Author(s): Jack Wilson
Mentor(s): John David Symons
Institution U of U
Acute ischemic stroke (AIS) elevates plasma ceramides that correlate with the prevalence and severity of neurological and motor deficits displayed by these patients. Our preclinical murine model of AIS [60-min transient middle cerebral artery occlusion (tMCAO) followed by 23-hr reperfusion (R)] increases ceramides (targeted mass spectrometry) in plasma and ipsilesional (ischemic) brain hemispheres compared to sham-operated controls. Notably, AIS-induced ceramides accumulated in cerebral endothelial cell (EC)-enriched but not non-EC (NEC)-enriched fractions (magnetic activated cell sorting). These findings inspired the hypothesis that preventing AIS-induced cerebral EC ceramide accrual mitigates infarct volume, neurological and motor deficits in mice. Dihydroceramide desaturase 1 (DES1, gene: Degs1) catalyzes toxic ceramide generation from inert dihydroceramide. We deployed a cerebral EC-specific adeno-associated virus BR1 serotype (AAV-BR1) to either carry Cre-recombinase (AAV-BR1-Cre) or Null virus (AAV-BR1-Null) to adult, male Degs1fl/fl mice, enabling selective depletion of Degs1 in cerebral ECs (aka Cre-Degs1) or not (Null-Degs1) [n=5 each]. Fourteen days after tMCAO+R, Degs1 gene expression was reduced (p<0.05) in ECs (but not NECs) isolated from ipsilesional hemispheres of Cre-Degs1 vs. Null-Degs1 mice. Further, AIS-induced infarct volume (TTC staining), neurological (Bederson score) and motor (Grip score) deficits were less severe (all p<0.05) in Cre-Degs1 vs. Null-Degs1 mice, suggesting that loss of cerebral EC-specific ceramide generation improves AIS outcomes. To substantiate these findings, we used a gain of cerebral EC ceramide approach. Serine palmitoyl transferase (SPT), the committed enzyme in ceramide biosynthesis, is comprised of 3-subunits. We deployed AAV-BR1-Cre (or Null vector) to adult, male mice enabling overexpression of a fusion (f) construct comprising the 3 SPT subunits preceded by STOPfl/fl, enabling rapid synthesis of cerebral EC ceramides (aka Cre-fSPT mice) or not (aka Null-fSPT mice) [n=5 each]. Fourteen days after tMCAO+R, we confirmed that SPTLC1 and SPTLC2 gene expression was elevated (p<0.05) in ECs (but not NECs) of ipsilesional hemispheres of Cre-fSPT vs. Null-fSPT mice. Importantly, AIS-induced infarct volume, neurological and motor deficits were greater (all p<0.05) in Cre-fSPT vs. Null-fSPT mice. Cerebral EC ceramide accrual might represent a new therapeutic target to combat the debilitating complications of AIS.