Cardiovascular Regulation by the Neuronal BBSome

Abstract

The BBSome, a complex of eight Bardet-Biedl syndrome (BBS) proteins known for its role in the control of cilia function and other cellular processes, has been implicated in blood pressure control, but the underlying mechanisms are not fully understood. Here, we show that neuronal BBSome plays an important role in blood pressure regulation. Targeted inactivation of the BBSome in the nervous system through Bbs1 gene deletion causes sympathetically-mediated increase in blood pressure in mice. This phenotype is reproduced by selective ablation of the Bbs1 gene from the leptin receptor (LRb)-expressing neurons. Strikingly, the well-known role of the BBSome in the regulation of cilia formation and function is unlikely to account for the prohypertensive effect of BBSome inactivation as disruption of the intraflagellar transport (IFT) machinery required for ciliogenesis by deleting the Ift88 gene in LRb neurons had no effect on arterial pressure and sympathetic nerve activity. Furthermore, we found that Bbs1 gene deletion from agouti-related protein (AgRP) neurons or proopiomelanocortin (POMC) neurons increased renal and splanchnic sympathetic nerve activity without altering blood pressure. This lack of blood pressure increase despite the sympathetic overdrive may be explained by vascular adrenergic desensitization as indicated by the reduced vascular contractile response evoked by phenylephrine and the decreased expression of adrenergic receptors. Our results identify the neuronal BBSome as a new player in hemodynamic, sympathetic and vascular regulation, in a manner independent of cilia.

Graphical Abstract

Introduction

The BBSome is a protein complex composed of eight conserved Bardet-Biedl syndrome (BBS) proteins (BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9 and BBS18)¹. This protein complex is best known for its role in the control of ciliary function by facilitating the anterograde and retrograde transport of key cargos within cilia through the various biochemical interactions that the BBSome makes with the intraflagellar transport (IFT) machinery including the IFT complex B (IFT-B)^2–4. It should be noted, however, that the BBSome has been involved in several other cellular functions not linked to cilia. For instance, BBS proteins have been implicated in the regulation of gene expression^{5, 6}, proteasomal activity⁷, melanosome transport in zebrafish⁸, and trafficking of various receptors to the plasma membrane^9–12.

Obesity and hypertension are common conditions in patients with BBS^13–15. Interestingly, BBS gene polymorphism have also been associated with obesity, hypertension and other cardiovascular risks in non-BBS individuals^14–16. Additional evidence highlighting the importance of BBS genes for metabolic and cardiovascular regulation derives from model organisms. For instance, several BBS mouse models which were generated by disrupting individual Bbs genes recapitulate the obesity and hypertension phenotypes observed in BBS patients^17–19. We previously determined that the hypertension in BBS mutant mice involve activation of the sympathetic nervous system²⁰. Moreover, our previous work showed that disruption of the BBSome throughout the nervous system or in specific neuronal populations is sufficient to cause obesity in mice^{11, 12} highlighting the importance of the neuronal BBSome for energy homeostasis. However, the contribution of the neuronal BBSome to blood pressure and sympathetic control remain unknown.

To gain insight into the role of neuronal BBSome in cardiovascular regulation we used conditional knockout mouse models to directly assess the effects on blood pressure of disrupting the BBSome through Bbs1 gene deletion in the nervous system or specific neuronal populations including neurons that express the long signaling form of the leptin receptor (LRb), agouti-related peptide (AgRP) or proopiomelanocortin (POMC). We also analyzed how loss of another cilia-related gene, Ift88, in the LRb-containing neurons affects arterial pressure. IFT88 protein is a key subunit of the IFT-B that is required for cilia formation^{11, 21}.

Methods

The data that support the findings of this study are available from the corresponding author on reasonable request.

Animals

All animal testing was performed based on guidelines set forth by the National Institutes of Health and approved by The University of Iowa Animal Care and Use Committee. Mice were housed in groups of 3-5 per cage and maintained on 12-h light-dark cycle with lights on at 6 am. Room temperature was maintained at 22°C. Food (normal chow) and water were available ad libitum. Mice homozygous for the conditional Bbs1 allele (Bbs1^fl/fl mice) were generated with two loxP sites outside of exon 3 of Bbs1 as described previously^{11, 22}. ROSA (td-Tomato) reporter transgenic mice has a stop codon flanked by loxP sites preceding the start position of a td-Tomato locus (Stop^fl/fl-tdTomato)^{11, 12}. Cre recombination removes the stop site, leading to the expression of the fluorescent td-Tomato protein.

To obtain selective Bbs1 gene deletion in the nervous system, LRb, AgRP or POMC-neurons, transgenic Nestin^Cre, LRb^Cre, AgRP^Cre or POMC^Cre male mice were crossed with Bbs1^fl/fl female mice as reported before^{11, 12}. For each strain, male offspring that are Cre⁺/Bbs1^fl/wt were subsequently crossed with female Bbs1^fl/wt mice to generate Cre positive Bbs1^fl/fl and Bbs1^wt/wt mice, respectively. A similar strategy was used to selectively delete the Ift88 gene in the LRb-expressing cells using Ift88^fl/fl mice. To visualize Cre recombinase, each mouse strain was further crossed with the td-Tomato reporter mice. Presence of the Cre transgene, Bbs1 and Ift88 alleles, and td-Tomato of conditional knockout mice were detected by PCR analysis of tail DNA using the following conditions: 95°C for 30 sec, 62°C for 30 sec, and 72°C for 30 sec for a total of 30 cycles. Primer sequences for genotyping are provided in Table S1 (see online-only Data Supplement). Cre-positive Bbs1^wt/wt and Ift88^wt/wt and/or Cre-negative Bbs1^fl/fl and Ift88^fl/fl littermates were used as controls in the studies. Mice (12-16 weeks of age) used in this study were on a mixed background (129SvEv and C57B/6J). It should be noted that the different mouse models used in the current studies were previously validated^{11, 12}.

Telemetric Measurement of Arterial Pressure and Heart Rate

Continuous radiotelemetric measurement of arterial pressure and heart rate was obtained using PA-C10 units (Data Science Instruments). Mice were anesthetized with Isoflurane (up to 5% for induction, 1.5-2% for maintenance). Under aseptic surgical conditions, the catheter of the telemeter was inserted in the left carotid artery and tied securely using 6-0 silk suture. The transmitter was tunneled sub-dermally from neck area until the unit has reached the mid-abdominal region. The neck incision was sutured closed with 4-0 absorbable cat gut and then further sealed with tissue adhesive (Vet-Bond) along the incision line.

Animals were allowed to recover for at least 10 days before arterial pressure and heart rate were recorded continuously in the conscious unrestrained state for 7 days. Hemodynamic parameters were recorded for 10 seconds every 5 minutes and stored on a personal computer using Data Science Dataquest software. The effect of ganglionic blockade on the hemodynamic parameters was tested using hexamethonium bromide (1 μg/g body weight, intraperitoneally).

Conscious Sympathetic Nerve Recording

Renal sympathetic nerve activity (SNA) was recorded in the conscious state as described previously^{23, 24}. Briefly, mice were anesthetized with Isoflurane (up to 5% for induction, 1.5-2% for maintenance) and equipped for hemodynamic recording using an MRE-40 catheter inserted into the left carotid artery. The nerve fascicule subserving the left kidney or the splanchnic bed was identified using a dissecting scope and then mounted on a bipolar 36-gauge platinum-iridium electrode (Cooner Wire Co.). Once optimal recording parameters were established, the nerve fibers were fixed to the electrode with Kwik-Sil silicone gel (World Precision Instruments Inc.). The electrode wires were exteriorized at the nape of the neck. After full recovery from anesthesia (~4 hrs), the recording electrodes were attached to a high-impedance probe (HIP-511, Grass Instruments Co.). The signal was amplified 10⁵ times with a Grass P5 AC preamplifier and filtered at both low (100 Hz) and high-frequency (1000 Hz) cut-off. This amplified, filtered signal was then sent to a speaker system and oscilloscope (model 54501A, Hewlett-Packard Co.). The signal was also routed to a MacLab analogue-digital converter (Ad Instruments) for recording and data analysis on a Macintosh computer. Background noise was excluded from the measurements of SNA by correcting for post-mortem activity.

Vascular Function

Vascular function was assessed using aortic, carotid and mesenteric rings as reported previously²⁵. In brief, the thoracic aorta (3mm length rings), carotid (2mm length rings) and second-order mesenteric arteries (2mm length rings) were dissected and cleaned of debris before they were mounted on a wire myograph (DMT; Model 620M or 610M). Aortic rings were mounted on steel pins and carotid and mesenteric arterial rings were mounted on 2 tungsten wires (25μm diameter) and placed in a 95%O₂/5%CO₂ Krebs solution containing the following (in mM): 118.3 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, 2.5 CaCl₂, 11 Glucose, pH 7.4 at 37°C. Resting tension was set at 0.5g for aortic rings, 0.25g for carotid rings, and IC₉₀ was applied to all mesenteric rings for 45 minutes. Cumulative contractile responses to phenylephrine (1nM-100μM) and KCl depolarization (100mM) were performed. Data are reported as contraction (% of maximal KCl) or maximum tension (mN/mm) generated. All reagents for vascular studies were purchased from Sigma.

Immunofluorescence and Confocal Microscopy.

Mice anesthetized with ketamine and xylazine cocktail were perfused with PBS (5 ml/min; 25 ml) followed by 4% paraformaldehyde/HistoChoice Tissue Fixative (Amresco) in PBS (3 ml/min; 40 ml) using Harvard PHD 22/2000 Syringe Pump. Entire brain was excised and incubated in the same fixative overnight at 4°C. Fixed brains were washed 3 times with PBS and incubated in 30% sucrose/PBS overnight with one change of solution after 4-6 hrs of initial incubation. Brains were vibratome-sectioned with 40 μm thickness. Immunochemistry was performed on brain sections to label cilia using Adenyl cyclase III antibody (Santa Cruz, sc-32113 (N-14), 1:250 dilution), followed with secondary anti-rabbit IgG antibody (Alexa Fluor goat-anti-rabbit IgG (H+L), Life Technologies Corporation, A11008, 1:2000 dilution). Processed brain sections were mounted using VectaShield mounting medium with DAPI. Images were visualized using confocal microscopy (Zeiss LSM710).

Gene expression.

RNA was isolated using RNeasy Plus Mini Kit from Qiagen. Total RNA from the mesenteric arteries (250 ng in final volume of 40 μl) or brain regions or kidney (500 ng in final volume of 20 μl) was used to synthesize first-strand cDNAs with the Super-Script pre-amplification system. Then, 10 μl of cDNA and 0.4 mmol/L of primers were added in a final volume of 25 μl PCR mixture (Fast SYBR Green Master Mix, Applied Biosystems), and amplified in a StepOnePlus Real Time PCR System (Applied Biosystems). S18 ribosomal RNA expression was used as internal control to normalize mRNA expression of these genes. For detection of Bbs1 and S18 gene expression in the brain tissues and kidney, PCR conditions were as follow: denaturation for 3 min at 95°C, then 30 cycles for 30 seconds at 95°C, 30 seconds at 60°C and 45 seconds at 72°C. The PCR conditions for adrenergic genes were as follow: denaturation for 3 min at 95°C, then 40 cycles for 30 seconds at 95°C and 30 seconds at 60°C. Primer sequences are provided in Table S1.

Data Analysis

Results are shown as mean±SEM. Data were analyzed using unpaired Student’s t-test or 2-way analysis of variance (ANOVA) with repeated measures. When ANOVA reached significance, a post-hoc comparison was made using Fisher test or Tukey’s test. A p value less than 0.05 was considered statistically significant.

Results

Nervous system Bbs1 gene ablation increase arterial pressure

To assess the cardiovascular consequence of BBSome inactivation in the nervous system we studied Nestin^Cre/Bbs1^fl/fl mice. We visualized Cre-mediated recombination by crossing the Nestin^Cre mice with the td-Tomato reporter mice. Analysis of expression of fluorescent td-Tomato driven by Nestin^Cre confirmed its restriction to the nervous system (Figure S1A available online). Gene expression measurement showed that Nestin^Cre/Bbs1^fl/fl mice have nervous system-selective Bbs1 gene deletion as revealed by the loss of Bbs1 mRNA in the brain, but not in the kidney (Figure 1A). It should be noted that we previously demonstrated that Bbs1 gene deletion in the nervous system lead to BBSome disruption¹¹. In line with our previous report¹¹, Nestin^Cre/Bbs1^fl/fl mice are obese as indicated by the significantly increased body weight relative to littermate controls (Figure 1B).

Figure 1.

(A) Nervous system-selective Bbs1 gene deletion in Nestin^Cre/Bbs1^fl/fl mice as indicated by the absence of Bbs1 gene expression in the brain (hypothalamus [Hypoth], hippocampus [Hippoc] and cortex), but not kidney, of Cre⁺ relative to Cre^- (controls). (A-F) Body weight (B), mean arterial pressure (C), depressor response to ganglionic blockade (D), and renal SNA (E-F: representative neurograms [E, 5 sec segments] and average data [F]) of Nestin^Cre/Bbs1^fl/fl mice and littermate controls (CTL, n=7 per group). *p<0.05, and **p<0.01 vs. CTL via unpaired t-test.

To evaluate the cardiovascular consequences of ablating the Bbs1 gene in the nervous system, we assessed arterial pressure and heart rate. We found significant differences in arterial pressure between Nestin^Cre/Bbs1^fl/fl and control mice. Mean arterial pressure was significantly elevated in the Nestin^Cre/Bbs1^fl/fl mice (Figure 1C). Systolic and diastolic arterial pressure were also elevated in Nestin^Cre/Bbs1^fl/fl mice compared to controls (Figure S1B–C). However, heart rate was not different between Nestin^Cre/Bbs1^fl/fl mice and controls (Figure S1D).

We investigated the contribution of neurogenic mechanisms to the hypertension observed in Nestin^Cre/Bbs1^fl/fl mice by assessing the arterial pressure response to ganglionic blockade with hexamethonium bromide. As shown in Figure 1D, the magnitude of the arterial pressure decrease evoked by ganglionic blockade was significantly greater in Nestin^Cre/Bbs1^fl/fl mice than controls pointing to the importance of sympathetic transmission for the arterial pressure elevation evoked by Bbs1 gene deletion in the nervous system. Consistent with this idea, Nestin^Cre/Bbs1^fl/fl mice displayed higher baseline renal SNA measured in the conscious state (Figure 1E–F). These data indicate that disruption of the BBSome in the nervous system is associated with obesity and hypertension.

Increased arterial pressure in mice lacking the Bbs1 gene in the LRb neurons

Next, we assessed the cardiovascular effects of ablating the Bbs1 gene from a subset of neurons, those expressing the LRb. For this, we crossed the Bbs1^fl/fl mice with mice expressing Cre-recombinase from an IRES element inserted into the 3’ untranslated region of the LRb gene²⁶. Using the td-Tomato reporter mice to visualize LRb-driven Cre recombinase showed expression of td-Tomato in nuclei where the LRb is normally located. This includes the arcuate nucleus, dorsomedial hypothalamus and ventromedial hypothalamus (Figure 2A). Body weight of LRb^Cre/Bbs1^fl/fl mice was significantly higher than control littermates (Figure 2B). Arterial pressure, but not heart rate, was also elevated in LRb^Cre/Bbs1^fl/fl mice relative to controls (Figure 2C and Figure S2). Moreover, LRb^Cre/Bbs1^fl/fl mice displayed an exaggerated arterial pressure-lowering response to ganglionic blockade (Figure 2D). In line with this finding, baseline renal SNA was higher in the LRb^Cre/Bbs1^fl/fl mice relative to controls (Figure 2E). Thus, inactivation of the BBSome in the LRb-expressing neurons is sufficient to cause obesity and hypertension.

Figure 2.

(A) Evidence of Cre recombinase (presence of fluorescent td-Tomato) in the hypothalamus of the reporter LRb^Cre/Stop^fl/fl-tdTomato mice. (B-E) Body weight (B), mean arterial pressure (C), depressor response to ganglionic blockade (D) and renal SNA (E) of LRb^Cre/Bbs1^fl/fl and control mice (n=6 for CTL and 7 for LRb^Cre/Bbs1^fl/fl mice). *p<0.05 and **p<0.01vs. CTL via unpaired t-test.

Ift88 gene deletion from the LRb neurons does not alter arterial pressure

To test whether ablation of another cilia-related gene yield similar cardiovascular outcomes as Bbs1 gene deletion, we studied mice lacking the Ift88 gene in the LRb-expressing cells (LRb^Cre/Bbs1^fl/fl mice). Ift88 gene deletion lead to absent or severely truncated cilia (Figure 3A) which is consistent with our previous report¹¹ and the requirement of the IFT88 protein for cilia formation²¹. Although body weight was higher in LRb^Cre/Ift88^fl/fl mice (Figure 3B), no difference in arterial pressure was observed between LRb^Cre/Ift88^fl/fl and control mice (Figure 3C and Figure S3A–B). Interestingly, heart rate was significantly lower in LRb^Cre/Ift88^fl/fl mice relative to controls (Figure S3C). The arterial pressure decrease evoked by ganglionic blockade was not different in LRb^Cre/Ift88^fl/fl mice versus controls (Figure 3D). Consistent with the lack of blood pressure phenotype, renal SNA was not altered by Ift88 gene deletion in the LRb-expressing cells (Figure 3E). These findings demonstrate that loss of the Ift88 gene does not recapitulate the cardiovascular effects evoked by Bbs1 gene deletion in the LRb-containing neurons.

Figure 3.

(A) Representative confocal images of the hypothalamic arcuate nucleus showing loss of cilia (ACIII immunostaining, green) in the LRb⁺ cells (arrows, red), but not in LRb^- cells (arrow heads) of LRb^Cre/Ift88^fl/fl mice. (B-E) Body weight (B), mean arterial pressure (C), depressor response to ganglionic blockade (D) and renal SNA (E) of LRb^Cre/Ift88^fl/fl mice and littermate controls (n=6 per group). *p<0.05 vs. CTL via unpaired t-test.

AgRP neuron Bbs1 gene deletion affect SNA, but not arterial pressure

To narrow down the neuronal populations that underlie the effects of BBSome disruption on arterial pressure, we determined the effects of Bbs1 gene deletion from select population of neurons. First, we examined the effects of ablating the Bbs1 gene from AgRP neurons. Cre recombinase under the control of AgRP promoter displayed td-Tomato expression in the arcuate nucleus of the hypothalamus where AgRP neurons reside (Figure 4A). Consistent with our previous report¹², AgRP^Cre/Bbs1^fl/fl mice displayed a slight, but significant increase in body weight when compared to littermate controls (Figure 4B). However, arterial pressure and heart rate were comparable between AgRP^Cre/Bbs1^fl/fl and control mice (Figure 4C and Figure S4A–C). Furthermore, the blood pressure lowering effect of ganglionic blockade was not altered in AgRP^Cre/Bbs1^fl/fl mice relative to controls (Figure 4D). Despite this, renal SNA was significantly higher in AgRP^Cre/Bbs1^fl/fl mice (Figure 4E).

Figure 4.

(A) Evidence of Cre recombinase (presence of fluorescent td-Tomato) in the arcuate nucleus of the hypothalamus of the reporter AgRP^cre/Stop^fl/fl-tdTomato mice. (B-E) Body weight (B), mean arterial pressure (C), depressor response to ganglionic blockade (D) and renal SNA (E) of AgRP^Cre/Bbs1^fl/fl and control mice (n=6 per group). *p<0.05 vs. CTL via unpaired t-test.

Bbs1 gene disruption in POMC neurons increase SNA, but not arterial pressure

We next determined the effects of ablating the Bbs1 gene from POMC neurons. We confirmed that Cre recombinase-mediated td-Tomato expression was present in the arcuate nucleus of the hypothalamus where POMC neurons are located (Figure 5A). Body weight of POMC^Cre/Bbs1^fl/fl mice was significantly higher than that of controls (Figure 5B). Like AgRP^Cre/Bbs1^fl/fl mice, POMC^Cre/Bbs1^fl/fl mice displayed no significant changes in arterial pressure or heart rate (Figure 5C and Figure S5A–C). Furthermore, the blood pressure lowering effect of ganglionic blockade was not altered in POMC^Cre/Bbs1^fl/fl mice relative to controls (Figure 5D). Nonetheless, renal SNA was elevated in POMC^Cre/Bbs1^fl/fl mice compared to controls (Figure 5E). Next, we assessed whether SNA subserving other beds was affected by POMC neuron Bbs1 gene deletion. Interestingly, splanchnic SNA that subserve the vasculature was elevated in POMC^Cre/Bbs1^fl/fl mice (Figure 5F). AgRP^Cre/Bbs1^fl/fl and LRb^re/Bbs1^fl/fl mice also exhibited higher splanchnic SNA than their littermate controls (Figure S6).

Figure 5.

(A) Evidence of Cre recombinase (presence of fluorescent td-Tomato) in the arcuate nucleus of the hypothalamus of the reporter POMC^cre/Stop^fl/fl-tdTomato mice. (B-E) Body weight (B), mean arterial pressure (C), depressor response to ganglionic blockade (D), renal SNA (E), and splanchnic SNA (F) of POMC^Cre/Bbs1^fl/fl and control mice (n=5 for CTL and 5-7 for POMC^Cre/Bbs1^fl/fl mice). *p<0.05 vs. CTL via unpaired t-test.

Vascular adrenergic receptor desensitization in POMC^Cre/Bbs1^fl/fl and AgRP^Cre/Bbs1^fl/fl mice

The normal blood pressure despite the higher renal and splanchnic sympathetic tone in AgRP^Cre/Bbs1^fl/fl and POMC^Cre/Bbs1^fl/fl mice point to possible vascular adrenergic tachyphylaxis in these animals. To test this possibility, we measured adrenergic reactivity in resistance vessel (mesenteric artery) as well as conduit vessels (aorta and carotid artery) of POMC^Cre/Bbs1^fl/fl and control mice. Consistent with our hypothesis, POMC^Cre/Bbs1^fl/fl mice showed significantly reduced phenylephrine-induced vascular contraction in mesenteric and aortic rings (Figure 6A–B). Vascular adrenergic reactivity of carotid rings tended to be lower in POMC^Cre/Bbs1^fl/fl mice, but this was not statistically significant (Figure 6C). Contraction induced by KCl was also decreased in mesenteric and carotid rings, but not aortic rings (Figure S7). In line with the vascular adrenergic desensitization, expression levels of α_1A and α_1D-adrenergic receptors which mediates contractile function in blood vessels^{27, 28} were significantly lower in the mesenteric artery of POMC^Cre/Bbs1^fl/fl mice compared to controls (Figure 6D). Interestingly, AgRP^Cre/Bbs1^fl/fl mice also exhibited decreased α_1A- and α_1D-adrenergic receptors expression in the mesenteric artery (Figure 6E). In contrast, mesenteric artery expression levels of α_1A- and α_1D-adrenergic receptors were elevated in the LRb^Cre/Bbs1^fl/fl mice although this was not statistically significant for the α_1D-receptor (Figure 6F). Together, these data suggest that sympathetically-mediated vascular adrenergic desensitization, that likely decrease total peripheral resistance, may offset the prohypertensive effects of increased sympathetic drive in POMC^Cre/Bbs1^fl/fl and AgRP^Cre/Bbs1^fl/fl mice.

Figure 6.

(A-C) Contractile responses of mesenteric (A), aortic (B), and carotid (C) rings from POMC^Cre/Bbs1^fl/fl and control mice to phenylephrine (PE). (D-F) Expression of α_1A- and α_1D-adrenergic receptors in the mesenteric artery of POMC^Cre/Bbs1^fl/fl (D), AgRP^Cre/Bbs1^fl/fl (E), and LRb^Cre/Bbs1^fl/fl mice (F) (n=5-6 per group). *p<0.05 vs. CTL with 2-way ANOVA with repeated measures (A-B) or via unpaired t-test (D-F).

Discussion

This study demonstrates the importance of the neuronal BBSome for blood pressure and sympathetic regulation in a manner independent of cilia. Indeed, we showed that mice bearing disruption of the BBSome by deleting the Bbs1 gene from the nervous system develop hypertension. This was associated with increased renal sympathetic outflow and exaggerated blood pressure decrease in response to ganglionic blockade indicating that the increase in blood pressure is sympathetically-mediated. Furthermore, we showed that this phenotype is reproduced in mice lacking the Bbs1 gene in the LRb-expressing neurons. However, mice bearing Ift88 gene deletion in the LRb-expressing neurons displayed normal blood pressure and renal sympathetic tone. Interestingly, mice lacking the Bbs1 gene in AgRP or POMC neurons have normal arterial pressure despite an elevated sympathetic tone. We show that vascular adrenergic tachyphylaxis may account for the dissociation between the sympathetic nerve activation and blood pressure in these mice.

Neuronal BBSome inactivation-induced excess weight gain is consistent with our previous studies that implicated this protein complex in the control of neural mechanisms that regulate energy balance^{11, 12}. The increased blood pressure and sympathetic nerve activity evoked by disruption of the BBSome in the nervous system and LRb-expressing neurons recapitulate the hypertension and sympathetic overdrive we previously reported in various global BBS null mouse models such as Bbs4^−/− mice²⁰. The critical role of the BBSome in ciliary function led us to consider the possible contribution of cilia to the increase in blood pressure and sympathetic tone we observed in mice lacking the BBSome in the LRb-expressing neurons. Interestingly, loss of cilia (Ift88 gene) in the LRb neurons was associated with a decrease in heart rate, but no change in blood pressure or renal sympathetic nerve traffic. These findings indicate that the role of the BBSome in blood pressure and sympathetic control is not linked to its involvement in ciliary functions. It should be noted that the BBSome is implicated in several cellular functions that are not related to cilia including gene expression^{5, 6}. For instance, we previously found that disruption of the BBSome in smooth muscle cells caused an increase in the expression of the angiotensinogen gene leading to activation of the renin-angiotensin system²⁵. However, the contribution of such mechanism to the increase in blood pressure and sympathetic activity in mice lacking the neuronal BBSome remain to be determined.

It is interesting that blood pressure was differentially affected by loss of the BBSome in the nervous system or LRb-expressing neurons versus AgRP or POMC neurons. These findings indicate that the hypertension evoked by loss of the BBSome is caused by neurons other than those expressing AgRP and POMC. It should be noted that a subset of AgRP or POMC neurons express the LRb and mediates many of the physiological actions of leptin²⁴. AgRP and POMC neurons are first-order neurons of the melanocortin system which is known to play a critical role in obesity-associated blood pressure increase²⁹. Indeed, patients or mice carrying loss of function mutations or gene deletion in the components of the brain melanocortin system such as the melanocortin-4 receptor are protected from the development of hypertension despite being obese^30–32. Previous studies have also shown that interference with various signaling pathways in POMC neurons prevents the hypertension commonly associated with obesity. For example, blockade of the proinflammatory pathways by ablating the gene encoding the activator IκB kinase-β prevents the development of hypertension caused by high fat diet in mice³³. Moreover, mice bearing POMC neuron-specific deletion of the transcription factor Stat3 develop obesity but remain normotensive³⁴. Our current findings provide further support for the notion that the brain melanocortinergic system is a key mediator of obesity-induced hypertension.

Our data implicating sympathetic activation in the blood pressure increase in mice lacking the Bbs1 gene in the nervous system or the LepRb-expressing neurons extend our previous demonstration that global BBS mice develop a sympathetically-mediated hypertension²⁰. These findings are also in line with the well-established role of heightened sympathetic nerve activity in mediating obesity-induced hypertension³⁵. However, mice bearing BBSome deficiency in AgRP or POMC neurons display elevated renal and splanchnic sympathetic traffic, but no change in blood pressure. These findings show that an increase in renal and splanchnic sympathetic tone does not necessarily translate into blood pressure elevation. Counterregulatory mechanisms can offset the increase in sympathetic tone to maintain normal blood pressure. Consistent with such possibility, we show impaired vascular contraction evoked by norepinephrine in mice lacking the Bbs1 gene in POMC neurons indicative of adrenergic receptor desensitization. This is further supported by the decreased vascular α_1A- and α_1D-adrenergic receptors expression in POMC^Cre/Bbs1^fl/fl and AgRP^Cre/Bbs1^fl/fl mice, but not in the LRb^Cre/Bbs1^fl/fl mice. A similar phenomenon of vascular adrenergic receptor desensitization was previously shown to be associated with diet-induced obese mice that fails to develop hypertension³⁶. Such uncoupling of sympathetic nerve activity and vascular tone has also been reported in normotensive obese patients³⁷. Additional studies are needed to determine how loss of the BBSome in POMC or AgRP neurons cause vascular adrenergic receptor desensitization.

Perspectives

The present study highlights the relevance of the neuronal BBSome for the control of blood pressure regulation through the control of sympathetic tone and vascular reactivity. Hypertension which is a frequent manifestation of BBS^{38, 39} and an important cardiovascular risk appears to be one of the leading causes of death in BBS patients⁴⁰. By demonstrating the significance of the neurogenic mechanisms in the development of hypertension in BBS, our findings provide insight into the pathophysiological processes involved in BBS-related cardiovascular risks. Harnessing these mechanisms may help improve the management of hypertension and cardiovascular disease in BBS patients. Further investigation of the contribution of the neuronal BBSome to blood pressure control may enhance our understanding of common forms of hypertension and associated cardiovascular disease.

Novelty and Significance.

What Is New?

• Disruption of the BBSome in the nervous system or leptin receptor (LRb)-containing neurons cause a sympathetically-mediated increase in blood pressure.

• Specific loss of the BBSome in AgRP or POMC neurons is associated with heightened sympathetic tone, but normal blood pressure.

• Desensitization of vascular adrenergic receptors appears to explain the uncoupling of sympathetic overdrive and blood pressure.

What Is Relevant?

• Neuronal BBSome is a novel regulator of sympathetic tone and blood pressure.

• Neurogenic mechanisms play a key role in driving the hypertension associated with Bardet-Biedl syndrome.

Summary.

Our findings show that conditional nervous system-specific inactivation of the BBSome through Bbs1 gene deletion results in a sympathetically-mediated hypertension. This phenotype is recapitulated in mice lacking the Bbs1 gene selectively in the LRb-containing neurons. On the other hand, Bbs1 gene deletion from AgRP or POMC neurons lead to an increase in sympathetic tone without changes in blood pressure. This uncoupling of sympathetic activity from blood pressure seems due to vascular adrenergic receptor desensitization. These results highlight the importance of the neuronal BBSome for the regulation of sympathetic nerve activity and blood pressure. Our findings may help improve the management of hypertension and cardiovascular disease in Bardet-Biedl syndrome patients.