Skip to main content

Increased Spontaneous Apoptosis of Rat Primary Neurospheres In Vitro After Experimental Autoimmune Encephalomyelitis

Abstract

Survival of neuronal progenitors (NPCs) is a critical determinant of the regenerative capacity of brain following cellular loss. Herein, we report for the first time, the increased spontaneous apoptosis of the first acute phase of Experimental Autoimmune Encephalomyelitis (EAE) derived neurospheres in vitro. Neuronal as well as oligodendroglial loss occurs during experimental autoimmune encephalomyelitis (EAE). This loss is replenished spontaneously by the concomitant increase in the NPC proliferation evidenced by the presence of thin myelin sheaths in the remodeled lesions. However, remyelination depends upon the survival of NPCs and their lineage specific differentiation. We observed significant increase (P < 0.001) in number of BrdU (+) cells in ependymal subventricular zone (SVZ) in EAE rats. EAE derived NPCs showed remarkable increase in S-phase population which was indeed due to the decrease in G-phase progeny suggesting activation of neuronal progenitor cells (NPCs) from quiescence. However, EAE derived neurospheres showed limited survival in vitro which was mediated by the significantly (P < 0.01) depolarized mitochondria, elevated Caspase-3 (P < 0.001) and fragmentation of nuclear DNA evidenced by single cell gel electrophoresis. Our results suggest EAE induced spontaneous apoptosis of NPCs in vitro which may increase the possibility of early stage cell death in the negative regulation of the proliferative cell number and may explain the failure of regeneration in human multiple sclerosis.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. 1.

    Bruck W (2005) Inflammatory demyelination is not central to the pathogenesis of multiple sclerosis. J Neurol 252:10–15

    Article  Google Scholar 

  2. 2.

    Huppert J, Closhen D, Croxford A et al. (2009) Cellular mechanisms of IL-17-induced blood-brain barrier disruption. FASEB J (in press)

  3. 3.

    Friese MA, Fugger L (2009) Pathogenic CD8 (+) T cells in multiple sclerosis. Ann Neurol 66:132–141

    PubMed  Article  CAS  Google Scholar 

  4. 4.

    Goverman J (2009) Autoimmune T cell responses in the central nervous system. Nat Rev Immunol 9:393–407

    PubMed  Article  CAS  Google Scholar 

  5. 5.

    Dhib-Jalbut S (2007) Pathogenesis of myelin/oligodendrocyte damage in multiple sclerosis. Neurology 68:S13–S21

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    Brosnan CF, Raine CS (1996) Mechanisms of immune injury in multiple sclerosis. Brain Pathol 62:43–57

    Google Scholar 

  7. 7.

    Probert L, Eugster HP, Akassoglou K et al (2000) TNFR1 signaling is critical for the development of demyelination and the limitation of T-cell responses during immune-mediated CNS disease. Brain 123:2005–2019

    PubMed  Article  Google Scholar 

  8. 8.

    Prineas JW, Kwon EE, Cho ES (1984) Continual breakdown and regeneration of myelin in progressive multiple sclerosis plaques. Ann N Y Acad Sci 436:11–32

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    Sajad M, Zargan J, Chawla R et al (2009) Hippocampal neurodegeneration in experimental autoimmune encephalomyelitis: potential role of inflammation activated myeloperoxidase. Mol Cell Biochem 328:183–188

    PubMed  Article  CAS  Google Scholar 

  10. 10.

    French HM, Reid M, Mamontov P et al (2009) Oxidative stress disrupts oligodendrocyte maturation. J Neurosci Res 87:3076–3087

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    Luskin MB (1993) Restricted proliferation and migration of postnatally generated neurons derived from the forebrain subventricular zone. Neuron 11:173–189

    PubMed  Article  CAS  Google Scholar 

  12. 12.

    Altman J (1969) Autoradiographic and histological studies of postnatal neurogenesis. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb. Comp Neurol 1374:33–457

    Google Scholar 

  13. 13.

    Lachapelle F, Avellana-Adalid V, Nait-Oumesmar B et al (2002) Fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor AB (PDGF AB) promote adult SVZ-derived oligodendrogenesis in vivo. Mol Cell Neurosci 20:390–403

    PubMed  Article  CAS  Google Scholar 

  14. 14.

    Pencea V, Bingaman KD, Freedman LJ et al (2001) Neurogenesis in the subventricular zone and rostral migratory stream of the neonatal and adult primate forebrain. Exp Neurol 72:1–16

    Article  Google Scholar 

  15. 15.

    Kornack DR, Rakic P (2001) Cell proliferation without neurogenesis in adult primate neocortex. Science 294:2127–2130

    PubMed  Article  CAS  Google Scholar 

  16. 16.

    Sanai N, Tramontin AD, Quiñones-Hinojosa A et al (2004) Unique astrocyte ribbon in adult human brain contains neural stem cells but lacks chain migration. Nature 427:740–744

    PubMed  Article  CAS  Google Scholar 

  17. 17.

    Lois C, Alvarez-Buylla A (1994) Long-distance neuronal migration in the adult mammalian brain. Science 264:1145–1148

    PubMed  Article  CAS  Google Scholar 

  18. 18.

    Picard-Riera N, Nait-Oumesmar B, Baron-Van Evercooren A (2004) Endogenous adult neural stem cells: limits and potential to repair the injured central nervous system. J Neurosci Res 76:223–231

    PubMed  Article  CAS  Google Scholar 

  19. 19.

    Sajad M, Chawla R, Zargan J, Umar S, Sadaqat M, Khan HA (2011) Cytokinetics of adult rat SVZ rat after EAE. Brain Res 1371:140–149

    PubMed  Article  CAS  Google Scholar 

  20. 20.

    Kuan CY, Roth KA, Flavell RA et al (2000) Mechanisms of programmed cell death in the developing brain. Trends Neurosci 23:291–297

    PubMed  Article  CAS  Google Scholar 

  21. 21.

    Roth KA, D’Sa C (2001) Apoptosis and brain development. Ment Retard Dev Disabil Res Rev 7:261–266

    PubMed  Article  CAS  Google Scholar 

  22. 22.

    Sommer L, Rao M (2002) Neural stem cells and regulation of cell number. Prog Neurobiol 66:1–18

    PubMed  Article  CAS  Google Scholar 

  23. 23.

    Luo Y, Cai J, Liu Y et al (2002) Microarray analysis of selected genes in neural stem and progenitor cells. J Neurochem 83:1481–1497

    PubMed  Article  CAS  Google Scholar 

  24. 24.

    Imitola J, Comabella M, Chandraker AK et al (2004) Neural stem/progenitor cells express costimulatory molecules that are differentially regulated by inflammatory and apoptotic stimuli. Am J Pathol 164:1615–1625

    PubMed  Article  CAS  Google Scholar 

  25. 25.

    Sajad M, Zargan J, Chawla R, Umar S, Khan HA (2011) Upregulation of CSPG3 accompanies neuronal progenitor proliferation and migration in EAE. J Mol Neurosci 43:531–540

    PubMed  Article  CAS  Google Scholar 

  26. 26.

    Issazadeh S, Navikas V, Schaub M et al (1998) Kinetics of expression of costimulatory molecules and their ligands in murine relapsing experimental autoimmune encephalomyelitis in vivo. J Immunol 161:1104–1112

    PubMed  CAS  Google Scholar 

  27. 27.

    Seiffert E, Dreier JP, Ivens S et al (2004) Lasting blood–brain barrier disruption induces epileptic focus in the rat somatosensory cortex. J Neurosci 24:7829–7836

    PubMed  Article  CAS  Google Scholar 

  28. 28.

    Prohaska J, Clark D, Wella W (1973) Improved rapidity and precision in the determination of brain 2′, 3′-cyclic nucleotide 3′-phosphohydrolase. Anal Biochem 56:275–282

    PubMed  Article  CAS  Google Scholar 

  29. 29.

    Agrawal AK, Shukla S, Chaturvedi RK et al (2004) Olfactory ensheathing cell transplantation restores functional deficits in rat model of Parkinson’s disease: a cotransplantation approach with fetal ventral mesencephalic cells. Neurobiol Dis 16:516–526

    PubMed  Article  CAS  Google Scholar 

  30. 30.

    Storch A, Lester HA, Boehm BO et al (2003) Functional characterization of dopaminergic neurons derived from rodent mesencephalic progenitor cells. J Chem Neuroanat 26:133–142

    PubMed  Article  CAS  Google Scholar 

  31. 31.

    Mosmann T et al (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63

    PubMed  Article  CAS  Google Scholar 

  32. 32.

    Kitazawa M, Anantharam V, Kanthasamy AG (2001) Dieldrin-induced oxidative stress and neurochemical changes contribute to apoptotic cell death in dopaminergic cells. Free Radic Biol Med 31:1473–1485

    PubMed  Article  CAS  Google Scholar 

  33. 33.

    Smiley S, Reers T, Mottola-Hartshorn M et al (1991) Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate forming lipophilic cation JC-1. Proc Natl Acad Sci USA 88:3671–3675

    PubMed  Article  CAS  Google Scholar 

  34. 34.

    Singh NP, McCoy MT, Tice RR et al (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191

    PubMed  Article  CAS  Google Scholar 

  35. 35.

    Bradford MM et al (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    PubMed  Article  CAS  Google Scholar 

  36. 36.

    Fallon J, Reid S, Kinyamu R et al (2000) In vivo induction of massive proliferation, directed migration, and differentiation of neural cells in the adult mammalian brain. Proc Natl Acad Sci USA 97:14686–14691

    PubMed  Article  CAS  Google Scholar 

  37. 37.

    Kasper LH, Shoemaker J et al (2010) Multiple sclerosis immunology: the healthy immune system vs. the MS immune system. Neurology 74:S2–S8

    PubMed  Article  CAS  Google Scholar 

  38. 38.

    Gay D, Esiri M et al (1991) Blood-brain barrier damage in acute multiple sclerosis. Brain 114:557–572

    PubMed  Article  Google Scholar 

  39. 39.

    Bahbouhi B, Berthelot L, Pettré S et al (2009) Peripheral blood CD4+ T lymphocytes from multiple sclerosis patients are characterized by higher PSGL-1 expression and transmigration capacity across a human blood-brain barrier-derived endothelial cell line. J Leukoc Biol 86:1049–1063

    PubMed  Article  CAS  Google Scholar 

  40. 40.

    Lefkowitz DL, Lefkowitz SS (2008) Microglia and myeloperoxidase: a deadly partnership in neurodegenerative disease. Free Radic Biol Med 45:726–731

    PubMed  Article  CAS  Google Scholar 

  41. 41.

    Bradl M, Lassmann H et al (2010) Oligodendrocytes: biology and pathology. Acta Neuropathol 119:37–53

    PubMed  Article  Google Scholar 

  42. 42.

    Milosevic J, Storch A, Schwarz J (2004) Spontaneous apoptosis in murine free-floating neurospheres. Exp Cell Res 294:9–17

    PubMed  Article  CAS  Google Scholar 

  43. 43.

    Mattson MP, Liu D (2002) Energetics and oxidative stress in synaptic plasticity and neurodegenerative disorders. Neuromol Med 2:215–231

    Article  CAS  Google Scholar 

  44. 44.

    Lemasters JJ, Nieminen AL, Qian T et al (1998) The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochim Biophys Acta 1366:177–196

    PubMed  Article  CAS  Google Scholar 

  45. 45.

    Zakeri Z, Lockshin RA (2008) Cell death: history and future. Adv Exp Med Biol 615:1–11

    PubMed  Article  Google Scholar 

Download references

Acknowledgments

Authors are thankful to Dr. G.N Qazi (Vice Chancellor, Jamia Hamdard) for moral support during the course of this study. Mir Sajad is recipient of Senior Research Fellowship (SRF) from Indian Council of Medical Research (File no. 81/4/09-BM-Stem Cell), Ministry of Health, Govt. of India, New Delhi.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Haider A. Khan.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Sajad, M., Zargan, J., Sharma, J. et al. Increased Spontaneous Apoptosis of Rat Primary Neurospheres In Vitro After Experimental Autoimmune Encephalomyelitis. Neurochem Res 36, 1017–1026 (2011). https://doi.org/10.1007/s11064-011-0441-2

Download citation

Keywords

  • EAE
  • Ventricular proliferation
  • NPC apoptosis
  • DNA fragmentation
  • Remyelination