Functional Defect Conferred by the Parkinson's Disease-Causing α-Synuclein (Ala30Pro) Mutation

  • Poul Henning Jensen
Protocol
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 62)

Abstract

The association of missense mutations in the α-synuclein gene to heritable Parkinson’s diesease (PD) indicates that dysfunction of normal α-synuclein metabolism, or novel gain-of-functions by the mutant peptides, can elicit early-onset PD (1,2). The accumulation of α-synuclein in nerve cell Lewy bodies of the common sporadic PD suggests a wider role for α-synuclein in the development of PD (3). However, investigations into the loss and gain of functions caused by the specific mutations have been sparse, although an increased fibrillogenic potential have been reported (4,5). We have shown that a fraction of α-synuclein is moved by fast axonal transport to the synaptic terminus (6,7). Fast axonal transport is mediated by the motor-driven movement of vesicular structures along microtubules. By studying the interaction between α-synuclein and brain vesicles we discovered that the Ala30Pro mutation inhibits the interaction between mutant a-synuclein and brain vesicles (6). Hypothetically, this inhibitory effect could perturb the fast axonal transport of α-synuclein thus increasing its concentration in the cell body where it may aggregate into Lewy bodies. The technique described in this chapter is modified from Brown & Rose (8) and allows the study of interactions between α-synuclein and brain vesicles and therefore the functional effects of pathogenic point mutations or deletions of specific domains. Information on the following points can be found in Jensen et al. (6,9) and references therein: (1) Mutagenesis, expression, and purification of recombinant human α-synucleins and (2) methodology for performing sodium dodecyl sulfate (SDS) gel electrophoresis and electroblotting.

Keywords

Vortex Sucrose Glycerol Acetone Urea 

References

  1. 1.
    Polymeropoulos, M. H., Lavedan, C., Leroy, E., Ide, S. E., Dehejia, A., Dutra, A., et al. (1997) Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease. Science 276, 2045–2047.CrossRefPubMedGoogle Scholar
  2. 2.
    Kruger, R., Kuhn, W., Muller, T., Woitalla, D., Graeber, M., Kosel, S., et al. (1998) Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson’s disease. Nat. Genet. 18, 106–108.CrossRefPubMedGoogle Scholar
  3. 3.
    Spillantini, M. G., Schmidt, M. L., Lee, V. M., Trojanowski, J. Q., Jakes, R., and Goedert, M. (1997) Alpha-synuclein in Lewy bodies. Nature 388, 839–840.CrossRefPubMedGoogle Scholar
  4. 4.
    Conway, K. A., Harper, J. D., and Lansbury, P. T. (1998) Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease. Nat. Med. 11, 1318–1320.CrossRefGoogle Scholar
  5. 5.
    El-Agnaf, O. M., Jakes, R., Curran, M. D., and Wallace, A. (1998) Effects of the mutations Ala30 to Pro and Ala53 to Thr on the physical and morphological prop-erties of alpha-synuclein protein implicated in Parkinson’s disease. FEBS Lett. 440, 67–70.CrossRefPubMedGoogle Scholar
  6. 6.
    Jensen, P. H., Nielsen, M. S., Jakes, R., Dotti, C. G., and Goedert, R. (1998) Binding of a-synuclein to brain vesicles is abolished by familial Parkinson’s disease mutation. J. Biol. Chem. 273, 26,292–26,294.CrossRefPubMedGoogle Scholar
  7. 7.
    Jensen, P. H., Li, J.-Y., Dahlström, A., and Dotti, C. G. (1999) Axonal transport of synucleins are mediated by all rate components. Eur. J. Neurosci. 11, 3369–3376.CrossRefPubMedGoogle Scholar
  8. 8.
    Brown, D. A. and Rose, J. K. (1992) Sorting of GPI-anchored proteins to gly-colipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68, 533–544.CrossRefPubMedGoogle Scholar
  9. 9.
    Jensen, P. H., Højrup, P., Hager, H., Nielsen, M. S., Jacobsen, L., Olesen, O. F., et al. (1997) Binding of Ab to α-and β-synucleins: identification of segments in α-synuclein/NAC precursor that bind Aβ and NAC. Biochem. J. 323, 539–546.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2001

Authors and Affiliations

  • Poul Henning Jensen
    • 1
  1. 1.Department of Medical BiochemistryUniversity of AarhusDenmark

Personalised recommendations