Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letters to the Editor
  • Published:

Lack of apoptosis in mice with ALS

Nature Medicine volume 5, pages 966–967 (1999)Cite this article

The role of apoptosis in neurodegenerative disorders remains controversial1. Most of the uncertainty derives from the fact that (i) since these diseases progress over years, the possibility of finding a single dying cell in vivo is extremely remote, (ii) tissues are from end-stage patients and are therefore already depleted of neurons, (iii) even if present, death changes may be due to premortem agonal status rather than to the disease itself. Such problems may be avoided by using appropriate animal models. Severe loss of motor neurons characterizes amyotrophic lateral sclerosis (ALS), yet there is no conclusive evidence that apoptosis is involved2. The etiology of sporadic ALS is unknown; in 20% of familial cases, point mutations in the superoxide dismutase 1 (SOD1) gene are present, and mutant SOD1 transgenic mice develop an ALS-like disease mediated by a toxic gain-of-function of the mutated enzyme3. Although mutant SOD1 induces apoptosis in vitro3, it is unclear whether the same happens in vivo. Overexpression of the antiapoptotic protein Bcl-24 or inhibition of proapoptotic interleukin-1β-converting enzyme (ICE)5 modestly slow disease progression in mutant SOD1 mice. These effects, however, do not necessarily imply apoptosis regulation, since Bcl-2 might protect cells from SOD1 toxicity through its antioxidant properties, and ICE has many activities unrelated to apoptosis. Moreover, detailed ultrastructural studies in mutant SOD1 mice did not report on the presence of morphological signs of apoptosis, although they did show a wide array of ongoing degenerative changes, ranging from early vacuolar changes due to mitochondrial damage, to late formation of cytoplasmic inclusions in remaining motor neurons6.

To clarify whether degenerating motor neurons in mutant SOD1 mice undergo apoptosis, we used transgenic mice derived from Jackson Laboratory (Bar Harbor, Maine), that carry about 20 copies of human SOD1 gene with a glycine-to-alanine substitution at position 93 (G93A)7. These mice become symptomatic at around 70 days of age and die by 142±4.3 days. Sixteen transgenic and six wild-type mice combined in three age groups (9-14-19 weeks) were studied. Following perfusion fixation with 4% paraformaldehyde and paraffin embedding, the cervical and lumbar spinal cord were serially cut. Intraneuronal inclusions were immunohistochemically revealed with antibodies to human SOD1 (Sigma) and ubiquitin (Chemicon). To detect DNA fragmentation, a hallmark of apoptosis, we performed an in situ end-labeling (ISEL) assay (Boehringer)2. Briefly, sections were pretreated with proteinase K (up to 50 μg/ml) and incubated with terminal deoxynucleotidyl transferase and fluorescein-11-dUTP, followed by peroxidase-conjugated anti-fluorescein sheep antisera. To evaluate the expression of apoptosis-related proteins, immunohistochemistry was performed with antibodies to c-Jun/AP-1 (Calbiochem) and PCNA (Dako), two proteins that accumulate in the nuclei of apoptotic neurons8, as well as with an antiserum that selectively recognizes the active form of apoptotic protease caspase-3 (MF397, kindly provided by Dr. Donald Nicholson). For each assay, at least eight sections/mouse were studied. Sections from the cerebellum of P7 mice carrying the weaver mutation, which causes massive apoptosis of granule cell precursors8, were stained as control.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: (a-c) Apoptotic changes are absent in mutant SOD1 mice.

References

  1. Perry, G., Nunomura, A. & Smith, M.A. A suicide note from Alzheimer disease neurons? Nature Med. 4, 897–898 ( 1998).

    Article  CAS  Google Scholar 

  2. Migheli, A., Piva, R., Atzori, C., Troost, D. & Schiffer, D. c-Jun, JNK/SAPK kinases and transcription factor NF-kB are selectively activated in astrocytes, but not motor neurons, in amyotrophic lateral sclerosis. J. Neuropathol. Exp. Neurol. 56, 1314–1322 (1997).

    Article  CAS  Google Scholar 

  3. Cookson, M.R. & Shaw, P.J. Oxidative stress and motor neuron disease. Brain Pathol. 9, 165– 186 (1999).

    Article  CAS  Google Scholar 

  4. Kostic, V., Jackson-Lewis, V., de Bilbao, F., Dubois-Dauphin, M. & Przedborski, S. Bcl-2: prolonging life in a transgenic mouse model of familial amyotrophic lateral sclerosis. Science 277, 559–562 (1997).

    Article  CAS  Google Scholar 

  5. Friedlander, R.M., Brown, R.H., Gagliardini, V., Wang, J. & Yuan, J. Inhibition of ICE slows ALS in mice. Nature 388, 31 (1997 ).

    Article  CAS  Google Scholar 

  6. Dal Canto, M.C. & Gurney, M.E. Development of central nervous system pathology in a murine transgenic model of human amyotrophic lateral sclerosis. Am. J. Pathol. 145, 1271 –1279 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Gurney, M.E. et al. Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Science 264, 1772–1775 (1994).

    Article  CAS  Google Scholar 

  8. Migheli, A. et al. Diverse cell death pathways result from a single missense mutation in weaver mouse. Am. J. Pathol. 151 , 1629–1638 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Zhang, B., Tu, P.-H., Abtahian, F., Trojanowski, J.Q. & Lee, V.M.-Y. Neurofilaments and orthograde transport are reduced in ventral root axons of transgenic mice that express human SOD1 with a G93A mutation. J. Cell Biol. 139, 1307– 1315 (1997).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Supported by Telethon, Italy (grants 878 and 1004). We are grateful to Dr. Carla Dossi and Ms. Tiziana Rotondo from Consorzio Mario Negri Sud, S. Maria Imbaro, Italy, for the development and care of transgenic mice colony.

Author information

Authors and Affiliations

  1. Department of Neuroscience, University of Turin, Laboratory of Neuropathology, Via Cherasco 15, Turin, 10126, Italy

    Antonio Migheli, Cristiana Atzori, Roberto Piva & Davide Schiffer

  2. Department of Neuroscience, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy

    Massimo Tortarolo, Marina Girelli & Caterina Bendotti

Authors
  1. Antonio Migheli
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cristiana Atzori
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Roberto Piva
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Massimo Tortarolo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Marina Girelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Davide Schiffer
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Caterina Bendotti
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Migheli, A., Atzori, C., Piva, R. et al. Lack of apoptosis in mice with ALS. Nat Med 5, 966–967 (1999). https://doi.org/10.1038/12381

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/12381

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing