Worm Breeder's Gazette 15(1): 37 (October 1, 1997)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Aggregation of beta amyloid peptide variants in transgenic worms.

Chris Link, Amy Fluet, David Fay

IBG, University of Colorado, Boulder, CO, 80309

  Numerous lines of evidence suggest that for beta amyloid peptide (and
other amyloidic proteins), the ability to aggregate into insoluble
amyloid fibrils is closely linked to pathogenic potential.  Transgenic
animals containing unc-54/signal peptide/beta 1-42 constructs show
putative amyloid deposits, as demonstrated by binding of the specific
dye thioflavin S.  We have recently succeeded in visualizing green
birefringent deposits using Congo Red (the classic characteristic of
amyloid deposits) in these animals, further supporting the proposal
that these animals are an appropriate model for studying amyloid
formation. We have now used this system to investigate beta peptide
sequence requirements for amyloid formation by generating transgenic
worms expressing 6 different beta peptide variants designed to test
models of beta peptide tertiary structure.

  In summary, we have identified two single amino acid substitutions,
L17P and M35C, that completely abrogate the formation of thioflavin
S-reactive deposits.  Conversely, conservative changes at these
positions (L17V and M35L) have no apparent effect on formation of
thioflavin S deposits.  A proline substitution for A30 significantly
reduces, but does not eliminate, thioflavin S deposits.  Animals
expressing a single chain head-to-tail beta dimer also fail to make
thioflavin S deposits.  These results support current models of beta
peptide folding, but contradict some in vitro studies performed on
small sub-peptides derived from the beta peptide sequence.

  Although the results described above have been consistently observed
in multiple independent extrachromosomal and integrated lines, and all
lines examined show similar levels of beta peptide by
immunohistochemistry, we are developing a quantitative Western blot
assay to rigorously compare these lines.  Initial experiments with 25
worm samples of an unc-54/signal peptide/ beta 1-42 (non-variant) line
indicate that these animals produce an appropriately sized product
(confirming signal peptide processing), and that these animals contain
as much as 1 ng beta peptide per adult animal.