PROJECT SUMMARY
Alzheimer's disease (AD) is the most common neurodegenerative disease that is characterized by pathological
protein aggregation and inflammation in the brain (neuroinflammation). Microglia are the key immune cells of
the brain that mediate neuroinflammation. Novel therapeutic strategies and AD biomarkers can be identified if
we can define the molecular changes occurring in microglia in AD at the protein level, and not just at the
mRNA (transcriptomic) level. Our knowledge about microglial disease mechanisms in AD are mostly shaped
by transcriptomic studies although proteins are the enactors of biological processes and the correlation
between mRNA and protein is poor. The major barrier to microglial proteomic studies is the dependence on
isolation strategies to purify microglia before analyses. Isolation of microglia from brain induces artefacts,
yields very little protein and provides highly biased sampling. To overcome this barrier, we will use a novel
strategy (ciBONCAT) for microglia-specific proteomic labeling in-vivo. CiBONCAT allows us to label newly
synthesized proteins in microglia with an azide tag (azidonorleucine). These azide-tagged proteins can be
easily isolated from the brain without need for cell isolation. We have demonstrated the feasibility of using
ciBONCAT to label neuronal and astrocytic proteomes in adult mice, and have optimized the in-vivo and mass
spectrometry pipelines for proteomics using brain, cerebrospinal fluid (CSF) and other biofluids. This novel
strategy, coupled with our extensive expertise in mass spectrometry (proteomics) methods, will allow us to test
our central hypothesis that amyloid beta (Aß) accumulation, APOEe4, and aging independently and
synergistically impact proteomic phenotypes of microglia, and that these microglia-mediated mechanisms are
reflected in the CSF via immune biomarkers of AD pathology. In Aim 1, we will use ciBONCAT to define
microglia-specific protein alterations occurring in AD using two models of progressive amyloid beta pathology
(APP-PS1 and 5xFAD). In Aim 2, we will use knock-in mouse models to determine the effect of human
APOEe4 and APOEe3 expression on microglial proteins changes and how APOEe4 impacts with Aß pathology.
In Aim 3, we will identify microglia-derived proteins in the cerebrospinal fluid in mouse models of AD pathology
to identify novel biomarkers of neuroinflammation that reflect activation or depletion of microglia in the brain in
AD mouse models. Through the successful completion of this R01 proposal, we will obtain novel molecular
insights into microglia-mediated AD mechanisms and will establish ciBONCAT as a powerful approach to
investigate cell type-specific mechanisms of neurodegeneration. Our multidisciplinary expertise in microglial
biology and transgenic models of AD pathology (Dr Rangaraju), and quantitative proteomics, systems biology
and CSF biomarker discovery (Dr Seyfried), uniquely position us to execute this innovative R01 proposal.