NeuroMolecular Medicine

, Volume 14, Issue 1, pp 15–29 | Cite as

Naturally Occurring Genetic Variability in Expression of Gsta4 is Associated with Differential Survival of Axotomized Rat Motoneurons

  • Mikael StrömEmail author
  • Faiez Al Nimer
  • Rickard Lindblom
  • Jens Randel Nyengaard
  • Fredrik Piehl
Original Paper


A large number of molecular pathways have been implicated in the degeneration of axotomized motoneurons. We previously have demonstrated substantial differences in the survival rate of axotomized motoneurons across different rat strains. Identification of genetic differences underlying such naturally occurring strain differences is a powerful approach, also known as forward genetics, to gain knowledge of mechanisms relevant for complex diseases, like injury-induced neurodegeneration. Overlapping congenic rat strains were used to fine map a gene region on rat chromosome eight previously shown to regulate motoneuron survival after ventral root avulsion. The smallest genetic fragment, R5, contains 35 genes and displays a highly significant regulatory effect on motoneuron survival. Furthermore, expression profiling in a F2(DAxPVG) intercross demonstrates one single cis-regulated gene within the R5 fragment; Gsta4, encoding glutathione S-transferase alpha-4. Confirmation with real-time PCR shows higher Gsta4 expression in PVG compared with DA both in naïve animals and at several time points after injury. Immunolabeling with a custom made rat Gsta4 antibody demonstrates a neuronal staining pattern, with a strong cytoplasmic labeling of motoneurons. These results demonstrate and map naturally occurring genetic differences in the expression of Gsta4 is associated both with a highly significant increase in the survival of axotomized motoneurons and with a trans-regulation of several molecular pathways involved in neurodegenerative processes. This adds to a large body of evidence implicating lipid peroxidation as an important pathway for neurodegeneration.


Motoneuron Glutathione S-transferase Gsta4 Axotomy 4-HNE Lipid peroxidation 





Alzheimer’s disease


Advanced intercross line


Amyotrophic lateral sclerosis


Bovine serum albumin


Central nervous system


Dark Agouti


Expression QTL


Glutathione S-transferase alpha4


Multiple sclerosis


Neuronal nuclei


Phosphate-buffered saline


Parkinson’s disease


Piebald Virol Glaxo


Reactive oxygen species


Superoxide dismutase 1


Quantitative trait locus


Ventral root avulsion



We thank Prof. Tomas Olsson for expert advice. Further, Matthias Heinig and Norbert Hübner at MDC, Berlin, for help with the eQTL data analysis and Peter Lundbäck at the Rheumatology unit, Karolinska Institutet, CMM, for help and expertise in Western Blot. This study was supported by the 6th Framework Program of the European Union, NeuroproMiSe, LSHM-CT-2005-018637, EURATools, LSHG-CT-2005019015, and the 7th Framework Program of the European Union, EURATrans, HEALTH-F4-2010-241504, by the Swedish Research Council, the Swedish Brain Foundation and the Swedish Association of Persons with Neurological Disabilities. Centre for Stochastic Geometry and advanced Bioimaging was supported by the Villum Foundation. The funders had no role in study design, data collection or analysis, manuscript preparation or decision to publish.

Conflict of interest

No competing interests are declared.


  1. Aitman, T. J., Critser, J. K., Cuppen, E., Dominiczak, A., Fernandez-Suarez, X. M., Flint, J., et al. (2008). Progress and prospects in rat genetics: A community view. Nature Genetics, 40(5), 516–522. doi: 10.1038/ng.147.PubMedCrossRefGoogle Scholar
  2. Aitman, T. J., Glazier, A. M., Wallace, C. A., Cooper, L. D., Norsworthy, P. J., Wahid, F. N., et al. (1999). Identification of Cd36 (fat) as an insulin-resistance gene causing defective fatty acid and glucose metabolism in hypertensive rats. Nature Genetics, 21(1), 76–83. doi: 10.1038/5013.PubMedCrossRefGoogle Scholar
  3. Aktas, O., Waiczies, S., & Zipp, F. (2007). Neurodegeneration in autoimmune demyelination: Recent mechanistic insights reveal novel therapeutic targets. Journal of Neuroimmunology, 184(1–2), 17–26. doi: 10.1016/j.jneuroim.2006.11.026.PubMedCrossRefGoogle Scholar
  4. Awasthi, Y. C., Sharma, R., Cheng, J. Z., Yang, Y., Sharma, A., Singhal, S. S., et al. (2003). Role of 4-hydroxynonenal in stress-mediated apoptosis signaling. Molecular Aspects of Medicine, 24(4–5), 219–230.PubMedCrossRefGoogle Scholar
  5. Balogh, L. M., & Atkins, W. M. (2011). Interactions of glutathione transferases with 4-hydroxynonenal. Drug Metabolism Reviews, 43(2), 165–178. doi: 10.3109/03602532.2011.558092.PubMedCrossRefGoogle Scholar
  6. Beckett, G. J., & Hayes, J. D. (1993). Glutathione S-transferases: Biomedical applications. Advances in Clinical Chemistry, 30, 281–380.PubMedCrossRefGoogle Scholar
  7. Behmoaras, J., Bhangal, G., Smith, J., McDonald, K., Mutch, B., Lai, P. C., et al. (2008). Jund is a determinant of macrophage activation and is associated with glomerulonephritis susceptibility. Nature Genetics, 40(5), 553–559.PubMedCrossRefGoogle Scholar
  8. Block, M. L., Zecca, L., & Hong, J. S. (2007). Microglia-mediated neurotoxicity: Uncovering the molecular mechanisms. Nature Reviews. Neuroscience, 8(1), 57–69. doi: 10.1038/nrn2038.PubMedCrossRefGoogle Scholar
  9. Broman, K. W., Wu, H., Sen, S., & Churchill, G. A. (2003). R/qtl: QTL mapping in experimental crosses. Bioinformatics, 19(7), 889–890.PubMedCrossRefGoogle Scholar
  10. Centonze, D., Muzio, L., Rossi, S., Furlan, R., Bernardi, G., & Martino, G. (2010). The link between inflammation, synaptic transmission and neurodegeneration in multiple sclerosis. Cell Death and Differentiation, 17(7), 1083–1091. doi: 10.1038/cdd.2009.179.PubMedCrossRefGoogle Scholar
  11. Cheng, J. Z., Singhal, S. S., Saini, M., Singhal, J., Piper, J. T., Van Kuijk, F. J., et al. (1999). Effects of mGST A4 transfection on 4-hydroxynonenal-mediated apoptosis and differentiation of K562 human erythroleukemia cells. Archives of Biochemistry and Biophysics, 372(1), 29–36. doi: 10.1006/abbi.1999.1479.PubMedCrossRefGoogle Scholar
  12. Coles, B. F., & Kadlubar, F. F. (2005). Human alpha class glutathione S-transferases: Genetic polymorphism, expression, and susceptibility to disease. Methods in Enzymology, 401, 9–42. doi: 10.1016/S0076-6879(05)01002-5.PubMedCrossRefGoogle Scholar
  13. Conforti, L., Tarlton, A., Mack, T. G., Mi, W., Buckmaster, E. A., Wagner, D., et al. (2000). A Ufd2/D4Cole1e chimeric protein and overexpression of Rbp7 in the slow Wallerian degeneration (WldS) mouse. Proceedings of the National Academy of Sciences of the United States of America, 97(21), 11377–11382. doi: 10.1073/pnas.97.21.11377.PubMedCrossRefGoogle Scholar
  14. Cutler, R. G., Kelly, J., Storie, K., Pedersen, W. A., Tammara, A., Hatanpaa, K., et al. (2004). Involvement of oxidative stress-induced abnormalities in ceramide and cholesterol metabolism in brain aging and Alzheimer’s disease. Proceedings of the National Academy of Sciences of the United States of America, 101(7), 2070–2075. doi: 10.1073/pnas.0305799101.PubMedCrossRefGoogle Scholar
  15. Darvasi, A., & Soller, M. (1994). Selective DNA pooling for determination of linkage between a molecular marker and a quantitative trait locus. Genetics, 138(4), 1365–1373.PubMedGoogle Scholar
  16. Darvasi, A., & Soller, M. (1995). Advanced intercross lines, an experimental population for fine genetic mapping. Genetics, 141(3), 1199–1207.PubMedGoogle Scholar
  17. Deng, H. X., Hentati, A., Tainer, J. A., Iqbal, Z., Cayabyab, A., Hung, W. Y., et al. (1993). Amyotrophic lateral sclerosis and structural defects in Cu, Zn superoxide dismutase. Science, 261(5124), 1047–1051.PubMedCrossRefGoogle Scholar
  18. Desmots, F., Rissel, M., Loyer, P., Turlin, B., & Guillouzo, A. (2001). Immunohistological analysis of glutathione transferase A4 distribution in several human tissues using a specific polyclonal antibody. Journal of Histochemistry and Cytochemistry, 49(12), 1573–1580.PubMedCrossRefGoogle Scholar
  19. Dorph-Petersen, K. A., Nyengaard, J. R., & Gundersen, H. J. (2001). Tissue shrinkage and unbiased stereological estimation of particle number and size. Journal of Microscopy, 204(Pt 3), 232–246.Google Scholar
  20. Engle, M. R., Singh, S. P., Czernik, P. J., Gaddy, D., Montague, D. C., Ceci, J. D., et al. (2004). Physiological role of mGSTA4-4, a glutathione S-transferase metabolizing 4-hydroxynonenal: Generation and analysis of mGsta4 null mouse. Toxicology and Applied Pharmacology, 194(3), 296–308. doi: 10.1016/j.taap.2003.10.001.PubMedCrossRefGoogle Scholar
  21. Esterbauer, H., Schaur, R. J., & Zollner, H. (1991). Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radical Biology and Medicine, 11(1), 81–128.PubMedCrossRefGoogle Scholar
  22. Geiger, L. K., Kortuem, K. R., Alexejun, C., & Levin, L. A. (2002). Reduced redox state allows prolonged survival of axotomized neonatal retinal ganglion cells. Neuroscience, 109(3), 635–642.PubMedCrossRefGoogle Scholar
  23. Giralt, A., Rodrigo, T., Martín, E. D., Gonzalez, J. R., Milà, M., Ceña, V., et al. (2009). Brain-derived neurotrophic factor modulates the severity of cognitive alterations induced by mutant huntingtin: Involvement of phospholipaseC[gamma] activity and glutamate receptor expression. Neuroscience, 158(4), 1234–1250.PubMedCrossRefGoogle Scholar
  24. Gundersen, H. J., Jensen, E. B., Kieu, K., & Nielsen, J. (1999). The efficiency of systematic sampling in stereology–reconsidered. Journal of Microscopy, 193(Pt 3), 199–211.Google Scholar
  25. Hubatsch, I., Ridderstrom, M., & Mannervik, B. (1998). Human glutathione transferase A4-4: An alpha class enzyme with high catalytic efficiency in the conjugation of 4-hydroxynonenal and other genotoxic products of lipid peroxidation. Biochemical Journal, 330(Pt 1), 175–179.PubMedGoogle Scholar
  26. Hubner, N., Wallace, C. A., Zimdahl, H., Petretto, E., Schulz, H., Maciver, F., et al. (2005). Integrated transcriptional profiling and linkage analysis for identification of genes underlying disease. Nature Genetics, 37(3), 243–253.PubMedCrossRefGoogle Scholar
  27. Irizarry, R. A., Hobbs, B., Collin, F., Beazer-Barclay, Y. D., Antonellis, K. J., Scherf, U., et al. (2003). Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics, 4(2), 249–264. doi: 10.1093/biostatistics/4.2.249.PubMedCrossRefGoogle Scholar
  28. Jagodic, M., Colacios, C., Nohra, R., Dejean, A. S., Beyeen, A. D., Khademi, M., et al. (2009). A role for VAV1 in experimental autoimmune encephalomyelitis and multiple sclerosis. Science Translational Medicine, 1(10), 10ra21. doi: 10.1126/scitranslmed.3000278.PubMedCrossRefGoogle Scholar
  29. Johnson, J. A., el Barbary, A., Kornguth, S. E., Brugge, J. F., & Siegel, F. L. (1993). Glutathione S-transferase isoenzymes in rat brain neurons and glia. Journal of Neuroscience, 13(5), 2013–2023.PubMedGoogle Scholar
  30. Kato, S. (2008). Amyotrophic lateral sclerosis models and human neuropathology: Similarities and differences. Acta Neuropathologica, 115(1), 97–114. doi: 10.1007/s00401-007-0308-4.PubMedCrossRefGoogle Scholar
  31. Leoni, V. (2009). Oxysterols as markers of neurological disease—A review. Scandinavian Journal of Clinical and Laboratory Investigation, 69(1), 22–25. doi: 10.1080/00365510802651858.PubMedCrossRefGoogle Scholar
  32. Lidman, O., Swanberg, M., Horvath, L., Broman, K. W., Olsson, T., & Piehl, F. (2003). Discrete gene loci regulate neurodegeneration, lymphocyte infiltration, and major histocompatibility complex class II expression in the CNS. Journal of Neuroscience, 23(30), 9817–9823.PubMedGoogle Scholar
  33. Lieven, C. J., Hoegger, M. J., Schlieve, C. R., & Levin, L. A. (2006). Retinal ganglion cell axotomy induces an increase in intracellular superoxide anion. Investigative Ophthalmology & Visual Science, 47(4), 1477–1485. doi: 10.1167/iovs.05-0921.CrossRefGoogle Scholar
  34. Li-Hawkins, J., Lund, E. G., Bronson, A. D., & Russell, D. W. (2000). Expression cloning of an oxysterol 7alpha-hydroxylase selective for 24-hydroxycholesterol. Journal of Biological Chemistry, 275(22), 16543–16549. doi: 10.1074/jbc.M001810200.PubMedCrossRefGoogle Scholar
  35. Lovell, M. A., Ehmann, W. D., Mattson, M. P., & Markesbery, W. R. (1997). Elevated 4-hydroxynonenal in ventricular fluid in Alzheimer’s disease. Neurobiology of Aging, 18(5), 457–461.PubMedCrossRefGoogle Scholar
  36. Lundberg, C., Lidman, O., Holmdahl, R., Olsson, T., & Piehl, F. (2001). Neurodegeneration and glial activation patterns after mechanical nerve injury are differentially regulated by non-MHC genes in congenic inbred rat strains. Journal of Comparative Neurology, 431(1), 75–87.PubMedCrossRefGoogle Scholar
  37. Mannervik, B., Board, P. G., Hayes, J. D., Listowsky, I., & Pearson, W. R. (2005). Nomenclature for mammalian soluble glutathione transferases. Methods in Enzymology, 401, 1–8. doi: 10.1016/S0076-6879(05)01001-3.PubMedCrossRefGoogle Scholar
  38. Martin, L. J., Kaiser, A., & Price, A. C. (1999). Motor neuron degeneration after sciatic nerve avulsion in adult rat evolves with oxidative stress and is apoptosis. Journal of Neurobiology, 40(2), 185–201. doi: 10.1002/(SICI)1097-4695(199908)40:2<185:AID-NEU5>3.0.CO;2-#.PubMedCrossRefGoogle Scholar
  39. Mitchell, J., Paul, P., Chen, H. J., Morris, A., Payling, M., Falchi, M., et al. (2010). Familial amyotrophic lateral sclerosis is associated with a mutation in d-amino acid oxidase. Proceedings of the National Academy of Sciences of the United States of America, 107(16), 7556–7561. doi: 10.1073/pnas.0914128107.PubMedCrossRefGoogle Scholar
  40. Olofsson, P., Holmberg, J., Tordsson, J., Lu, S., Akerstrom, B., & Holmdahl, R. (2003). Positional identification of Ncf1 as a gene that regulates arthritis severity in rats. Nature Genetics, 33(1), 25–32. doi: 10.1038/ng1058.PubMedCrossRefGoogle Scholar
  41. Pedersen, W. A., Fu, W., Keller, J. N., Markesbery, W. R., Appel, S., Smith, R. G., et al. (1998). Protein modification by the lipid peroxidation product 4-hydroxynonenal in the spinal cords of amyotrophic lateral sclerosis patients. Annals of Neurology, 44(5), 819–824. doi: 10.1002/ana.410440518.PubMedCrossRefGoogle Scholar
  42. Piehl, F., Lundberg, C., Khademi, M., Bucht, A., Dahlman, I., Lorentzen, J. C., et al. (1999). Non-MHC gene regulation of nerve root injury induced spinal cord inflammation and neuron death. Journal of Neuroimmunology, 101(1), 87–97.PubMedCrossRefGoogle Scholar
  43. Pitkänen, A., Longhi, L., Marklund, N., Morales, D. M., & McIntosh, T. K. (2005). Neurodegeneration and neuroprotective strategies after traumatic brain injury. Drug Discovery Today: Disease Mechanisms, 2(4), 409–418.CrossRefGoogle Scholar
  44. Requena, J. R., Fu, M. X., Ahmed, M. U., Jenkins, A. J., Lyons, T. J., & Thorpe, S. R. (1996). Lipoxidation products as biomarkers of oxidative damage to proteins during lipid peroxidation reactions. Nephrology, Dialysis, Transplantation, 11(Suppl 5), 48–53.PubMedGoogle Scholar
  45. Samuelson, D. J., Hesselson, S. E., Aperavich, B. A., Zan, Y., Haag, J. D., Trentham-Dietz, A., et al. (2007). Rat Mcs5a is a compound quantitative trait locus with orthologous human loci that associate with breast cancer risk. Proceedings of the National Academy of Sciences of the United States of America, 104(15), 6299–6304. doi: 10.1073/pnas.0701687104.PubMedCrossRefGoogle Scholar
  46. Sasaki, T., Kitagawa, K., Yagita, Y., Sugiura, S., Omura-Matsuoka, E., Tanaka, S., et al. (2006). Bcl2 enhances survival of newborn neurons in the normal and ischemic hippocampus. Journal of Neuroscience Research, 84(6), 1187–1196. doi: 10.1002/jnr.21036.PubMedCrossRefGoogle Scholar
  47. Schmidt, N., & Ferger, B. (2001). Neurochemical findings in the MPTP model of Parkinson’s disease. Journal of Neural Transmission, 108(11), 1263–1282.PubMedCrossRefGoogle Scholar
  48. Selley, M. L. (1998). (E)-4-Hydroxy-2-nonenal may be involved in the pathogenesis of Parkinson’s disease. Free Radical Biology and Medicine, 25(2), 169–174.PubMedCrossRefGoogle Scholar
  49. Singh, S. P., Niemczyk, M., Saini, D., Awasthi, Y. C., Zimniak, L., & Zimniak, P. (2008). Role of the electrophilic lipid peroxidation product 4-hydroxynonenal in the development and maintenance of obesity in mice. Biochemistry, 47(12), 3900–3911.Google Scholar
  50. Singh, S. P., Niemczyk, M., Saini, D., Sadovov, V., Zimniak, L., & Zimniak, P. (2010). Disruption of the mGsta4 gene increases life span of C57BL mice. The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, 65(1), 14–23.Google Scholar
  51. Sharma, A., Sharma, R., Chaudhary, P., Vatsyayan, R., Pearce, V., Jeyabal, P. V., et al. (2008). 4-Hydroxynonenal induces p53-mediated apoptosis in retinal pigment epithelial cells. Archives of Biochemistry and Biophysics, 480(2), 85–94. doi: 10.1016/ Scholar
  52. Swanberg, M., Harnesk, K., Strom, M., Diez, M., Lidman, O., & Piehl, F. (2009). Fine mapping of gene regions regulating neurodegeneration. PLoS One, 4(6), e5906. doi: 10.1371/journal.pone.0005906.PubMedCrossRefGoogle Scholar
  53. Swanberg, M., Lidman, O., Padyukov, L., Eriksson, P., Akesson, E., Jagodic, M., et al. (2005). MHC2TA is associated with differential MHC molecule expression and susceptibility to rheumatoid arthritis, multiple sclerosis and myocardial infarction. Nature Genetics, 37(5), 486–494. doi: 10.1038/ng1544.PubMedCrossRefGoogle Scholar
  54. Tanner, C. M., Kamel, F., Ross, G. W., Hoppin, J. A., Goldman, S. M., Korell, M., et al. (2011). Rotenone, paraquat, and Parkinson’s disease. Environmental Health Perspectives, 119(6), 866–872.Google Scholar
  55. Trancikova, A., Ramonet, D., & Moore, D. J. (2011). Genetic mouse models of neurodegenerative diseases. In K. T. Chang & K.-T. Min (Eds.), Animal models of human disease, Progress in molecular biology and translational science (Vol. 100, pp. 1–526). London: Academic Press. ISBN: 978-0-12-384878-9.Google Scholar
  56. Tsuji, S. (2010). Genetics of neurodegenerative diseases: insights from high-throughput resequencing. Human Molecular Genetics, 19(R1), R65–R70. doi: 10.1093/hmg/ddq162.PubMedCrossRefGoogle Scholar
  57. Twine, N. A., Janitz, K., Wilkins, M. R., & Janitz, M. (2011). Whole transcriptome sequencing reveals gene expression and splicing differences in brain regions affected by Alzheimer’s disease. PLoS One, 6(1), e16266. doi: 10.1371/journal.pone.0016266.PubMedCrossRefGoogle Scholar
  58. Wang, J., Williams, R. W., & Manly, K. F. (2003). WebQTL: Web-based complex trait analysis. Neuroinformatics, 1(4), 299–308. doi: 10.1385/NI:1:4:299.PubMedCrossRefGoogle Scholar
  59. West, M. J., & Gundersen, H. J. (1990). Unbiased stereological estimation of the number of neurons in the human hippocampus. Journal of Comparative Neurology, 296(1), 1–22. doi: 10.1002/cne.902960102.PubMedCrossRefGoogle Scholar
  60. Yoritaka, A., Hattori, N., Uchida, K., Tanaka, M., Stadtman, E. R., & Mizuno, Y. (1996). Immunohistochemical detection of 4-hydroxynonenal protein adducts in Parkinson disease. Proceedings of the National Academy of Sciences of the United States of America, 93(7), 2696–2701.PubMedCrossRefGoogle Scholar
  61. Yoshihara, D., Fujiwara, N., Ookawara, T., Kato, S., Sakiyama, H., Yokoe, S., et al. (2009). Protective role of glutathione S-transferase A4 induced in copper/zinc-superoxide dismutase knockout mice. Free Radical Biology and Medicine, 47(5), 559–567. doi: 10.1016/j.freeradbiomed.2009.05.022.PubMedCrossRefGoogle Scholar
  62. Zhao, T., Singhal, S. S., Piper, J. T., Cheng, J., Pandya, U., Clark-Wronski, J., et al. (1999). The role of human glutathione S-transferases hGSTA1-1 and hGSTA2-2 in protection against oxidative stress. Archives of Biochemistry and Biophysics, 367(2), 216–224.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Mikael Ström
    • 1
    Email author
  • Faiez Al Nimer
    • 1
  • Rickard Lindblom
    • 1
  • Jens Randel Nyengaard
    • 2
  • Fredrik Piehl
    • 1
  1. 1.Department of Clinical Neuroscience, Karolinska InstitutetKarolinska University HospitalStockholmSweden
  2. 2.Stereology and Electron Microscopy Laboratory, Centre for Stochastic Geometry and Advanced BioimagingAarhus University HospitalAarhusDenmark

Personalised recommendations