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KCTD proteins regulate morphine dependence via heterologous sensitization of adenylyl cyclase 1 in mice [1]

['Zhong Ding', 'Jiangsu Key Laboratory Of Neuropsychiatric Diseases', 'Department Of Pharmacology', 'College Of Pharmaceutical Sciences', 'Soochow University', 'Suzhou', 'Chunsheng Zhang', 'Huicui Yang', 'Jiaojiao Chen', 'Zhiruo Sun']

Date: 2024-07

Abstract Heterologous sensitization of adenylyl cyclase (AC) results in elevated cAMP signaling transduction that contributes to drug dependence. Inhibiting cullin3-RING ligases by blocking the neddylation of cullin3 abolishes heterologous sensitization, however, the modulating mechanism remains uncharted. Here, we report an essential role of the potassium channel tetramerization domain (KCTD) protein 2, 5, and 17, especially the dominant isoform KCTD5 in regulating heterologous sensitization of AC1 and morphine dependence via working with cullin3 and the cullin-associated and neddylation-dissociated 1 (CAND1) protein. In cellular models, we observed enhanced association of KCTD5 with Gβ and cullin3, along with elevated dissociation of Gβ from AC1 as well as of CAND1 from cullin3 in heterologous sensitization of AC1. Given binding of CAND1 inhibits the neddylation of cullin3, we further elucidated that the enhanced interaction of KCTD5 with both Gβ and cullin3 promoted the dissociation of CAND1 from cullin3, attenuated the inhibitory effect of CAND1 on cullin3 neddylation, ultimately resulted in heterologous sensitization of AC1. The paraventricular thalamic nucleus (PVT) plays an important role in mediating morphine dependence. Through pharmacological and biochemical approaches, we then demonstrated that KCTD5/cullin3 regulates morphine dependence via modulating heterologous sensitization of AC, likely AC1 in PVT in mice. In summary, the present study revealed the underlying mechanism of heterologous sensitization of AC1 mediated by cullin3 and discovered the role of KCTD proteins in regulating morphine dependence in mice.

Citation: Ding Z, Zhang C, Yang H, Chen J, Sun Z, Zhen X (2024) KCTD proteins regulate morphine dependence via heterologous sensitization of adenylyl cyclase 1 in mice. PLoS Biol 22(7): e3002716. https://doi.org/10.1371/journal.pbio.3002716 Academic Editor: Chris Pierce, Rutgers Robert Wood Johnson Medical School, UNITED STATES OF AMERICA Received: September 26, 2023; Accepted: June 18, 2024; Published: July 15, 2024 Copyright: © 2024 Ding et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All data underlying the main and supplementary figures can be found at supplementary S1 Data. Funding: This work was supported by the National Innovation of Science and Technology-2030 (Program of Brain Science and Brain-Inspired Intelligence Technology) Grant (2021ZD0204004, XCZ); National Key Research and Development Program of China (2021YFE0206000, XCZ), https://www.most.gov.cn/index.html; Priority Academic Program Development of the Jiangsu Higher Education Institutes (PAPD, XCZ), https://jyt.jiangsu.gov.cn/; National Natural Science Foundation of China (No. 82003737, ZD), https://www.nsfc.gov.cn/; National Science Foundation of the Jiangsu Higher Education Institutions of China (20KJD310001, ZD), and Suzhou International Joint Laboratory for Diagnosis and Treatment of Brain Diseases (ZD), https://kjj.suzhou.gov.cn/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: AC, adenylyl cyclase; BLA, basolateral amygdala; BTB, bric-à-brac, tramtrack, and broad complex; CAND1, cullin-associated and neddylation-dissociated 1; KCTD, potassium channel tetramerization domain; NAc, nucleus accumbens; PFC, prefrontal cortex; PVT, paraventricular thalamic nucleus; vHipp, ventral hippocampus

Introduction There are 10 isoforms of adenylyl cyclase (AC) encoded in mammalian cells, AC1 to AC10. AC1 to AC9 are membrane bonded. AC1, AC3, and AC8 are classified as Ca2+/calmodulin stimulated group, while AC5 and AC6 are inhibited by physiological concentrations of Ca2+. All these isoforms are expressed in rodent brain with isoform specific patterns [1–3]. Heterologous sensitization of AC (aka AC super-activation or cAMP overshoot) refers to a compensatory increase in AC activity upon persistence activation of Gαi/o-coupled GPCRs [4–6]. This phenomenon has long been proposed as a cellular mechanism underlying opioids dependence [4,5,7]. Opioids dependence is characterized as unpleasant physical and emotional responses after drug withdrawal [8] and increased cAMP signaling including PKA, CREB, and NMDARs plays a critical role in this process [7]. Chronic morphine exposure significantly enhanced the AC responses and cAMP signaling transduction in a series of brain regions such as nucleus accumbens (NAc), prefrontal cortex (PFC), and the thalamus nucleus [7,9–12]. Moreover, genetic knockout of AC1, AC5, or AC8 significantly attenuated physical signs of the mice in morphine withdrawal [13,14]. These observations revealed the significance of increased AC/cAMP signaling in morphine dependence; however, the detailed molecular mechanism associated with morphine-induced heterologous sensitization of AC and its significance in the development of morphine dependence is largely uncharted. Activation of a Gαi/o coupled GPCR promotes the dissociation of Gαi/o subunit from Gβγ and persistent Gβγ signaling is essential for the development of heterologous sensitization of AC, for instance, in the cases of AC5 and AC6 [15–18]. The K+ channel tetramerization domain (KCTD) family consists of 25 members, which share a structurally similar bric-à-brac, tramtrack, and broad complex (BTB) domain [19]. At least half of the KCTD family has the capacity for Gβγ interaction [20]. Through interaction with Gβγ, KCTDs 8, 12, and 16 are involved in regulating GABA B receptor signaling [21,22], while KCTDs 2, 5, and 17 regulate the cAMP signaling [23]. Biochemical and structural studies revealed that the C-terminal domains of KCTDs are mainly involved in binding Gβγ [20,24,25]. Expression respective KCTDs 2, 5, 17, which acts as Gβγ scavenger, strongly blocked heterologous sensitization of AC5 [20]. Besides Gβγ sequestrating, the KCTD also could act as substrate receptor of cullin3-RING ligases (CRL3s) for ubiquitination and degradation of substrate proteins including Gβγ [26,27]. Gβγ is reported to inhibit ACs 1, 3, 8 [28–30] but conditionally stimulates AC5 and AC6 [31], while knockdown respective KCTDs 2, 5, 17 elevated the expression of Gβγ and enhanced the activation of AC5 [23], revealing that KCTD family members may modulate AC activity through targeting Gβγ. The cullin3 is a member of the cullin protein family, which scaffolds BTB and RING-box proteins to form fully assembled CRL3s [32]. The fully assembled CRL3s is functionally inactive and requires neddylation to be activated [33]. It is interesting to note that genetic knockdown of CRL3s or blocking the neddylation of CRL3s by MLN4924 completely blocks heterologous sensitization of ACs 1, 5, 6 [34], revealing the importance of CRL3s system in regulating heterologous sensitization of AC. Binding of the cullin-associated and neddylation-dissociated 1 (CAND1) protein to cullin is known to prevent the neddylation of cullin and subsequent ubiquitination of substrate proteins [35,36]. Interestingly, increased availability of substrate proteins may promote a dissociation of the CAND1-cullin complex and diminish the inhibitory effect of CAND1 on the neddylation of cullin [37]. These reports imply that CAND1 may be involved in heterologous sensitization of AC via mediating the neddylation of CRL3s. The paraventricular thalamic nucleus (PVT), one of the major inputs of NAc, selectively mediates behavioral aversion [38]. AC1 is the major isoform of ACs in PVT [1,2]. Thus, it would be interesting to investigate how AC1 sensitization is regulated in PVT and its role in the development of morphine dependence. In this study, we first examined the role and underlying mechanisms of the KCTD in heterologous sensitization of AC, particularly Ca2+/calmodulin stimulated ACs, in cellular models. We further revealed that heterologous sensitization of AC1 in PVT may contribute to the development of morphine dependence.

Discussion Heterologous sensitization of AC results in elevated cAMP signaling transduction that contributes to drug dependence. The cullin3 has recently been demonstrated as an essential regulator for AC sensitization, but the underlying mechanisms are undefined [34]. In the current study, we revealed the involvement of KCTD and CAND1, working in complex with cullin3, in mediating heterologous sensitization of AC, especially AC1. Moreover, we also provided the first evidence that KCTD5/cullin3 may regulate morphine dependence via modulating heterologous sensitization of AC1 in PVT. One of the major findings of the current study is the elucidation of KCTD as critical regulator of heterologous sensitization of AC1. We found that overexpression of respective of KCTDs 2, 5, 17 significantly increased heterologous sensitization of AC1, while knockdown of the respective KCTDs largely blocked AC1 sensitization. Moreover, our findings suggested that the KCTDs may regulate AC1 sensitization through Gβγ sequestration. This conclusion is supported by the following observations: (1) neither overexpression of KCTD5 nor chronic morphine treatment resulted in accelerated turnover of Gβ in AC1 sensitization; (2) the enhanced association between KCTD5 and Gβ was companied by an increase in dissociation of Gβ from AC1 during heterologous sensitization; (3) the AC sensitization blocker MLN4924 significantly attenuated morphine-induced increase on the association between Gβ and KCTD5 as well as the dissociation of Gβ from AC1; (4) the boosted AC1 sensitization in KCTD5 overexpression cells was attenuated by complementary of the Gβ subunit; and (5) inhibiting proteasome-mediated protein degradation did not suppress AC1 sensitization. Except AC1, we observed that either overexpression or knockdown of respective of KCTDs 2, 5, 17 inhibited heterologous sensitization of endogenous ACs, mainly AC6, in HEK-293 cells. These observations are partially in agreement with a previous report that overexpression of KCTDs 2, 5, or 17, through Gβγ sequestration, strongly blocked heterologous sensitization of AC5 in HEK-293 cells [20]. Nevertheless, it should be noticed that although AC6 contributes approximately 85% ATP catalyzing activity, AC1 and AC3 are also expressed in HEK-293 cells [39], which could be inhibited by increased Gβγ after KCTDs knockdown. Therefore, we could not completely exclude the potential roles of other AC isoforms, since it was shown that knockdown of KCTDs 2, 5, or 17 elevated the activity of AC5 in mice primary neurons [23]. CAND1 was previously shown to regulate the neddylation of cullin [36,37,45]. We found that CAND1 expression was reversely associated with the neddylation of cullin3 as well as AC1/6 sensitization. Moreover, we observed that increased association of Gβ/KCTD5/cullin3 promoted the dissociation of CAND1 from cullin3 and enhanced cullin3 neddylation in heterologous sensitization of AC1/6. These findings are consistent with previous reports that binding of the CAND1 with cullin1/2 blocks their neddylation, while increased availability of substrates/substrate receptors promotes the dissociation of cullin/CAND1 complex and increases the neddylation of cullin1/2 [36,37,45]. In addition, in agreement with previous reports [28–30], our data also showed that the abundance of Gβ may be negatively correlated with AC1 activation and sensitization. The Lys48 (K48)-linked polyubiquitination typically results in protein degradation, whereas monoubiquitylation or multi-ubiquitination may alter functions of the substrate without resulting a degradation [46]. For example, monoubiquitylated Gβ in yeast limited polarized growth of the cell but exhibited a normal stability [47]. In the present study, we cannot exclude the possibility that the CRL3s may regulate the development of heterologous sensitization through ubiquitinating the Gβ in a nondegradable way. However, the relation between the type of ubiquitination and degradation for Gβ is rather complex, since the degradation of monoubiquitinated Gβ has also been reported recently in HEK-293 cells [23]. Information on the functions of nondegradable ubiquitination of Gβ subunit are however limited, which requires more investigations to reveal their roles including that in heterologous sensitization of AC. Another important finding of the present study is to uncover the roles of CUL3/KCTD5-mediated AC1 sensitization in morphine dependence. Knockdown or overexpression of the KCTD5 in PVT significantly attenuated or sensitized local AC responses in morphine-dependent mice, respectively. Similarly, knockdown of cullin3 in PVT robustly blocked local AC sensitization as well. These in vivo data is in line with our observations with regard on the role of KCTD5/cullin3 in regulating AC1 sensitization in cellular models. Importantly, our data showed that the alterations of AC responses in PVT were coordinated with the development of morphine dependence. Moreover, we observed that local infusion of selective AC1 inhibitor into PVT was sufficient to attenuate the development of morphine dependence, indicating AC1 could be the dominate isoform in PVT involved in heterologous sensitization of AC that was responsible for the development of morphine dependence. It is worth noting that heterologous sensitization of AC has been reported in different brain regions including PFC, basolateral amygdala (BLA), and ventral hippocampus (vHipp) [9–12]. In contrast to that of PVT, their projections to NAc are mainly related to rewarding [38,48]. Therefore, it will be interestingly to examine the role of the KCTD5/cullin3-mediated AC sensitization in these brain regions in drug rewarding in the future. In summary, our findings may shed a light on our understanding of CRL3s-mediated heterologous sensitization of AC and provide the first direct evidence that heterologous sensitization of AC1 in PVT gates aversive states that associated with opiate dependence. Moreover, heterologous sensitization of AC is shared by most Gαi/o-coupled receptors including mu/kappa/delta opioid, D2/4 dopamine, alpha2 adrenergic, M2/4 muscarinic, and 5HT1A serotonin, which may render our findings of general physiological and therapeutic importance in different AC-linked GPCR systems.

Methods and materials Cell lines and animals HEK-293 cells stably expressing the μ-receptor (HEK-μR and HEK-μR/AC1) and SH-SY5Y cells stably expressing the μ-receptor (SH-SY5Y-μR) were constructed and maintained in DMEM (Gibco, Thermo Fisher Scientific) supplemented with 5% fetal clone I serum (Hyclone, Logan, Utah, United States of America), 1% Antibiotic-Antimycoctic (Life Technologies), and 1 mg/ml puromycin (Sigma). All cell lines were cultured at 37°C with 5% CO 2 . Male adult C57BL/6 mice (10 to 14 weeks of age) were used for behavioral tests (purchased from Shanghai SLAC Laboratory Animal Co.). Mice were group-housed four/cage under a 12-h light-dark cycle (light on from 6 AM to 6 PM) in stable conditions with food and water ad libitum. All animal studies and experimental procedures were approved by the Animal Care and Use Committee of Soochow University and were following Guidelines for the Care and Use of Laboratory Animals (Chinese National Research Council, 2006, ethical approval number: 202208A0688) and the “ARRIVE” (Animals in Research: Reporting In Vivo Experiments) guidelines. Antibodies The following antibodies were used: anti-CUL3 (SAB4200180, Sigma), anti-KCTD2/5/17 (15553-1-AP, Proteintech), anti-CAND1 (#8759, Cell Signaling Technology), anti-β-actin (#3700, Cell Signaling Technology), anti-tubulin (T5168, Sigma), anti-GAPDH (ab9485, Abcam), anti-Nedd8 (ab81264, Abcam), anti-CREB (#9197, Cell Signaling Technology), anti-pCREB (#9198, Cell Signaling Technology), anti-HA (51064-2-AP, Proteintech), anti-Flag (F7425, Sigma), anti-Gβ (sc-166123, Santa Cruz), anti-Gβ1 (10247-2-AP, Proteintech), and anti-Gβ2 (16090-1-AP, Proteintech). DNA plasmid and siRNA transfections Briefly, DNA plasmids were first diluted in DMEM to the desired concentrations. Lipofectamine 2000 (Thermo Fisher Scientific) was diluted in Opti-MEM according to the manufacturer’s protocol and incubated for 5 min. DNA and Lipofectamine solutions were mixed, followed by incubation at room temperature for 30 min, and added to the cells dropwise. Cells were transfected for approximately 48 h prior to being used for the cAMP/immunoblot assays. Similarly, siRNAs were diluted in DMEM to the desired concentrations (30 pmol final for 6-well plate). RNAiMAX (Thermo Fisher Scientific) was diluted in DMEM media, incubated for 5 min, and mixed with siRNA. Mixture of Lipofectamine and siRNAs was incubated at room temperature for 30 min before adding to the cells dropwise. Immunoprecipitation and western blot For immunoprecipitation assays, cell extracts or brain tissue homogenates were prepared in lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM EDTA, 0.1% Triton, X-100) containing 1 mM DTT and protease inhibitor cocktail (Sigma), and 150~200 μg of lysate were incubated with 1.5 μg of antibodies overnight at 4°C, followed by incubation with Protein A/G Magnetic Beads (Selleck) for 4 h at room temperature. The precipitates were washed 3 times with a washing buffer (50 mM Tris, 150 mM NaCl, 0.1% Triton, X-100, pH 7.5). The immunoprecipitated proteins were eluted in 1× loading buffer (NCM Biotech) and boiled for 5 min before applying for electrophoresis. For western blot, 15~25 μg proteins were loaded and separated by SDS-PAGE gel (10%) electrophoresis before transferring onto a PVDF membrane. The primary and secondary antibodies were used under the instructions of manufacturers with a dilution rate from 1:1,000 to 1:10,000. ECL reagents were applied to visualize bands with ChemiScope 3300 Mini (CLINX, Shanghai, China). cAMP assays in cells and brain tissues To induce heterologous sensitization, the cells were subjected to either vehicle or μ-receptor agonist morphine (10 μm final concentration) for 2 h at 37°C. The culture plate was equilibrated to room temperature for 30 min before addition of forskolin or A23187 with 500 μm IBMX and 10 μm μ-receptor antagonist naloxone for 1 h. The reaction was stopped by the addition of the homogenous time-resolved fluorescence cAMP detection reagents, D2-labeled cAMP, and Cryptate labeled anti-cAMP antibody (Cisbio, Bedford, Massachusetts, USA). Fluorescence (Ex 330/80, Em 615/10 and 665/7) was measured on the EnVision 2104 Multilabel Reader (PerkinElmer) under the instructions of manufacturers. To determine the relative cAMP accumulation per well, the ratio of 665 nm/615 nm fluorescence values was calculated. For cAMP assays in mice brain tissues, the brains of the mice with morphine dependence or controls were quickly removed on ice. Then, the desired brain regions were dissected in cold PBS under the stereoscope and stored in −80°C before the assay. The tissue was thawed on ice and homogenized ultrasonically in membrane preparation buffer (10 mM Tris, 5 mM EDTA, pH 7.4) and the protein concentrations were determined using BCA assay kit. The cAMP level in respective brain regions was directly measured in 384-well plate using Cisbio cAMP assay kit for 20 μg tissue lysates. Alternatively, crude membranes were prepared after homogenizing the tissue in membrane preparation buffer by centrifugation at 16,000g for 15 min at 4°C. Then, the membranes were resuspended in assay buffer (75 mM Tris-HCl (pH 7.4), 15 mM MgCl 2 , 2 mM EDTA, 500 mM IBMX), and the protein concentration was determined using BCA assay kit. Membranes pellet (15 μg per well) was added to a 384-well plate and 3 μm calmodulin (final concentration) was added in stimulation buffer (75 mM Tris-HCl (pH 7.4), 15 mM MgCl 2 , 250 μm ATP, 1 μm GppNHp, 500 μm IBMX, and 500 μm CaCl 2 −10 μm free Ca2+) and incubated at room temperature for 1 h. The cAMP concentration was measured according to the manufacture instruction using Cisbio cAMP assay kit at last [11,49]. Quantitative real time-PCR Total RNA was extracted from mouse PVT tissues using RNAiso Plus (TaKaRa, Tokyo, Japan) according to the manufacturer’s instructions. The RNA (1 μg) was reverse transcribed into cDNA using the TaRaKa reverse transcription kit with Oligo (dT) primer and cDNA was amplified using the specific primers (Mouse) listed in S1 Table. A StepOnePlus Real-Time PCR System (Applied Biosystems, Carlsbad, California, USA) was used to quantify mRNA expression using SYBR Premix II (TaKaRa, Tokyo, Japan). The parameters for quantitative real-time PCR were 30 s at 95°C, 5 s at 95°C, and 30 s at 60°C for 40 cycles. GAPDH was used as a reference gene. Stereotactic injection and histology The AAV-KCTD5-P2A-mCherry, Lenti-shRNA (KCTD5)-mCherry, and AAV-shRNA (CUL3)-EGFP viruses were purchased from Obio Technology. For AAV vector, serotype 9 was used in this study and all the plasmids were designed and constructed by standard methods. For virus injection, mice were anesthetized with ketamine (100 mg/kg of body weight) and xylazine (8 mg/kg) by i.p. injection and placed in a stereotactic frame. Mice were injected with 0.6 μl of AAV virus (approximately 1012 infection units per ml) or 1 μl of lentivirus (approximately 108 infection units per ml) into the PVT (coordinates from bregma: −1.1 mm AP, 0 mm ML, −3.2 mm DV) [43] using glass microelectrodes at a slow rate (approximately 25 nl/min). The injection microelectrode was slowly withdrawn 5 min after the virus infusion. Behavior experiments were performed 2 to 3 weeks after surgery. The injection sites were examined at the end of the tests and only data obtained from animals with correct injections were included. Brain slices from mice were counterstained with Hoechst and directly examined under fluorescent microscope after mounting on the slides. Naloxone-induced morphine withdrawal and conditioned place aversion Mice after AAV injection were allowed to freely explore both sides of a CPP training apparatus for 15 min to assess their baseline place preference. Mice with AAV injection received a single daily injection of morphine (i.p.) for 6 consecutive days with doses escalating at 10, 20, 30, 40, 50, and 50 mg/kg in their home cage to develop morphine dependence. For another group of animals, MLN4924 were administrated 30 min before each dose of the morphine injections. Two hours after the last morphine injection, mice received either a 5 mg/kg naloxone (i.p.) injection to induce withdrawal responses or 1 mg/kg naloxone injection to induce CPA. The mice were placed in a transparent cylinder and withdrawal responses were videotaped for 20 min. Jumping, paw tremor, teeth-chartering, wet-dog shakes were recorded and scored from 0 to 3 based on behavioral bouts (0 = absent; 1, 1–3 bouts; 2, 4–6 bouts; 3, ≥7) at 5-min intervals. The total bouts of each behavior and the global withdrawal scores were calculated [50]. The body weight loss was calculated in the following formula: (weight before withdrawal but immediately after naloxone injection–weight immediately after withdrawal)/weight before withdrawal but immediately after naloxone injection*100%. Alternatively, the mice were confined in the counterbalanced side of the CPP chamber for 45 min post 1 mg/kg naloxone injection. One day after naloxone injection, withdrawal mice were re-exposed to the CPP chamber and allowed to explore both sides of the CPP chamber for 15 min. The CPA score was calculated by subtracting the time spent in the aversive stimulus-paired side of the chamber during baseline from the time spent in the same side of the chamber during the test. Statistical analysis All data are presented as mean ± SEM (n ≥ 3). Comparisons between 2 groups were done using Student’s t test. Comparisons among multiple groups under 1 condition were done using one-way ANOVA and comparisons among multiple groups under 2 different conditions were performed using two-way ANOVA. The Tukey’s and Bonferroni’s multiple-comparison test were used as indicated in the legends for post hoc analyses. For quantification of immunofluorescence double staining, Mander’s coefficient value was analyzed using Fiji software.

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