Longitudinal Study of Differential Protein Expression in an Alzheimer’s Mouse Model Lacking Inducible Nitric Oxide Synthase

Alzheimer’s
disease (AD) is a complex neurodegenerative
process that involves altered brain immune, neuronal and metabolic
functions. Understanding the underlying mechanisms has relied on mouse
models that mimic components of AD pathology. We used gel-free, label-free
LC–MS/MS to quantify protein and phosphopeptide levels in brains
of APPSwDI/NOS2–/– (CVN–AD) mice. CVN–AD
mice show a full spectrum of AD-like pathology, including amyloid
deposition, hyperphosphorylated and aggregated tau, and neuronal loss
that worsens with age. Tryptic digests, with or without phosphopeptide
enrichment on an automated titanium dioxide LC system, were separated
by online two-dimensional LC and analyzed on a Waters Synapt G2 HDMS,
yielding relative expression for >950 proteins and >1100 phosphopeptides.
Among differentially expressed proteins were known markers found in
humans with AD, including GFAP and C1Q. Phosphorylation of connexin
43, not previously described in AD, was increased at 42 weeks, consistent
with dysregulation of gap junctions and activation of astrocytes.
Additional alterations in phosphoproteins suggests dysregulation of
mitochondria, synaptic transmission, vesicle trafficking, and innate
immune pathways. These data validate the CVN–AD mouse model
of AD, identify novel disease and age-related changes in the brain
during disease progression, and demonstrate the utility of integrating
unbiased and phosphoproteomics for understanding disease processes
in AD.