Integrated Approaches for Analyzing U1-70K Cleavage in Alzheimer’s Disease

The
accumulation of pathologic protein fragments is common in neurodegenerative
disorders. We have recently identified in Alzheimer’s disease
(AD) the aggregation of the U1-70K splicing factor and abnormal RNA
processing. Here, we present that U1-70K can be cleaved into an N-terminal
truncation (N40K) in ∼50% of AD cases, and the N40K abundance
is inversely proportional to the total level of U1-70K. To map the
cleavage site, we compared tryptic peptides of N40K and stable isotope
labeled U1-70K by liquid chromatography–tandem mass spectrometry
(MS), revealing that the proteolysis site is located in a highly repetitive
and hydrophilic domain of U1-70K. We then adapted Western blotting
to map the cleavage site in two steps: (i) mass spectrometric analysis
revealing that U1-70K and N40K share the same N-termini and contain
no major modifications; (ii) matching N40K with a series of six recombinant
U1-70K truncations to define the cleavage site within a small region
(Arg300 ± 6 residues). Finally, N40K expression led to substantial
degeneration of rat primary hippocampal neurons. In summary, we combined
multiple approaches to identify the U1-70K proteolytic site and found
that the N40K fragment might contribute to neuronal toxicity in Alzheimer’s
disease.