Necroptosis drives motor neuron death in models of both sporadic and familial ALS

doi:10.1016/j.neuron.2014.01.011

. 2014 Mar 5;81(5):1001-1008.

doi: 10.1016/j.neuron.2014.01.011. Epub 2014 Feb 6.

Necroptosis drives motor neuron death in models of both sporadic and familial ALS

Diane B Re¹, Virginia Le Verche¹, Changhao Yu¹, Mackenzie W Amoroso², Kristin A Politi¹, Sudarshan Phani¹, Burcin Ikiz¹, Lucas Hoffmann³, Martijn Koolen⁴, Tetsuya Nagata¹, Dimitra Papadimitriou¹, Peter Nagy¹, Hiroshi Mitsumoto⁵, Shingo Kariya¹, Hynek Wichterle⁶, Christopher E Henderson⁷, Serge Przedborski⁸

Affiliations

¹ Center for Motor Neuron Biology and Disease, the Columbia Translational Neuroscience Initiative, and the Columbia Stem Cell Initiative, Columbia University, New York, NY 10032, USA; Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA.
² Center for Motor Neuron Biology and Disease, the Columbia Translational Neuroscience Initiative, and the Columbia Stem Cell Initiative, Columbia University, New York, NY 10032, USA; Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA; Department of Rehabilitation and Regenerative Medicine, Columbia University, New York, NY 10032, USA; Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, New York, NY 10032, USA.
³ Center for Motor Neuron Biology and Disease, the Columbia Translational Neuroscience Initiative, and the Columbia Stem Cell Initiative, Columbia University, New York, NY 10032, USA.
⁴ Center for Motor Neuron Biology and Disease, the Columbia Translational Neuroscience Initiative, and the Columbia Stem Cell Initiative, Columbia University, New York, NY 10032, USA; Academisch Medisch Centrum, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands.
⁵ Center for Motor Neuron Biology and Disease, the Columbia Translational Neuroscience Initiative, and the Columbia Stem Cell Initiative, Columbia University, New York, NY 10032, USA; Department of Neurology, Columbia University, New York, NY 10032, USA.
⁶ Center for Motor Neuron Biology and Disease, the Columbia Translational Neuroscience Initiative, and the Columbia Stem Cell Initiative, Columbia University, New York, NY 10032, USA; Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA; Department of Neuroscience, Columbia University, New York, NY 10032, USA; Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, New York, NY 10032, USA.
⁷ Center for Motor Neuron Biology and Disease, the Columbia Translational Neuroscience Initiative, and the Columbia Stem Cell Initiative, Columbia University, New York, NY 10032, USA; Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA; Department of Neurology, Columbia University, New York, NY 10032, USA; Department of Neuroscience, Columbia University, New York, NY 10032, USA; Department of Rehabilitation and Regenerative Medicine, Columbia University, New York, NY 10032, USA; Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, New York, NY 10032, USA.
⁸ Center for Motor Neuron Biology and Disease, the Columbia Translational Neuroscience Initiative, and the Columbia Stem Cell Initiative, Columbia University, New York, NY 10032, USA; Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA; Department of Neurology, Columbia University, New York, NY 10032, USA. Electronic address: sp30@columbia.edu.

PMID: 24508385
PMCID: PMC3951532
DOI: 10.1016/j.neuron.2014.01.011

Necroptosis drives motor neuron death in models of both sporadic and familial ALS

Diane B Re et al. Neuron. 2014.

. 2014 Mar 5;81(5):1001-1008.

doi: 10.1016/j.neuron.2014.01.011. Epub 2014 Feb 6.

Authors

Affiliations

¹ Center for Motor Neuron Biology and Disease, the Columbia Translational Neuroscience Initiative, and the Columbia Stem Cell Initiative, Columbia University, New York, NY 10032, USA; Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA.
² Center for Motor Neuron Biology and Disease, the Columbia Translational Neuroscience Initiative, and the Columbia Stem Cell Initiative, Columbia University, New York, NY 10032, USA; Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA; Department of Rehabilitation and Regenerative Medicine, Columbia University, New York, NY 10032, USA; Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, New York, NY 10032, USA.
³ Center for Motor Neuron Biology and Disease, the Columbia Translational Neuroscience Initiative, and the Columbia Stem Cell Initiative, Columbia University, New York, NY 10032, USA.
⁴ Center for Motor Neuron Biology and Disease, the Columbia Translational Neuroscience Initiative, and the Columbia Stem Cell Initiative, Columbia University, New York, NY 10032, USA; Academisch Medisch Centrum, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands.
⁵ Center for Motor Neuron Biology and Disease, the Columbia Translational Neuroscience Initiative, and the Columbia Stem Cell Initiative, Columbia University, New York, NY 10032, USA; Department of Neurology, Columbia University, New York, NY 10032, USA.
⁶ Center for Motor Neuron Biology and Disease, the Columbia Translational Neuroscience Initiative, and the Columbia Stem Cell Initiative, Columbia University, New York, NY 10032, USA; Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA; Department of Neuroscience, Columbia University, New York, NY 10032, USA; Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, New York, NY 10032, USA.
⁷ Center for Motor Neuron Biology and Disease, the Columbia Translational Neuroscience Initiative, and the Columbia Stem Cell Initiative, Columbia University, New York, NY 10032, USA; Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA; Department of Neurology, Columbia University, New York, NY 10032, USA; Department of Neuroscience, Columbia University, New York, NY 10032, USA; Department of Rehabilitation and Regenerative Medicine, Columbia University, New York, NY 10032, USA; Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, New York, NY 10032, USA.
⁸ Center for Motor Neuron Biology and Disease, the Columbia Translational Neuroscience Initiative, and the Columbia Stem Cell Initiative, Columbia University, New York, NY 10032, USA; Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA; Department of Neurology, Columbia University, New York, NY 10032, USA. Electronic address: sp30@columbia.edu.

PMID: 24508385
PMCID: PMC3951532
DOI: 10.1016/j.neuron.2014.01.011

Abstract

Most cases of neurodegenerative diseases are sporadic, hindering the use of genetic mouse models to analyze disease mechanisms. Focusing on the motor neuron (MN) disease amyotrophic lateral sclerosis (ALS), we therefore devised a fully humanized coculture model composed of human adult primary sporadic ALS (sALS) astrocytes and human embryonic stem-cell-derived MNs. The model reproduces the cardinal features of human ALS: sALS astrocytes, but not those from control patients, trigger selective death of MNs. The mechanisms underlying this non-cell-autonomous toxicity were investigated in both astrocytes and MNs. Although causal in familial ALS (fALS), SOD1 does not contribute to the toxicity of sALS astrocytes. Death of MNs triggered by either sALS or fALS astrocytes occurs through necroptosis, a form of programmed necrosis involving receptor-interacting protein 1 and the mixed lineage kinase domain-like protein. The necroptotic pathway therefore constitutes a potential therapeutic target for this incurable disease.

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Figures

**Figure 1. Primary sporadic ALS astrocytes specifically kill human ES-MNs**
**(A)** GFAP and vimentin immunostaining of primary astrocyte monolayers (astro) from sALS patients. (B) GFP and MAP2 immunostaining of hES-MN. (**C–F**) Representative images of hES-MNs cultured for 7 d on astro produced from NDC, COPD, AD, or sALS subjects. White arrowheads indicate GFP⁺/MAP2⁺ MNs. (G) Quantification of hES-MN numbers over 14 d on NDC, COPD, AD, or sALS astro. Extra-sum-of-squares ANOVA (Bates and Watts, 1988) indicates that NDC, COPD, and AD curves do not differ (F_[3,33] < 2.44; P > 0.075), but that these curves are different from the sALS curve (F_[3,33] > 19.12, P < 0.001). (H) The number of GFP⁺/MAP2⁺ MNs is reduced when co-cultured for 7 d with sALS astro compared to Ctrs astro (Ctrs = NDC, and/or COPD and/or AD) (t_[4] = 5.498E-15), but the number of non-MN neurons (MAP2⁺/GFP⁻ or GABA⁺/MAP2⁺) does not differ between the two conditions (t_[4] = −0.523, p = 0.63). (I) The number of hES-MNs is not different between co-cultures with NDC or sALS fibroblasts after 7 d (t_[16] = 0.023, p = 0.98). Values represent means ± SEM (n=3–9 per group). *Scale bars*: 20 µm (A, B), 40 µm (C–F).

**Figure 2. SOD1 knockdown in mouse, but not in human astrocytes, mitigates MN loss**
(**A–D**) Representative images of mES-MNs cultured for 7 d on mouse non-transgenic (NTg) or transgenic SOD1^G93A (fALS) astro infected with lentiviral empty vector (EV), or a lentiviral shRNA hairpin to knockdown human SOD1 (clone #39812). (E) Quantification of mES-MN number after 7 d on NTg or fALS astro infected with EV or one of the four shRNA clones against human SOD1 (#39812, #18344, #39808, or #09869). mES-MN numbers on the fALS/EV astro are lower than on NTg/EV astro (*p<0.001). However, mES-MN numbers on the fALS/shSOD1 astro are not different than those on the NTg/shSOD1 astro (p>0.05). In addition, when compared within the NTg condition, mES-MN numbers are not different between EV and shRNA infected astro. Within the fALS condition, mES-MN numbers are higher in shRNA as compared to EV infected astro p<0.001. (**F–I**) Representative images of hES-MNs cultured for 7 d on astro prepared from Ctrs or sALS subjects. Astro were infected with EV or the shRNA clone #39812. (J) Quantification of hES-MN number after 7 d on Ctrs or sALS astro infected with EV or one of the four shRNA clones. hES-MN numbers are lower when co-cultured with sALS astro as compared to Ctrs astro under all lentiviral conditions (*p<0.001). In addition, when compared within Ctrs or sALS conditions, hES-MN numbers are not different between EV and shRNA infected astro. (**E, J**) Data are expressed as percent of MN number on NTg or Ctrs astro infected with EV and represent means ± SEM (n=3–9). (**A–D, F–I**) White arrowheads indicate GFP⁺ MNs. *Scale bars*: 40 µm.

**Figure 3. Human and mouse astrocytes are toxic through similar mechanisms**
**(A)** Like hES-MNs (Figure 1H), mouse primary MN number is lower when co-cultured with sALS astro as compared to Ctrs astro after 7 d (left panel, t_[22] = 7.619, *p = 1.32×10−8). In the same co-culture wells, the number of non-MN neurons do not differ between sALS or Ctrs astro (MAP2⁺/GFP⁻, right panel, t-test: t_[17] = −0.892, p = 0.385). (B) hES-MN number is lower when grown in media conditioned by sALS astrocytes as compared to Ctrs astrocytes (ACM) (left panel, *p<0.001). Non-MN neuron numbers do not differ between these two conditions (MAP2⁺/GFP⁻, right panel, p=0.626). (C) Numbers of hES-MNs fractin⁺ (*p = 2.63×10⁻⁴), EthD⁺ (*p = 1.48×10⁻³) or TUNEL⁺ (*p = 9.11×10⁻⁴) MNs are higher after 3 d exposure to sALS astro compared to Ctrs astro. (D) The reduction in hES-MN number when co-cultured with sALS astro as compared to Ctrs astro (DMSO vehicle [Veh], *p<0.001) is not mitigated by the addition of the pan-caspase inhibitor zVAD-FMK (zVAD, 20 µM, *p×0.001). Inhibition of Bax by the pentapeptide V5 (50 µM) abrogates the difference in hES-MN numbers when co-cultured with sALS or Ctrs astro. Data are expressed as percent of MN or neuron number on Ctrs astro and represent means ± SEM (n=4–7 per group).

**Figure 4. ALS astrocytes trigger necroptosis in MNs, in a RIP1/MLKL-dependent manner**
**(A)** Mouse primary or hES-MN MN number is reduced when co-cultured for 7 d with fALS or sALS astro as compared to non-ALS astro (NTg, Ctrs) in the presence of vehicle (DMSO, *p<0.001, left panel). The RIP1 inhibitor necrostatin-1 (Nec1, 5 µM) abrogates the MN loss observed in both mouse and human co-cultures, respectively (p=0.532, p=0.829, right panel). (B) There are fewer TUNEL⁺ and EthD⁺ (*p<0.001) hES-MNs but not fractin⁺ (p=0.182) MNs in sALS astro co-cultures incubated with Nec1 compared to Veh. (C) There are more mouse primary MNs in co-cultures with fALS astro where MNs are transduced with the viral vector containing the shRNA against RIP1 (#22465) compared to EV or non-mammalian targeted scrambled (SC) transduced MNs (*p<0.001). **(D)** There are more hES-MNs in co-cultures with sALS astro where MNs are transduced with the viral vector containing the shRNA against RIP1 (#200006) compared to EV-transduced hES-MNs (*p<0.001). (E) In presence of Veh (DMSO), there are fewer (*p<0.001) MNs on sALS astro compared to Ctrs astro. 250 nM of the MLKL inhibitor necrosulfonamide (NSA) prevents the loss of hES-MNs co-cultured for 7 d on sALS astro, as hES-MN numbers no longer differ (p>0.123). Data are expressed as percent of MN number on Ctrs astro (A, E), or NTg astro (A), as percent of total MN population (B), or as percent of clone-transduced MN number on NTg astro (C, D) and represent means ± SEM (n=3–5 per group).

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Comment in

Motor neuron death in ALS: programmed by astrocytes?
Pirooznia SK, Dawson VL, Dawson TM. Pirooznia SK, et al. Neuron. 2014 Mar 5;81(5):961-963. doi: 10.1016/j.neuron.2014.02.024. Neuron. 2014. PMID: 24607221 Free PMC article.

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References

1. Amoroso MW, Croft GF, Williams DJ, O’Keeffe S, Carrasco MA, Davis AR, Roybon L, Oakley DH, Maniatis T, Henderson CE, et al. Accelerated high-yield generation of limb-innervating motor neurons from human stem cells. J. Neurosci. 2013;33:574–586. - PMC - PubMed
1. Artus C, Boujrad H, Bouharrour A, Brunelle M-N, Hoos S, Yuste VJ, Lenormand P, Rousselle J-C, Namane A, England P, et al. AIF promotes chromatinolysis and caspase-independent programmed necrosis by interacting with histone H2AX. EMBO J. 2010;29:1585–1599. - PMC - PubMed
1. Bates DM, Watts DG. Nonlinear Regression Analysis and Its Applications. Hoboken, NJ, USA: John Wiley & Sons, Inc.; 1988.
1. Bosco DA, Morfini G, Karabacak NM, Song Y, Gros-Louis F, Pasinelli P, Goolsby H, Fontaine BA, Lemay N, McKenna-Yasek D, et al. Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS. Nat. Neurosci. 2010;13:1396–1403. - PMC - PubMed
1. Cassina P, Pehar M, Vargas MR, Castellanos R, Barbeito AG, Estevez AG, Thompson JA, Beckman JS, Barbeito L. Astrocyte activation by fibroblast growth factor-1 and motor neuron apoptosis: implications for amyotrophic lateral sclerosis. J. Neurochem. 2005;93:38–46. - PubMed

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[1] Amoroso MW, Croft GF, Williams DJ, O’Keeffe S, Carrasco MA, Davis AR, Roybon L, Oakley DH, Maniatis T, Henderson CE, et al. Accelerated high-yield generation of limb-innervating motor neurons from human stem cells. J. Neurosci. 2013;33:574–586. - PMC - PubMed

[2] Amoroso MW, Croft GF, Williams DJ, O’Keeffe S, Carrasco MA, Davis AR, Roybon L, Oakley DH, Maniatis T, Henderson CE, et al. Accelerated high-yield generation of limb-innervating motor neurons from human stem cells. J. Neurosci. 2013;33:574–586. - PMC - PubMed

[3] Artus C, Boujrad H, Bouharrour A, Brunelle M-N, Hoos S, Yuste VJ, Lenormand P, Rousselle J-C, Namane A, England P, et al. AIF promotes chromatinolysis and caspase-independent programmed necrosis by interacting with histone H2AX. EMBO J. 2010;29:1585–1599. - PMC - PubMed

[4] Artus C, Boujrad H, Bouharrour A, Brunelle M-N, Hoos S, Yuste VJ, Lenormand P, Rousselle J-C, Namane A, England P, et al. AIF promotes chromatinolysis and caspase-independent programmed necrosis by interacting with histone H2AX. EMBO J. 2010;29:1585–1599. - PMC - PubMed

[5] Bates DM, Watts DG. Nonlinear Regression Analysis and Its Applications. Hoboken, NJ, USA: John Wiley & Sons, Inc.; 1988.

[6] Bates DM, Watts DG. Nonlinear Regression Analysis and Its Applications. Hoboken, NJ, USA: John Wiley & Sons, Inc.; 1988.

[7] Bosco DA, Morfini G, Karabacak NM, Song Y, Gros-Louis F, Pasinelli P, Goolsby H, Fontaine BA, Lemay N, McKenna-Yasek D, et al. Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS. Nat. Neurosci. 2010;13:1396–1403. - PMC - PubMed

[8] Bosco DA, Morfini G, Karabacak NM, Song Y, Gros-Louis F, Pasinelli P, Goolsby H, Fontaine BA, Lemay N, McKenna-Yasek D, et al. Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS. Nat. Neurosci. 2010;13:1396–1403. - PMC - PubMed

[9] Cassina P, Pehar M, Vargas MR, Castellanos R, Barbeito AG, Estevez AG, Thompson JA, Beckman JS, Barbeito L. Astrocyte activation by fibroblast growth factor-1 and motor neuron apoptosis: implications for amyotrophic lateral sclerosis. J. Neurochem. 2005;93:38–46. - PubMed

[10] Cassina P, Pehar M, Vargas MR, Castellanos R, Barbeito AG, Estevez AG, Thompson JA, Beckman JS, Barbeito L. Astrocyte activation by fibroblast growth factor-1 and motor neuron apoptosis: implications for amyotrophic lateral sclerosis. J. Neurochem. 2005;93:38–46. - PubMed

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Necroptosis drives motor neuron death in models of both sporadic and familial ALS

Affiliations

Necroptosis drives motor neuron death in models of both sporadic and familial ALS

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