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.

junction 39 . The sequences of remaining previously predicated splice variants (AKAP7x1, AKAP7x2, AKAP7x4, AKAP7x5) suggest they have the capacity to code for proteins. The protein isoforms which would be generated by these splice variants would be highly homologous to AKAP7γ, however lack the presence of the PKA binding domain. This suggests it is likely that AKAP7 protein isoforms exist in nature which lack A-kinase binding ability. It is possible that these isoforms could act as decoy proteins which occupy AKAP7 anchoring sites to inhibit the scaffolding of A-kinases. Alternatively, because three of these four potential protein isoforms are predicted to contain an untruncated PDE domain as their predominant feature, it is also plausible that they have some other biological function directly inferred by the PDE domain. As future studies provide more insight regarding the role of the PDE domain, the function of these previously predicated splice variants may become more clear. Our results imply that these previously predicted splice variants are expressed at much lower levels in the peripheral immune system than the established splice variants which code for PKA-binding protein isoforms, however, the dynamic intra-variant regulation we observed suggests they are of biological significance. Expectedly, due to their seemingly opposing biological function within the context of A-kinase signaling, we observed inversely regulated expression levels between the previously predicted splices variants and the established splice variants which code for PKA-binding protein isoforms. Thus, AKAP7 alternative splicing may play a much larger role in regulation of cAMP signaling than previously anticipated. Due to the preliminary nature of this study, it is important to note that it is not without limitations. The sample size in this study was not large enough to draw definitive conclusions regarding the potential use of AKAP7 as a clinical biomarker for prediction of post-stroke BBB complications. However, we feel the results of this this preliminary analysis are promising and investigation into the prognostic value of AKAP7 in a larger clinical cohort is warranted. In terms of understanding the mechanistic role of AKAP7 in the peripheral immune system with regards to stroke pathophysiology, much of the analysis in this study was associative in nature and performed only at the level of transcription. The biological mechanisms proposed based on the associative observations of this study should be validated in future pre-clinical work which takes a more direct experimental approach, potentially via genetic manipulation of AKAP7 in-vivo. Furthermore, our assumption that the association between whole blood AKAP7 levels and post-stroke BBB disruption is driven by a lymphocyte-dependent mechanism was based on the observation of lymphocyte-specific AKAP7 expression on isolated leukocyte subpopulations; however, this analysis was restricted to a limited number of healthy individuals and only surveyed the three most predominant subpopulations of leukocytes. Thus, future work may want to address the possibility that non-lymphoid sources of AKAP7 may contribute to the whole blood elevation in expression levels observed with post-stroke BBB disruption. Despite the aforementioned limitations, we feel that this work provides compelling evidence that AKAP7 in the peripheral immune system plays a role in post-stroke BBB pathology. We feel these results provide valuab