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 SOD1G85R mutation as well as on those derived from genetically corrected iPSCs (SOD1G85G). Our results demonstrated that the motor neurons differentiated from iPSC with SOD1G85R 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 (SOD1G85G). 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.

Fig 1. Differentiation of human motor neurons (MNs) from the induced pluripotent stem cells (iPSCs) of an amyotrophic lateral sclerosis (ALS) patient.

(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.

Fig 2. Treatment with BP restores voltage-gated calcium channel function and reduces excessive calcium influx in ALS-SOD1 mutant MNs.

(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.

Fig 3. Treatment with BP inhibits AMPAR and NMDAR glutamate receptor overexpression in ALS-SOD1 mutant MNs.

(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.

Fig 4. Treatment with BP can effectively attenuate excessive calcium influx triggered by S-AMPA and NMDA in ALS-SOD1 mutant MNs.

(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.

Fig 5. Treatment with BP attenuates neurite degeneration and apoptosis in MNs harboring the ALS-associated SOD1^G85R mutation.

(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.

Fig 6. Treatment with BP activates the autophagic pathway in ALS-SOD1 mutant MNs.

(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.

Fig 7. Schematic illustrating the mechanisms by which BP treatment may reduce glutamate excitotoxicity in SOD1^G85R motor neurons.

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.