ABSTRACT Amyotrophic lateral sclerosis (ALS) is a late-onset progressive neurodegenerative disease characterized by the loss of motor neurons in the spinal cord and brain. Mutations in Cu/Zn

superoxide dismutase 1 (SOD1) are known to induce ALS. Although many research models have been developed, the exact pathological mechanism of ALS remains unknown. The recently developed

induced pluripotent stem (iPS) cell technology is expected to illuminate the pathological mechanisms and new means of treatment for neurodegenerative diseases. To determine the pathological

mechanism of ALS, we generated mouse iPS (miPS) cells from experimental ALS transgenic mice and control mice and characterized the cells using molecular biological methods. The generated

will be helpful in revealing the mechanism of motor neuronal cell death in ALS. SIMILAR CONTENT BEING VIEWED BY OTHERS NEURONAL MITOCHONDRIAL DYSFUNCTION IN SPORADIC AMYOTROPHIC LATERAL

SCLEROSIS IS DEVELOPMENTALLY REGULATED Article Open access 23 September 2021 CELLULAR ANALYSIS OF SOD1 PROTEIN-AGGREGATION PROPENSITY AND TOXICITY: A CASE OF ALS WITH SLOW PROGRESSION

HARBORING HOMOZYGOUS _SOD1-D92G_ MUTATION Article Open access 25 July 2022 HETEROZYGOUS KNOCKOUT OF SYNAPTOTAGMIN13 PHENOCOPIES ALS FEATURES AND TP53 ACTIVATION IN HUMAN MOTOR NEURONS

Article Open access 03 August 2024 INTRODUCTION Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disease characterized by the loss of motor neurons in the spinal cord

and brain.1 Progressive paralysis of voluntary muscles and progressive spread of symptoms are typical features of ALS. Respiratory failure with denervation of the respiratory muscles and

diaphragm is the last symptom in ALS. Most cases of ALS (~90%) are sporadic, and the remaining cases (~10%) are familial.2 Mutations of Cu/Zn superoxide dismutase 1 (SOD1) are related to the

development of ~20% of familial ALS cases.3 SOD1 is a predominantly cytoplasmic protein that consists of 153 amino acids. SOD1 converts superoxide anion to hydrogen peroxide to protect

cells. It was reported that while SOD1 null mice do not develop motor neuron death, mutant SOD1 transgenic mice recapitulate ALS symptoms.4 It is thought that mutant SOD1 induces cell death

by a gain of function, although the precise pathologic mechanism remains unknown. There are many theories about the cause of motor neuronal cell death in ALS. These include genetic factors,5

oxidative stress,6 mitochondrial dysfunction,7 ER stress,8 excitotoxicity,9 proteasome inhibition,10 axonal transport defeat,11 dysregulation of RNA processing12 and formation of protein

aggregates.13 ALS transgenic mice carrying the human mutant _SOD1 (G93A)_ gene provide a common research model for ALS.14 These mice present a pathology similar to that of human ALS

patients, such as motor neuronal loss in the brain and spinal cord, the presence of aggregates, inflammation and death.15 In particular, these mice present hind limb weakness and tremor

around postnatal day 90 and then die at approximately postnatal day 120. Degenerative processes in the motor neurons are observed in the early stages of the development of symptoms, and

degeneration of neuromuscular junctions may precede the loss of motor neurons. Pathological characteristics, such as mitochondrial vacuolization, Golgi fragmentation or

neurofilament-positive inclusions, are present in the motor neurons of ALS transgenic mice. Motor neurons of these mice are also affected by inflammation that causes astrocytosis and

microgliosis. Recently, somatic reprogramming technology was used to produce induced pluripotent stem (iPS) cells by applying four pluripotent genes, namely, Oct4, Sox2, Klf4 and c-Myc.16

Researchers discovered these key pluripotent genes using differentiated mouse embryonic fibroblasts and tested the expression of these genes and the differentiation ability of iPS cells.

There are many advantages to using iPS cells. For example, they are easy to create, can be applied in patient-specific cell therapy and research, and require no special ethical

considerations. In particular, iPS cells are expected to help identify drugs for the treatment of patients with neurodegenerative disease.17 For these reasons, many iPS cell lines have been

produced, using human or animal models, for research on ALS.18, 19 In the present study, we report pathological differences between iPS cell-derived motor neurons from ALS mice and those

from control mice; these differences include neural dendrites, aggregates and cell death. Our results demonstrate that motor neurons derived from ALS-related mouse iPS cells recapitulate the

pathological features of ALS. MATERIALS AND METHODS ANIMALS ALS transgenic mice expressing the human mutant _SOD1 (G93A)_ gene (B6SJL-Tg[SOD1-G93A]1Gur/J) and their non-transgenic

littermates (B6SJLF1/J)—the latter used as controls—were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). All mouse care and experiments were agreed upon by the Institutional

Animal Care and Use Committee of Korea University. TAIL-TIP FIBROBLAST CULTURE FROM MOUSE Tail-tip fibroblasts (TTFs) were prepared from the transgenic and control mice as previously

described.20 The TTFs were maintained in Dulbecco’s modified minimal essential medium (DMEM; Gibco, Life Technologies, Grand Island, NY, USA) supplemented with 10% FBS (Gibco). RETROVIRAL

PRODUCTION AND TITRATION Retroviral production and titration were conducted as described elsewhere.21 GENERATION OF MOUSE IPS CELLS To generate the mouse iPS cells, 5 × 104 mouse TTFs were

seeded on a six-well tissue culture plate, and retroviral infection was performed for 3 days on the mouse TTFs with polybrene (4 μg ml−1). After 3 days, the infected TTFs were transferred to

Mouse Embryonic Fibroblast (MEF) feeder cells to produce mouse iPS cells with mouse iPS medium (DMEM containing 10% horse serum (Sigma-Aldrich, St Louis, MO, USA), 2 mM L-glutamine (Gibco),

0.1 mM MEM NEAA (Gibco), 10 mM HEPES (Gibco), 10 mM β-mercaptoethanol (Gibco), 500 U ml−1 LIF (Millipore, Billerica, MA, USA) and penicillin/streptomycin (Gibco)). ALKALINE PHOSPHATASE

STAINING For the alkaline phosphatase staining, we used the Alkaline Phosphatase Staining Kit (Stemgent, Lexington, MA, USA) according to the manufacturer’s protocol. RT-PCR Total RNA was

extracted from the established mouse iPS cells with the easy-Blue Total RNA Extraction Kit (Intron, Seongnam, Korea) and then reverse-transcribed into first-strand cDNA using the RevertAid H

Minus First Strand cDNA Synthesis Kit (Fermentas, Thermo Fischer Scientific, Carlsbad, CA, USA). The primers against each of the pluripotent genes were described.20 IMMUNOFLUORESCENCE The

mouse iPS colonies were fixed with 4% formaldehyde for 15 min and permeabilized with 0.1% Triton X-100. After incubation with 2% BSA, the cells were incubated overnight at 4 °C with a

primary antibody against Oct4 (Abcam, Cambridge, MA, USA), Sox2 (Millipore), Nanog (Millipore) and SSEA-1 (Santa Cruz, Dallas, TX, USA). The cells were then washed three times in

phosphate-buffered saline and incubated with Alexa Fluor 488 and 594 (Life Technologies, Thermo Fisher Scientific, Carlsbad, CA, USA) for 1 h at room temperature. After washing, the nuclei

were stained with DAPI (Sigma-Aldrich). The stained mouse iPS colonies were mounted on glass slides using Fluorescent Mounting Medium (Dako, Agilent Technologies, Denmark). The fluorescent

images were captured using a LSM-700 confocal microscope (Carl Zeiss, Oberkochen, Germany). _IN VITRO_ DIFFERENTIATION Mouse iPS cells were directly differentiated into three germ layers on

a gelatin-coated dish for 10 days in a medium without a leukemia inhibitory factor. TERATOMA FORMATION For the teratoma formation, we harvested mouse iPS cells and subcutaneously injected 1

× 106 mouse iPS cells into SCID mice. After 6 weeks, the formed teratomas were analyzed by hematoxylin and eosin (H&E) staining. BISULFITE GENOMIC DNA SEQUENCING Genomic DNA was isolated

from mouse embryonic stem cells (mESs), MEFs and miPS cells using the QIAmp DNA Mini Kit (Qiagen, Valencia, CA, USA) for bisulfite modification. Bisulfite modification was performed using a

CpGenome Modification Kit (Millipore) according to the manufacturer’s protocol. Modified genomic DNA was amplified by PCR using a mouse Nanog gene promoter primer.16 The amplified PCR

products were cloned into a T&A Cloning Vector Kit (RBC, New Taipei City, Taiwan) and sequenced using the BIQ Analyzer software (Max Planck Institute, Saarbrucken, Germany). MOTOR NEURON

DIFFERENTIATION OF MIPS CELLS For the motor neuron differentiation, a procedure modified from a previous study was used.22 First, embryoid bodies (EBs) were formed in a suspension culture

for 4 days using an EB medium (DMEM containing 10% FBS (Gibco), 2 mM L-glutamine (Gibco), 0.1 mM MEM NEAA (Gibco), 10 mM HEPES (Gibco), 10 mM β-mercaptoethanol (Gibco) and

penicillin/streptomycin (Gibco)). After 4 days, the EBs were cultured for ~10 days in a tissue culture dish using an insulin-transferrin-selenium (ITS) medium (DMEM/F12 (Gibco) containing

ITS supplement (Gibco) and penicillin/streptomycin (Gibco)) to select neural precursor cells. The ITS medium was replaced every 2 or 3 days. To promote the proliferation of neural precursor

cells, selected neural precursor cells were detached by Trypsin-EDTA and maintained in an N2 medium (DMEM/F12 (Gibco) containing N2 supplement (Gibco) and penicillin/streptomycin (Gibco))

with bFGF (10 ng ml−1). Enriched neural precursor cells were differentiated into motor neurons using retinoic acid (1 μM) and Shh (200 ng ml−1) in an N2 medium. After differentiation, the

motor neurons were cultured in an N2 medium containing ascorbic acid for their maturation and survival. TUNEL ASSAY For the terminal deoxynucleotidyl transferase dUTP nick end labeling

(TUNEL) assay, the DeadEnd Fluorometric TUNEL System Kit (Promega, Madison, WI, USA) was used according to the manufacturer’s protocol. MEASUREMENT OF NEURITE LENGTH To measure the neurites

of differentiated motor neurons, differentiated motor neurons were immunostained with Tuj1 and Islet-1 antibodies. As Tuj1 was expressed in the cytosol, we measured neurite length by

immunostaining Tuj1 from cell body to end of neurite in co-immunostained motor neurons. The length of the neurite was calculated using the measurement function (using ‘Open Bezier’) of the

ZEN analysis program (Carl Zeiss, Oberkochen, Germany). STATISTICAL ANALYSIS The differences between the various experimental groups were calculated using Student’s two-tailed _t_-test.

_P_-values of <0.05 were considered to be statistically significant. RESULTS ESTABLISHMENT OF MIPS CELLS FROM ALS-RELATED TRANSGENIC MICE TTFS We produced mouse iPS cells from primary

mouse TTFs derived from ALS transgenic mice and control mice. First, we obtained fibroblasts from mouse tail-tip tissue and cultured them for 2 weeks for sufficient viral infection (Figure

1b). We reprogrammed the mouse TTFs by infection with four pluripotent genes (Oct4, Sox2, Klf4 and c-Myc) using a retrovirus for 3 days. We then seeded the infected TTFs with MEF feeder

cells to maintain the generation of miPS cells. After 6 days, the first miPS colony was observed in the infected ALS-TTFs (Figure 1a). On day 15 (D15), we picked multiple miPS colonies and

transferred them to other MEF feeder cells (Figure 1c). Next, miPS colonies were counted during D14 to D22 to measure the differences between WT-iPS and ALS-iPS cell lines. Although there

were a few differences in the total number of colonies on a single day, the rate of increase between WT- and ALS-iPS cells was almost equal during the counting period (WT-iPS; 18 and

ALS-iPS; 15 colonies per day) (Figure 1d). Thus, we can conclude that the mutant SOD1 gene did not affect the production of miPS cells. ESTABLISHED MIPS CELLS FROM ALS-MODEL MICE HAD

PLURIPOTENT PROPERTIES Many molecular biological experiments were conducted to characterize the established WT-iPS and ALS-iPS cell lines. To evaluate the iPS properties of these established

miPS cells, we investigated alkaline phosphatase activity (Figure 2a). Alkaline phosphatase staining experiments revealed that the WT-iPS and ALS-iPS cell lines were positive for alkaline

phosphatase activity. Next, we analyzed the mRNA expression of pluripotent genes, such as Ecat1, Nanog, Gdf3, Oct4, Sox2, Rex1 and Zfp296 by RT-PCR (Figure 2b). All cell lines of WT-iPS and

ALS-iPS expressed these genes. Moreover, all cell lines showed immunostaining for pluripotent markers, such as Oct4, Sox2, Nanog and SSEA-1 (Figures 2c and d). To test the _in

vitro_-differentiation ability of the established miPS cells, WT-iPS and ALS-iPS were differentiated in a LIF-free medium. The differentiated WT-iPS and ALS-iPS cells were immunostained with

Tuj-1 (ectoderm), AFP (endoderm) and Desmin (mesoderm) antibodies. The results demonstrate that the established miPS cells differentiated to three germ layers _in vitro_ (Figure 2e).

Furthermore, we also tested the _in vivo_-differentiation potential of the established miPS cells by observing teratoma formation. Teratomas formed within 6 weeks of the injection of the

miPS cells. The teratomas were analyzed by H&E staining (Figure 2f). The H&E staining revealed that the teratomas included three germ layers: epidermis and neural tissue (ectoderm),

muscle and blood (mesoderm), and intestine and gland (endoderm). These results demonstrate that the established miPS cells have the potential to differentiate into three germ layers _in

vivo_. Finally, we investigated epigenetic changes in the Nanog promoter in the established miPS cells (Figure 2g). Bisulfite sequencing analysis showed that the methylation patterns of

WT-iPS and ALS-iPS were similar to the mES cell pattern. Taken together, the results demonstrate that the established miPS had properties of general iPS cells and that mutant SOD1 gene did

not affect pluripotency, differentiation ability and epigenetic change. ALS-IPS CELLS DIFFERENTIATED INTO MOTOR NEURONS _IN VITRO_ Because the features of ALS were confirmed in motor

neurons, we differentiated WT-iPS and ALS-iPS cells into motor neurons using the five-stage differentiation method with minor modifications (Figure 3a). First, we induced EB formation to

differentiate the miPS cells without LIF for 4 days. Well-shaped EBs were observed in the culture dish with ITS medium for 10 days. Selected neural precursor cells were cultured with bFGF to

promote the proliferation of neural precursor cells. Finally, enriched neural precursor cells were differentiated into motor neurons with RA and Shh. We monitored all of the differentiation

steps by observing the differentiating cells (Figure 3b). To test the differentiation of the motor neurons, Islet-1 and Tuj-1 immunostaining experiments were performed. Figure 3c shows

successfully differentiated motor neurons. Moreover, other motor neuron markers, such as HB9 and ChAT, were also observed in the differentiated motor neurons (Figure 3d). We measured the

Islet-1 positive/Tuj-1 positive cells to evaluate the differentiation rate of neural precursor cells into motor neurons. Approximately 86.1 and 87.3% of the neural precursor cells

differentiated into motor neurons among the WT- and ALS-iPS cells, respectively (Figure 3e). Therefore, we can conclude that there was no difference in the motor neuron differentiation

between WT- and ALS-iPS cells and that the mutant SOD1 gene does not affect motor neuron differentiation. ALS-MOTOR NEURONS RECAPITULATE PATHOLOGICAL FEATURES OF ALS Considering that a

decrease in motor neuron neurite length is one of the major characteristics of ALS pathology, we evaluated the motor neuron neurite lengths of induced WT- and ALS-MNs. We immunostained the

cells with Islet-1 and Tuj-1 antibodies to distinguish motor neurons from other cells. To estimate the differences in neurite length between the induced WT- and ALS-MNs, we selected Islet-1

and Tuj-1 double-positive cells and measured neurite lengths using the Tuj-1 immunostaining (Figure 4a). A week after differentiation, the relative neurite length of ALS-MNs (0.68) decreased

more than the neurites of WT-MNs (1) (Figure 4b). Moreover, 3 weeks after differentiation, the relative neurite length of ALS-MNs (0.56) decreased to a greater extent (Figure 4c). These

results demonstrate that relative neurite lengths of ALS-motor neurons continuously decrease in the process of motor neuron maturation. The mutant SOD1 aggregate is the principal

pathological feature of ALS. Intracellular SOD1 aggregates were observed in ALS-MNs (Figure 4d), showing that the mutant SOD1 gene affects the physiology of the motor neuron state. Taken

together, these results let us conclude that the induced ALS-motor neurons recapitulate the pathological features of ALS. CELL SURVIVAL OF ALS MOTOR NEURONS IS DECREASED The most important

characteristic of ALS is death of motor neurons. We investigated motor neuron death using the TUNEL assay. We counted cells that were double positive for Islet-1 and TUNEL to distinguish

motor neuron cells and determine motor neuronal death (Figure 5a). The results showed that the cell survival rate of ALS-MNs was ~47.9% compared with that of WT-MNs (Figure 5b). Furthermore,

we assessed cell death in the iPS stage using the TUNEL assay (Figure 5c). There were 3.4 and 4.1 dead cells per iPS colony in the WT- and ALS-iPS colonies, respectively; thus, WT- and

ALS-iPS colonies appeared to have similar proportions of cell death (Figure 5d). Thus, we can conclude that induced ALS-MNs and WT-MNs did not show any difference of cell death rate in the

embryonic stage, but showed a drastic difference in the motor neuron stage. DISCUSSION The pathological mechanism and medical treatment of many neurodegenerative diseases, including ALS, are

uncertain. Because neurodegenerative diseases have a late onset, the investigation of their causes and processes is very difficult. Although many animal models have been developed, there is

no system for studying the development of ALS from the embryonic to the adult stage. The use of iPS cells has many advantages in neurodegenerative disease research, such as avoiding ethical

considerations and providing patient-specific therapy.17 In the present study, we used iPS cells derived from a transgenic mouse model to reveal the pathological mechanism of ALS. The

results of our study will be helpful in revealing the mechanism of motor neuronal cell death in ALS. We produced mouse iPS cells using TTFs derived from ALS transgenic mice (Figure 1). The

generated miPS cells developed a rounded and brightened appearance on the MEF feeder cells. In particular, we investigated the number of miPS colonies produced after viral infection to

reveal the relationship between the transduced mutant SOD1 gene and miPS-cell production. The results demonstrate that overexpressed mutant SOD1 did not affect the production of the miPS

cells. Moreover, the characterization of the miPS cells suggests that overexpressed mutant SOD1 does not affect the miPS cells. No differences were found between WT-iPS and ALS-iPS cells in

alkaline phosphatase staining, RT-PCR, immunostaining, _in vitro_ differentiation, or _in vivo_ differentiation (Figure 2). Therefore, we can assume that the pathological symptoms of ALS

would not occur in the embryonic stage. To elucidate the differences in the developmental stage of the ALS model, we used a five-stage method to differentiate motor neuron cells from the

miPS cells (Figure 3). We divided motor neuron differentiation into the iPS stage, the neural precursor stage, and the motor neuron stage, similar to that of embryonic development _in vivo_.

Studies of motor neuron differentiation showed that the mutant SOD1 gene did not affect the rate of differentiation of neural precursor cells into motor neurons derived from ALS-iPS. The

major pathology of ALS includes motor neuronal cell death, a decrease in motor neuron neurites, and the presence of mutant SOD1 aggregates.23, 24, 25 In previous studies, these ALS

pathologies were presented in G93A SOD1 transgenic mice, an ALS model. Therefore, we investigated WT- and ALS-motor neurons derived from the miPS cells to confirm whether these pathologies

were reproducible (Figure 4). In this study, the length of ALS-MN neurites decreased more than the neurites from the WT-MNs, and mutant SOD1 aggregates were present in ALS-MNs but not in

WT-MNs. Moreover, the cell survival rate of ALS-MNs also decreased (Figure 5). In particular, the difference in the cell death rate between WT and ALS was present in the motor neuron stage.

Thus, ALS-motor neurons derived from the miPS cells recapitulated various pathologies found in the G93A transgenic mouse model of ALS. Although G93A mutant SOD1 retains native enzyme

activity, oxidative stress may accelerate the aggregation of G93A SOD1.26 Since SOD1 is an anti-oxidant enzyme and converts superoxide to hydrogen peroxide, G93A SOD1 suffers from oxidative

damage more easily than other proteins.6 Moreover, oxidation of SOD1 is caused by hydrogen peroxide, and mutant SOD1 has a more increased affinity for hydrogen peroxide than wild-type

SOD1.27, 28 Thus, oxidized G93A SOD1 proteins have conformational changes that are strongly prone to becoming insoluble aggregates.29 These insoluble aggregates may be the reason for the

fragmented neural fibers observed in the differentiated motor neurons from ALS-iPS cells (Figure 4d). In a previous study,30 researchers generated motor neurons from ALS transgenic

mouse-derived miPS cells, but they did not use the miPS-derived motor neurons in further studies. In the present study, we carried out functional studies of the properties of miPS-derived

motor neurons, showing that miPS cells recapitulate various pathologies in the ALS model. Therefore, ALS-MNs derived from miPS cells could provide an effective model for drug screening or

experimental subjects, although our study was limited to molecular biological research. Further studies should investigate genetic changes of iPS cells and motor neurons. Our study results

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2013; 8: e64720. Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by the Korea Healthcare Technology R&D Project,

Ministry for Health, Welfare & Family Affairs, Republic of Korea (no. A120340), a grant from the National R&D Program for Cancer Control, the Ministry of Health & Welfare,

Republic of Korea (no. 1320010), the National Research Foundation of Korea Grants, the Ministry of Science, ICT and Future Planning, Republic of Korea (NRF-2015R1A4A1041919) and the National

Research Foundation of Korea (NRF) grant (MEST) (NRF-2015R1A2A2A01003516). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Division of Life Sciences, College of Life Sciences and

Biotechnology, Korea University, Seoul, Republic of Korea Ju-Hwang Park & Seongman Kang * Department of Integrated Biomedical and Life Science, College of Health Science, Korea

University, Seoul, Republic of Korea Hang-Soo Park & Sunghoi Hong Authors * Ju-Hwang Park View author publications You can also search for this author inPubMed Google Scholar * Hang-Soo

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mouse iPS cells recapitulate pathological features of ALS. _Exp Mol Med_ 48, e276 (2016). https://doi.org/10.1038/emm.2016.113 Download citation * Received: 07 March 2016 * Revised: 06 July

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