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The addiction-susceptibility TaqIA/Ankyrin repeat and kinase domain containing 1 kinase (ANKK1) controls reward and metabolism through dopamine receptor type 2 (D2R)-expressing neurons
Institute for Diabetes and Obesity, Helmholtz Diabetes Center (HDC), Helmholtz Zentrum Munchen, 85764 Neuherberg, GermanyGerman Center for Diabetes Research (DZD), 85764 Neuherberg, Germany
Correspondence. Enrica Montalban (E.M): , UMR 1286 NutriNeuro - INRAE / Université de Bordeaux / INP, 146, rue Léo Saignat. 33076 Bordeaux cedex – France, Tel :+33 6 60 33 93 24, Serge Luquet (S.H.L) : , BFA, Université Paris Cité, CNRS UMR 8251, 4 rue Marie-Andrée Lagroua Weill-Hallé, Case courrier 7126, 75205 Paris Cedex 13, France, Tel : +33 1 57 27 77 93
Footnotes
8 Lead contact
Affiliations
Université Paris Cité, CNRS, Unité de Biologie Fonctionnelle et Adaptative, F-75013 Paris, FranceThe Modern Diet and Physiology Research Center, New Haven CT, USA
A large body of evidence highlights the importance of genetic variants in the development of psychiatric and metabolic conditions. Among these, the TaqIA polymorphism is one of the most commonly studied in psychiatry. TaqIA is located in the gene that codes for the Ankyrin repeat and kinase domain containing 1 kinase (Ankk1) near the dopamine D2 dopamine receptor (D2R) gene. Homozygous expression of the A1 allele correlates with a 30 to 40% reduction of striatal D2R, a typical feature of addiction, over-eating and other psychiatric pathologies. The mechanisms by which the variant influences dopamine signaling and behavior are unknown.
METHODS
Here we used transgenic and viral-mediated strategies to reveal the role of Ankk1 in the regulation of activity and functions of the striatum.
RESULTS
We found that Ankk1 is preferentially enriched in striatal D2R-expressing neurons and that Ankk1 loss-of-function in dorsal and ventral striatum leads to alteration in learning, impulsivity and flexibibility resembling endophenotypes described in A1 carriers. We also observed an unsuspected role of Ankk1 in striatal D2R-expressing neurons of the ventral striatum in the regulation of energy homeostasis and documented differential nutrient partitioning in humans with or without the A1 allele.
CONCLUSIONS
Overall, our data demonstrate that the Ankk1 gene is necessary for the integrity of striatal functions and reveal a new role for Ankk1 in the regulation of body metabolism.
Psychiatric diseases are multifactorial disorders, the risk of which is influenced by both genetic and environmental factors. Even though classically considered as distinct pathologies, various psychiatric disorders share common symptomatic dimensions such as alterations of mood, cognitive functions or reward processing, suggesting similar pathophysiological mechanisms. In line with tis, the Research Domain Criteria (RDoC) classifies psychiatric illnesses based on common neurobiological, behavioral or genetic dimensions, aiming at identifying both the mechanisms that are shared across multiple psychiatric disorders, as well as the processes that are unique to specific psychiatric symptoms (
). Interestingly, psychiatric disorders are often accompanied by disturbances in energy metabolism and a higher risk of developing metabolic syndrome, with appetite changes as core feature of multiple diseases (
). Among SNPs, TaqIA polymorphisms have attracted growing attention. TaqIA polymorphism was initially believed to be in the D2R gene but was later mapped to the neighboring gene that codes for the Ankyrin repeat, and kinase domain containing 1 kinase (Ankk1) and corresponds to the single nucleotide polymorphism A2 (T→C) in the position 2137 of the Ankk1 transcript (
). The TaqIA variant results in an amino acid change (E[GAG]→K [AAG],Glu→Lys) in position 713 of Ankk1 protein in humans. While the minor A1 variant is the ancestral polymorphism, the A2 variant has only recently appeared in primate evolution (
). Ankk1 maps onto chromosome 11 in humans and onto chromosome 9 in mice, which includes the dopamine (DA) receptor D2 (D2R). TaqIA corresponds to three variants, A1/A1, A1/A2 and A2/A2. Approximately 30% of European, 80% of Asian, and 40% of African populations possess one or two copies of the A1 allele.
Strikingly, in humans the A1 allele is associated with psychiatric and neurological disorders such as attention deficit, hyperactivity disorder (
D2 dopamine receptor (DRD2) gene Taq1A polymorphism and the eating-related psychological traits in eating disorders (anorexia nervosa and bulimia) and obesity.
D2 dopamine receptor (DRD2) gene Taq1A polymorphism and the eating-related psychological traits in eating disorders (anorexia nervosa and bulimia) and obesity.
). Altogether, this suggests that A1 might be a genetic node for the convergence of neuropsychiatric and metabolic symptoms.
Alterations in reward processing, motivation, working memory and cognitive flexibility, all characterized by dysregulation of the cortico-limbic system, and its regulation by DA neurotransmission (
Evaluation of genetic variability in the dopamine receptor D2 in relation to behavioral inhibition and impulsivity/sensation seeking: an exploratory study with d-amphetamine in healthy participants.
) or steeper delayed discounting, which are typical features of several psychiatric symptoms and eating disorders. Moreover, homozygous dosage of the A1 allele correlates with a 30∼40% reduction of striatal D2Rabundance (
). Yet, such a decrease in D2R availability is a typical feature of addiction and believed to be a core endophenotype responsible for compulsive drug consumption (
These observations strongly suggest that; i) the interaction between environment and Ankk1 is critical in the susceptibility to reward-related and metabolic-based dysfunctions, and that ii) perturbations of various components of ingestive behavior in A1 carriers may result from a dysregulation of D2R-dependent DA transmission. To date, the molecular and cellular functions of Ankk1 remain largely unknown primarily due to the lack of animal models. As a consequence, very little is known regarding the mechanisms by which A1 and A2 variants of Ankk1 alter DA signaling and, in general, in which direction those variants affect Ankk1 activity and D2R-related function to contribute to the protection or the vulnerability to psychiatric and metabolic diseases.
MATERIAL AND METHODS
Animals
Both male and female Ankk1lox/lox, Ankk1Δ-D2R N and Drd2-Cre mice were used. Animal protocols were performed in accordance with the regulations and approved by the relevant committee: Paris, guidelines of the French Agriculture and Forestry Ministry for handling animals (decree 87-848) under the approval of the “Direction Départementale de la Protection des Populations de Paris” (authorization number C-75-828, license B75-05-22), Animal Care Committee of the University of Paris (APAFIS # 2015062611174320), Institut de Biologie Paris Seine of Sorbonne University (C75-05-24) (See supplementary Methods).
Human participants
Thirty-six subjects were recruited from the greater New Haven, Connecticut area via flyers or social media advertisements. Subjects were enrolled in this pilot study based on BMI (<26) and underwent indirect calorimetry measurement and genotyping on separate days. All subjects provided written informed consent at the first visit and the study was approved by the Yale Human Investigation Committee. (See supplementary Methods).
Total RNA purification, cDNA preparation and real-time PCR
Real-Time quantitative PCR (RT-qPCR) was normalized to a house-keeping gene using the delta-delta-CT (ddCT) method. (See supplementary Methods).
Pharmacological treatments
For acute treatments, apomorphine (Tocris) was dissolved in phosphate-buffered saline (PBS) and injected i.p. (3 mg.kg-1). PBS was used as vehicle treatment in control conditions. Haloperidol (Tocris) was dissolved in saline and injected i.p. (0.5 mg.kg-1).
Histology
Mice were anaesthetized with pentobarbital (500 mg/kg, i.p., Sanofi-Aventis) and transcardially perfused with 4 °C PFA 4% for 5 minutes. Sections were processed as in (
Patch-clamp recordings of D2R-SPNs neurons in the NAc
Mice were anesthetized with isoflurane, decapitated and coronal brain slices sections were prepared. D2R-SPNs identified in the NAc were patch-clamped and recorded as described in supplementary methods.
Stereotaxic injections
Annk1lox/lox animals were anaesthetized with isoflurane and received 10 mg.kg-1 intraperitoneal injection (i.p.) of Buprécare® (Buprenorphine 0.3 mg) diluted 1/100 in NaCl 9 g.L-1 and 10 mg.kg-1 of Ketofen® (Ketoprofen 100 mg) diluted 1/100 in NaCl 9 g.L-1, and placed on a stereotactic frame (Model 940, David Kopf Instruments, California). We bilaterally injected 0.6 (DS) or 0.3 (NAc) μl of virus (AAV9.CMV.HI.eGFP-Cre.WPRE.SV40 or AAV5.CMV.HI.eGFP-Cre.WPRE.SV40 and GFP controls, (titer 1013 vg.mL-1, working dilution 1:10 into the DS (L = +/-1.75; AP = +0.6; V = -3.5, and -3 in mm) or the NAc (L=+/- 1; AP=+1.55, V=-4.5) at a rate of 100 nl.min-1. The injection needle was carefully removed after 5 min waiting at the injection site and 2 min waiting half way to the top.
Behavioral & metabolic characterization
Haloperidol-induced catalepsy was measure 45-180 min after haloperidol injection. The behavior of mice was explored using a food cued T-Maze paradigm, operant conditioning and binge eating protocols (See supplementary Methods).
Binge feeding experiment
Intermittent access to a palatable food (High Fat Diet, ResearchDiet D12492i) was provided 1-hour/day during 4 consecutive days at 10-11 am. During binge sessions, chow pellets were not removed. The amount of the consumed palatable food was measured at the end of each session, data were presented as kcal/BW.
Metabolic efficiency analysis
All mice were monitored for metabolic efficiency (Labmaster, TSE Systems GmbH, Bad Homburg, Germany). After an initial period of acclimation in the calorimetry cages of at least two days, food and water intake, whole energy expenditure (EE), oxygen consumption and carbon dioxide production, respiratory quotient (RQ=VCO2/VO2, where V is volume) and locomotor activity were recorded as previously described. Additionally, fatty acid oxidation was calculated as previously reported. Reported (
). Data are the result of the average of the last three days of recording. Before and after indirect calorimetry assessment, body mass composition was analyzed using an Echo Medical systems’ EchoMRI (Whole Body Composition Analyzers, EchoMRI, Houston, USA).
Statistical analyses
Compiled data are reported as mean ± SEM., with single data points plotted. Data were analyzed with GraphPad Prism 9. Normal distribution was tested with Anderson-Darling, D'agostino Pearson test, Shapiro-Wilk test and Kolmorogrov-Smirnov. Data were analyzed with Two-tailed Mann-Whitney, unpaired Student’s T-Test, one-way ANOVA, two-way ANOVA or repeated-measures ANOVA, as applicable and Holm-Sidak’s post-hoc test for two by two comparisons. All tests were two-tailed. Significance was considered as p<0.05. Detailed statistical results are reported in Supplementary Table 3.
RESULTS
Striatal regional distribution and regulation of Ankk1 mRNA
We first showed an enrichment of Ankk1 mRNA expression in the DS as compared to the NAc (Fig.1-A, AI). We next re-analyzed available RNA-seq from striatal extracts based on Translating Ribosome Affinity Purification (TRAP) technology (Fig.1-B) (
) and showed that Ankk1 mRNA is virtually absent in D1R-SPNs and selectively expressed in D2R-SPNs (Fig.1-BI). We experimentally confirmed Ankk1 mRNA enrichment in D2R-expressing neurons (Fig.1-B) by performing quantitative RT-PCR on mRNA isolated by TRAP selectively from D1R- and D2R-SPNs (Fig.1-C). Next, we showed that stimulation of DA receptors by apomorphine treatment induced downregulation of Ankk1 mRNA in both NAc and DS 1h and 3h after injection (Fig.1-D, E).
Figure 1Ankk1 mRNA is expressed in the D2-SPNs of the DS and NAc and its downregulation leads to a decrease of D2r mRNA. (A) Schematic representation of the dissected tissue samples on coronal sections of striatum. Brains were rapidly dissected and placed in a stainless-steel matrix with 0.5 mm coronal section interval. A thick slice containing the striatum (3mm-thick) was obtained. The dorsal striatum (DS, green) and the nucleus accumbens (NAc, light blue) were punched out on ice. (AI) mRNA was purified from NAc and DS of BL-6 mice and analyzed by qRT-PCR. The expression levels were calculated by the comparative ddCt method with RPL19 as an internal control. Data points are individual results from different mice (n=5 per group). Means ± SEM are indicated. Statistical analyses are performed with two-tailed Mann-Whitney’s test, **p=0.0079. (B) Schematic representation of the translating ribosome affinity purification (TRAP) technique. Briefly, mRNA from either D2R- (orange) or D1R- (grey) SPNs were immunoprecipitated by using antibodies against the EGFP protein expressed in D2R or D1R ribosomes. (BI) Analysis of available RNAseq from TRAP data reveals a specific enrichment of Ankk1 mRNA in D2R-SPNs as compared to D1R-SPNs in both NAc and DS. Count per millions in each immunoprecipitation are reported. Data points are individual results from different pools of mice. Means ± SEM are indicated. Statistical analyses are performed with two-way ANOVA: interaction F(1,56)=8,643, p=0.0048, NA/DS=F(1,56)= 8,25, p=0.0057, D1/D2 SPNs: F(1,56)=319,5 p<0.0001 Tukey’s post hoc test ****p<0.0001, *** p=0.0007. (C) mRNA level of Ankk1 in isolated D1R- or D2R-SPNs populations, analyzed by qRT-PCR from male and female transgenic D1- and D2-TRAP mice. Statistical analysis are performed with two-tailed Mann Withney unpaired T-Test: p<0.0001. (D-E) mRNA level of Ankk1 in the NAc (D) and the DS (E) of mice injected with either saline or apomorphine (3 mg/Kg) and sacrificed 1h and 3h after injections. Data points are individual results from different mice (n= 5-6 per group). Means ± SEM are indicated. (D) Statistical analyses NAc: one-way ANOVA: F=11.47 p=0.0013 followed byDunnett’s multiple comparison p=0.0012, and p=0.0045. (E) DS, One-way ANOVA, F=20,87 p<0,0001 followed by Dunnett’s multiple comparison ****p<0,0001, **p=.0046. (F) Strategy of production of Ankk1 floxed mice. (G) Ankk1 mRNA levels from NAc of Ankk1lox/lox and Ankk1Δ-D2R N mice. Data are reported as means ± SEM of results of individual mice (n=11), statistical analysis are performed with two-tailed Mann Withney unpaired T-Test: p=0.0473. (H) D2r mRNA level from NAc of Drd2-Cre and Ankk1Δ-D2R N mice. Data are reported as means ± SEM of results of individual mice (n=7-9). Statistical analyses are performed with non-parametric Mann-Whitney test, D2r p=0.0418. (I) The effects of Ankk1 and D2r mRNA downregulation on D2R function were investigated by evaluating catalepsy after haloperidol injection (0.1 mg.kg-1, i.p.). Statistical analyses are performed with two-way ANOVA (18 mice per group): interaction p=0.0008, genotype p=0.0020, followed by Sidak’s post-hoc test: line 6 p=0.018.
We then assessed whether Ankk1 loss-of-function selectively in D2R-expressing neurons could recapitulate some of the phenotypes of A1 carriers. To do so, we generated Ankk1lox/lox mice in which exon 3-8 are flanked with LoxP sites allowing for Cre-targeted deletion of most of the Ankk1 gene (Fig.1-F and Supp.1A), that we crossed with a Drd2-Cre line. Specificity of the recombination of the Annk1 floxed locus was assessed by PCR on brain punches and peripheral tissues (Supp.1B). Cre-mediated decrease in Ankk1 mRNA was also validated in the NAc of Ankk1lox/lox::Drd2-Cre+/- (Ankk1Δ-D2R Neurons (Ankk1Δ-D2R N)) compared to control Ankk1lox/lox::Drd2-Cre-/- (Ankk1lox/lox) (Fig.1-G). However, due to the genetic construction of the Drd2-Cre BAC, which bears an additional copy of Ankk1 (
Schmidt EF, Kus L, Gong S, Heintz N (2013): BAC transgenic mice and the GENSAT database of engineered mouse strains. Cold Spring Harb Protoc 2013: pdb.top073692.
), crossing leads to a downregulation of Ankk1 rather than a full knock out in D2R-expressing neurons. Interestingly, we found a downregulation of ∼24% of the D2r mRNA in Ankk1Δ-D2R N (Fig.1-H), which resembles the ∼30% reduction of striatal D2R availability in homozygous A1 allele carriers (
). Decreased D2R activity by downregulation of Ankk1 in D2R-expressing neurons was further supported by blunted cataleptic effects of haloperidol in Ankk1Δ-D2R N mice (Fig.1-I) in both males and females (Supp.2 A-B). These results suggest that the A1 variant in humans might be associated with a loss of function for the Ankk1 protein.
Specific invalidation of Ankk1 in D2R neurons impacts the integrative properties of D2R-SPNs
We next performed whole-cell patch clamp recording in brain slices of Ankk1Δ-D2R N and Drd2-Cre mice locally injected with a viral vector bearing a Cre-dependent m-Cherry reporter. First, we confirmed the downregulation of Ankk1 mRNA in Ankk1Δ-D2R N as compared to Drd2-Cre mice (Fig.2-A). D2R-SPNs neurons were identified based on mCherry fluorescence (Fig.2-B). Ankk1 downregulation did not affect basic membrane and synaptic properties in D2R-SPNs, as shown by resting membrane potential, resistance and rheobase (Fig.2-C, Supp. Table1), amplitude and frequency of spontaneous excitatory (sEPSCs, Fig.2-D) and inhibitory (sIPSCs, Fig. 2-E) postsynaptic currents. By contrast, paired-pulse response (50 ms interval) of excitatory inputs onto D2R-SPNs were reduced in Ankk1Δ-D2R N mice, compared to Drd2-Cre mice (Fig.2-F), suggesting an enhanced presynaptic probability of glutamatergic inputs onto D2R-SPNs (
). Accordingly, spiking probability in response to electrical stimulation of excitatory afferents to the NAc was significantly enhanced in D2R-SPNs of Ankk1Δ-D2R N mice (Fig.2-G), reflecting an increase in excitability (
). Overall, these data demonstrate that Ankk1 downregulation in D2R-expressing neurons enhances glutamatergic transmission onto D2R-SPNs leading to increased excitability (
Figure 2Ankk1 downregulation in D2R-expressing neurons increases their excitability. (A) mRNA level of Ankk1 in the NAc of Drd2-Cre and Ankk1Δ-D2R N mice. (B) Males and females Drd2-Cre and Ankk1Δ-D2R N mice were stereotaxically injected with a viral vector carrying a flex AAV-mcherry. D2R-SPNs in the NAc were identified based on red fluorescence and patched. (C) Illustrative voltage responses of D2R-SPNs recorded in Drd2-Cre (grey, left) and Ankk1Δ-D2R N (orange, right) mice in response to series of 600ms current pulses starting at -150 pA with 20 pA increment. (D) Ankk1 downregulation does not alter frequency (left) or amplitude (right) of spontaneous EPSC (sEPSC). Mann Whitney unpaired T-Test p=0.39 and p=0.09 respectively (Drd2-Cre: n=15 neurons in 9 mice, Ankk1Δ-D2R N: n=14 neurons in 9 mice). (E) Ankk1 downregulation does not alter frequency (left) or amplitude (right) of spontaneous IPSC (sIPSC). Mann-Whitney unpaired T-Test p=0.59 (Drd2-Cre: n=6 neurons in 3 mice, Ankk1Δ-D2R N: n=8 neurons in 3 mice). (F) In paired pulse ratio (PPR) experiments, excitatory fibers were stimulated twice with an interval of 50ms, while EPSC were monitored in voltage clamp. In D2R-SPNs, Ankk1 downregulation resulted in a decrease of PPR. Two-tailed Mann-Whitney unpaired T-Test, p=0.0171 (Drd2-Cre: n=17 neurons in 10 mice, Ankk1Δ-D2R N: n=15 neurons in 9 mice). (G) The excitability of D2R-SPNs in the NAc was measured by quantifying the spiking probability with increasing electrical stimulation of excitatory inputs. The spiking probability is represented as a function of EPSP slope (mV/ms). Excitability of D2R-SPNs is increased in Ankk1Δ-D2R N mice. p<0.0001 (Drd2-Cre: n=18 neurons in 12 mice, Ankk1Δ-D2R N: n=10 neurons in 8 mice).
Effect of Ankk1 downregulation in D2R-expressing neurons on striatal-related learning
We next assessed the effect of Ankk1 downregulation in tasks known to strongly depend on striatal integrity. We first used striatal-dependent procedural learning based on an egocentric strategy to learn to locate, without any external cues, the baited arm in a T-maze (
) (Fig.3-A, right drawing). Ankk1Δ-D2RN mice displayed a strong impairment in the ability to learn the location of the reward in the maze (Fig.3-A) supporting a key role of Ankk1 in procedural learning. We next investigated performance of controls and Ankk1Δ-D2R N mice in an operant conditioning paradigm (Fig.3-B, right drawing), another striatal-related associative task (
). Both males and females Ankk1lox/lox and Ankk1Δ-D2RN displayed similar discriminatory performances when analyzing the percentage of lever presses on the reinforced lever (Supp.2 C-F and Supp.3-A, C, E, F). Ankk1Δ-D2RN displayed enhanced active lever pressing when operant ratios were increased (Fig.3-B). Although this result suggests enhanced motivational component in Ankk1Δ-D2RN mice, performances were similar to controls in a progressive ratio task (Supp.3 A-D). We also analyzed the time to initiate and complete the operant task and found no significant difference between controls and Ankk1Δ-D2RN mice. Our findings show that downregulation of Ankk1 in D2R-expressing neurons leads to deficits in an egocentric strategy-based procedural learning task, as well as in reward-driven operant conditioning paradigms.
Figure 3Consequences of Ankk1 downregulation in D2R-expressing neurons on striatal-dependent behaviors and energy metabolism. (A) Effect of downregulation of Ankk1 on procedural learning. Acquisition of the food-rewarded arm choice in a T maze is impaired in Ankk1Δ-D2R N mice. Statistical analysis were performed with two-way ANOVA: interaction p=0.12, genotype p<0.0001. N=12, (males 6-8, females 6-4) (B) Average of active lever press across fixed ratios (FR1 and 5) of instrumental conditioning, statistical analysis are performed with two-way ANOVA: interaction p=0.0115, genotype p=0.0964, time (Learning) p<0.0001, followed by Sidak’s multiple comparison *=0.014 n=18 (males 12-13, females 6-4).
), we next explored the consequence of Ankk1 knockdown on metabolic efficiency in mice fed ad libitum with a standard diet. Food intake, locomotor activity, metabolic efficiency and selective carbohydrates versus lipids substrate utilization in Ankk1lox/lox and Ankk1Δ-D2RN mice were measured as previously described (
). Both Ankk1lox/lox and Ankk1Δ-D2RN mice were comparable for body weight (Supp. 4-A), body composition (Supp 4-B, C, D), locomotor activity (Supp..4-E), energy intake (Supp..4-F), energy expenditure (EE) (Supp.4-G), or whole-body fatty acid oxidation (Supp. 4-H). Two-way ANOVA analysis of food intake showed a significant interaction in genotype per time, with no significant genotype differences (Supp. 4-H). Similar results were obtained when male and female were analyzed separately (Supp.5 A-P).
D2R-neurons specific Ankk1 knock down leads to changes in nutrient partitioning and protection from diet-induced obesity
Various studies have associated variations of Ankk1 with metabolic changes and more specifically with obesity (
). Hence, we next explored whether the interaction between genotype and obesogenic environment would unveil alterations in energy homeostasis regulation in Ankk1Δ-D2RN. Therefore, we subjected Ankk1lox/lox and Ankk1Δ-D2RN to 3 months of a high fat high sucrose diet (HFHS) prior to metabolic characterization. Obese mice (Ob-Ankk1Δ-D2RN and Ob-Ankk1lox/lox) showed comparable body weight (Fig.4-A) and lean mass (Fig.4-B), however, fat mass was decreased in Ob-Ankk1Δ-D2RN mice (Fig.4-C, D). Ob- Ankk1Δ-D2RN and control mice also displayed similar locomotor activity (Fig.4-E), caloric intake (Fig.4-F) and a trend towards decreased EE (p=0.12) (Fig.4-G). Interestingly, upon consumption of the obesogenic diet, Ankk1Δ-D2RN displayed a decreased level in fatty acid oxidation (Fig.4-H). Such a decrease does not seem to depend on the kilocalorie intake, as shown by the lack of correlation between food intake and fatty acid oxidation (Fig.4-I, J). This finding reveals that in an obesogenic downregulation of Ankk1 in D2R-expressing neurons is sufficient to induce alterations in peripheral substrate utilization and nutrient partitioning (
Figure 4Consequences on metabolism of Ankk1 downregulation in D2R-expressing neurons in a DIO paradigm. Ankk1Δ-D2R N do not show alteration in body weight (A) and lean mass (B), however significantly decreases fat mass p=0.037 (C) fat mass % (D) p=0.0430 (Two-tailed Mann-Whitney n=11-10, Males 5-6, Females 6-4). Ankk1Δ-D2R N and controls show similar locomotor activity (E) food intake (F), and energy expenditure (G). Ankk1Δ-D2R N showed decreased fatty acid oxidation (H), two-way ANOVA: Interaction p=0.4487, genotype p=0.0221, time p<0.0001. Fatty acid oxidation does not correlate with kcal intake in either light (I) or dark phase (J).
Region-specific invalidation of Ankk1 in the ventral or dorsal part of the striatum differentially affect reward-driven behavior
In order to unveil the neuroanatomical specificity of Ankk1 loss-of-function within the striatum we compared the consequence of viral-mediated knockdown of Ankk1 in the DS or NAc. Littermate Ankk1lox/lox mice received stereotactic injection of AAV-GFP or AAV-Cre in the NAc or in DS to produce controls (Ankk1GFP-NAc and Ankk1GFP-DS) or Ankk1 knockdown (Ankk1Δ-NAc and Ankk1Δ-DS). Accuracy of injections site and consequent change in Ankk1 levels were assessed through viral-mediated expression of GFP and q-RT-PCR (Supp.6-A, B for DS and Supp.6, E-F for NAc). Downregulation of Ankk1 in the DS did not affect the levels of D2r mRNA (Supp.6-C) nor haloperidol-induced catalepsy (Supp6-D), knockdown of Ankk1 in the NAc significantly decreased D2r mRNA levels (Supp.6-G) and blunted haloperidol-induced cataleptic events similar to Ankk1Δ-D2RN mice (Supp.6-H). Further, as for Ankk1Δ-D2RN, Ankk1Δ-NAc mice displayed an impairment in learning the egocentric strategy in the T-maze task (Fig.5-A), together with enhanced lever pressing from lower ratio requirement in the operant conditioning paradigm (Fig.5-B). In this latter task, Ankk1Δ-NAc mice exerted significantly more lever presses on the non-reinforced lever, while performance in the progressive ratio task was unchanged (Supp.7-A, B). Ankk1Δ-NAc mice also produced significantly more lever presses on the active lever during the inactive phase, a proxy for impulsive behavior (
). These behavioral alterations were observed in both ad libitum and fasting, indicating that this phenotype is not strictly dependent of hunger state (Supp. 7-C, D). We also analyzed the time to initiate and complete the operant task and found no significant difference between controls and Ankk1Δ-NAc mice.
Figure 5Consequences of Ankk1 downregulation in NAc on striatal-dependent behaviors and energy metabolism. (A) Effect of Ankk1 loss-of-function in the NAc on procedural learning. Acquisition of the food-rewarded arm choice in a T-maze is impaired in Ankk1Δ-NAc mice. Statistical analyses are performed with two-way ANOVA: interaction p=0.0004, time p<0.0001, genotype p<0.0001. Sidak’s multiple comparison *=0,0176 **=0,0055 ***=0,0002 N=9-13 (Males 5-8, females 4-5). (B) Average of active lever press across fixed ratios (FR1 and 5) of instrumental conditioning is increased in Ankk1Δ-NAc as compared to Ankk1GFP-NAc. Statistical analyses are performed with two-way ANOVA: interaction p=0.776, learning p=0.0006, genotype p=0.0162. N=8-11 (Males 4-6, females 4-5) C. Ankk1 loss-of-function in the NAc does not alter body weight (C) and lean mass (D) but decreases fat mass (E) and fat mass % (F), statistical analyses are performed with two tailed T-test: t=2,284 df=20 p=0.0335, fat mass t=2,696, df=20 p=0.0139, respectively. Data are expressed as mean ± SEM. n=10-13 (males 5-7, females 5-5). Ankk1Δ-NAc and Ankk1GFP-NAc showed comparable locomotor activity (G) and food intake (H), however Ankk1Δ-NAc display decreased energy expenditure (I),statistical analysis two-way ANOVA: interaction p=0,034 time, p<0.0001, genotype= F (
Altogether, our findings demonstrate that Ankk1 loss-of-function in the NAc is sufficient to recapitulate some of the behavioral phenotypes of Ankk1Δ-D2RN mice. By contrast, Ankk1Δ-DS mice had similar performance as controls in both the learning phase of the T-maze paradigm, as well as in operant conditioning for both ratio requirements. However, Ankk1Δ-DS mice displayed a selective deficit in the reversal phase of the T-maze (Supp.8-A, B). This latter finding resembles impaired cognitive flexibility described in Taq1A carriers (
Striatal deletion of Ankk1 alters energy homeostasis
We next assessed metabolic parameters in Ankk1Δ-DS and Ankk1Δ-NAc mice. Ankk1 loss-of-function in the NAc did not result in any significant changes of body weight (Fig5-C) or lean mass (Fig5-D) but decreased fat mass (Fig5-E) and fat mass % (Fig5-F). As for the Ankk1Δ-D2RN groups, Ankk1Δ-NAc mice showed unaltered locomotor activity (Fig.5-G) and caloric intake (Fig.5-H), although a trend was detected (Genotype p=0.056, Interaction p=0.06). Moreover, Ankk1Δ-NAc displayed a decrease in energy expenditure (Fig.5-I) and fatty acid oxidation (Fig.5-J) indicating that loss of function of Ankk1 specifically influences peripheral nutrient utilization. Change in fatty acid oxidation correlated with caloric intake (Fig.5-K, L), but was independent from body weight and lean mass, which were comparable between the two genotypes (Fig.5-C, D). Interestingly, fatty acid oxidation mostly decreased during the light phase (Fig.5-L), which might indicate a dissociation between circadian-entrained-rhythm and whole body fatty acid oxidation (
). Importantly, when tested on a binge eating paradigm with HFHS, a test aiming at evaluating uncontrolled voracious eating, Ankk1Δ-NAc mice showed enhanced food consumption during the binge period (Supp.5-E). We next explored if the obesogenic environment could magnify the metabolic consequence of Ankk1 knockdown. Mice were fed a HFHS diet for 3 months prior to the replication of the metabolic efficiency assessment. As compared to obese (Ob) controls (Ob-Ankk1GFP-NAc), Ob-Ankk1Δ-NAc mice showed a decrease in body weight (Fig.6-A), comparable lean mass (Fig.6-B), a decrease in fat mass (Fig.6-C) and a tendency of decreasing fat mass % (Fig.6-D). Ob-Ankk1Δ-NAc mice showed comparable caloric intake (Fig.6-E) but higher locomotor activity (Fig.6-F) and decreased energy expenditure (Fig.6-G). As for Ob-Ankk1Δ-D2RN, Ob-Ankk1Δ-NAc mice displayed lower fatty acid oxidation (Fig.6-H), which was independent from food intake (Fig.6-I, J). Overall, these data indicate that Ankk1 loss-of-function in the NAc exerts some protective effect from HFHS diet-induced disturbance in nutrient intake and partitioning. On the contrary, Ankk1 loss-of-function in the DS did not result in any relevant alteration in body weight and composition (Supp.6-D, G), locomotor activity (Supp.6-H), feeding (Supp.6-I) or energy expenditure (Supp.6-J). However, we could observe a small difference in the light-dark phase distribution of whole fatty acid in oxidation Ankk1Δ-DS mice (Supp.6-H). No difference between groups were observed in the binge eating paradigm (Supp.6-C). These results underscore the neuroanatomical discrimination of Ankk1-dependent regulation of metabolism in the striatum.
Figure 6Consequences of Ankk1 loss-of-function in the NAc on metabolism in a DIO paradigm. Ankk1Δ-NAc display decreased body weight (A) two-tailed Mann-Whitney’s test, p = 0.0455, but not lean mass (B), as well as decreased fat mass (C) two-tailed Mann-Whitney’s test, p=0.0411, but not fat mass percentage (D) two-tailed Mann-Whitney’s test, p=0.0931. (E) Caloric intake is comparable between Ankk1GFP-NAc and Ankk1Δ-NAc but the Ankk1Δ-NAc group showed increased locomotor activity (F) and decreased energy expenditure (G). Statistical analyses in F are performed with two-way ANOVA, Interaction p<0,0001 Time, p<0.0001, genotype p=0.0138. Statistical analyses in G are performed withtwo-way ANOVA: interaction p<0.0001, time, p<0.0001, genotype p=0.0222. (H) Downregulation of Ankk1 in the NAc decreases fatty acid oxidation, statistical analyses are performed with two-way ANOVA: interaction p<0.0001, time p<0.0001, genotype p=0.0074. Sidak’s post hoc test, *=p<0.05, **=p<0.001 ***=p<0.0001. (I-J) Fatty acid oxidation does not significantly correlate with food intake in both light (I) and dark (J) phases, however, in the dark phase slopes of the regression lines are significantly different between the two groups p=0,0129 (J). Data are expressed as mean ± SEM. n=6 (Male 2-4, female 4-2).
Differential respiratory quotient as a function of Taq1A A1 allele status in human participants
Given the change in nutrient partitioning associated with Ankk1 loss of function in mice, we hypothesized that a qualitatively similar phenotype could arise from A1/A2 variant in humans. We used indirect calorimetry during resting state to calculate the respiratory quotient (RQ), the ratio between carbon dioxide (CO2) and oxygen (O2) indicative of substrate utilization (RQ=1 for carbohydrate and RQ=0.7 for lipid) in 32 healthy human participants (19 A1- and 13 A1+)(Fig.7). Age, fat mass and other anthropometric measures were similar for both genotypes (Supp. Table 2). The groups also did not differ in hours of sleep and hours since last meal prior to the metabolic measure (Supp. Table 2). However, there was a significant difference in sex distribution between A1+ and A1 groups (p = 0.0014) (Supp. Table 2) so this factor is included in statistical models. Consistent with the observations in mice, A1+ individuals showed a significantly higher resting respiratory quotient (RQ) compared to A1- individuals (R2 = .41, F(6) = 7.31, p = 0.012). This result suggests a shift towards carbohydrate use as a primary energy source in A1+ and a shift towards fat in A1- at rest. This indicates that the polymorphism affecting the Ankk1 gene in human does alter peripheral nutrient utilization. These data support our reverse translational approach and show that the metabolic phenotype observed in mice translates to humans.
Figure 7Resting respiratory quotient (RQ) between A1 allele carriers (A1+) and non-A1 carriers (A1-) of Taq1A polymorphism (rs1800497). 32 participants with healthy weight (BMI<26) underwent metabolic measurements using indirect calorimetry and genotyping from saliva. Data points are individual results from different participants (n=19 in A1- and n=13 in A1+). Means + SEM are indicated. Statistical analysis is performed with an independent t-test and controlled for BMI, sex, age, and study. *p=0.012.
In this study, we showed for the first time that Ankk1 mRNA is enriched in striatal D2R-SPNs, that its downregulation in D2R-expressing neurons is sufficient alter their activity, and to decrease D2r mRNA expression and D2R-mediated response. These changes were associated with altered performance in striatal-dependent tasks such as procedural learning and reward-driven operant conditioning. Both D2R-specific and accumbal-restricted knockdown of Ankk1 were similarly associated with change in nutrient partitioning, suggesting a role for Ankk1 in striatal control of energy homeostasis. Finally, we performed a translational study that, in accordance with the mouse data, revealed differential whole-body metabolism in A1 carriers versus noncarriers.
The reduction of D2r mRNA expression transcript as a consequence of Ankk1 knockdown are congruent with published studies pointing at the consequences of TaqIA variants on D2R abundance, D2R-dependent function (
In fact, viral-mediated deletion of Ankk1 selectively in the NAc at adulthood recapitulates and even amplifies some of the behavioral phenotypes obtained in Ankk1Δ-D2R N, such as alteration in procedural learning and operant behavior. The consistent deficits we observed in the T-maze task for both Ankk1Δ-D2R N and Ankk1Δ-NAc are unlikely to be solely related to learning inabilities. In fact, even though it has been shown that T-maze task relies of propriocentric and egocentric strategy that depend on the integrity of the striatum (
), manipulations of the NAc can spare the acquisition of action-outcome associations, while impairing flexible adaptation of previously learned rules (
). In accordance, both Ankk1Δ-D2R N and Ankk1Δ-NAc were capable of learning the association between lever pressing and reward obtainment. However, Ankk1Δ-NAc also displayed an increased number of lever presses on the non-rewarded lever, as well as enhanced active lever pressing during the time out period, a feature considered as a proxy for impulsivity (
This suggests that the increase operant conditioning responding in Ankk1Δ-NAc as well as Ankk1Δ-D2RN mice might result from increased impulsivity. Importantly, impulsivity has been associated with decreased D2R availability in the NAc (
Evaluation of genetic variability in the dopamine receptor D2 in relation to behavioral inhibition and impulsivity/sensation seeking: an exploratory study with d-amphetamine in healthy participants.
). In addition, Selective impairment in the reversal phase of the T-maze in Ankk1Δ-DS reassembles decreased cognitive flexibility observed in TaqIA individuals (
Evaluation of genetic variability in the dopamine receptor D2 in relation to behavioral inhibition and impulsivity/sensation seeking: an exploratory study with d-amphetamine in healthy participants.
Of note, in addition to D2R-SPNs, cholinergic interneurons neurons also express D2r and therefore could be affected by Drd2-Cre mediated Ankk1 knockdown. While the presence of Ankk1 in cholinergic interneurons neurons remains elusive, one cannot rule out that loss of Ankk1 in CINs using a Drd2-Cre driver could contribute to the behavioral and metabolic outputs we observed given the prominent role of CIN in various striatal function (
In addition to the behavioral consequences of Ankk1 knockdown on reward-related behaviors, the present study demonstrates a role for Ankk1 integrity in the control of peripheral substrate utilization in both rodent and human. In both rodent models of Ankk1 knockdown, the metabolic changes were magnified. This is particularly relevant in the context of interactions of genetic polymorphisms, including Taq1A, and the modern food environment (for review (
)) as a risk factor for pathological conditions. Furthermore, while vulnerability for the metabolic defect in Taq1A carriers has been largely attributed to altered reward-feeding and overconsumption, the link we established between Ankk1 integrity and nutrient partitioning suggests an additional component of central control of energy homeostasis might be at play, interpedently from caloric intake. This is in line with the associations with TaqIA and insulin sensitivity (
In our mice models Ankk1 loss-of-function paradoxically appears to protect against increases in body weight and fat mass. This could be the result of an overall change in inter-organ communication and metabolic fluxes (
D2 dopamine receptor (DRD2) gene Taq1A polymorphism and the eating-related psychological traits in eating disorders (anorexia nervosa and bulimia) and obesity.
), which share common symptomatic dimensions with compulsive eating such as deficit in cognitive flexibility, impaired reward processing and impulsivity (
). However, while the decreased D2r mRNA levels under Ankk1 loss of function together with diminished adiposity appears counterintuitive since a decrease in D2r in the DS has been associated with obesity (
) and various studies suggest the decrease in D2r levels could correlate with other dimensions linked to obesity that are independent from BMI, such as opportunistic eating or decreased locomotor activity (
). Moreover, although fewer studies are available regarding the NAc, findings in humans and rodents reveal a negative association between ventral striatal D2r and BMI (
High-fat diet exposure increases dopamine D2 receptor and decreases dopamine transporter receptor binding density in the nucleus accumbens and caudate putamen of mice.
Several molecular mechanisms could account for the decrease in D2r and defective D2R-neuorns in our model of Ankk1 loss of function or in the TaqIA carrier. Ankk1 has been found to exert transcriptional control of the nuclear factor-kappa B (NF-κB)-regulated gene (
) it is possible that a reduced dosage of Ankk1 in heterozygous human carriers and in our animal model of loss of function, could lead to decreased D2r abundance and altered D2R-SPNs functions (
In conclusion, this work provides the first reverse translational approach exploring the biological functions of Ankk1 in the central regulation of both metabolic and reward functions and further translates the metabolic phenotype discovered in mice to humans. Collectively, our data show that Ankk1 loss-of-function is sufficient to mimic some of the phenotypic characteristics of Taq1A individuals and point at Ankk1 as potential molecular hub connecting striatal D2R-SPN to the control of energy homeostasis.
A limitation of our study is the lack of precise molecular mechanisms. We cannot rule out the possibility that D2 receptor abundance is the sole mechanism by which ANKK1 alters D2R-neurons physiology. Future studies are warrant to explore if and how ANKK1 could be targeted for the treatment of psychiatric and metabolic diseases.
AUTHORS CONTRIBUTION
EM, conceived the project, designed and performed most of the experiments, analyzed and interpreted the data, wrote the manuscript. RW, performed the electrophysiology experiments. JC performed the NAc surgeries, EF performed surgeries in DS, AA, RH, AP, JB performed experiments. JB, ACS edited the manuscript, XF, ZH, SM and EP performed human experiments. GG discussed the data and provided input to experiments and manuscript, CM supervised experiments, PT supervised the electrophysiology experiments, provided input and improvements to behavioral experiments and manuscript, CBB supervised and performed the electrophysiology experiments, D.M.S participated in the initial conception of the project and conceived and supervised the clinical study and provided input and corrections on the manuscript. SL conceived and supervised the overall project, analyzed and interpreted the data, secured funding provided input and corrections to the manuscript with the help of co-authors.
ACKNOWLEDGEMENTS
This work was funded by FRM Project #EQU202003010155. We acknowledge funding supports from the Modern Diet and Physiology Research Center (MDPRC), the Centre National de la Recherche Scientifique (CNRS) through the International Research Project (IRP) « BrainHealth », L’agence Nationale de la Recherche (ANR) ANR-19-CE37-0020-02, ANR-20-CE14-0020, The Université Paris Cité, INRAE, Université de Bordeaux, the project has been supported by the « Fédération pour la recherche sur le cerveau (FRC) » and the « Fondation des Treilles » « Fondation des Treilles créée par Anne Gruner Schlumberger, a notamment pour vocation d’ouvrir et de nourrir le dialogue entre les sciences et les arts afin de faire progresser la création et la recherche contemporaines. Elle accueille également des chercheurs et des écrivains dans le domaine des Treilles (Var) www.les-treilles.com » EM was supported by a post doctoral fellowship from the FRM. We thank Dr Thomas S. Hnasko and Professor Ralph DiLeone for valuable inputs on the study. We thank Olja Kacanski for administrative support, Isabelle Le Parco, Aurélie Djemat, Daniel Quintas, Magguy Boa and Ludovic Maingault and Angélique Dauvin for animals’ care and Florianne Michel for genotyping. We acknowledge the technical platform Functional and Physiological Exploration platform (FPE) of the Université Paris Cité, CNRS, Unité de Biologie Fonctionnelle et Adaptative, F-75013 Paris, France, the viral production facility of the UMR INSERM 1089 and the animal core facility “Buffon” of the Université Paris Cité/Institut Jacques Monod. We thank the animal facility of IBPS of Sorbonne Université, Paris.
D2 dopamine receptor (DRD2) gene Taq1A polymorphism and the eating-related psychological traits in eating disorders (anorexia nervosa and bulimia) and obesity.
Evaluation of genetic variability in the dopamine receptor D2 in relation to behavioral inhibition and impulsivity/sensation seeking: an exploratory study with d-amphetamine in healthy participants.
Schmidt EF, Kus L, Gong S, Heintz N (2013): BAC transgenic mice and the GENSAT database of engineered mouse strains. Cold Spring Harb Protoc 2013: pdb.top073692.
High-fat diet exposure increases dopamine D2 receptor and decreases dopamine transporter receptor binding density in the nucleus accumbens and caudate putamen of mice.
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