Acta Neuropathologica

, Volume 115, Issue 3, pp 313–326 | Cite as

Abnormal motoneuron migration, differentiation, and axon outgrowth in spinal muscular atrophy

  • Goran Simic
  • Mihovil Mladinov
  • Durdica Seso Simic
  • Natasa Jovanov Milosevic
  • Atiqul Islam
  • Alen Pajtak
  • Nina Barisic
  • Jadranka Sertic
  • Paul J. Lucassen
  • Patrick R. Hof
  • Bozo Kruslin
Original Paper


The role of heterotopic (migratory) motoneurons (HMN) in the pathogenesis of spinal muscular atrophy (SMA) is still controversial. We examined the occurrence and amount of HMN in spinal cord tissue from eight children with SMA (six with SMA-I and two with SMA-II). All affected subjects were carrying a homozygous deletion of exon 7 in the SMN1 gene. Unlike controls, virtually free from HMN, all SMA subjects showed a significant number of HMN at all levels of the spinal cord. Heterotopic neurons were hyperchromatic, located mostly in the ventral white matter and had no axon or dendrites. More than half of the HMN were very undifferentiated, as judged from their lack of immunoreactivity for NeuN and MAP2 proteins. Small numbers of more differentiated heterotopic neurons were also found in the dorsal and lateral white matter region. As confirmed by ultrastructural analysis, in situ end labeling (ISEL) and CD68 immunoreactivity, HMN in the ventral outflow were found to have no synapses, to activate microglial cells, and to eventually die by necrosis. An unbiased quantitative analysis showed a significant negative correlation between age of SMA subjects (a reflection of the clinical severity) and the number of HMN. Subjects who died at older ages had increased number of GFAP-positive astrocytes. Complementing our previous report on motoneuron apoptosis within the ventral horns in SMA, we now propose that abnormal migration, differentiation, and lack of axonal outgrowth may induce motoneuron apoptosis predominantly during early stages, whereas a slower necrosis-like cell death of displaced motoneurons which “escaped” apoptosis characterizes later stages of SMA.


Motoneurons Migration Pathogenesis Spinal muscular atrophy SMN1 gene 



This work was supported by grants from the Ministry of Science, Education and Sports of Republic of Croatia (108-1081870-1884 to BK and 108-1081870-1942 to GS). We thank Z. Cmuk, D. Budinscak, B. Popovic (Department of Neuroscience, Croatian Institute for Brain Research), D. Poljan (Department of Pathology, Medical School Zagreb), and I. Jusinsky (Clinical Research Center – Electron Microscopy Unit, Huddinge University Hospital, Karolinska Institute, Stockholm) for excellent technical help.


  1. 1.
    Battaglia G, Princivalle A, Forti F, Lizier C, Zeviani M (1997) Expression of the SMN gene, the spinal muscular atrophy determining gene, in the mammalian central nervous system. Hum Mol Genet 6:1961–1971PubMedCrossRefGoogle Scholar
  2. 2.
    Béchade C, Rostaing P, Cisterni C, Kalisch R, La Bella V, Pettmann B, Triller A (1999) Subcellular distribution of SMN protein: possible involvement in nucleoplasmic and dendritic transport. Eur J Neurosci 11:293–304PubMedCrossRefGoogle Scholar
  3. 3.
    Botta A, Tacconelli A, Bagni I, Giardina E, Bonifazi E, Pietropolli A, Clementi M, Novelli G (2005) Transmission ration distortion in the spinal muscular atrophy locus: data from 314 prenatal tests. Neurology 65:1631–1635PubMedCrossRefGoogle Scholar
  4. 4.
    Brock TO, McIlwain DL (1984) Astrocytic proteins in the dorsal and ventral roots in amyotrophic lateral sclerosis and Werdnig–Hoffmann disease. J Neuropahol Exp Neurol 43:609–619Google Scholar
  5. 5.
    Burlet P, Burglen L, Clermont O, Lefebvre S, Viollet L, Munnich A, Melki J (1996) Large scale deletions of the 5q13 region are specific to Werdnig–Hoffmann disease. J Med Genet 33:281–283PubMedCrossRefGoogle Scholar
  6. 6.
    Campbell L, Hunter KM, Mohaghegh P, Tinsley JM Brasch MA, Davies KE (2000) Direct interaction of SMN with dp103, a putative RNA helicase: a role for SMN in transcription regulation? Hum Mol Genet 9:1093–1100PubMedCrossRefGoogle Scholar
  7. 7.
    Camu W, Billiard M (1993) Coexistence of amyotrophic lateral sclerosis and Werdnig–Hoffmann disease within a family. Muscle Nerve 16:569–570PubMedGoogle Scholar
  8. 8.
    Carrel TL, McWhorter ML, Workman E, Zhang H, Wolstencroft EC, Lorson C, Bassell GJ, Burghes AH, Beattie CE (2006) Survival motor neuron function in motor axons is independent of functions required for small nuclear ribonucleoprotein biogenesis. J Neurosci 26:11014–11022PubMedCrossRefGoogle Scholar
  9. 9.
    Chou SM, Wang HS (1997) Aberrant glycosylation/phoshorylation in chromatolytic motoneurons of Werdnig–Hoffmann disease. J Neurol Sci 152:198–209PubMedCrossRefGoogle Scholar
  10. 10.
    Clarke PGH (1990) Developmental cell death: morphological diversity and multiple mechanisms. Anat Embryol 181:195–213PubMedCrossRefGoogle Scholar
  11. 11.
    Corcia P, Camu W, Halimi JM, Vourc’h P, Antar C, Vedrine S, Giraudeau B, de Toffol B, Andres CR (2006) SMN1 gene, but not SMN2, is a risk factor for sporadic ALS. Neurology 67:1147–1150PubMedCrossRefGoogle Scholar
  12. 12.
    Cuscó I, Barceló MJ, Rojas-García R, Illa I, Gamez J, Cervera C, Pou A, Izquierdo G, Baiget M, Tizzano EF (2006) SMN copy number predicts acute or chronic spinal muscular atrophy but does not account for intrafamilial variability in siblings. J Neurol 253:21–25PubMedCrossRefGoogle Scholar
  13. 13.
    Dubowitz V (1995) Disorders of the lower motor neuron, the spinal muscular atrophy. In: Dubowitz V (ed) Muscle disorders in childhood. Saunders, London, pp 325–369Google Scholar
  14. 14.
    Feldkötter M, Schwarzer V, Wirth R, Wienker TF, Wirth B (2002) Quantitative analyses of SMN1 and SMN2 based on real-time light-cycler PCR: fast and highly reliable carrier testing and prediction of severity of spinal muscular atrophy. Am J Hum Genet 70:358–368PubMedCrossRefGoogle Scholar
  15. 15.
    Fidziańska A, Rafalowska J (2002) Motoneuron death in normal and spinal muscular atrophy-affected human fetuses. Acta Neuropathol 104:363–368PubMedGoogle Scholar
  16. 16.
    Fischer U, Liu Q, Dreyfuss G (1997) The SMN-SIP1 complex has an essential role in spliceosome biogenesis. Cell 90:1023–1029PubMedCrossRefGoogle Scholar
  17. 17.
    Galluzzi L, Maiuri MC, Vitale I, Zischka H, Castedo M, Zitvoogel L, Kroemer G (2007) Cell death modalities: classification and pathophysiological implications. Cell Death Diff 14:1237–1243CrossRefGoogle Scholar
  18. 18.
    Ghatak NR (1978) Spinal roots in Werdnig–Hoffmann disease. Acta Neuropathol 41:1–7PubMedCrossRefGoogle Scholar
  19. 19.
    Giavazzi A, Setola V, Simonati A, Battaglia G (2006) Neuronal-specific roles of the survival motor neuron protein: evidence from survival motor neuron expression patterns in the developing human central nervous system. J Neuropathol Exp Neurol 65:267–277PubMedGoogle Scholar
  20. 20.
    Gunthinas-Lichius O, Mockenhaupt J, Stennert E, Neiss WF (1993) Simplified nerve cell counting in the rat brainstem with the physical disector using a drawing microscope. J Microsci 172:177–180Google Scholar
  21. 21.
    Iwahashi H, Eguchi Y, Yasuhara N, Hanafusa T, Matsuzawa Y, Tsujimoto Y (1997) Synergistic antiapoptotic activity between bcl-2 and SMN implicated in spinal muscular atrophy. Nature 390:413–417PubMedCrossRefGoogle Scholar
  22. 22.
    Kerr DA, Nery JP, Traystman RJ, Chau BN, Hardwick JM (2000) Survival motor neuron protein modulates neuron-specific apoptosis. Proc Natl Acad Sci USA 97:13312–13317PubMedCrossRefGoogle Scholar
  23. 23.
    Kesari A, Idris MM, Chandak GR, Mittal B (2005) Genotype–phenotype correlation of SMN locus genes in spinal muscular atrophy patients from India. Exp Mol Med 37:147–154PubMedGoogle Scholar
  24. 24.
    Kimura T, Budka H (1984) Glial bundles in spinal nerve roots. An immunocytochemical study stressing their nonspecificity in various spinal cord and peripheral nerve diseases. Acta Neuropathol 65:46–52PubMedCrossRefGoogle Scholar
  25. 25.
    Kozlowski MA, Williams C, Hinton DR, Miller CA (1989) Heterotopic neurons in spinal cord of patients with ALS. Neurology 39:644–648PubMedGoogle Scholar
  26. 26.
    Lefebvre S, Burglen L, Reboullet S, Clermont O, Burlet P, Viollet L, Benichou B, Cruaud C, Millasseau P, Zeviani M, Le Paslier D, Frézal J, Cohen D, Weissenbach J, Munnich A, Melki J (1995) Identification and characterization of a spinal muscular atrophy-determining gene. Cell 80:155–165PubMedCrossRefGoogle Scholar
  27. 27.
    Lind D, Franken S, Kappler J, Jankowski J, Schilling J (2005) Characterization of the neuronal marker NeuN as a multiply phosphorylated antigen with discrete subcellular localization. J Neurosci Res 79:295–302PubMedCrossRefGoogle Scholar
  28. 28.
    Lorson CL, Strasswimmer J, Yao J-M, Baleja JD Hahnen E, Wirth B, Le T, Burghes AH, Androphy EJ (1998) SMN oligomerization defect correlates with SMA severity. Nat Genet 19:63–66PubMedCrossRefGoogle Scholar
  29. 29.
    Lorson CL, Hahnen E, Androphy EJ, Wirth B (1999) A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proc Natl Acad Sci 96:6307–6311PubMedCrossRefGoogle Scholar
  30. 30.
    Lucassen PJ, Chung WCJ, Vermeulen JP, Van Lookeren Campagne M, Van Dierendonck JH, Swaab DF (1995) Microwave-enhanced in situ end-labeling of fragmented DNA: parametric studies in relation to post mortem delay and fixation of rat and human brain. J Histochem Cytochem 43:1163–1171PubMedGoogle Scholar
  31. 31.
    Mailman MD, Heinz JW, Papp AC, Snyder PJ, Sedra MS, Wirth B, Burghes AH, Prior TW (2002) Molecular analysis of spinal muscular atrophy and modification of the phenotype by SMN2. Genet Med 4:20–26PubMedCrossRefGoogle Scholar
  32. 32.
    Marschall A, Duchen LW (1975) Sensory system involvement in infantile spinal muscular atrophy. J Neurol Sci 26:349–359CrossRefGoogle Scholar
  33. 33.
    Martin JE, Mather K, Swash M (1993) Heterotopic neurons in amyotrophic lateral sclerosis. Neurology 43:1420–1422PubMedGoogle Scholar
  34. 34.
    McWhorter ML, Monani UR, Burghes AH, Beattie CE (2003) Knockdown of the survival motor neuron (Smn) protein in zebrafish causes defects in motor axon outgrowth and pathfinding. J Cell Biol 162:919–931PubMedCrossRefGoogle Scholar
  35. 35.
    Monani UR, Lorson CL, Parsons DW, Prior TW, Androphy EJ, Burghes AH, McPherson JD (1999) A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2. Hum Mol Genet 8:1177–1183PubMedCrossRefGoogle Scholar
  36. 36.
    Oskoui M, Levy G, Garland CJ, Gray JM, O′Hagen J, De Vivo DC, Kaufmann P (2007) The changing natural history of spinal muscular atrophy type 1. Neurology 69:1931–1936PubMedCrossRefGoogle Scholar
  37. 37.
    Pagliardini S, Giavazzi A, Setola V, Lizier C, Di Luca M, DeBiasi S, Battaglia G (2000) Subcellular localization and axonal transport of the survival motor neuron (SMN) in the developing rat spinal cord. Hum Mol Genet 9:47–56PubMedCrossRefGoogle Scholar
  38. 38.
    Parsons DW, McAndrew PE, Iannaccone ST, Mendell JR, Burghes AH, Prior TW (1998) Intragenic telSMN mutations: frequency, distribution, evidence of a founder effect, and modification of the spinal muscular atrophy phenotype by cenSMN copy number. Am J Hum Genet 63:1712–1723PubMedCrossRefGoogle Scholar
  39. 39.
    Rossoll W, Jablonka S, Andreassi C, Kroning AK, Karle K, Monani UR, Sendtner M (2003) Smn, the spinal muscular atrophy-determining gene product, modulates axon growth and localization of beta-actin mRNA in growth cones of motoneurons. J Cell Biol 163:801–812PubMedCrossRefGoogle Scholar
  40. 40.
    Roy N, Mahadevan MS, McLean M, Shutler G, Yaraghi Z, Farahani R, Baird S, Besner-Johnston A, Lefebvre C, Kang X, Salih M, Aubry H, Tamai K, Guan X, Ioannou P, Crawford TO, de Jong PJ, Surh L, Ikeda JE, Korneluk RG, MacKenzie A (1995) The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy. Cell 80:167–178PubMedCrossRefGoogle Scholar
  41. 41.
    Sasaki S, Iwata M (2004) Characterization of heterotopic neurons in the spinal cord of amyotrophic lateral sclerosis patients. Acta Neuropathol 95:367–372CrossRefGoogle Scholar
  42. 42.
    Sato K, Eguchi Y, Kodama TS, Tsujimoto Y (2000) Regions essential for the interaction between Bcl-2 and SMN, the spinal muscular atrophy disease gene product. Cell Death Differ 7:374–383PubMedCrossRefGoogle Scholar
  43. 43.
    Scharf JM, Endrizzi MG, Wetter A, Huang S Thompson TG, Zerres K, Dietrich WF, Wirth B, Kunkel LM (1998) Identification of a candidate modifying gene for spinal muscular atrophy by comparative genomics. Nat Genet 20:83–86PubMedCrossRefGoogle Scholar
  44. 44.
    Selig S, Bruno S, Scharf JM, Wang CH Vitale E, Gilliam TC, Kunkel LM (1995) Expressed cadherin pseudogenes are localized to the critical region of the spinal muscular atrophy gene. Proc Natl Acad Sci 92:3702–3706PubMedCrossRefGoogle Scholar
  45. 45.
    Sertic J, Barisic N, Sostarko M, Bosnjak N, Culic V, Cvitanovic L, Ferencak G, Brzovic Z, Stavljenic-Rukavina A (1997) Deletions in the SMN and NAIP genes in patients with spinal muscular atrophy in Croatia. Coll Antropol 21:487–492PubMedGoogle Scholar
  46. 46.
    Setola V, Terao M, Locatelli D, Bassanini S Garattini E, Battaglia G (2007) Axonal-SMN (a-SMN), a protein isoform of the survival motor neuron gene, is specifically involved in axonogenesis. Proc Natl Acad Sci USA 104:1959–1964PubMedCrossRefGoogle Scholar
  47. 47.
    Shishikura K, Hara M, Sasaki Y, Misugi K (1983) A neuropathologic study of Werdnig–Hoffmann disease with special reference to the thalamus and posterior roots. Acta Neuropathol 60:99–106PubMedCrossRefGoogle Scholar
  48. 48.
    Simic G, Seso-Simic D, Lucassen P, Islam A, Krsnik Z, Cviko A Jelasic D, Barisic N, Winblad B, Kostovic I, Kruslin B (2000) Ultrastructural analysis and TUNEL demonstrate motor neuron apoptosis in Werdnig–Hoffmann disease. J Neuropathol Exp Neurol 59:398–407PubMedGoogle Scholar
  49. 49.
    Veldink JH, Kalmijn S, Van der Hout AH, Lemmink HH, Groeneveld GJ, Lummen C, Scheffer H, Wokke JH, Van den Berg LH (2005) SMN genotypes producing less SMN protein increase susceptibility to and severity of sporadic ALS. Neurology 65:820–825PubMedCrossRefGoogle Scholar
  50. 50.
    Werdnig G (1981) Zwei frühinfantile hereditäre Fälle von progressiver Muskelatrophie unter dem Bilde der Dystrophie, aber auf neurotischer Grundlage. Arch Psychiatr Nervenkr 22:437–481CrossRefGoogle Scholar
  51. 51.
    Wharton S, Ince PG (2003) Pathology of motor neuron disorders. In: Shaw PJ, Strong MJ (eds) Motor neuron disorders. Blue books of practical neurology, book 28. Butterworth-Heineman, Elsevier Science, Philadelphia, pp 17–49Google Scholar
  52. 52.
    Wijsman JH, Jonker RR, Keijzer R, Van de Velde CJH, Cornelisse CJ, Van Dierendonck JH (1993) A new method to detect apoptosis in paraffin sections: in situ end labeling of fragmented DNA. J Histochem Cytochem 41:7–12PubMedGoogle Scholar
  53. 53.
    Young P, Le TT, Dunckley M, Nguyen TM Burghes AH, Morris GE (2001) Nuclear gems and Cajal (coiled) bodies in fetal tissues: nucleolar distribution of the spinal muscular atrophy protein, SMN. Exp Cell Res 265:252–261PubMedCrossRefGoogle Scholar
  54. 54.
    Young PJ, Day PM, Androphy EJ, Morris GE Morris GE, Lorson CL (2002) A direct interaction between survival motor neuron protein and p53 and its relationship to spinal muscular atrophy. J Biol Chem 277:2852–2859PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Goran Simic
    • 1
  • Mihovil Mladinov
    • 1
  • Durdica Seso Simic
    • 2
  • Natasa Jovanov Milosevic
    • 1
  • Atiqul Islam
    • 3
  • Alen Pajtak
    • 1
  • Nina Barisic
    • 2
  • Jadranka Sertic
    • 2
  • Paul J. Lucassen
    • 4
  • Patrick R. Hof
    • 5
  • Bozo Kruslin
    • 1
  1. 1.Department of Neuroscience, School of Medicine, Croatian Institute for Brain Research, Medical School ZagrebZagreb UniversityZagrebCroatia
  2. 2.University Hospital Center ZagrebZagrebCroatia
  3. 3.Karolinska InstituteStockholmSweden
  4. 4.Centre for Neuroscience, Swammerdam Institute for Life SciencesUniversity of AmsterdamAmsterdamThe Netherlands
  5. 5.Department of NeuroscienceMount Sinai School of MedicineNew YorkUSA

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