2 research outputs found
Elucidating Molecular Mass and Shape of a Neurotoxic Aβ Oligomer
Alzheimer's
disease (AD), the most prevalent type of dementia,
has been associated with the accumulation of amyloid β oligomers
(AβOs) in the central nervous system. AβOs vary widely
in size, ranging from dimers to larger than 100 kDa. Evidence indicates
that not all oligomers are toxic, and there is yet no consensus on
the size of the actual toxic oligomer. Here we used NU4, a conformation-dependent
anti-AβO monoclonal antibody, to investigate size and shape
of a toxic AβO assembly. By using size-exclusion chromatography
and immuno-based detection, we isolated an AβO-NU4 complex amenable
for biochemical and morphological studies. The apparent molecular
mass of the NU4-targeted oligomer was 80 kDa. Atomic force microscopy
imaging of the AβO-NU4 complex showed a size distribution centered
at 5.37 nm, an increment of 1.5 nm compared to the size of AβOs
(3.85 nm). This increment was compatible with the size of NU4 (1.3
nm), suggesting a 1:1 oligomer to NU4 ratio. NU4-reactive oligomers
extracted from AD human brain concentrated in a molecular mass range
similar to that found for in vitro prepared oligomers, supporting
the relevance of the species herein studied. These results represent
an important step toward understanding the connection between AβO
size and toxicity
Synapse-Binding Subpopulations of Aβ Oligomers Sensitive to Peptide Assembly Blockers and scFv Antibodies
Amyloid β42 self-assembly is complex, with multiple
pathways
leading to large insoluble fibrils or soluble oligomers. Oligomers
are now regarded as most germane to Alzheimer’s pathogenesis.
We have investigated the hypothesis that oligomer formation itself
occurs through alternative pathways, with some leading to synapse-binding
toxins. Immediately after adding synthetic peptide to buffer, solutions
of Aβ42 were separated by a 50 kDa filter and fractions assessed
by SDS-PAGE silver stain, Western blot, immunoprecipitation, and capacity
for synaptic binding. Aβ42 rapidly assembled into aqueous-stable
oligomers, with similar protein abundance in small (<50 kDa) and
large (>50 kDa) oligomer fractions. Initially, both fractions were
SDS-labile and resolved into tetramers, trimers, and monomers by SDS-PAGE.
Upon continued incubation, the larger oligomers developed a small
population of SDS-stable 10–16mers, and the smaller oligomers
generated gel-impermeant complexes. The two fractions associated differently
with neurons, with prominent synaptic binding limited to larger oligomers.
Even within the family of larger oligomers, synaptic binding was associated
with only a subset of these species, as a new scFv antibody (NUsc1)
immunoprecipitated only a small portion of the oligomers while eliminating
synaptic binding. Interestingly, low doses of the peptide KLVFFA blocked
assembly of the 10–16mers, and this result was associated with
loss of the smaller clusters of oligomers observed at synaptic sites.
What distinguishes these smaller clusters from the unaffected larger
clusters is not yet known. Results indicate that distinct species
of Aβ oligomers are generated by alternative assembly pathways
and that synapse-binding subpopulations of Aβ oligomers could
be specifically targeted for Alzheimer’s therapeutics