ABSTRACT. Our collaboration builds on the protein kinase expertise of the Taylor lab and the expertise of the Chang lab to isolate Synthetically-evolved and engineered Nanobodies (SENs). Our goal is to isolate SENs that will discriminate between the isoforms of the catalytic (C) subunits of cAMP-dependent protein kinase (PKA). This builds on the realization that >50% of PKA signaling in the brain is mediated by C subunits. Although the C subunit of PKA was the second to be discovered, the first to be sequenced, and the first to be crystallized, virtually nothing is known about the C proteins, which represent a family of proteins that differ only in their N-terminal sequence encoded for by Exon 1. The C3/4 isoforms are expressed exclusively in brain while C2 is expressed in lymphoid tissues and T cells. Using the retina as a “Window into the Brain” Taylor showed that C is highly expressed in these terminally differentiated neurons and that it localizes differently from C, which supports the functional non-redundancy of these isoforms. The surprising discovery that C, but not C, RII, or RII, localizes to mitochondria adds further credence to the importance of C and its link to neurodegenerative diseases. To validate the hypothesis that imaging of the retina could serve as a window into the more complex signaling in brain and to determine if PKA signaling changes as a function of the onset and progression of AD, Taylor, in collaboration with the Alzheimer Disease Research Center at UCSD (UCSD ADRC) Neuropathology Core and Robert Rissman who directs this core is now comparing AD brains from individuals with mild and advanced AD. The preliminary results, coupled with imaging of pTau, a hallmark of advanced AD, confirm the hypothesis and highlight the importance of having tools that will discriminate between the various C isoforms. To achieve this essential next step we turned to the newly developed SENS technology. Using C-subunit proteins, purified in the Taylor lab, the Chang lab will fluorescently tag the proteins and then select for SENs that will discriminate between the different C-subunit isoforms. Initially they will focus on C4/C4ab as these, based on RNA scope, appeared to be the best candidates for association with mitochondria, and C2 which is expressed in T cells and has the potential to serve as a Biomarker for early onset PD. In Aim I Taylor will express C-subunit isoforms as well as peptide fragments that correspond to the N-terminal tails of each subunit. In Aim II Chang will fluorescently tag these proteins and through multiple rounds of screening will isolate selective high affinity SENs. These SENs will be analyzed for their ability to detect specific isoforms by Western blots and by imaging in both human and mouse tissues. Inhibition of kinase activity will also be assessed. A long-term goal will be to crystallize SENs: C-subunit complexes. Because this toolbox of reagents can be widely used by both clinicians and ...