Modeling hallmark pathology using motor neurons derived from the family and sporadic amyotrophic lateral sclerosis patient-specific iPS cells

Abstract

Background: Amyotrophic lateral sclerosis (ALS) represents a devastating, progressive, heterogeneous, and the most common motor neuron (MN) disease. To date, no cure has been available for the condition. Studies with transgenic mice have yielded significant results that help us understand the underlying mechanisms of ALS. Nonetheless, none of more than 30 large clinical trials over the past 20 years proved successful, which led some researchers to challenge the validity of the preclinical models.

Methods: Human-induced pluripotent cells (iPSCs) were established by introducing Sendai virus into fibroblast cells. We established TDP-43 HES by inserting CAG-TDP43 (G298S) cassette or the CAG-EGFP cassette into PPP1R12C-locus of human embryonic stem cells (ESC, H9) by TALEN-mediated homologous recombination. iPSCs or HESC were differentiated to motor neurons and non-motor neuron as control. Relevant biomarkers were detected in different differentiated stages. TDP-43 aggregates, neurofilament, and mitochondria analyses were performed.

Results: In this study, using iPSCs-derived human MN from an ALS patient with a TDP43 G298S mutation and two sporadic ALS patients, we showed that both sporadic and familial ALS were characterized by TDP-43 aggregates in the surviving MN. Significantly higher neurofilament (NF) inclusion was also found in ALS MN compared with wild-type (WT) GM15 controls (P < 0.05). The neurite mitochondria density was significantly lower in ALS MN than that in the control MNs. Transgenesis of TDP-43 G298S into AAVS locus in human embryonic stem cells reproduced phenotype of patient-derived G289S MN. By challenging MNs with a proteasome inhibitor, we found that MNs were more vulnerable to MG132, with some accompanying phenotype changes, such as TDP43 translocation, NF inclusion, mitochondria distribution impairment, and activation of caspase3.

Conclusions: Our results suggested that changes in TDP43 protein, NF inclusion, and distribution impairment of mitochondria are common early pathology both in familial and sporadic ALS. These findings will help us gain insight into the pathogenesis of the condition and screen relevant drugs for the disease.

Keywords: Amyotrophic lateral sclerosis; Induced pluripotent cells; Mitochondria; Neurofilament; TDP-43.

Fig. 1

iPSC generation. a Contrast image of iPSC colonies generated by Sendai virus. Scale bar = 50 μm. b Alkaline phosphatase (AKP) staining of iPSC. c–f Immunofluorescent image of SOX2 (c), NANOG (d), OCT4 (e), and SSEA-4 (f) expression in ALS iPSCs. g–i HE staining of teratoma from ALS iPSCs showing gut epithelia (endoderm), cartilage (mesoderm), and epiderm (ectoderm) like structures. j Karyotyping of iPSCs. (k) DNA sequencing showing heterozygous nucleotides (C/T) in TDP43, but not sporadic and WT iPSCs

Fig. 2

Neuron differentiation of iPSCs. a Schematic protocol for MN and non-MN differentiation. 3c, 3 small molecular compounds; Pur, purmorphamine; Cyclo, cyclopamine. b Immunofluorescent images of OLIG2⁺ MN progenitors, MNX1⁺postmitotic MNs, CHAT⁺ maturing MNs, SOX2⁺ progenitors, MNX1⁻ and CHAT⁻ spinal non-MNs. c Quantification of TUJ⁺ neuronal population among total Hoechst-labeled (HO) cells and MNX1⁺ MNs among neurons. Scale bar = 50 μm

Fig. 3

TDP43 expression and aggregation in iPSC-derived neurons. a Immunofluorescent image of TDP43 expression in WT, TDP43, and sporadic MN. Translocation of TDP43 was found in G298S TDP43 MN. Scale bar = 50 μm. b TDP43 immuno-EM in neurites, cytoplasm and nuclei of MN cultures. TDP43 MN had a lot of small gold particles in the neurites and cytoplasm, while sporadic MNs had gold particle clusters. Arrows = clusters of gold particles. No contrast staining for ALS MNs for better view of fine gold particles. Scale bar = 2 μm. c Representative Western blots and relative TDP43 expression levels (to GAPDH) in MNs

Fig. 4

NF inclusions in ALS iPSC-derived neurons. a Immunofluorescent images of NF-H, NF-M, and NF-L in CHAT+ MNs. NF staining in the insets is magnified on the right panel. Arrows indicate NF inclusions in the cell body; arrowheads indicate NF inclusions in neurites. Scale bar = 50 μm. b Quantification of NF inclusion-containing cell bodies and neurites in MNs and non-MNs at days 4, 7, and 10 after plating. *p < 0.05. c EM showing NF arrangement in cell body and neuritis of MN cultures. Scale bar = 2 μm. d Immunofluorescent images of NF-H, NF-M, and NF-L in non-MNs. Scale bar = 50 μm

Fig. 5

Mitochondria density decreased on the neurite in ALS MN. a Schematic diagram of conditional expression of NL-EGFP fusion protein in the AAVS1 sites via TALEN. b Phase contrast image of NF inclusion in neurites. Scale bar = 50 μm. c Neurofilament inclusion blocks the mitochondria movement on the neurites. Arrow head indicates colocalization of NF inclusion and mitochondria on neuritis. d Mitotracker staining of mitochondria shows colocalization of NF inclusions and mitochondria in cell body. e, f Mitochondria density decreased in ALS MN, but not in non-MN (*P < 0.05)

Fig. 6

NF aggregation and mitochondria density in WT neurons expressing G298S TDP43. a Schematic diagram showing TALEN-mediated insertion of G298S TDP43 or EGFP into the AAVS1 locus. b Western blots and relative expression of TDP43 (to GPDH) in MNs and non-MN derived from hESCs expressing G298S TDP43 or EGFP. c Phase contrast and corresponding GFP fluorescent images of MNs and non-MNs from the transgenic hESCs. d Immunofluorescent images of NF-H, NF-M, and NF-L in CHAT+ cells from TDP43- and EGFP-expressing hESCs. NF staining in the insets is magnified on the right panel. Arrows indicate NF inclusions in the cell body; arrowheads indicate NF inclusions in neurites. Scale bar = 50 μm. e Quantification of NF inclusion-containing cell bodies (c) and neurites (d) at days 4, 7, and 10 after plating neurons. f Mitochondria number decreased in TDP43-expressing MNs as compared to EGFP-expressing MNs. *p < 0.05. g Immunofluorescent images of NF-H, NF-M, and NF-L in non-MN cells from TDP43- and EGFP-expressing hESCs. Scale bar = 50 μm

Fig. 7

ALS MNs are vulnerable to MG132 challenge. a TDP43 translocation in MN after challenge with MG132. Scale bar = 20 μm. b NF inclusion significantly increased in ALS MNs as compared with WT MN but not in non-MNs after challenge with MG132. Scale bar = 50 μm. c Caspase 3-positive cells increased significantly in ALS MNs compared with WT MN but not in non-MNs after challenge with MG132. (*P < 0.05). Scale bar = 50 μm. d LDH concentration increased in ALS MN compared with WT MN but not non-MN after challenge with MG132. (*P < 0.05). e Immunofluorescent images of NF-H and Caspase3 in non-MN cells from TDP43- and EGFP-expressing hESCs after challenge with MG132. Scale bar = 50 μm