Peripheral blood AKAP7 expression as an early marker for lymphocyte-mediated post-stroke blood brain barrier disruption

O’Connell, Grant C.; Treadway, Madison B.; Petrone, Ashley B.; Tennant, Connie S.; Lucke-Wold, Noelle; Chantler, Paul D.; Barr, Taura L.

The objective of this study was to determine whether the early expression levels of any of 16 candidate genes which we previously established as differentially regulated in AIS could be used to identify patients at high risk for post-stroke BBB disruption. Our results suggest that elevated expression levels of AKAP7 during the early acute phase of care may be predicative for the development of BBB-disruption in the days following injury. Furthermore, we show evidence that AKAP7 expression may be elevated in patients who later develop post-stroke BBB disruption as a result of the presence of an invasive lymphocyte population in peripheral circulation. AKAP7 is a widely expressed scaffolding protein whose primary cellular function is spatial regulation of cyclic adenosine monophosphate (cAMP) signaling via cytoskeletal anchoring of PKA [22] [23] [24] . Alternative splicing results in multiple AKAP7 isoforms which are functionally diverse within the context of A-kinase signaling. The AKAP7 gene codes for three splice variants (AKAP7α, AKAP7β, AKAP7γ) which are known to be expressed and produce functional protein isoforms (Supplementary Figure 3) 20 . These AKAP7 isoforms all contain a conserved PKA-binding domain, but distinctive tethering domains and targeting motifs which are associated with differing intracellular localization 20, 25 . AKAP7γ is unique with regards to AKAP7α and AKAP7β in that it contains a phosphodiesterase (PDE) domain 26 . The function of this domain is currently unclear; structural similarity to bacterial 2′5′ RNA ligases has led to the hypothesis that it may have RNA ligase activity 27 , while studies which have shown AMP binding ability have led to the suggestion that it serves as an AMP sensor 26 . In addition to the splice variants which are known to code for proteins, the National Center for Biotechnology Information (NCBI) eukaryotic genome pipeline had previously predicted that the AKAP7 gene may also code for as many as six additional splice variants 21 . The sequences of these previously predicated splice variants suggest some would be targeted for non-sense mediated decay while others would generate protein isoforms containing the PDE domain but lacking the PKA binding domain (Supplementary Figure 3) . While AKAP7 has been most widely studied in the skeletal muscle, cardiac tissue, and the brain, little is known about the physiological role of AKAP7 in the peripheral immune system. In order to gain insight into the possible function of leukocyte AKAP7 in the context of post-stroke BBB disruption, we aimed to establish which AKAP7 splice variants are expressed in the peripheral immune system following stroke, determine what cell populations they are expressed on, as well as identify molecules which exhibit a pattern of co-regulation. Our results provided evidence that all previously established protein coding AKAP7 splice variants, as well as all previously predicted AKAP7 splice variants, are expressed in the peripheral immune system. Our results further suggest that the PKA-binding isoforms of AKAP7 are predominantly expressed on cells of lymphoid origin, and that their expression levels are highly correlated with those of ITGA3, a cell adhesion molecule known to interact with various extracellular matrix proteins including with laminin, collagen, and fibronectin 28 . ITGA3 has recently been shown to signal via cAMP and PKA upon ligand binding 29 ; thus, it is highly plausible that the strong relationship