Angiotensin‐II, insulin, and NELL2 involvement in forebrain synaptic circuit modifications during hypertension
Authors: Jeong Jin Kwon , Kim Dong Hee , Lee Byung Ju , Young Colin N. 
Published: May 2022

The central nervous system (CNS) is strongly implicated in cardiovascular disease development including hypertension. In particular, alterations in the subfornical organ (SFO), a forebrain sensory circumventricular nucleus located outside of the blood‐brain‐barrier, is directly associated with hypertension in response to elevations in the hormone angiotensin‐II (Ang‐II). From a circuitry perspective, SFO excitatory neurons that project to the hypothalamic paraventricular nucleus (PVN; hereafter referred to as SFO to PVN) have been suggested to be critical in the development of Ang‐II hypertension. While most investigations have focused on functional mechanisms, little attention has been paid to anatomical synaptic reorganization of SFO to PVN circuits during hypertension development. Thus, we aimed to characterize SFO to PVN synaptic input during hypertension development and further delineate molecular mechanisms involved. Male C57Bl/6J mice were infused with Ang‐II (600 ng/kg/min) for 14 days. Immunohistochemistry in the PVN following Ang‐II infusion revealed elevations in synaptophysin, pointing to overall increases in synaptic innervation of this nucleus (100 ± 1 vs. 112 ± 2 density/area, day 0 vs day 14, n=3/group, p<0.05). Given the strong link between SFO to PVN neurons in cardiovascular regulation, we next focused on the SFO. Our recent work demonstrated a key role for SFO insulin receptors in blood pressure regulation, and therefore, we determined if insulin receptors are expressed on SFO to PVN neurons using retrograde cholera toxin B tracing from the PVN. Immunohistochemistry revealed clear expression of insulin receptors on SFO to PVN neurons. Building on this, we and others have characterized a novel neural tissue‐enriched epidermal growth factor repeat domain containing protein (NELL2) that is involved in axonal projections and synaptic reorganization. Importantly, NELL2 has been suggested to be a downstream signaling molecule of neurotropic receptors, such as insulin receptors. In situ hybridization and immunohistochemistry confirmed the expression of NELL2 in the SFO, and further demonstrated elevated SFO NELL2 during Ang‐II hypertension (100 ± 5 vs. 137 ± 15 density/area, day 0 vs day 14, n=3‐4, p<0.05). Interestingly, co‐immunohistochemistry indicated that the majority of SFO insulin receptor cells express NELL2. Finally, to determine functional crosstalk between insulin receptors and NELL2, we utilized in vitro cell culture experiments. Hippocampal HT22 cells were transfected with a NELL2‐luciferase dependent vector and insulin was administrated into the medium. Insulin stimulated NELL2 gene transcription in a dose‐dependent manner (e.g. 174 and 196% increase relative to vehicle, 100 and 1000 nM insulin respectively, n=3/group, p<0.05). Collectively, these results indicate increased PVN synaptic innervation during hypertension. Moreover, they point to an SFO insulin receptor‐NELL2 network that may be involved in Ang‐II hypertension development, potentially via reorganization of SFO to PVN synaptic circuits.