n-Butylidenephthalide recovered calcium homeostasis to ameliorate neurodegeneration of motor neurons derived from amyotrophic lateral sclerosis iPSCs

Abstract

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that causes muscle atrophy and primarily targets motor neurons (MNs). Approximately 20% of familial ALS cases are caused by gain-of-function mutations in superoxide dismutase 1 (SOD1), leading to MN degeneration and ion channel dysfunction. Previous studies have shown that n-Butylidenephthalide (BP) delays disease progression and prolongs survival in animal models of ALS. However, no studies have been conducted on models from human sources. Herein, we examined the protective efficacy of BP on MNs derived from induced pluripotent stem cells (iPSCs) of an ALS patient harboring the SOD1^G85R mutation as well as on those derived from genetically corrected iPSCs (SOD1^G85G). Our results demonstrated that the motor neurons differentiated from iPSC with SOD1^G85R mutation exhibited characteristics of neuron degeneration (as indicated by the reduction of neurofilament expression) and ion channel dysfunction (in response to potassium chloride (KCl) and L-glutamate stimulation), in contrast to those derived from the gene corrected iPSC (SOD1^G85G). Meanwhile, BP treatment effectively restored calcium ion channel function by reducing the expression of glutamate receptors including glutamate ionotropic receptor AMPA type subunit 3 (GluR3) and glutamate ionotropic receptor NMDA type subunit 1 (NMDAR1). Additionally, BP treatment activated autophagic pathway to attenuate neuron degeneration. Overall, this study supports the therapeutic effects of BP on ALS patient-derived neuron cells, and suggests that BP may be a promising candidate for future drug development.

Introduction

Amyotrophic lateral sclerosis (ALS) is one of the most fatal motor neuron (MN) degenerative diseases characterized by the progressive loss of MNs in the spinal cord and brain [1,2]. Within 3–5 years of diagnosis, the disease destroys the MNs that control voluntary muscle movement, causing paralysis and death [3,4]. The estimated global prevalence and incidence of ALS are 4.42 per 100,000 and 1.59 per 100,000, respectively, and there is currently no effective treatment established for it [5].

Approximately 90% of ALS cases are sporadic, and approximately 10% are familial. Of these familial cases, 20% result from mutations in the gene encoding superoxide dismutase 1 (SOD1), an antioxidant enzyme that accounts for 1%–2% of the total soluble protein in the central nervous system (CNS) [6]. Wild type (WT) SOD1 protects neurons against oxidative stress from excessive reactive oxygen species (ROS) generation by converting superoxide anions into oxygen and hydrogen peroxide, whereas SOD1 mutants associated with neurodegenerative disease induce cell death through functional gain, although the exact pathological mechanism remains unclear [1]. Mice expressing the SOD1^G85R mutation are a widely studied ALS disease model as the G85R substitution induces protein misfolding and aggregation, yielding inclusion bodies that cause MN neurotoxicity resembling that of the human disorder [7,8]. It is believed that the SOD1^G85R variant influences copper and zinc binding characteristics at the catalytic site, allowing these metal ions to adversely influence other cellular processes, and conferring other conformational changes that promote protein aggregation and dysfunction [8,9].

There are many factors considered to be the cause of motor neuronal cell death in ALS. The clinical pathology of ALS includes neurofilament (NF) degeneration and loss of essential cytoskeletal functions in MNs [10,11]. Induced pluripotent stem cells (iPSCs) from familial ALS patients produce MNs in culture with reduced neurite length and limited survival [1] due in part to a greater apoptosis rate [12] compared to MNs derived from WT iPSCs. In addition to cytoskeletal dysfunction, MNs harboring ALS-associated mutations exhibit impaired calcium homeostasis, leading to changes in excitability, failure of action potential conduction, and activation of various calcium-dependent degenerative pathways [13–15]. For example, excessive presynaptic glutamate release and overactivation of postsynaptic glutamate receptors result in pathological intracellular calcium accumulation known as excitotoxicity [16,17].

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR) are the primary glutamate receptor subtypes mediating fast excitatory synaptic transmission in the mammalian CNS [18]. These receptors also modulate synaptic strength, especially the NMDAR [19]. The AMPAR is a heteromultimer consisting of various combinations of GluR1, GluR2, GluR3, and GluR4 subunits. Channels consisting of GluR1, GluR3, and GluR4 are permeable to calcium in addition to sodium and potassium, while GluR2 blocks or permits calcium ion impermeability depending on a Q/R polymorphism. Most AMPARs include GluR2, thus the Q/R status frequently determines the calcium permeability. Overexpression of the calcium-impermeable variant in ALS SOD1 transgenic mice reduces MN calcium influx, promotes MN survival, and delays the onset of disease [20,21]. The GluR3 subunit also confers calcium permeability, and expression levels are upregulated at both mRNA and protein levels in SOD1^G93A mutant mice, causing excessive calcium influx and glutamate excitotoxicity [22,23]. However, NMDARs have the greatest calcium permeability among the ionotropic glutamate receptors, and hyperactivation is a major inducer of calcium-dependent neuronal death [24]. These receptors are also heterotetramers, in this case composed of one or more obligatory NR1 subunits plus NR2A–D and (in some cases) NR3A/B. Adding to this complexity, these subunits have alternatively spliced variants [25]. Marcuzzo and colleagues (2019) reported that a SOD1^G93A transgenic mouse exhibited significantly higher levels of NR1 protein and mRNA expression compared to WT mice, while Fuller and colleagues (2006) reported that NR1 is overexpressed in motor nuclei, suggesting contributions to MN loss [26,27]. Consistent with this notion, drugs that can antagonize glutamate receptor activation such as Riluzole can delay the progression of ALS [28]. The glutamate receptor antagonists ketamine, amantadine, memantine, and dextromethorphan are also approved by the United States Food and Drug Administration (FDA) for various indications, including neurodegenerative diseases. Thus, targeting the glutamate receptor may prevent intracellular calcium dysregulation and ensuing excitotoxicity in ALS.

n-Butylidenephthalide (BP) is among the main components extracted from Angelica sinensis oil, an herb used in traditional Chinese medicine for the treatment angina and atherosclerosis, antagonizes numerous common pathogenic processes such as platelet agglutination, inflammation, and apoptosis [29–33]. Furthermore, BP inhibits autophagic activity, inflammation, and oxidative stress to reduce MN loss and prolong the survival of ALS mice [34]. Additionally, BP effectively reduces β-amyloid accumulation and improve short-term memory in an Alzheimer’s disease (AD) mouse model by modulating the LncCYP3A43-2/miR29-2-5p/PSEN1 network [35]. Furthermore, BP reduces neuron cell loss in an animal model of spinocerebellar ataxia (SCA) by regulating autophagy, and reduce the excitotoxicity of ataxin-3 (ATXN3) on Purkinje progenitor cells from SCA3 patient-derived iPSCs by inhibiting the activity of the calcium-activated proteolytic enzyme calpain [36,37].

Although genetically modified animals and cell lines have provided crucial insights into ALS pathogenesis, there are clinically significant anatomical, physiological, and genetic differences between humans and animals that influence ALS pathogenesis and symptom progression [38]. Moreover, there are over 30 familial ALS subtypes, each with a distinctive molecular pathology and constellation of clinical phenotypes, and preclinical models do not fully recapitulate key pathological pathways [39]. Specifically, mutated SOD1 transgenic mice usually require 20–40-fold protein overexpression to exhibit symptoms [40], whereas only a single mutated gene copy confers familial ALS. Moreover, the pathology and sequence induced by mutant overexpression may be distinct despite similar symptoms [41]. In recent years, patient-derived iPSCs have contributed to the development of drugs for neurodegenerative disease treatment [42]. These cells can be applied for cell therapy and research on specific patients without the ethical issues associated with fetal cells, thereby increasing the probability of successful translation from research to clinical trials. In the current study, we used human iPSCs (SOD1^G85R) from ALS patients to derive MNs, and examined the efficacy of BP to improve stability of neurofilament, calcium homeostasis, and ion channel expression in iPSC-derived MNs.

Methods

Human-induced pluripotent stem cell culture

This study used two iPSC lines (SOD1^G85R and SOD1^G85G) derived from ALS patient provided by Dr. C.Y. Chang (Buddhist Tzu Chi Medical Foundation). Mutant iPSCs were isolated from the peripheral blood mononuclear cells of ALS-related patient through reprograming and clonal selection, and CRISPR/Cas9 gene editing was used to correct the mutation for the control cell line SOD1^G85G [3]. Both iPSC populations were incubated in Essential 8™ medium (Thermo Fisher Scientific Waltham, MA, USA), subcultured for approximately 3–4 days until 70%–80% confluent, and detached cells with accutase (Merck-Millipore), subsequently reseeded on plates precoated with Geltrex (Thermo Fisher Scientific). The rho-associated protein kinase (ROCK) inhibitor Y27632 was then added to enhance progenitor cell survival.

MNs differentiation and characterization

The MN differentiation procedure used here was based on Ting’s report [3]. Briefly, 1 mg/ml dispase II (Sigma-Aldrich) was added to iPSC cultures at 80% confluence for 5–10 min until cell colonies rounded and were less adherent. These colonies were then scraped off and incubated in petri dishes (Alpha Plus) containing Essential 6™ medium (Thermo Fisher Scientific) and refreshed 4–6 h after seeding. Subsequently, cultures were incubated for one day to form embryoid bodies (EBs), and then incubated in neural induction medium consisting of DMEM-F12 and DMEM/HG with 1% N-2 Supplement, 1 mM nonessential amino acids (NEAAs), 2 mM glutamine (all procured from Thermo Fisher Scientific), 10 μM SB431542 (Sigma-Aldrich), 10 ng/mL recombinant human FGF-basic (Peprotech), and 3 μM CHIR99021 (Cayman). After 4 days in induction medium, cells were cultured in Neurobasal™ Medium with 1% N-2 Supplement, 2 mM L-glutamine, 1 mM NEAAs, 2 μM SB431542 (Sigma-Aldrich), 0.2 μM LDN193189 (Cayman), 3 μM CHIR99021, 0.5 μM SAG (Cayman), and 0.5 μM retinoic acid (Sigma-Aldrich) to promote neuronal differentiation. During the differentiation period, various biomarkers were analyzed by immunofluorescence staining, as detailed in the next section, with DAPI (Thermo Fisher Scientific) counterstaining to assess differentiation status. Cells were immunostained for Olig2 (Novus Biologicals, Littleton, CO, USA) and Sox1 (R&D Systems, Minneapolis, MN, USA) on day 15 of differentiation, and for HB9 (DSHB) and NF-H (Sigma-Aldrich) on day 28–30. The HB9/DAPI ratio was calculated using ImageJ to quantify the final proportion of differentiated MNs.

Immunofluorescence staining and quantification

Cells seeded on cover glasses (Deckgläser, Thermo Fisher Scientific) were washed with PBS, fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 min at room temperature (RT); washed three times with PBS; incubated on ice with 0.3% Triton-100 (J. T. Baker, Phillipsburg, NJ, USA) for 15 min; blocked using 5% horse serum (Thermo Fisher) at RT for 1 h; and incubated in antibodies against Olig2 (1:500), Sox1 (1:200), HB9 (1:100), and NF-H (1:1000) at 4°C for 16 h. Immunostained cultures were washed with PBST and incubated with Alexa Fluor-conjugated secondary antibodies (donkey anti-mouse IgG (H+L)-Alexa Fluor™ 488, donkey anti-rabbit IgG (H+L)-Alexa Fluor™ 594, donkey anti-goat IgG (H+L)-Alexa Fluor 594, and goat anti-rabbit IgG (H+L)-Alexa Fluor 546; all procured from Thermo Fisher) at RT for 1 h under darkness. Finally, cells were washed with PBST, counterstained with DAPI, and imaged through fluorescence microscopy (Imager Z1, Zeiss, Oberkochen, Germany). Both MN differentiation and neurite area were quantified using ImageJ.

Calcium imaging

Cells seeded on coverslips (Deckgläser) were rinsed with physiological buffer (130 mM NaCl, 5 mM KCl, 2 mM CaCl2, 10 mM HEPES, pH 7.4) containing 10 mM glucose, and incubated with 5 μM Fluo-4 (Thermo Fisher) at 37°C for 40 min under darkness. Then, Fluo-4-loaded cells were transferred to a calcium imaging chamber and washed with physiological buffer under darkness for20 min. The ratio of △F/F₀ was then measured during the indicated treatments for estimation of intracellular calcium. For these experiments, Fluo-4 loaded cells were treated with 60 mM KCl for 30 s, reperfused with physiological buffer for 300 s, and treated with 1 mM L-glutamate for 30 s. On the other hand, using agonist (10 μM S-AMPA, 40 μM NMDA and 10 μM S-AMPA + 40 μM NMDA) to treat Fluo-4-loaded cells for 30 s, and the cells will be washed with physiological buffer for 300 s between each agonist treatment. Fluo-4 emission ratio images were recorded using an ECLIPSE Ti2-E microscope (Nikon, Tokyo, Japan) and analyzed using NIS-Elements AR (Nikon).

Western blotting analysis

Total protein was extracted from cells using RIPA lysis reagent (iNtRON Biotechnology) containing protease inhibitor (Thermo Fisher), separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to PVDF membranes (Merk Millipore) at 4°C for 18 h. Subsequently, membranes were blocked in TBST containing 5% FBS (Thermo Fisher) at RT for 1 hour, and then probed with primary antibodies against GluR3 (GeneTex), NR1 (GeneTex), LC3BII (Abcam), p62 (Abcam), activated/ cleaved caspase-3 (GeneTex), and GAPDH (Merk Millipore) in TBST containing 3% FBS at 4°C for 16 h. Membranes were washed three times with TBST at RT (10 min each), incubated with mouse and rabbit secondary antibodies (Merk Millipore) at RT for 1 h, rewashed three times in PBS, and incubated with horseradish peroxidase chemiluminescent substrate (Merk Millipore). Finally, signals were detected using the iBright CL1000 Imaging System (Thermo Fisher Scientific).

Treatment of BP on MNs

After 35 days of differentiation, SOD1^G85R and SOD1^G85G MNs were treated with 0, 10, or 20 μM BP (Alfa Aesar) for 72 h (according to effective dose reported previously in an AD iPSC model [29]). BP was dissolved in DMSO (Sigma-Aldrich) as the 10% (w/v) BP/DMSO stock. Following the BP treatment, we conducted immunofluorescence staining, calcium imaging, and Western blot analysis to test efficacy and investigate mechanisms.

Statistical analysis

Results are presented as the mean ± standard deviation (SD) of at least three independent experiments. Statistical differences between groups were tested using one-way ANOVA with Dunnet’s test. For the comparison between only two groups (Figs 1E and 2C), a Student’s t-test was used. p-value of <0.05 was considered statistically significant for all tests.

[Figure omitted. See PDF.]

(A) Schematic depicting the MN differentiation protocol. (B) Images showing changes in the morphology of MNs derived from SOD1^G85R iPSCs (mutant) and SOD1^G85G iPSCs (genetically corrected controls) during the differentiation process; scale bar, 300 μm. (C and D) Phenotype characterization of SOD1^G85R and SOD1^G85G iPSC-derived cells on days 15 and 30 of differentiation by immunostaining for the MN progenitor markers SOX1 and Olig2 (red) and the mature MN markers (HB9, green) and NF-H (red), all with DAPI counterstaining (blue). (E) Quantification of the HB9-positive to DAPI (total cell) ratio. Mean ± SD from n = 3 independently treated cultures per treatment group; scale bar, 200 μm.

[Figure omitted. See PDF.]

(A, B and C) Analysis of basal intracellular calcium levels and spontaneous calcium transients in untreated SOD1^G85R iPSC-derived MNs (n = 67) (A and C) and SOD1^G85G iPSC-derived MNs (controls, n = 46) (B and C). (D, E and F) Microfluorometric measurements of intracellular calcium transients in untreated SOD1^G85R iPSC-derived MNs (D), SOD1^G85R iPSC-derived MNs treated with 10 μM BP (E), and SOD1^G85G iPSC-derived MNs (controls) (F) loaded with Fluo-4 and exposed to 60 mM KCl at 60 s and 1 mM L-glutamate at 390 s. (G and H) Average △F/F₀ ratios during peak calcium induced by 60 mM KCl (G) and 1 mM L-glutamate (H). n = 95, 55 and 49 MNs for G85R, BP-G85R and G85G group, respectively. ***p < 0.005 vs. the untreated SOD1^G85R group.

Results

The MN Differentiation ratio of both SOD1^G85R iPSCs and SOD1^G85G iPSCs exceeded 85%

SOD1^G85R iPSCs and SOD1^G85G iPSCs (genetically corrected controls) were established. The neuron differentiation protocol adapted from Ting et al. (2021) was performed [3] (Fig 1A). As depicted in Fig 1B, morphology observation in sequential neural differentiation was performed. On the first day (D0, defined as the EB stage), iPSCs were seeded in Essential 6™ medium, which resulted in the formation of cell clusters with the characteristics of ectoderm, mesoderm, and endoderm (embryoid bodies, EBs). On the following day, EBs were cultured in neuron induction medium for 4 days (days D1 to D4), during which neuroepithelial cells formed a tighter and denser structure around the cell sphere. These neuronal progenitors were then grown in Neurobasal™ Medium to promote MN differentiation. By day 15 of the differentiation period (D15), cells were significantly enlarged and hypertrophic, and exhibited a more compact structure by D28.

To confirm the progression to neuronal differentiation, cultures were analyzed at each stage of the process (EB formation, induction, differentiation) [43]. The cultures were immunostained for SOX1 and Olig2 on D15, and both were found to be widely expressed in SOD1^G85R and SOD1^G85G iPSC-derived progenitor cells (Fig 1C and 1D). Subsequently, cultures were stained for the MN-specific homeobox transcription factor HB9 (Fig 1C and 1D). Calculation of the HB9-positive to DAPI-positive ratio indicated that 85.36% ± 1.290% of SOD1^G85R iPSC-derived cells (ALS) and 86.51% ± 2.433% of SOD1^G85G iPSC-derived cells (controls) were MNs by D28 in differentiation culture (Fig 1E). It revealed no significant difference between MNs from SOD1^G85R iPSCs and SOD1^G85G iPSCs. Thus, mutation in SOD1 ^G85R did not influence that capacity of iPSCs to differentiate into MNs.

Treatment of SOD1^G85R iPSC-derived MNs with BP prevented glutamate-induced excitotoxicity

Calcium dysregulation and intracellular overload is a major contributor of progressive MN loss in ALS [15,44]. Fluo-4-based Ca²⁺ microfluorometry was performed on MNs derived from SOD1^G85R-mutated iPSCs (G85R) and genetically corrected SOD1^G85G iPSCs (G85G). The spontaneous calcium transients of MNs in the G85R and G85G were shown in Fig 2A and 2B. The results showed no significant difference in basal calcium content and spontaneous calcium transients between SOD1^G85R-derived (ALS) and SOD1^G85G-derived (control) groups (Fig 2C, G85R:0.196 ± 0.1251, G85G: 0.154 ± 0.1035). To examine whether BP can improve calcium homeostasis in ALS patient-derived MNs, we compared intracellular calcium responses among untreated and BP-treated SOD1^G85R-derived (ALS) and SOD1^G85G-derived (control). Furthermore, cells were treated with 60 mM KCl to stimulate voltage-gated calcium channels, yielding △F/F₀ intensity ratios (positively correlated with intracellular calcium concentration) of 0.518 ± 0.3356 in untreated SOD1^G85R MNs, 1.063 ± 1.4433 in BP-treated SOD1^G85R MNs, and 1.258 ± 0.8873 in SOD1^G85G MNs (Fig 2D–2F). Thus, voltage-gated calcium influx was 2.4-fold lower in SOD1^G85R (ALS model) MNs compared to genetically corrected controls, indicating dysfunctional voltage-gated calcium channels or excitability mechanisms in ALS. However, voltage-gated calcium influx was restored by BP treatment (Fig 2G). Conversely, stimulation with L-glutamate evoked substantially greater calcium influx into untreated SOD1^G85R MNs (△F/F₀ = 3.130 ± 1.3304) compared to BP-treated SOD1^G85R MNs and control SOD1^G85G MNs (-0.044 ± 0.3724 and 0.265 ± 0.1904, respectively). Thus, while voltage-gated calcium influx was deficient, glutamate-induced influx was excessive in SOD1^G85R MNs, and this abnormality was recovered by BP (Fig 2D–2H). These findings revealed that BP treatment can restore voltage-gated influx and prevent glutamate-induced excitotoxicity in MNs carrying an ALS-associated SOD1^G85R mutation.

BP downregulated glutamate receptor overexpression in SOD1 mutant MNs

Calcium influx induced by glutamatergic synaptic transmission is mediated mainly by AMPARs and NMDARs, and overactivation can cause selective death of MNs [45]. Therefore, we investigated whether BP (10 and 20 μM) could improve calcium ion overload by regulating the expression of AMPARs and NMDARs. The Western blotting results revealed that BP reduced NMDAR1expression (Fig 3A and 3B) and GluR3 expression (Fig 3C and 3D). In the 20 μM BP treatment group, expression levels of GluR3 were reduced to the level of SOD1^G85G MNs. BP treatment reversed the overexpression of GluR3 (an AMPAR subunit) and NMDAR1 (an NMDAR subunit). This downregulation may explain how BP mitigated excessive glutamate-induced calcium influx and ameliorates excitotoxicity.

[Figure omitted. See PDF.]

(A and C) Western blotting for NMDAR-NR1 (A) and AMPAR-GluR3 (C) expression in untreated SOD1^G85R iPSC-derived MNs, SOD1^G85R iPSC-derived MNs treated with BP (10 and 20 μM), and SOD1^G85G iPSC-derived MNs (controls). (B and D) Quantification of the signal was performed using ImageJ.

Treatment of SOD1^G85R MNs with BP suppressed AMPAR and NMDAR agonist-induced excitotoxicity

The effects of BP in preventing glutamate-induced excitotoxicity and downregulating glutamate receptor overexpression in SOD1^G85R MNs were confirmed in this study. We also examined the influence of BP on calcium flux through specific glutamate receptors by stimulating them with receptor-specific agonists, including S-AMPA (an AMPAR-specific agonist) and NMDA (an NMDAR-specific agonist). Cells treated with S-AMPA, NMDA, or S-AMPA + NMDA were examined through calcium imaging, which showed that SOD1^G85R MNs exhibited stronger calcium flux in response to all agonist stimulations (S-AMPA: 0.491 ± 0.4262, NMDA: 0.147 ± 0.1312; S-AMPA + NMDA: 0.596 ± 0.5328) (Fig 4A) compared to SOD1^G85G MNs (S-AMPA: 0.044 ± 0.0724; NMDA: 0.093 ± 0.0508; S-AMPA+NMDA: 0.017 ± 0.0888) (Fig 4C). Furthermore, after BP treatment, the calcium flux in SOD1^G85R MNs decreased, returning to a level similar to that of SOD1^G85G MNs (S-AMPA: 0.130 ± 0.0617; NMDA: 0.055 ± 0.0773; S-AMPA+NMDA: 0.247 ± 0.1486) (Fig 4B). Calculations of the calcium flux induced by S-AMPA, NMDA, and S-AMPA + NMDA in all groups were presented in Fig 4D–4F. The calcium flux induced by S-AMPA (Fig 4D), NMDA (Fig 4E), and S-AMPA + NMDA (Fig 4F) were significantly lower in SOD1^G85G MNs compared to that of SOD1^G85R MNs. It was further revealed that BP treatment significantly reduced the calcium flux in SOD1^G85R MNs, bringing it to a level comparable to that of SOD1^G85G MNs. Additionally, BP was found to be more effective at inhibiting excessive S-AMPA-induced calcium influx compared to NMDA-induced calcium influx (Fig 4D and 4E). Collectively, our Western blot and calcium imaging results confirmed that BP restored normal calcium influx by inhibiting the expression of AMPAR and NMDAR in SOD1 mutant MNs.

[Figure omitted. See PDF.]

(A, B and C) Microfluorometric measurements of intracellular calcium transients in untreated SOD1^G85R iPSC-derived MNs (A), SOD1^G85R iPSC-derived MNs treated with 10 μM BP (B), and SOD1^G85G iPSC-derived MNs (controls) (C) loaded with Fluo-4 and exposed to S-AMPA at 120 s, NMDA at 450 s and S-AMPA + NMDA at 780 s. (D, E and F) Average △F/F₀ ratios during peak calcium induced by S-AMPA (D), NMDA (E) and S-AMPA + 40 μM NMDA (F). n = 30, 41 and 32 MNs for G85R, BP-G85R and G85G group, respectively. * p < 0.05 and ***p < 0.005 vs. the untreated SOD1^G85R group.

BP inhibited cell apoptosis and attenuated neurite degeneration in SOD1 mutant MNs

Accumulation of misfolded proteins and ensuing structural damage to neurites and axons is a pathological hallmark of ALS [10,46]. To study whether BP can diminish neurite degeneration and reduce motor neurons loss, SOD1^G85R and SOD1^G85G MNs treated with 10 μM BP for 72 h were immunostained for NF-H to present their neurite areas. The NF-H expression rate of control SOD1^G85G MNs was 100%, whereas that of SOD1^G85R MNs was merely 55.91% (Fig 5B). However, BP treatment increased this ratio to 84.62%, and neurite morphology was more robust than that in untreated SOD1^G85R MNs (Fig 5A and 5B), revealing that BP can inhibit NF degeneration associated with the SOD1^G85R mutation. The expression levels of activated caspase-3 were examined because it has been reported that this apoptosis effector protease is strongly associated with MN loss and damage due to ALS-related SOD1 mutations in cultured mouse spinal cord MNs and iPSC models [1,47,48]. Expression levels of activated caspase-3 was 27% higher in untreated ALS-SOD1^G85R MNs compared to SOD1^G85G MNs, but was reduced almost to the level of the SOD1^G85G group by BP treatment (Fig 5C and 5D). Thus, we demonstrated that BP prevented MN death in the iPSC model of ALS, possibly by blocking the apoptosis pathway.

[Figure omitted. See PDF.]

(A) Neurite density of untreated SOD1^G85R iPSC-derived MNs (left panel), SOD1^G85R iPSC-derived MNs treated with 10 μM BP (middle panel), and genetically corrected SOD1^G85G iPSC-derived MNs (right panel) as revealed by NF-H immunofluorescence staining (typical results from n = 5 cultures per group). Scale bar = 200 μm. (B) Quantification of neurite area/cell based on NF-H immunofluorescence staining using ImageJ; 60 to 195 cells per view were counted. * p < 0.05 and ** p < 0.01 vs. the untreated SOD1^G85R group. (C and D) Western blotting for the apoptosis marker activated (cleaved) caspase-3. Expression levels in SOD1^G85R, BP treatment (10 and 20 μM), and SOD1^G85G (control) groups in iPSC-derived MNs were quantified using ImageJ.

BP could activate autophagy in SOD1 mutant MNs

Calcium homeostasis influences apoptosis by regulating various proteolytic processes as well as autophagy, which is a biologically conserved pathway for the elimination of damaged cellular macromolecules and recycling of the constituents [49]. Therefore, we investigated whether BP can reduce MNs loss and restore calcium homeostasis related to autophagic pathway using the human iPSCs model. Expression of the early stage autophagy marker LC3BII was substantially lower in untreated ALS-SOD1^G85R MNs compared to SOD1^G85G MNs but was enhanced to the level of SOD1^G85G MNs by BP (Fig 6A and 6B). Conversely, expression of the late stage autophagy marker p62 was substantially higher in SOD1^G85R MNs than SOD1^G85G MNs, but again was reduced to near SOD1^G85G MN levels by 20 μM BP (Fig 6C and 6D). Collectively, our results indicated that the autophagy pathway was induced after BP treatment, which further resulted in the prevention of apoptotic MN death in SOD1^G85R MNs.

[Figure omitted. See PDF.]

(A and C) Western blots of the early autophagy marker LC3BII (A) and late-stage marker p62 (C) from untreated SOD1^G85R iPSC-derived MNs, SOD1^G85R iPSC-derived MNs treated with BP (10 and 20 μM), and SOD1^G85G iPSC-derived MNs. (B and D) Quantification of the signal was performed using ImageJ.

Discussion

Dysregulation of intracellular calcium homeostasis is widely regarded as a key pathomechanism common to multiple neurodegenerative diseases, including ALS [44]. A sustained elevation in intracellular calcium concentration can induce endoplasmic reticulum (ER) stress and disruption of post-translational protein processing, failure of mitochondrial ATP generation, activation of catabolic enzymes that damage cellular macromolecules, and generation of ROS leading to oxidative stress [16,24,27,50,51]. Preventing unregulated calcium increases or restoring cellular calcium homeostasis is thus a promising therapeutic strategy. Here we demonstrate that BP treatment can reduce glutamate-induced calcium influx in ALS patient-derived MNs, likely by reversing overexpression of NMDA NMDAR1 and AMPA GluR3 subunits. Similarly, BP was reported to reduce activities of the calcium-activated proteases calpastatin and calpain in a SCA model, leading to a reduction in toxic protein fragment formation and ensuing neurotoxicity [36,52]. Thus, treatment with BP may be generally effective for restoring neuronal calcium homeostasis in neurodegenerative diseases of distinct etiology.

In contrast to this reduction in glutamate-induced calcium influx, BP enhanced KCl-induced influx. Superfusion of high-KCl is commonly used in physiological research to enhance membrane excitability, promote nerve conduction [53], and activate voltage-gated calcium channels (VGCCs) [54]. We suggest that this restoration of VGCC-mediated influx may also contribute to a therapeutic effect by restoring normal membrane excitability and synaptic communication.

Based on evidence for excitotoxicity in neurodegenerative diseases, several glutamate receptor antagonists have been developed as potential therapeutics. For example, the competitive NMDAR antagonist Riluzole is FDA-approved for reducing neuronal excitotoxicity in ALS. In addition to inhibiting NMDAR overexpression and potential hyperactivity; however, BP can also reduce the expression of AMPARs, ionotropic receptor-channels that can both enhance neuronal excitability and in some molecular conformations directly mediate calcium influx. Therefore, BP may act as a multimodal protective agent against calcium overload and neuronal excitotoxicity.

In addition to regulating the expression of glutamate receptors, BP enhanced the autophagic pathway (Fig 5), consistent with the BP treatment response reported in a SCA-iPSC model and with these studies suggesting that BP enhances autophagy and influences autophagosome formation [37]. However, the autophagic pathway was inhibited by BP treatment in a SOD1^G93A mutant mouse model of ALS, and the effect actually prolonged mouse survival [34]. This discrepancy may be explained by different mutations and distinct downstream pathogenic mechanisms. Also, it is speculated that gene copy number varies between iPSCs and animal models, again leading to distinct pathogenesis [40,41]. Compared to the MNs derived from normal iPSCs, down-regulation of autophagy markers was observed in the MNs derived from C9ORF72 mutated iPSCs [55]. However, compared to WT mice, up-regulation of autophagy markers was observed in C9ORF72 Transgenic Mice [56]. These discrepancies may reflect differences in the duration of drug treatment. For example, the antihistaminergic clemastine differentially regulated autophagy in an ALS model depending on duration [57], with short-term treatment increasing LC3BII marker expression but long-term administration reducing LC3BII expression. In the current study as well, a 72-h treated enhanced LC3BII expression (Fig 5A) while a 3-h treatment was found to inhibit LC3BII expression in the SOD1^G93A mutant mouse model of ALS [34]. The duration of BP treatment may thus differentially influence the expression of other proteins, potentially including VGCCs and glutamate receptor subunits. Additional studies are needed to examine the effects of different BP doses and durations on various ALS models.

Although our iPSC-based ALS model provided a precise representation that closely mimics real patient conditions, and we successfully identified potential compounds and related mechanisms for ALS therapy, there are several limitations to this study. First, we did not conduct extensive testing of BP’s effective concentration and treatment duration. However, BP has been previously applied in neurological models, including both animal and cell models [29,34–37], which may allow us to predict the potential effective concentration and treatment duration in our model based on prior reports. The second limitation lies in the strategies used to uncover the potential mechanisms of BP in ALS. In this study, we did not employ systematic approaches such as RNA sequencing or proteomic analysis; therefore, the detailed mechanisms of BP remain to be further explored in future research. Additionally, our ALS motor neuron (MN) model has its own limitations. We utilized G85R SOD1 mutant MNs for further studies, which restricts the findings to a specific mutant type of familial ALS. The applicability of our findings to multiple genetic mutations and sporadic ALS needs further investigation. Moreover, our model is a simplified, single-cell type model that lacks interactions between different cell types, such as glial cells, muscles, and immune cells. Therefore, a comprehensive 3D organoid or animal model is necessary to evaluate the clinical potential of our findings.

In the current model, increased autophagy may have contributed to the improvement in calcium homeostasis and the reduction in apoptosis signaling. In addition, BP increased the neurite density of SOD1^G85R iPSC-derived MNs compared to the untreated group, suggesting potential benefits for information processing. Previous studies have suggested that BP may stimulate neurogenesis [37], and Chi and coworkers (2018) reported that BP increased the neural differentiation of adipose-derived stem cells [58]. We suggest that BP may protect neurons both by preventing apoptosis and by promoting nerve regeneration from progenitors.

Conclusion

As summarized in Fig 7, we demonstrated that BP can prevent or reverse neuron degeneration, reduce the overexpression of glutamate receptor subunits GluR3 and NMDAR1, inhibit excessive glutamate-induced calcium influx, and prohibit hyperactivated apoptosis signaling in motor neurons derived from ALS patient iPSCs harboring the SOD1^G85R mutation. Moreover, enhanced autophagy may contribute to these effects, although further experimental verification is required. These findings highlight the therapeutic potential of BP for ALS and the utility of this experimental system as a drug-screening platform for neurodegenerative diseases.

[Figure omitted. See PDF.]

In ALS, excess glutamate release overstimulates calcium-permeable postsynaptic AMPARs and NMDARs. In addition, reversal of the glutamate transporter adds to this synaptic glutamate and stimulates further calcium influx into postsynaptic neurons. Calcium overload induces cell damage and eventually apoptosis and neuronal degeneration. The results of this study suggest that BP can reverse GluR3 and NR1 overexpression, thereby reducing excessive calcium accumulation in postsynaptic motor neurons. Furthermore, BP can also activate the autophagic pathway to prevent MN apoptosis.

Supporting information

S1 Fig. Original blot images in this study.

https://doi.org/10.1371/journal.pone.0311573.s001

S2 Fig. Original immunofluorescent images in this study.

https://doi.org/10.1371/journal.pone.0311573.s002

S1 File.

https://doi.org/10.1371/journal.pone.0311573.s003

(XLSX)

S1 Raw images.

https://doi.org/10.1371/journal.pone.0311573.s004

References

1. 1. Park JH, Park HS, Hong S, Kang S. Motor neurons derived from ALS-related mouse iPS cells recapitulate pathological features of ALS. Exp Mol Med. 2016;48(12):e276. Epub 20161209. pmid:27932790.

* View Article

* PubMed/NCBI

* Google Scholar

2. 2. Zhao S, Chen R, Gao Y, Lu Y, Bai X, Zhang J. Fundamental roles of the Optineurin gene in the molecular pathology of Amyotrophic Lateral Sclerosis. Front Neurosci. 2023;17:1319706. Epub 20231221. pmid:38178841.

* View Article

* PubMed/NCBI

* Google Scholar

3. 3. Ting HC, Yang HI, Harn HJ, Chiu IM, Su HL, Li X, et al. Coactivation of GSK3beta and IGF-1 Attenuates Amyotrophic Lateral Sclerosis Nerve Fiber Cytopathies in SOD1 Mutant Patient-Derived Motor Neurons. Cells. 2021;10(10). Epub 20211016. pmid:34685754.

* View Article

* PubMed/NCBI

* Google Scholar

4. 4. Robberecht W, Philips T. The changing scene of amyotrophic lateral sclerosis. Nat Rev Neurosci. 2013;14(4):248–64. Epub 20130306. pmid:23463272.

* View Article

* PubMed/NCBI

* Google Scholar

5. 5. Xu L, Liu T, Liu L, Yao X, Chen L, Fan D, et al. Global variation in prevalence and incidence of amyotrophic lateral sclerosis: a systematic review and meta-analysis. J Neurol. 2020;267(4):944–53. Epub 20191203. pmid:31797084.

* View Article

* PubMed/NCBI

* Google Scholar

6. 6. Gill C, Phelan JP, Hatzipetros T, Kidd JD, Tassinari VR, Levine B, et al. SOD1-positive aggregate accumulation in the CNS predicts slower disease progression and increased longevity in a mutant SOD1 mouse model of ALS. Sci Rep. 2019;9(1):6724. Epub 20190430. pmid:31040321.

* View Article

* PubMed/NCBI

* Google Scholar

7. 7. Wang J, Farr GW, Hall DH, Li F, Furtak K, Dreier L, et al. An ALS-linked mutant SOD1 produces a locomotor defect associated with aggregation and synaptic dysfunction when expressed in neurons of Caenorhabditis elegans. PLoS Genet. 2009;5(1):e1000350. Epub 20090123. pmid:19165329.

* View Article

* PubMed/NCBI

* Google Scholar

8. 8. Boyd SD, Ullrich MS, Calvo JS, Behnia F, Meloni G, Winkler DD. Mutations in Superoxide Dismutase 1 (Sod1) Linked to Familial Amyotrophic Lateral Sclerosis Can Disrupt High-Affinity Zinc-Binding Promoted by the Copper Chaperone for Sod1 (Ccs). Molecules. 2020;25(5). Epub 20200228. pmid:32121118.

* View Article

* PubMed/NCBI

* Google Scholar

9. 9. Cao X, Antonyuk SV, Seetharaman SV, Whitson LJ, Taylor AB, Holloway SP, et al. Structures of the G85R variant of SOD1 in familial amyotrophic lateral sclerosis. J Biol Chem. 2008;283(23):16169–77. Epub 20080331. pmid:18378676.

* View Article

* PubMed/NCBI

* Google Scholar

10. 10. Carpenter S. Proximal axonal enlargement in motor neuron disease. Neurology. 1968;18(9):841–51. pmid:4176657.

* View Article

* PubMed/NCBI

* Google Scholar

11. 11. Buck E, Oeckl P, Grozdanov V, Bopp V, Kuhlwein JK, Ruf WP, et al. Increased NF-L levels in the TDP-43(G298S) ALS mouse model resemble NF-L levels in ALS patients. Acta Neuropathol. 2022;144(1):161–4. Epub 20220518. pmid:35585288.

* View Article

* PubMed/NCBI

* Google Scholar

12. 12. Kiskinis E, Sandoe J, Williams Luis A, Boulting Gabriella L, Moccia R, Wainger Brian J, et al. Pathways Disrupted in Human ALS Motor Neurons Identified through Genetic Correction of Mutant SOD1. Cell Stem Cell. 2014;14(6). pmid:24704492

* View Article

* PubMed/NCBI

* Google Scholar

13. 13. Yang M, Liu M, Sanchez YF, Avazzadeh S, Quinlan LR, Liu G, et al. A novel protocol to derive cervical motor neurons from induced pluripotent stem cells for amyotrophic lateral sclerosis. Stem Cell Reports. 2023;18(9):1870–83. Epub 20230817. pmid:37595581.

* View Article

* PubMed/NCBI

* Google Scholar

14. 14. Griffioen G. Calcium Dyshomeostasis Drives Pathophysiology and Neuronal Demise in Age-Related Neurodegenerative Diseases. Int J Mol Sci. 2023;24(17). Epub 20230826. pmid:37686048.

* View Article

* PubMed/NCBI

* Google Scholar

15. 15. Verma M, Lizama BN, Chu CT. Excitotoxicity, calcium and mitochondria: a triad in synaptic neurodegeneration. Transl Neurodegener. 2022;11(1):3. Epub 20220125. pmid:35078537.

* View Article

* PubMed/NCBI

* Google Scholar

16. 16. Hayashi R, Ishikawa Y, Sasamoto Y, Katori R, Nomura N, Ichikawa T, et al. Co-ordinated ocular development from human iPS cells and recovery of corneal function. Nature. 2016;531(7594):376–80. Epub 20160309. pmid:26958835.

* View Article

* PubMed/NCBI

* Google Scholar

17. 17. Satarker S, Bojja SL, Gurram PC, Mudgal J, Arora D, Nampoothiri M. Astrocytic Glutamatergic Transmission and Its Implications in Neurodegenerative Disorders. Cells. 2022;11(7). Epub 20220328. pmid:35406702.

* View Article

* PubMed/NCBI

* Google Scholar

18. 18. Hansen KB, Wollmuth LP, Bowie D, Furukawa H, Menniti FS, Sobolevsky AI, et al. Structure, Function, and Pharmacology of Glutamate Receptor Ion Channels. Pharmacol Rev. 2021;73(4):298–487. pmid:34753794.

* View Article

* PubMed/NCBI

* Google Scholar

19. 19. Italia M, Ferrari E, Di Luca M, Gardoni F. GluA3-containing AMPA receptors: From physiology to synaptic dysfunction in brain disorders. Neurobiol Dis. 2021;161:105539. Epub 20211029. pmid:34743951.

* View Article

* PubMed/NCBI

* Google Scholar

20. 20. Bailey JM, Colón-Rodríguez A, Atchison WD. Evaluating a Gene-Environment Interaction in Amyotrophic Lateral Sclerosis: Methylmercury Exposure and Mutated SOD1. Curr Environ Health Rep. 2017;4(2):200–7. pmid:28397096.

* View Article

* PubMed/NCBI

* Google Scholar

21. 21. Tateno M, Sadakata H, Tanaka M, Itohara S, Shin RM, Miura M, et al. Calcium-permeable AMPA receptors promote misfolding of mutant SOD1 protein and development of amyotrophic lateral sclerosis in a transgenic mouse model. Hum Mol Genet. 2004;13(19):2183–96. Epub 20040804. pmid:15294873.

* View Article

* PubMed/NCBI

* Google Scholar

22. 22. Tortarolo M, Grignaschi G, Calvaresi N, Zennaro E, Spaltro G, Colovic M, et al. Glutamate AMPA receptors change in motor neurons of SOD1G93A transgenic mice and their inhibition by a noncompetitive antagonist ameliorates the progression of amytrophic lateral sclerosis-like disease. J Neurosci Res. 2006;83(1):134–46. pmid:16323214.

* View Article

* PubMed/NCBI

* Google Scholar

23. 23. Spalloni A, Albo F, Ferrari F, Mercuri N, Bernardi G, Zona C, et al. Cu/Zn-superoxide dismutase (GLY93—>ALA) mutation alters AMPA receptor subunit expression and function and potentiates kainate-mediated toxicity in motor neurons in culture. Neurobiol Dis. 2004;15(2):340–50. pmid:15006704.

* View Article

* PubMed/NCBI

* Google Scholar

24. 24. Sibarov DA, Antonov SM. Calcium-Dependent Desensitization of NMDA Receptors. Biochemistry (Mosc). 2018;83(10):1173–83. pmid:30472955.

* View Article

* PubMed/NCBI

* Google Scholar

25. 25. Duffield GE, Mikkelsen JD, Ebling FJ. Conserved expression of the glutamate NMDA receptor 1 subunit splice variants during the development of the Siberian hamster suprachiasmatic nucleus. PLoS One. 2012;7(5):e37496. Epub 20120531. pmid:22675426.

* View Article

* PubMed/NCBI

* Google Scholar

26. 26. Marcuzzo S, Terragni B, Bonanno S, Isaia D, Cavalcante P, Cappelletti C, et al. Hyperexcitability in Cultured Cortical Neuron Networks from the G93A-SOD1 Amyotrophic Lateral Sclerosis Model Mouse and its Molecular Correlates. Neuroscience. 2019;416:88–99. Epub 20190807. pmid:31400485.

* View Article

* PubMed/NCBI

* Google Scholar

27. 27. Fuller PI, Reddrop C, Rodger J, Bellingham MC, Phillips JK. Differential expression of the NMDA NR2B receptor subunit in motoneuron populations susceptible and resistant to amyotrophic lateral sclerosis. Neurosci Lett. 2006;399(1–2):157–61. Epub 20060220. pmid:16490316.

* View Article

* PubMed/NCBI

* Google Scholar

28. 28. Tzeplaeff L, Wilfling S, Requardt MV, Herdick M. Current State and Future Directions in the Therapy of ALS. Cells. 2023;12(11). Epub 20230531. pmid:37296644.

* View Article

* PubMed/NCBI

* Google Scholar

29. 29. Chang CY, Chen SM, Lu HE, Lai SM, Lai PS, Shen PW, et al. N-butylidenephthalide attenuates Alzheimer’s disease-like cytopathy in Down syndrome induced pluripotent stem cell-derived neurons. Sci Rep. 2015;5:8744. Epub 20150304. pmid:25735452.

* View Article

* PubMed/NCBI

* Google Scholar

30. 30. Fu RH, Harn HJ, Liu SP, Chen CS, Chang WL, Chen YM, et al. n-butylidenephthalide protects against dopaminergic neuron degeneration and α-synuclein accumulation in Caenorhabditis elegans models of Parkinson’s disease. PLoS One. 2014;9(1):e85305. Epub 20140108. pmid:24416384.

* View Article

* PubMed/NCBI

* Google Scholar

31. 31. Liu PY, Sheu JJ, Lin PC, Lin CT, Liu YJ, Ho LI, et al. Expression of Nur77 induced by an n-butylidenephthalide derivative promotes apoptosis and inhibits cell growth in oral squamous cell carcinoma. Invest New Drugs. 2012;30(1):79–89. Epub 20100901. pmid:20809206.

* View Article

* PubMed/NCBI

* Google Scholar

32. 32. Tsai NM, Chen YL, Lee CC, Lin PC, Cheng YL, Chang WL, et al. The natural compound n-butylidenephthalide derived from Angelica sinensis inhibits malignant brain tumor growth in vitro and in vivo. J Neurochem. 2006;99(4):1251–62. Epub 20060920. pmid:16987298.

* View Article

* PubMed/NCBI

* Google Scholar

33. 33. Chan SS, Choi AO, Jones RL, Lin G. Mechanisms underlying the vasorelaxing effects of butylidenephthalide, an active constituent of Ligusticum chuanxiong, in rat isolated aorta. Eur J Pharmacol. 2006;537(1–3):111–7. Epub 20060320. pmid:16624277.

* View Article

* PubMed/NCBI

* Google Scholar

34. 34. Hsueh KW, Chiou TW, Chiang SF, Yamashita T, Abe K, Borlongan CV, et al. Autophagic down-regulation in motor neurons remarkably prolongs the survival of ALS mice. Neuropharmacology. 2016;108:152–60. Epub 20160406. pmid:27059126.

* View Article

* PubMed/NCBI

* Google Scholar

35. 35. Wuli W, Lin SZ, Chen SP, Tannous BA, Huang WS, Woon PY, et al. Targeting PSEN1 by lnc-CYP3A43-2/miR-29b-2-5p to Reduce beta Amyloid Plaque Formation and Improve Cognition Function. Int J Mol Sci. 2022;23(18). Epub 20220911. pmid:36142465.

* View Article

* PubMed/NCBI

* Google Scholar

36. 36. Yang HH, Chiang IT, Liu JW, Hsieh J, Lee JH, Lu HE, et al. Anti-Excitotoxic Effects of N-Butylidenephthalide Revealed by Chemically Insulted Purkinje Progenitor Cells Derived from SCA3 iPSCs. Int J Mol Sci. 2022;23(3). Epub 20220126. pmid:35163312.

* View Article

* PubMed/NCBI

* Google Scholar

37. 37. Lee JH, Lin SY, Liu JW, Lin SZ, Harn HJ, Chiou TW. n-Butylidenephthalide Modulates Autophagy to Ameliorate Neuropathological Progress of Spinocerebellar Ataxia Type 3 through mTOR Pathway. Int J Mol Sci. 2021;22(12). Epub 20210613. pmid:34199295.

* View Article

* PubMed/NCBI

* Google Scholar

38. 38. Lutz C. Mouse models of ALS: Past, present and future. Brain Res. 2018;1693(Pt A):1–10. Epub 20180322. pmid:29577886.

* View Article

* PubMed/NCBI

* Google Scholar

39. 39. Fisher EMC, Greensmith L, Malaspina A, Fratta P, Hanna MG, Schiavo G, et al. Opinion: more mouse models and more translation needed for ALS. Mol Neurodegener. 2023;18(1):30. Epub 20230504. pmid:37143081.

* View Article

* PubMed/NCBI

* Google Scholar

40. 40. Julien JP, Kriz J. Transgenic mouse models of amyotrophic lateral sclerosis. Biochim Biophys Acta. 2006;1762(11–12):1013–24. Epub 20060421. pmid:16675207.

* View Article

* PubMed/NCBI

* Google Scholar

41. 41. Chen H, Qian K, Du Z, Cao J, Petersen A, Liu H, et al. Modeling ALS with iPSCs reveals that mutant SOD1 misregulates neurofilament balance in motor neurons. Cell Stem Cell. 2014;14(6):796–809. Epub 20140403. pmid:24704493.

* View Article

* PubMed/NCBI

* Google Scholar

42. 42. Okano H, Morimoto S. iPSC-based disease modeling and drug discovery in cardinal neurodegenerative disorders. Cell Stem Cell. 2022;29(2):189–208. pmid:35120619.

* View Article

* PubMed/NCBI

* Google Scholar

43. 43. Shimojo D, Onodera K, Doi-Torii Y, Ishihara Y, Hattori C, Miwa Y, et al. Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells. Mol Brain. 2015;8(1):79. Epub 20151201. pmid:26626025.

* View Article

* PubMed/NCBI

* Google Scholar

44. 44. Tadic V, Prell T, Lautenschlaeger J, Grosskreutz J. The ER mitochondria calcium cycle and ER stress response as therapeutic targets in amyotrophic lateral sclerosis. Front Cell Neurosci. 2014;8:147. Epub 20140530. pmid:24910594.

* View Article

* PubMed/NCBI

* Google Scholar

45. 45. Guo C, Ma YY. Calcium Permeable-AMPA Receptors and Excitotoxicity in Neurological Disorders. Front Neural Circuits. 2021;15:711564. Epub 20210817. pmid:34483848.

* View Article

* PubMed/NCBI

* Google Scholar

46. 46. Hirano A, Nakano I, Kurland LT, Mulder DW, Holley PW, Saccomanno G. Fine structural study of neurofibrillary changes in a family with amyotrophic lateral sclerosis. J Neuropathol Exp Neurol. 1984;43(5):471–80. pmid:6540800.

* View Article

* PubMed/NCBI

* Google Scholar

47. 47. Garcera A, Bahi N, Periyakaruppiah A, Arumugam S, Soler RM. Survival motor neuron protein reduction deregulates autophagy in spinal cord motoneurons in vitro. Cell Death Dis. 2013;4(6):e686. Epub 20130620. pmid:23788043

* View Article

* PubMed/NCBI

* Google Scholar

48. 48. Pasinelli P, Houseweart MK, Brown RH Jr., Cleveland DW. Caspase-1 and -3 are sequentially activated in motor neuron death in Cu,Zn superoxide dismutase-mediated familial amyotrophic lateral sclerosis. Proc Natl Acad Sci U S A. 2000;97(25):13901–6. pmid:11095709.

* View Article

* PubMed/NCBI

* Google Scholar

49. 49. De Marco G, Lomartire A, Manera U, Canosa A, Grassano M, Casale F, et al. Effects of intracellular calcium accumulation on proteins encoded by the major genes underlying amyotrophic lateral sclerosis. Sci Rep. 2022;12(1):395. Epub 20220110. pmid:35013445.

* View Article

* PubMed/NCBI

* Google Scholar

50. 50. Bootman MD, Bultynck G. Fundamentals of Cellular Calcium Signaling: A Primer. Cold Spring Harb Perspect Biol. 2020;12(1). Epub 20200102. pmid:31427372.

* View Article

* PubMed/NCBI

* Google Scholar

51. 51. Bruijn LI, Becher MW, Lee MK, Anderson KL, Jenkins NA, Copeland NG, et al. ALS-linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD1-containing inclusions. Neuron. 1997;18(2):327–38. pmid:9052802.

* View Article

* PubMed/NCBI

* Google Scholar

52. 52. Rajamani K, Liu JW, Wu CH, Chiang IT, You DH, Lin SY, et al. n-Butylidenephthalide exhibits protection against neurotoxicity through regulation of tryptophan 2, 3 dioxygenase in spinocerebellar ataxia type 3. Neuropharmacology. 2017;117:434–46. Epub 20170220. pmid:28223212.

* View Article

* PubMed/NCBI

* Google Scholar

53. 53. Rienecker KDA, Poston RG, Saha RN. Merits and Limitations of Studying Neuronal Depolarization-Dependent Processes Using Elevated External Potassium. ASN Neuro. 2020;12:1759091420974807. pmid:33256465.

* View Article

* PubMed/NCBI

* Google Scholar

54. 54. Mueller BH, 2nd, Park Y, Daudt DR, 3rd, Ma HY, Akopova I, Stankowska DL, et al. Sigma-1 receptor stimulation attenuates calcium influx through activated L-type Voltage Gated Calcium Channels in purified retinal ganglion cells. Exp Eye Res. 2013;107:21–31. Epub 20121123. pmid:23183135.

* View Article

* PubMed/NCBI

* Google Scholar

55. 55. Madill M, McDonagh K, Ma J, Vajda A, McLoughlin P, O’Brien T, et al. Amyotrophic lateral sclerosis patient iPSC-derived astrocytes impair autophagy via non-cell autonomous mechanisms. Mol Brain. 2017;10(1):22. Epub 20170613. pmid:28610619.

* View Article

* PubMed/NCBI

* Google Scholar

56. 56. Zhu Q, Jiang J, Gendron TF, McAlonis-Downes M, Jiang L, Taylor A, et al. Reduced C9ORF72 function exacerbates gain of toxicity from ALS/FTD-causing repeat expansion in C9orf72. Nat Neurosci. 2020;23(5):615–24. Epub 20200413. pmid:32284607.

* View Article

* PubMed/NCBI

* Google Scholar

57. 57. Apolloni S, Fabbrizio P, Amadio S, Volonté C. Actions of the antihistaminergic clemastine on presymptomatic SOD1-G93A mice ameliorate ALS disease progression. J Neuroinflammation. 2016;13(1):191. Epub 20160822. pmid:27549088.

* View Article

* PubMed/NCBI

* Google Scholar

58. 58. Chi K, Fu RH, Huang YC, Chen SY, Hsu CJ, Lin SZ, et al. Adipose-derived Stem Cells Stimulated with n-Butylidenephthalide Exhibit Therapeutic Effects in a Mouse Model of Parkinson’s Disease. Cell Transplant. 2018;27(3):456–70. Epub 20180514. pmid:29756519.

* View Article

* PubMed/NCBI

* Google Scholar

Citation: Deng Y-C, Liu J-W, Ting H-C, Kuo T-C, Chiang C-H, Lin E-Y, et al. (2024) n-Butylidenephthalide recovered calcium homeostasis to ameliorate neurodegeneration of motor neurons derived from amyotrophic lateral sclerosis iPSCs. PLoS ONE 19(11): e0311573. https://doi.org/10.1371/journal.pone.0311573

About the Authors:

Yu-Chen Deng

Roles: Conceptualization, Data curation, Formal analysis, Investigation, Validation, Writing – original draft, Writing – review & editing

Affiliations: Department of Biochemical and Molecular Medical Sciences, National Dong Hwa University, Hualien, Taiwan, Everfront Biotech Inc., Taipei, Taiwan

ORICD: https://orcid.org/0009-0001-3392-0765

Jen-Wei Liu

Roles: Formal analysis, Methodology, Writing – original draft

Affiliation: Everfront Biotech Inc., Taipei, Taiwan

Hsiao-Chien Ting

Roles: Methodology

Affiliation: Bioinnovation Center, Buddhist Tzu Chi Medical Foundation, Hualien, Taiwan

Tzu-Chen Kuo

Roles: Investigation, Validation, Visualization

Affiliation: Department of Biochemical and Molecular Medical Sciences, National Dong Hwa University, Hualien, Taiwan

Chia-Hung Chiang

Roles: Formal analysis, Investigation

Affiliation: Department of Biochemical and Molecular Medical Sciences, National Dong Hwa University, Hualien, Taiwan

En-Yi Lin

Roles: Investigation, Methodology

Affiliations: Department of Biochemical and Molecular Medical Sciences, National Dong Hwa University, Hualien, Taiwan, Bioinnovation Center, Buddhist Tzu Chi Medical Foundation, Hualien, Taiwan

Horng-Jyh Harn

Roles: Conceptualization, Supervision, Writing – review & editing

Affiliations: Bioinnovation Center, Buddhist Tzu Chi Medical Foundation, Hualien, Taiwan, Department of Pathology, Hualien Tzu Chi Hospital, Hualien, Taiwan

Shinn-Zong Lin

Roles: Resources

Affiliations: Bioinnovation Center, Buddhist Tzu Chi Medical Foundation, Hualien, Taiwan, Department of Neurosurgery, Hualien Tzu Chi Hospital, Hualien, Taiwan

ORICD: https://orcid.org/0000-0002-4601-9933

Chia-Yu Chang

Roles: Conceptualization, Funding acquisition, Resources, Supervision, Writing – review & editing

E-mail: [email protected] (T-WC); [email protected] (C-YC)

Affiliations: Bioinnovation Center, Buddhist Tzu Chi Medical Foundation, Hualien, Taiwan, Department of Nursing, Tzu Chi University of Science and Technology, Hualien, Taiwan, Department of Medical Research, Hualien Tzu Chi Hospital, Hualien, Taiwan

Tzyy-Wen Chiou

Roles: Conceptualization, Funding acquisition, Resources, Supervision, Writing – review & editing

E-mail: [email protected] (T-WC); [email protected] (C-YC)

Affiliation: Department of Biochemical and Molecular Medical Sciences, National Dong Hwa University, Hualien, Taiwan

References

1. Park JH, Park HS, Hong S, Kang S. Motor neurons derived from ALS-related mouse iPS cells recapitulate pathological features of ALS. Exp Mol Med. 2016;48(12):e276. Epub 20161209. pmid:27932790.

2. Zhao S, Chen R, Gao Y, Lu Y, Bai X, Zhang J. Fundamental roles of the Optineurin gene in the molecular pathology of Amyotrophic Lateral Sclerosis. Front Neurosci. 2023;17:1319706. Epub 20231221. pmid:38178841.

3. Ting HC, Yang HI, Harn HJ, Chiu IM, Su HL, Li X, et al. Coactivation of GSK3beta and IGF-1 Attenuates Amyotrophic Lateral Sclerosis Nerve Fiber Cytopathies in SOD1 Mutant Patient-Derived Motor Neurons. Cells. 2021;10(10). Epub 20211016. pmid:34685754.

4. Robberecht W, Philips T. The changing scene of amyotrophic lateral sclerosis. Nat Rev Neurosci. 2013;14(4):248–64. Epub 20130306. pmid:23463272.

5. Xu L, Liu T, Liu L, Yao X, Chen L, Fan D, et al. Global variation in prevalence and incidence of amyotrophic lateral sclerosis: a systematic review and meta-analysis. J Neurol. 2020;267(4):944–53. Epub 20191203. pmid:31797084.

6. Gill C, Phelan JP, Hatzipetros T, Kidd JD, Tassinari VR, Levine B, et al. SOD1-positive aggregate accumulation in the CNS predicts slower disease progression and increased longevity in a mutant SOD1 mouse model of ALS. Sci Rep. 2019;9(1):6724. Epub 20190430. pmid:31040321.

7. Wang J, Farr GW, Hall DH, Li F, Furtak K, Dreier L, et al. An ALS-linked mutant SOD1 produces a locomotor defect associated with aggregation and synaptic dysfunction when expressed in neurons of Caenorhabditis elegans. PLoS Genet. 2009;5(1):e1000350. Epub 20090123. pmid:19165329.

8. Boyd SD, Ullrich MS, Calvo JS, Behnia F, Meloni G, Winkler DD. Mutations in Superoxide Dismutase 1 (Sod1) Linked to Familial Amyotrophic Lateral Sclerosis Can Disrupt High-Affinity Zinc-Binding Promoted by the Copper Chaperone for Sod1 (Ccs). Molecules. 2020;25(5). Epub 20200228. pmid:32121118.

9. Cao X, Antonyuk SV, Seetharaman SV, Whitson LJ, Taylor AB, Holloway SP, et al. Structures of the G85R variant of SOD1 in familial amyotrophic lateral sclerosis. J Biol Chem. 2008;283(23):16169–77. Epub 20080331. pmid:18378676.

10. Carpenter S. Proximal axonal enlargement in motor neuron disease. Neurology. 1968;18(9):841–51. pmid:4176657.

11. Buck E, Oeckl P, Grozdanov V, Bopp V, Kuhlwein JK, Ruf WP, et al. Increased NF-L levels in the TDP-43(G298S) ALS mouse model resemble NF-L levels in ALS patients. Acta Neuropathol. 2022;144(1):161–4. Epub 20220518. pmid:35585288.

12. Kiskinis E, Sandoe J, Williams Luis A, Boulting Gabriella L, Moccia R, Wainger Brian J, et al. Pathways Disrupted in Human ALS Motor Neurons Identified through Genetic Correction of Mutant SOD1. Cell Stem Cell. 2014;14(6). pmid:24704492

13. Yang M, Liu M, Sanchez YF, Avazzadeh S, Quinlan LR, Liu G, et al. A novel protocol to derive cervical motor neurons from induced pluripotent stem cells for amyotrophic lateral sclerosis. Stem Cell Reports. 2023;18(9):1870–83. Epub 20230817. pmid:37595581.

14. Griffioen G. Calcium Dyshomeostasis Drives Pathophysiology and Neuronal Demise in Age-Related Neurodegenerative Diseases. Int J Mol Sci. 2023;24(17). Epub 20230826. pmid:37686048.

15. Verma M, Lizama BN, Chu CT. Excitotoxicity, calcium and mitochondria: a triad in synaptic neurodegeneration. Transl Neurodegener. 2022;11(1):3. Epub 20220125. pmid:35078537.

16. Hayashi R, Ishikawa Y, Sasamoto Y, Katori R, Nomura N, Ichikawa T, et al. Co-ordinated ocular development from human iPS cells and recovery of corneal function. Nature. 2016;531(7594):376–80. Epub 20160309. pmid:26958835.

17. Satarker S, Bojja SL, Gurram PC, Mudgal J, Arora D, Nampoothiri M. Astrocytic Glutamatergic Transmission and Its Implications in Neurodegenerative Disorders. Cells. 2022;11(7). Epub 20220328. pmid:35406702.

18. Hansen KB, Wollmuth LP, Bowie D, Furukawa H, Menniti FS, Sobolevsky AI, et al. Structure, Function, and Pharmacology of Glutamate Receptor Ion Channels. Pharmacol Rev. 2021;73(4):298–487. pmid:34753794.

19. Italia M, Ferrari E, Di Luca M, Gardoni F. GluA3-containing AMPA receptors: From physiology to synaptic dysfunction in brain disorders. Neurobiol Dis. 2021;161:105539. Epub 20211029. pmid:34743951.

20. Bailey JM, Colón-Rodríguez A, Atchison WD. Evaluating a Gene-Environment Interaction in Amyotrophic Lateral Sclerosis: Methylmercury Exposure and Mutated SOD1. Curr Environ Health Rep. 2017;4(2):200–7. pmid:28397096.

21. Tateno M, Sadakata H, Tanaka M, Itohara S, Shin RM, Miura M, et al. Calcium-permeable AMPA receptors promote misfolding of mutant SOD1 protein and development of amyotrophic lateral sclerosis in a transgenic mouse model. Hum Mol Genet. 2004;13(19):2183–96. Epub 20040804. pmid:15294873.

22. Tortarolo M, Grignaschi G, Calvaresi N, Zennaro E, Spaltro G, Colovic M, et al. Glutamate AMPA receptors change in motor neurons of SOD1G93A transgenic mice and their inhibition by a noncompetitive antagonist ameliorates the progression of amytrophic lateral sclerosis-like disease. J Neurosci Res. 2006;83(1):134–46. pmid:16323214.

23. Spalloni A, Albo F, Ferrari F, Mercuri N, Bernardi G, Zona C, et al. Cu/Zn-superoxide dismutase (GLY93—>ALA) mutation alters AMPA receptor subunit expression and function and potentiates kainate-mediated toxicity in motor neurons in culture. Neurobiol Dis. 2004;15(2):340–50. pmid:15006704.

24. Sibarov DA, Antonov SM. Calcium-Dependent Desensitization of NMDA Receptors. Biochemistry (Mosc). 2018;83(10):1173–83. pmid:30472955.

25. Duffield GE, Mikkelsen JD, Ebling FJ. Conserved expression of the glutamate NMDA receptor 1 subunit splice variants during the development of the Siberian hamster suprachiasmatic nucleus. PLoS One. 2012;7(5):e37496. Epub 20120531. pmid:22675426.

26. Marcuzzo S, Terragni B, Bonanno S, Isaia D, Cavalcante P, Cappelletti C, et al. Hyperexcitability in Cultured Cortical Neuron Networks from the G93A-SOD1 Amyotrophic Lateral Sclerosis Model Mouse and its Molecular Correlates. Neuroscience. 2019;416:88–99. Epub 20190807. pmid:31400485.

27. Fuller PI, Reddrop C, Rodger J, Bellingham MC, Phillips JK. Differential expression of the NMDA NR2B receptor subunit in motoneuron populations susceptible and resistant to amyotrophic lateral sclerosis. Neurosci Lett. 2006;399(1–2):157–61. Epub 20060220. pmid:16490316.

28. Tzeplaeff L, Wilfling S, Requardt MV, Herdick M. Current State and Future Directions in the Therapy of ALS. Cells. 2023;12(11). Epub 20230531. pmid:37296644.

29. Chang CY, Chen SM, Lu HE, Lai SM, Lai PS, Shen PW, et al. N-butylidenephthalide attenuates Alzheimer’s disease-like cytopathy in Down syndrome induced pluripotent stem cell-derived neurons. Sci Rep. 2015;5:8744. Epub 20150304. pmid:25735452.

30. Fu RH, Harn HJ, Liu SP, Chen CS, Chang WL, Chen YM, et al. n-butylidenephthalide protects against dopaminergic neuron degeneration and α-synuclein accumulation in Caenorhabditis elegans models of Parkinson’s disease. PLoS One. 2014;9(1):e85305. Epub 20140108. pmid:24416384.

31. Liu PY, Sheu JJ, Lin PC, Lin CT, Liu YJ, Ho LI, et al. Expression of Nur77 induced by an n-butylidenephthalide derivative promotes apoptosis and inhibits cell growth in oral squamous cell carcinoma. Invest New Drugs. 2012;30(1):79–89. Epub 20100901. pmid:20809206.

32. Tsai NM, Chen YL, Lee CC, Lin PC, Cheng YL, Chang WL, et al. The natural compound n-butylidenephthalide derived from Angelica sinensis inhibits malignant brain tumor growth in vitro and in vivo. J Neurochem. 2006;99(4):1251–62. Epub 20060920. pmid:16987298.

33. Chan SS, Choi AO, Jones RL, Lin G. Mechanisms underlying the vasorelaxing effects of butylidenephthalide, an active constituent of Ligusticum chuanxiong, in rat isolated aorta. Eur J Pharmacol. 2006;537(1–3):111–7. Epub 20060320. pmid:16624277.

34. Hsueh KW, Chiou TW, Chiang SF, Yamashita T, Abe K, Borlongan CV, et al. Autophagic down-regulation in motor neurons remarkably prolongs the survival of ALS mice. Neuropharmacology. 2016;108:152–60. Epub 20160406. pmid:27059126.

35. Wuli W, Lin SZ, Chen SP, Tannous BA, Huang WS, Woon PY, et al. Targeting PSEN1 by lnc-CYP3A43-2/miR-29b-2-5p to Reduce beta Amyloid Plaque Formation and Improve Cognition Function. Int J Mol Sci. 2022;23(18). Epub 20220911. pmid:36142465.

36. Yang HH, Chiang IT, Liu JW, Hsieh J, Lee JH, Lu HE, et al. Anti-Excitotoxic Effects of N-Butylidenephthalide Revealed by Chemically Insulted Purkinje Progenitor Cells Derived from SCA3 iPSCs. Int J Mol Sci. 2022;23(3). Epub 20220126. pmid:35163312.

37. Lee JH, Lin SY, Liu JW, Lin SZ, Harn HJ, Chiou TW. n-Butylidenephthalide Modulates Autophagy to Ameliorate Neuropathological Progress of Spinocerebellar Ataxia Type 3 through mTOR Pathway. Int J Mol Sci. 2021;22(12). Epub 20210613. pmid:34199295.

38. Lutz C. Mouse models of ALS: Past, present and future. Brain Res. 2018;1693(Pt A):1–10. Epub 20180322. pmid:29577886.

39. Fisher EMC, Greensmith L, Malaspina A, Fratta P, Hanna MG, Schiavo G, et al. Opinion: more mouse models and more translation needed for ALS. Mol Neurodegener. 2023;18(1):30. Epub 20230504. pmid:37143081.

40. Julien JP, Kriz J. Transgenic mouse models of amyotrophic lateral sclerosis. Biochim Biophys Acta. 2006;1762(11–12):1013–24. Epub 20060421. pmid:16675207.

41. Chen H, Qian K, Du Z, Cao J, Petersen A, Liu H, et al. Modeling ALS with iPSCs reveals that mutant SOD1 misregulates neurofilament balance in motor neurons. Cell Stem Cell. 2014;14(6):796–809. Epub 20140403. pmid:24704493.

42. Okano H, Morimoto S. iPSC-based disease modeling and drug discovery in cardinal neurodegenerative disorders. Cell Stem Cell. 2022;29(2):189–208. pmid:35120619.

43. Shimojo D, Onodera K, Doi-Torii Y, Ishihara Y, Hattori C, Miwa Y, et al. Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells. Mol Brain. 2015;8(1):79. Epub 20151201. pmid:26626025.

44. Tadic V, Prell T, Lautenschlaeger J, Grosskreutz J. The ER mitochondria calcium cycle and ER stress response as therapeutic targets in amyotrophic lateral sclerosis. Front Cell Neurosci. 2014;8:147. Epub 20140530. pmid:24910594.

45. Guo C, Ma YY. Calcium Permeable-AMPA Receptors and Excitotoxicity in Neurological Disorders. Front Neural Circuits. 2021;15:711564. Epub 20210817. pmid:34483848.

46. Hirano A, Nakano I, Kurland LT, Mulder DW, Holley PW, Saccomanno G. Fine structural study of neurofibrillary changes in a family with amyotrophic lateral sclerosis. J Neuropathol Exp Neurol. 1984;43(5):471–80. pmid:6540800.

47. Garcera A, Bahi N, Periyakaruppiah A, Arumugam S, Soler RM. Survival motor neuron protein reduction deregulates autophagy in spinal cord motoneurons in vitro. Cell Death Dis. 2013;4(6):e686. Epub 20130620. pmid:23788043

48. Pasinelli P, Houseweart MK, Brown RH Jr., Cleveland DW. Caspase-1 and -3 are sequentially activated in motor neuron death in Cu,Zn superoxide dismutase-mediated familial amyotrophic lateral sclerosis. Proc Natl Acad Sci U S A. 2000;97(25):13901–6. pmid:11095709.

49. De Marco G, Lomartire A, Manera U, Canosa A, Grassano M, Casale F, et al. Effects of intracellular calcium accumulation on proteins encoded by the major genes underlying amyotrophic lateral sclerosis. Sci Rep. 2022;12(1):395. Epub 20220110. pmid:35013445.

50. Bootman MD, Bultynck G. Fundamentals of Cellular Calcium Signaling: A Primer. Cold Spring Harb Perspect Biol. 2020;12(1). Epub 20200102. pmid:31427372.

51. Bruijn LI, Becher MW, Lee MK, Anderson KL, Jenkins NA, Copeland NG, et al. ALS-linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD1-containing inclusions. Neuron. 1997;18(2):327–38. pmid:9052802.

52. Rajamani K, Liu JW, Wu CH, Chiang IT, You DH, Lin SY, et al. n-Butylidenephthalide exhibits protection against neurotoxicity through regulation of tryptophan 2, 3 dioxygenase in spinocerebellar ataxia type 3. Neuropharmacology. 2017;117:434–46. Epub 20170220. pmid:28223212.

53. Rienecker KDA, Poston RG, Saha RN. Merits and Limitations of Studying Neuronal Depolarization-Dependent Processes Using Elevated External Potassium. ASN Neuro. 2020;12:1759091420974807. pmid:33256465.

54. Mueller BH, 2nd, Park Y, Daudt DR, 3rd, Ma HY, Akopova I, Stankowska DL, et al. Sigma-1 receptor stimulation attenuates calcium influx through activated L-type Voltage Gated Calcium Channels in purified retinal ganglion cells. Exp Eye Res. 2013;107:21–31. Epub 20121123. pmid:23183135.

55. Madill M, McDonagh K, Ma J, Vajda A, McLoughlin P, O’Brien T, et al. Amyotrophic lateral sclerosis patient iPSC-derived astrocytes impair autophagy via non-cell autonomous mechanisms. Mol Brain. 2017;10(1):22. Epub 20170613. pmid:28610619.

56. Zhu Q, Jiang J, Gendron TF, McAlonis-Downes M, Jiang L, Taylor A, et al. Reduced C9ORF72 function exacerbates gain of toxicity from ALS/FTD-causing repeat expansion in C9orf72. Nat Neurosci. 2020;23(5):615–24. Epub 20200413. pmid:32284607.

57. Apolloni S, Fabbrizio P, Amadio S, Volonté C. Actions of the antihistaminergic clemastine on presymptomatic SOD1-G93A mice ameliorate ALS disease progression. J Neuroinflammation. 2016;13(1):191. Epub 20160822. pmid:27549088.

58. Chi K, Fu RH, Huang YC, Chen SY, Hsu CJ, Lin SZ, et al. Adipose-derived Stem Cells Stimulated with n-Butylidenephthalide Exhibit Therapeutic Effects in a Mouse Model of Parkinson’s Disease. Cell Transplant. 2018;27(3):456–70. Epub 20180514. pmid:29756519.