Motor Neuron Generation from iPSCs from Identical Twins Discordant for Amyotrophic Lateral Sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder characterized by the loss of the upper and lower motor neurons. Approximately 10% of cases are caused by specific mutations in known genes, with the remaining cases having no known genetic link. As such, sporadic cases have been more difficult to model experimentally. Here, we describe the generation and differentiation of ALS induced pluripotent stem cells reprogrammed from discordant identical twins. Whole genome sequencing revealed no relevant mutations in known ALS-causing genes that differ between the twins. As protein aggregation is found in all ALS patients and is thought to contribute to motor neuron death, we sought to characterize the aggregation phenotype of the sporadic ALS induced pluripotent stem cells (iPSCs). Motor neurons from both twins had high levels of insoluble proteins that commonly aggregate in ALS that did not robustly change in response to exogenous glutamate. In contrast, established genetic ALS iPSC lines demonstrated insolubility in a protein- and genotype-dependent manner. Moreover, whereas the genetic ALS lines failed to induce autophagy after glutamate stress, motor neurons from both twins and independent controls did activate this protective pathway. Together, these data indicate that our unique model of sporadic ALS may provide key insights into disease pathology and highlight potential differences between sporadic and familial ALS.

Keywords: C9orf72; SOD1; familial ALS; glutamate toxicity; induced pluripotent stem cells; protein aggregation; sporadic ALS.

Figure 1

Successful reprogramming of induced pluripotent stem cells (iPSCs) from discordant identical twins. Two independent clones from each patient express the pluripotency markers Oct4 (A) and Sox2 and Tra 1-81 (B), detected by immunocytochemistry. Scale bar = 50 µm. (C) The transcript levels of Sox2, Oct4, and Nanog are similar to an established iPSC line by qPCR. (D) Embryoid body sections from each patient express markers of the three germ layers, including β-III tubulin (Tuj1, ectoderm), smooth muscle actin (SMA, mesoderm), and α-fetoprotein (AFP, endoderm) detected by immunocytochemistry. Scale bar = 25 µm.

Figure 2

iPSC-derived motor neurons (MNs) from discordant identical twins show no viability or morphological differences. (A) Representative images of MNs from healthy and affected lines stained for ChAT (red) and Tuj1 (green). Scale bar = 25 µm. (B) Representative images show nuclear expression of the MN marker islet 1 (green) in both healthy and affected MN cultures. Western Blot for islet 1 also shows similar protein expression across the healthy and affected MN cultures. An unrelated control line was used as an additional control. Protein was normalized to total protein. (C) The quantification of A shows no difference in the number of ChAT+ MNs at four weeks of total differentiation. n = 3, not significant by Student’s t-test. (D) There is no significant alteration in the length of the neurites at 4 weeks of total differentiation. n = 3, not significant by Student’s t-test.

Figure 3

MNs from the discordant identical twins have similar levels of insoluble protein. (A) Western Blots of soluble protein samples show similar levels of SOD1, optineurin (OPTN), p62, or TDP-43 in the affected and unaffected twin MNs at baseline or after treatment with 100 µM glutamate for 96 h. n = 4–7. (B) There is no appreciable difference in the levels of insoluble SOD1 in the affected MNs compared to unaffected MNs at baseline. Treatment with 100 µM glutamate has a variable effect on aggregation in both cultures. n = 4–7. (C) Insoluble TDP-43 is similar in the affected and unaffected MNs that does not consistently increase or decrease after treatment with 100 µM glutamate. n = 4–7. (D) The levels of insoluble OPTN and p62 in the affected MNs are not significantly different compared to the unaffected MNs. Glutamate treatment has no consistent effect on insolubility. n = 4–7.

Figure 4

SOD1 and C9orf72 motor neurons have variable levels of insoluble protein. (A) SOD1 and C9orf72 MNs have similar levels of soluble SOD1, TDP-43, OPTN, and p62 compared to controls. n = 5–7. (B) Insoluble SOD1 is elevated primarily in SOD1 MNs, but not in C9orf72 MNs. These levels do not change after treatment with 100 µM glutamate. n = 5–7. (C) Western Blot of insoluble TDP-43 shows increased aggregation in SOD1 and C9orf72 MNs. SOD1 and control MNs tend to increase insoluble levels after glutamate stress, while the levels in C9orf72 MNs tend to decrease. n = 5–7. (D) SOD1 MNs tend to show higher levels of insoluble OPTN, whereas C9orf72 MNs show no apparent difference from controls. Insoluble p62 levels are unchanged in SOD1 and C9orf72 MNs compared to controls. n = 5–7.

Figure 5

Alterations in autophagy in familial ALS (fALS) motor neurons. (A) The Western blot for LC3 shows similar levels of autophagy in affected and unaffected MNs at baseline, and MNs from both twins increase autophagic flux after treatment with 100 µM glutamate for 96 h. n = 4–7. (B) SOD1 MNs have an increase in the LC3-II/LC3-I ratio at baseline compared to control MNs, indicating a higher autophagic flux in these cells. Control MNs increase autophagy after a 96-h treatment with 100 µM glutamate, while the SOD1 and C9orf72 MNs do not. n = 5–7.