Abstract 058: ACE2 Ubiquitination Regulates Neuronal Activity In Hypertension

Abstract

ACE2 expression on GABAergic neurons could maintain the inhibitory tone over pre-sympathetic neurons in the paraventricular nucleus (PVN) and thus contribute to the regulation of blood pressure (BP). Here we investigated the role of ACE2 ubiquitination in the bed nucleus of the stria terminalis (BNST), a region containing GABAergic neurons and involved in cardiovascular responses to stress. C57BL6/J male mice were injected in the BNST with a ubiquitination-resistant human ACE2 mutant (AAV-hACE2-5R). A subset of mice was also injected with AAV-GCaMP6s-eYFP in the PVN and BNST to record intracellular Ca 2+ activity via fiber photometry. After 6 weeks, the mice were chronically infused with Ang-II (600 ng/kg/min) for 2 weeks while BP was monitored by telemetry. Ang-II-infusion resulted in a significant increase in mean BP compared to controls (152±7 vs. 123±4 mmHg, respectively; n=6, p<0.05) that was blunted in mice expressing hACE2-5R (133±5 mmHg, n=6, p<0.05). Injection of bicuculline (GABA A antagonist, 1 mmol/L in 100nL) in the PVN produced a blunted BP rise in Ang-II-infused mice compared to controls (AUC: 261±36 vs. 392±45, respectively; n=4, p<0.05) and an enhanced pressor response in the hACE2-5R+Ang-II group (AUC: 690±81, n=5, p<0.05). Acute injection of Ang-II (250 ng iv) caused an immediate increase in Ca 2+ mediated fluorescence in the PVN and a parallel reduction of neuronal activity in the BNST (ΔF/F 0 : +0.9±0.02 and -0.1±0.03; respectively). Surprisingly, hACE2-5R expression in the BNST produced a rapid and persistent inhibition of neuronal activity. Capillary Western analysis showed a 3-fold increase in NEDD4-2 (n=4, p<0.05) with a parallel reduction of ACE2 levels (-50%, n=4, p<0.05) in the BNST of Ang-II-infused mice, while hACE2-5R prevented NEDD4-2 upregulation and ACE2 downregulation (n=4, p<0.05). Our data show that increases in peripheral Ang-II levels result in an immediate regulation of neuronal activity in the PVN and BNST. Hypertension is associated with NEDD4-2 mediated ubiquitination of ACE2 blunting GABAergic input to the PVN and potentially increasing the activity of pre-sympathetic neurons. ACE2 ubiquitination by NEDD4-2 in key brain regions is a new mechanism contributing to the development of neurogenic hypertension.