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. 2019 Nov;74(5):1181-1191.
doi: 10.1161/HYPERTENSIONAHA.119.13133. Epub 2019 Sep 30.

ACE2 and ADAM17 Interaction Regulates the Activity of Presympathetic Neurons

Snigdha Mukerjee  1   2   3 , Hong Gao  4   5 , Jiaxi Xu  1   2   3   6 , Ryosuke Sato  4 , Andrea Zsombok  4   5 , Eric Lazartigues  1   2   3   6
Affiliations

ACE2 and ADAM17 Interaction Regulates the Activity of Presympathetic Neurons

Snigdha Mukerjee et al. Hypertension. 2019 Nov.

Abstract

Brain renin angiotensin system within the paraventricular nucleus plays a critical role in balancing excitatory and inhibitory inputs to modulate sympathetic output and blood pressure regulation. We previously identified ACE2 and ADAM17 as a compensatory enzyme and a sheddase, respectively, involved in brain renin angiotensin system regulation. Here, we investigated the opposing contribution of ACE2 and ADAM17 to hypothalamic presympathetic activity and ultimately neurogenic hypertension. New mouse models were generated where ACE2 and ADAM17 were selectively knocked down from all neurons (AC-N) or Sim1 neurons (SAT), respectively. Neuronal ACE2 deletion revealed a reduction of inhibitory inputs to AC-N presympathetic neurons relevant to blood pressure regulation. Primary neuron cultures confirmed ACE2 expression on GABAergic neurons synapsing onto excitatory neurons within the hypothalamus but not on glutamatergic neurons. ADAM17 expression was shown to colocalize with angiotensin-II type 1 receptors on Sim1 neurons, and the pressor relevance of this neuronal population was demonstrated by photoactivation. Selective knockdown of ADAM17 was associated with a reduction of FosB gene expression, increased vagal tone, and prevented the acute pressor response to centrally administered angiotensin-II. Chronically, SAT mice exhibited a blunted blood pressure elevation and preserved ACE2 activity during development of salt-sensitive hypertension. Bicuculline injection in those models confirmed the supporting role of ACE2 on GABAergic tone to the paraventricular nucleus. Together, our study demonstrates the contrasting impact of ACE2 and ADAM17 on neuronal excitability of presympathetic neurons within the paraventricular nucleus and the consequences of this mutual regulation in the context of neurogenic hypertension.

Keywords: autonomic nervous system; hypertension; neurons; optogenetics; renin-angiotensin system.

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Conflict of interest statement

Disclosure

None, the authors declare no potential conflict of interest.

Figures

Figure 1.
Figure 1.. Synaptic activity of kidney-related PVN neurons in control and ACE2 knockout mice.
(A) Design of the neuronal ACE2 knockout mouse (AC-N) using CreLoxP system. ACE2 floxed mice, with LoxP sites flanking exon 4, were crossed with mice expressing cre recombinase driven by the synapsin1 promoter (specific to neurons). As a result, exon 4 of ACE2 was deleted from neurons in the progeny. (B) PVN pre-sympathetic neurons (green) following kidney PRV-eGFP injection. Post-recording staining with biocytin-avidin-Texas Red, in a GFP tagged kidney related neuron. Scale bar= 25 μm. (C) Example of a current-clamp recording from a kidney-related PVN neuron in CL mouse. Depolarizing current steps were applied following hyperpolarization to approximately −80 mV (insert illustrates the step protocol). Low-threshold spike (LTS) was observed in response to depolarizing steps. The red segment of the trace shown in the top panel is presented in the lower panel. Arrow points to LTS. (D) Recorded kidney-related neurons located in the PVN of CL (circle), AC-G (square) and AC-N (triangle) mice (E) Representative recordings of mIPSC at a holding potential of −10mV from a kidney-related PVN neuron in control (CL, top), AC-G (middle) and AC-N (bottom) mice. (F) Combined data showing the comparisons of frequency and amplitude for miniature and spontaneous IPSC (ANOVA *p<0.05, **p<0.01, n=13-19/group).
Figure 2.
Figure 2.. Prevalence of ACE2 within hypothalamic neuronal subpopulations.
(A-D) Immunohistochemistry of primary neuron cultures obtained from the hypothalamus of C57Bl6/J neonates. (A) Gad67-labeled (green) GABAergic neurons and ACE2-labeled (red) neurons overlap completely. (B) Oxytocinergic neurons (green) and ACE2-labeled neurons (red) overlap completely. (C) vGluT2-labeled glutamatergic neurons (green) and ACE2-labeled neurons (red) do not overlap but the excitatory neurons seem to synapse onto ACE2-expressing neurons. (D) Vasopressinergic neurons (green) co-localize with ACE2-expressing (red). (E) vGlut2-reporter mice express red fluorescence on excitatory neurons in the PVN. ACE2 expression (cyan) does not overlap with the glutamatergic neurons (purple) Scale Bar= 10 μm (primary neurons) and 25 μm (tissue)
Figure 3.
Figure 3.. ADAM17 expression within the Sim1-neurons.
(A) Design of the Sim1-tomato reporter mice (S-T) using CreLoxP system. (B) Inset: Sim1-promoter-driven cre-recombinase expression leads to tdTomato expression in all Sim1-neurons (red) around the third ventricle, scale bar= 30 μm. ADAM17 immunofluorescence (green) co-localizes (yellow) with Sim1 neurons (red), scale bar= 50 μm. Fluorescent in situ hybridization on PVN tissue from control mice, probing for: (C) vGluT2 mRNA (red) in tissue sections with ADAM17 mRNA (green), scale bar= 10 μm and (D) Gad67 mRNA (green) in tissue sections with ADAM17 mRNA (blue) reveals that ADAM17 is co-expressed on both excitatory and inhibitory neurons. Scale bar= 10 μm (E) Sim1-positive tomato neurons co-localize (yellow) with kidney projecting pre-sympathetic neurons labeled with PRV-eGFP (green), scale bar= 30 μm. (F) Digital droplet PCR data from sorted Sim-1 tomato neurons showing AT1 receptor and ADAM17 co-expression. ADAM17 expression is higher than AT1A receptor expression in these cells (PC: positive control [kidney], NC: negative control [water], Student’s unpaired t test, ***p<0.001, n=3).
Figure 4.
Figure 4.. Cardiovascular effect of stimulating Sim1-PVN neurons.
(A) AAV-DIO-ChR2-eYFP expression in PVN Sim1-neurons following virus injection. Scale bar= 20 μm. (B) BP recording from a mouse previously injected with AAV-DIO-ChR2-eYFP. Unilateral photo-excitation (20 Hz) of PVN neurons (blue shade) led to a rapid BP elevation. (C) Photo-activation in presence of a ganglionic blocker, Chlorisondamine, inhibited the BP response. (D) Summary data comparing the effect of light activation in presence and absence of chlorisondamine (Student’s unpaired t test, **p<0.01, n=3).
Figure 5.
Figure 5.. Selective deletion of ADAM17 from Sim1-PVN neurons.
(A) Design of the triple transgenic mouse (SAT) exhibiting a Sim1-Cre-driven ADAM17 deletion and tdTomato reporter expression. (B) Representative sorting strategy for fluorescence-activated cell sorting (FACS) in hypothalamic cells. The histogram (DAPI+ cells) was created based on the forward scatter heights (FSC-H) and side scatter heights (SSC-H) of the sorted cells (DAPI+ events). Respective gates for neurons (tdTomato+ cells) and non-neuron/non-astrocyte cells were created based on the PE heights (PE-A). qRT-PCR measurement of ADAM17 (C) and FosB (D) was performed in sorted hypothalamic cell populations from control and SAT mice (n=4/group). (E) Atropine i.p. led to an exaggerated tachycardia in SAT mice (Student’s unpaired t test, **p<0.01, n=7-12/group).
Figure 6.
Figure 6.. ADAM17 mediates RAS-induced BP rise.
Representative BP traces in S-T (A) and SAT mice (B) following unilateral PVN injection of Ang-II (200 ng/ 400 nl). (C) Summary data of Ang-II injection in the PVN of control (S-T, circle) and SAT mice (diamond), (inset, scale bar= 30 μm) location of the cannula placement for Ang-II injections in the PVN. (D) Summary data for mean arterial pressure (MAP) at baseline in S-T (closed circle) and SAT (closed diamond) mice and following 18 days of DOCA-salt treatment, showing a strong BP increase in S-T (open circle) and a blunted effect in SAT mice (open diamond) (1-way Kruskal-Wallis test, #p<0.05 vs. SAT, ***p<0.001 vs. S-T, $ $ $p<0.001 vs. S-T+DOCA, n=4-7/group). (E) DOCA-salt treatment reduced ACE2 activity in the control. It was preserved in SAT mice on DOCA-salt (1-way Kruskal-Wallis test, *p<0.05, n=8-12/group). (F) GABAA receptor antagonist, bicuculline, in the PVN induced change in MAP of CL (circle), AC-G (square) and SAT (Diamond) mice (1-way ANOVA, Tukey’s Multiple Comparison Test, **p<0.01, n=6-9/group)
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References

    1. Ambrosius WT, Sink KM, Foy CG, Berlowitz DR, Cheung AK, Cushman WC, Fine LJ, Goff DC Jr., Johnson KC, Killeen AA, Lewis CE, Oparil S, Reboussin DM, Rocco MV, Snyder JK, Williamson JD, Wright JT Jr., Whelton PK. The design and rationale of a multicenter clinical trial comparing two strategies for control of systolic blood pressure: The systolic blood pressure intervention trial (SPRINT). Clin Trials. 2014;11:532–546 - PMC - PubMed
    1. Leenen FH. Actions of circulating angiotensin II and aldosterone in the brain contributing to hypertension. Am J Hypertens. 2014;27:1024–1032 - PubMed
    1. Xia H, Lazartigues E. Angiotensin-converting enzyme 2: Central regulator for cardiovascular function. Curr Hypertens Rep. 2010;12:170–175 - PMC - PubMed
    1. Xia H, Sriramula S, Chhabra K, Lazartigues E. Brain ACE2 shedding contributes to the development of neurogenic hypertension. Circ Res. 2013;113:1087–1096 - PMC - PubMed
    1. Xu J, Sriramula S, Xia H, Moreno-Walton L, Culicchia F, Domenig O, Poglitsch M, Lazartigues E. Clinical relevance and role of neuronal AT1 receptors in ADAM17-mediated ACE2 shedding in neurogenic hypertension. Circ Res. 2017;121:43–55 - PMC - PubMed

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