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.

Established and bioinformatically predicted AKAP7 splice variants are expressed in the peripheral immune system and appear to be inversely regulated. It is well established that AKAP7 is subject to alternative splicing, however, little is known about the expression of these splice variants in the peripheral immune system. AKAP7 codes for three splice variants which are known to generate functional scaffolding proteins (AKAP7α, AKAP7β, AKAP7γ) with varying biological activity within the context of PKA signaling 20 . In addition to these established splice variants, AKAP7 may also code for as many as six bioinformatically predicated alternatively spliced transcripts (AKAP7x1, AKAP7x2, AKAP7x3, AKAP7x4, AKAP7x5, AKAP7x6) 21 . In order to determine which AKAP7 splice variants were responsible for the coregulatory relationship we observed with ITGA3, we first aimed to identify which AKAP7 splice variants are expressed in the peripheral immune system. Primers specific to each established and predicated splice variant were designed based on variant-specific priming sites (Fig. 4A ) and RT-PCR was used to probe for the presence of expression in peripheral whole blood samples obtained from two stroke patients and two healthy subjects. PCR products were detected for all tested AKAP7 splice variants in all samples (Fig. 4B) , providing direct evidence that all established and previously bioinformatically predicted AKAP7 splice variants are expressed in the peripheral immune system. Where possible, a second set of primers was used to validate the expression of previously predicted variants (Supplementary Figure 1A) . Once we identified that all established and previously predicted AKAP7 splice variants are expressed in the peripheral immune system, the expression levels of each splice variant were measured in parallel with those of ITGA3 in the peripheral blood of an identically recruited independent validation cohort of AIS patients using qRT-PCR (Table 2 ). In this second cohort of patients, early expression levels of AKAP7α, AKAP7β, and AKAP7γ exhibited significant positive correlations with expression levels of ITGA3 ( Fig. 5A-C) . Interestingly, all previously predicted AKAP7 splice variants displayed inversely related expression levels with regards to ITGA3 ( Fig. 5D -H) as well as the established AKAP7 splice variants (Supplementary Figure 2) . These data suggest that the strong positive relationship between AKAP7 and ITGA3 expression observed in the first cohort of patients was primarily driven by AKAP7α, AKAP7β, and AKAP7γ. This is feasible due to the fact that the previously predicted AKAP7 splice variants appeared to be expressed at much lower levels than those of the established splice variants, as indicated by higher CT values in qRT-PCR (data not shown) and the production of significantly less PCR product in standard RT-PCR when using primers designed for co-amplification (Supplementary Figure 1B) . Interestingly, AKAP7α, AKAP7β, and AKAP7γ all encode for scaffolding proteins with the ability to bind PKA 20 , while the remaining splice variants are predicted to undergo non-sense mediated decay or generate scaffolding proteins which lack a PKA binding domain (Supplementary Figure 3) . Collectively, the data validate the relationship between ITGA3 and AKAP7 observed in the discovery cohort, and suggest dynamic regulation between the PKA-binding and non-PKA-binding AKAP7 variants. Thus, the ratio between the expression levels of these sp