top candidate pathways [
]. In addition, aberrant expression and activation of
p38 MAPK in motor neurons and microglia may play a role in the development and
progression of ALS [
]. In a recent study [
], TAR DNA-binding protein 43 (TDP-43) depletion in
microglia was shown to upregulate COX-2 expression and prostaglandin E2 (PGE2)
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production through activation of MAPK/ERK signaling. MAPK9 and CXCL1 have not been
clearly associated with ALS, and there are no studies on their relationship to ALS. Upregulation
of these two genes provides new clues for the better understanding of the mechanism of ALS.
TOLLIP, a member of the TLR signaling pathway, acts as a negative regulator of the TLR
signaling pathway. TOLLIP associates with IL-1R and TLR4 after LPS activation, inhibiting the
immune response mediated by TLR. In addition, TOLLIP binds directly to IRAK-1 and
prevents NF-κB activation by inhibiting autophosphorylation of IRAK-1 [
]. Both NF-κB
and MAPK pathways can be activated when endogenous IRAK-4 is overexpressed and
interacts with IRAK-1 and TRAF6 in an IL-1-dependent manner [
]. Overexpression of TOLLIP
leads to inhibition of the TLR signaling pathway. However, TOLLIP does not seem to play a
neuroprotective role. When stimulated by IL-1β and LPS, TOLLIP-deficient mice showed
normal activation of NF-κB and the MAPK signaling pathway, but significantly reduced
production of proinflammatory cytokines. Therefore, TOLLIP is considered as a modulatory role,
controlling the induction of inflammatory cytokines [
]. A previous study analyzing brain
gene expression profiles of immune-related genes in aging and Alzheimer’s disease (AD)
showed that TOLLIP was downregulated in the brains of aged subjects and AD compared to
the brains of young subjects, whereas genes reflecting activation of microglia such as CD14,
TLR2, and TLR4 were upregulated in the brains of aged subjects and AD. The researchers
concluded that downregulation of TOLLIP attenuates TLR signaling and microglial activation
]. In the previous study, the expression of TOLLIP was highest in the brains of young
subjects, followed by the aged brain, and was lowest in the brains of subjects with AD. We carried
out additional experiments for a better understanding of why TOLLIP was upregulated in
passages 4 of ALS fibroblasts in our studies. Based on results from the research mentioned above,
we suggested that the expression of TOLLIP might change temporally according to the cellular
aging process, and found that expression of TOLLIP thus decreased with aging of ALS cells.
The similar results were further confirmed with LPS treatment for stimulation of
inflammation. We postulate that overexpression of TOLLIP in the early stages of passages represents the
compensatory activity of the cells, and that expression of TOLLIP decreases with additional
passages because of loss of compensation. A previous study reported that microglia and
inflammatory cells are highly dynamic over the course of the disease and time-dependent
modification is important for the development of ALS [
]. Expression profiles of cytokines such as
TNF-α, transforming growth factor-β1 (TGF-β1), and macrophage colony-stimulating factor
(M-CSF) in the spinal cord of the mouse model of ALS showed upregulation according to
aging, suggesting that this temporal profile of expression might contribute to the disease
]. In this study, we confirmed the low proliferation rate and advanced senescence of
fibroblasts in ALS according to the progression of cellular passages. This cellular aging process
may contribute to change TOLLIP expression according to the passages. We suggest that
TOLLIP expression reflects the temporally controlled neuroprotective and neurotoxic presentation
RNA for gene expression profiling was isolated from the fibroblasts of subjects. The
hallmark of ALS pathogenesis is motor neuronal death in the spinal cord and motor cortex,
whereas fibroblasts are non-neuronal cells. Although investigation of neuronal cells might
provide more information on the pathogenesis of ALS, biopsy of neuronal tissues is invasive and
not an easy technique. In contrast, fibroblasts are gladly accessible and may hold potential for
short-term, rapid diagnostic and prognostic plans. Several studies have examined pathologic
features in fibroblasts from ALS patients [
]. They showed that fibroblasts recapitulate
some of the hallmark abnormalities in TDP-43 observed in neuronal cells [
] and reported
findings of increased membrane potential and decreased mitochondrial content in ALS
]. Additionally, gene expression profiling of fibroblasts from ALS demonstrated
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differential expression of genes related in RNA processing and the stress response, a
noteworthy decrease in miRNA production, and a reduced response to hypoxia, suggesting that
fibroblasts can act as cellular models for ALS [
Since ALS is a CNS disease, study of gene expressions related to inflammation in fibroblasts
directly could not represent the inflammatory pathway in ALS, and it would be the best if we
could obtain spinal fluid or post mortem tissues from patients. Nevertheless, the simple way to
get patients’ samples is primary culture of fibroblasts via skin biopsy. Therefore, we have
strived to established neural lineage cells instead of getting the samples of spinal fluid or post
mortem tissues. We have established iPSCs derived from normal and patients’ fibroblasts and
generated neural rosettes from the iPSCs. The study of cells that differentiated into neural
lineage cells could be an alternative proposal. In this study, we studied the certain
inflammationrelated genes in three different types of cells such as fibroblasts, iPSCs, and neural rosettes, and
confirmed the genes that considered as representing the certain inflammatory pathways.
Taken together, besides other studies, this study showed the results in three different types
of cells to overcome the limitation of small sample size and the type of the original sample.
Since the original sample was the patient-derived fibroblasts and there were only three patients,
these were considered to be the limitation for the CNS disease research. However, while this
study established iPSCs and neural rosettes from the fibroblasts, these three different types of
cell lines added more various on sample research, and showed CNS related result. Nonetheless,
the results of our study should be confirmed by gene expression profiling of motor neuron cells
in the future.
Inflammatory responses might play a vital role in the pathogenesis of motor neuron damage in
ALS. Gene expression profiling and pathway analysis showed that the TLR and NLR signaling
pathways are related in the pathogenesis of ALS through fibroblasts, iPSCs and neural rosettes.
These pathways are related to pathological innate immunity and neuroinflammation.
Overexpression of genes related to inflammation, such as TOLLIP, MAPK9, IL-1β, IL-8, and CXCL1,
was validated and changes in TOLLIP expression associated with cellular aging were observed.
A future research will be focused on the possible contribution factors that related to the
etiology of ALS, so might be identified the therapeutic targets.
S1 Fig. Sequence analysis of C9ORF7 gene. (A) The sequencing result of C9ORF7 in ALS
patient number 1 (B) The sequencing result of C9ORF7 in ALS patient number 2 (C) The
sequencing result of C9ORF7 in ALS patient number 3
S1 Table. Raw data for 17,025 differentially expressed transcripts. Raw data of RNA
sequencing to identify genes differentially expressed between ALS patients and controls; 17,025
differentially expressed transcripts were obtained.
S2 Table. Raw data for 1215 differentially expressed transcripts. Among the total of 17,025
differentially expressed transcripts, 1215 transcripts were showing 2.0-fold up- or
down-regulation. The expression levels of 626 transcripts were downregulated and those of 589 transcripts
were upregulated in preeclampsia patients compared to controls.
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We would like to give honest and sincere appreciation to Suk-Young Song, for providing a
result of RT-PCR. The authors have declared that no competing interests exist.
Conceptualization: SRC SWK MYL YHW.
Data curation: MYL YHW.
Formal analysis: MYL.
Funding acquisition: SRC YHW.
Investigation: MYL YHW.
Methodology: MYL MSK JHY YHW JHS.
Project administration: SRC SWK.
Resources: SRC YHW.
Supervision: SRC SWK.
Visualization: MYL JHY MGK.
Writing – original draft: MYL YHW.
Writing – review & editing: SRC SWK YHW MYL YCC YH HBK DYK MSK JHY JHS MGK.
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