Knock-out of the critical nitric oxide synthase regulator DDAH1 in mice impacts amphetamine sensitivity and dopamine metabolism.

The enzyme dimethylarginine dimethylaminohydrolase 1 (DDAH1) plays a pivotal role in the regulation of nitric oxide levels by degrading the main endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA). Growing evidence highlight the potential implication of DDAH/ADMA axis in the etiopathogenesis of several neuropsychiatric and neurological disorders, yet the underlying molecular mechanisms remain elusive. In this study, we sought to investigate the role of DDAH1 in behavioral endophenotypes with neuropsychiatric relevance. To achieve this, a global DDAH1 knock-out (DDAH1-ko) mouse strain was employed. Behavioral testing and brain region-specific neurotransmitter profiling have been conducted to assess the effect of both genotype and sex. DDAH1-ko mice exhibited increased exploratory behavior toward novel objects, altered amphetamine response kinetics and decreased dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) level in the piriform cortex and striatum. Females of both genotypes showed the most robust amphetamine response. These results support the potential implication of the DDAH/ADMA pathway in central nervous system processes shaping the behavioral outcome. Yet, further experiments are required to complement the picture and define the specific brain-regions and mechanisms involved.

Divergent Dimethylarginine Dimethylaminohydrolase Isoenzyme Expression in the Central Nervous System.

The endogenous methylated derivative of ʟ-arginine, Nω,Nω'-dimethyl-ʟ-arginine (asymmetric dimethylarginine, ADMA), an independent risk factor in many diseases, inhibits the activity of nitric oxide synthases and, consequently, modulates the availability of nitric oxide. While most studies on the biological role of ADMA have focused on endothelial and inducible nitric oxide synthases modulation and its contribution to cardiovascular, metabolic, and renal diseases, a role in regulating neuronal nitric oxide synthases and pathologies of the central nervous system is less understood. The two isoforms of dimethylarginine dimethylaminohydrolase (DDAH), DDAH1 and DDAH2, are thought to be the main enzymes responsible for ADMA catabolism. A current impediment is limited knowledge on specific tissue and cellular distribution of DDAH enzymes within the brain. In this study, we provide a detailed characterization of the regional and cellular distribution of DDAH1 and DDAH2 proteins in the adult murine and human brain. Immunohistochemical analysis showed a wide distribution of DDAH1, mapping to multiple cell types, while DDAH2 was detected in a limited number of brain regions and exclusively in neurons. Our results provide key information for the investigation of the pathophysiological roles of the ADMA/DDAH system in neuropsychiatric diseases and pave the way for the development of novel selective therapeutic approaches.

Unleashing floret fertility in wheat through the mutation of a homeobox gene.

Floret fertility is a key determinant of the number of grains per inflorescence in cereals. During the evolution of wheat (Triticum sp.), floret fertility has increased, such that current bread wheat (Triticum aestivum) cultivars set three to five grains per spikelet. However, little is known regarding the genetic basis of floret fertility. The locus Grain Number Increase 1 (GNI1) is shown here to be an important contributor to floret fertility. GNI1 evolved in the Triticeae through gene duplication. The gene, which encodes a homeodomain leucine zipper class I (HD-Zip I) transcription factor, was expressed most abundantly in the most apical floret primordia and in parts of the rachilla, suggesting that it acts to inhibit rachilla growth and development. The level of GNI1 expression has decreased over the course of wheat evolution under domestication, leading to the production of spikes bearing more fertile florets and setting more grains per spikelet. Genetic analysis has revealed that the reduced-function allele GNI-A1 contributes to the increased number of fertile florets per spikelet. The RNAi-based knockdown of GNI1 led to an increase in the number of both fertile florets and grains in hexaploid wheat. Mutants carrying an impaired GNI-A1 allele out-yielded WT allele carriers under field conditions. The data show that gene duplication generated evolutionary novelty affecting floret fertility while mutations favoring increased grain production have been under selection during wheat evolution under domestication.

Assessment of DDAH1 and DDAH2 Contributions to Psychiatric Disorders via In Silico Methods.

The contribution of nitric oxide synthases (NOSs) to the pathophysiology of several neuropsychiatric disorders is recognized, but the role of their regulators, dimethylarginine dimethylaminohydrolases (DDAHs), is less understood. This study's objective was to estimate DDAH1 and DDAH2 associations with biological processes implicated in major psychiatric disorders using publicly accessible expression databases. Since co-expressed genes are more likely to be involved in the same biologic processes, we investigated co-expression patterns with DDAH1 and DDAH2 in the dorsolateral prefrontal cortex in psychiatric patients and control subjects. There were no significant differences in DDAH1 and DDAH2 expression levels in schizophrenia or bipolar disorder patients compared to controls. Meanwhile, the data suggest that in patients, DDAH1 and DDHA2 undergo a functional shift mirrored in changes in co-expressed gene patterns. This disarrangement appears in the loss of expression level correlations between DDAH1 or DDAH2 and genes associated with psychiatric disorders and reduced functional similarity of DDAH1 or DDAH2 co-expressed genes in the patient groups. Our findings evidence the possible involvement of DDAH1 and DDAH2 in neuropsychiatric disorder development, but the underlying mechanisms need experimental validation.

Genome-wide sequence information reveals recurrent hybridization among diploid wheat wild relatives.

Many conflicting hypotheses regarding the relationships among crops and wild species closely related to wheat (the genera Aegilops, Amblyopyrum, and Triticum) have been postulated. The contribution of hybridization to the evolution of these taxa is intensely discussed. To determine possible causes for this, and provide a phylogeny of the diploid taxa based on genome-wide sequence information, independent data were obtained from genotyping-by-sequencing and a target-enrichment experiment that returned 244 low-copy nuclear loci. The data were analyzed using Bayesian, likelihood and coalescent-based methods. D statistics were used to test if incomplete lineage sorting alone or together with hybridization is the source for incongruent gene trees. Here we present the phylogeny of all diploid species of the wheat wild relatives. We hypothesize that most of the wheat-group species were shaped by a primordial homoploid hybrid speciation event involving the ancestral Triticum and Am. muticum lineages to form all other species except Ae. speltoides. This hybridization event was followed by multiple introgressions affecting all taxa except Triticum. Mostly progenitors of the extant species were involved in these processes, while recent interspecific gene flow seems insignificant. The composite nature of many genomes of wheat-group taxa results in complicated patterns of diploid contributions when these lineages are involved in polyploid formation, which is, for example, the case for tetraploid and hexaploid wheats. Our analysis provides phylogenetic relationships and a testable hypothesis for the genome compositions in the basic evolutionary units within the wheat group of Triticeae.

Prevention of schizophrenia deficits via non-invasive adolescent frontal cortex stimulation in rats.

Schizophrenia is a severe neurodevelopmental psychiatric affliction manifested behaviorally at late adolescence/early adulthood. Current treatments comprise antipsychotics which act solely symptomatic, are limited in their effectiveness and often associated with side-effects. We here report that application of non-invasive transcranial direct current stimulation (tDCS) during adolescence, prior to schizophrenia-relevant behavioral manifestation, prevents the development of positive symptoms and related neurobiological alterations in the maternal immune stimulation (MIS) model of schizophrenia.

Boron demanding tissues of Brassica napus express specific sets of functional Nodulin26-like Intrinsic Proteins and BOR1 transporters.

The sophisticated uptake and translocation regulation of the essential element boron (B) in plants is ensured by two transmembrane transporter families: the Nodulin26-like Intrinsic Protein (NIP) and BOR transporter family. Though the agriculturally important crop Brassica napus is highly sensitive to B deficiency, and NIPs and BORs have been suggested to be responsible for B efficiency in this species, functional information of these transporter subfamilies is extremely rare. Here, we molecularly characterized the NIP and BOR1 transporter family in the European winter-type cv. Darmor-PBY018. Our transport assays in the heterologous oocyte and yeast expression systems as well as in growth complementation assays in planta demonstrated B transport activity of NIP5, NIP6, NIP7 and BOR1 isoforms. Moreover, we provided functional and quantitative evidence that also members of the NIP2, NIP3 and NIP4 groups facilitate the transport of B. A detailed B- and tissue-dependent B-transporter expression map was generated by quantitative polymerase chain reaction. We showed that NIP5 isoforms are highly upregulated under B-deficient conditions in roots, but also in shoot tissues. Moreover, we detected transcripts of several B-permeable NIPs from various groups in floral tissues that contribute to the B distribution within the highly B deficiency-sensitive flowers.

Functional evolution of nodulin 26-like intrinsic proteins: from bacterial arsenic detoxification to plant nutrient transport.

Nodulin 26-like intrinsic proteins (NIPs) play essential roles in transporting the nutrients silicon and boron in seed plants, but the evolutionary origin of this transport function and the co-permeability to toxic arsenic remains enigmatic. Horizontal gene transfer of a yet uncharacterised bacterial AqpN-aquaporin group was the starting-point for plant NIP evolution. We combined intense sequence, phylogenetic and genetic context analyses and a mutational approach with various transport assays in oocytes and plants to resolve the transorganismal and functional evolution of bacterial and algal and terrestrial plant NIPs and to reveal their molecular transport specificity features. We discovered that aqpN genes are prevalently located in arsenic resistance operons of various prokaryotic phyla. We provided genetic and functional evidence that these proteins contribute to the arsenic detoxification machinery. We identified NIPs with the ancestral bacterial AqpN selectivity filter composition in algae, liverworts, moss, hornworts and ferns and demonstrated that these archetype plant NIPs and their prokaryotic progenitors are almost impermeable to water and silicon but transport arsenic and boron. With a mutational approach, we demonstrated that during evolution, ancestral NIP selectivity shifted to allow subfunctionalisations. Together, our data provided evidence that evolution converted bacterial arsenic efflux channels into essential seed plant nutrient transporters.

Metabolic state and value-based decision-making in acute and recovered female patients with anorexia nervosa

Background: Patients with anorexia nervosa forgo eating despite emaciation and severe health consequences. Such dysfunctional decision-making might be explained by an excessive level of self-control, alterations in homeostatic and hedonic regulation, or an interplay between these processes. We aimed to understand value-based decision-making in anorexia nervosa and its association with the gut hormone ghrelin. Besides its homeostatic function, ghrelin has been implicated in the hedonic regulation of appetite and reward via the modulation of phasic dopamine signalling. Methods: In a cross-sectional design, we studied acutely underweight (n = 94) and recovered (n = 37) patients with anorexia nervosa of the restrictive subtype, as well as healthy control participants (n = 119). We assessed plasma concentrations of desacyl ghrelin and parameters of delay discounting, probability discounting for gains and losses, and loss aversion. Results: Recovered patients displayed higher risk aversion for gains, but we observed no group differences for the remaining decision-making parameters. Desacyl ghrelin was higher in acutely underweight and recovered participants with anorexia nervosa relative to healthy controls. Moreover, we found a significant group × desacyl ghrelin interaction in delay discounting, indicating that in contrast to healthy controls, acutely underweight patients with anorexia nervosa who had high desacyl ghrelin concentrations preferably chose the delayed reward option. Limitations: We probed decision-making using monetary rewards, but patients with anorexia nervosa may react differently to disorder-relevant stimuli. Furthermore, in contrast to acyl ghrelin, the functions of desacyl ghrelin are unclear. Therefore, the interpretation of the results is preliminary. Conclusion: The propensity for risk aversion as found in recovered patients with anorexia nervosa could help them successfully complete therapy, or it could reflect sequelae of the disorder. Conversely, ghrelin findings might be related to a mechanism contributing to disease maintenance; that is, in acutely underweight anorexia nervosa, a hungry state may facilitate the ability to forgo an immediate reward to achieve a (dysfunctional) long-term goal.

Dysfunctional approach behavior triggered by alcohol-unrelated Pavlovian cues predicts long-term relapse in alcohol dependence.

We demonstrated that alcohol-dependent patients who relapsed within 1 year after detoxification showed stronger PIT effects compared with abstainers and controls. Relapsers particularly failed to correctly perform in trials where an instrumental stimulus required inhibition while a Pavlovian background cue indicated a monetary gain. Under that condition, relapsers approached the instrumental stimulus, independent of the expected punishment. The failure of inhibiting an aversive stimulus in favor of approaching an appetitive context cue reflects dysfunctional altered learning mechanisms in relapsers.

Neural correlates of instrumental responding in the context of alcohol-related cues index disorder severity and relapse risk.

The influence of Pavlovian conditioned stimuli on ongoing behavior may contribute to explaining how alcohol cues stimulate drug seeking and intake. Using a Pavlovian-instrumental transfer task, we investigated the effects of alcohol-related cues on approach behavior (i.e., instrumental response behavior) and its neural correlates, and related both to the relapse after detoxification in alcohol-dependent patients. Thirty-one recently detoxified alcohol-dependent patients and 24 healthy controls underwent instrumental training, where approach or non-approach towards initially neutral stimuli was reinforced by monetary incentives. Approach behavior was tested during extinction with either alcohol-related or neutral stimuli (as Pavlovian cues) presented in the background during functional magnetic resonance imaging (fMRI). Patients were subsequently followed up for 6 months. We observed that alcohol-related background stimuli inhibited the approach behavior in detoxified alcohol-dependent patients (t = - 3.86, p < .001), but not in healthy controls (t = - 0.92, p = .36). This behavioral inhibition was associated with neural activation in the nucleus accumbens (NAcc) (t(30) = 2.06, p < .05). Interestingly, both the effects were only present in subsequent abstainers, but not relapsers and in those with mild but not severe dependence. Our data show that alcohol-related cues can acquire inhibitory behavioral features typical of aversive stimuli despite being accompanied by a stronger NAcc activation, suggesting salience attribution. The fact that these findings are restricted to abstinence and milder illness suggests that they may be potential resilience factors.Clinical trial: LeAD study, http://www.lead-studie.de , NCT01679145.

No association of goal-directed and habitual control with alcohol consumption in young adults.

Alcohol dependence is a mental disorder that has been associated with an imbalance in behavioral control favoring model-free habitual over model-based goal-directed strategies. It is as yet unknown, however, whether such an imbalance reflects a predisposing vulnerability or results as a consequence of repeated and/or excessive alcohol exposure. We, therefore, examined the association of alcohol consumption with model-based goal-directed and model-free habitual control in 188 18-year-old social drinkers in a two-step sequential decision-making task while undergoing functional magnetic resonance imaging before prolonged alcohol misuse could have led to severe neurobiological adaptations. Behaviorally, participants showed a mixture of model-free and model-based decision-making as observed previously. Measures of impulsivity were positively related to alcohol consumption. In contrast, neither model-free nor model-based decision weights nor the trade-off between them were associated with alcohol consumption. There were also no significant associations between alcohol consumption and neural correlates of model-free or model-based decision quantities in either ventral striatum or ventromedial prefrontal cortex. Exploratory whole-brain functional magnetic resonance imaging analyses with a lenient threshold revealed early onset of drinking to be associated with an enhanced representation of model-free reward prediction errors in the posterior putamen. These results suggest that an imbalance between model-based goal-directed and model-free habitual control might rather not be a trait marker of alcohol intake per se.

Value-based decision-making battery: A Bayesian adaptive approach to assess impulsive and risky behavior.

Using simple mathematical models of choice behavior, we present a Bayesian adaptive algorithm to assess measures of impulsive and risky decision making. Practically, these measures are characterized by discounting rates and are used to classify individuals or population groups, to distinguish unhealthy behavior, and to predict developmental courses. However, a constant demand for improved tools to assess these constructs remains unanswered. The algorithm is based on trial-by-trial observations. At each step, a choice is made between immediate (certain) and delayed (risky) options. Then the current parameter estimates are updated by the likelihood of observing the choice, and the next offers are provided from the indifference point, so that they will acquire the most informative data based on the current parameter estimates. The procedure continues for a certain number of trials in order to reach a stable estimation. The algorithm is discussed in detail for the delay discounting case, and results from decision making under risk for gains, losses, and mixed prospects are also provided. Simulated experiments using prescribed parameter values were performed to justify the algorithm in terms of the reproducibility of its parameters for individual assessments, and to test the reliability of the estimation procedure in a group-level analysis. The algorithm was implemented as an experimental battery to measure temporal and probability discounting rates together with loss aversion, and was tested on a healthy participant sample.

Dated tribe-wide whole chloroplast genome phylogeny indicates recurrent hybridizations within Triticeae.

BACKGROUND: Triticeae, the tribe of wheat grasses, harbours the cereals barley, rye and wheat and their wild relatives. Although economically important, relationships within the tribe are still not understood. We analysed the phylogeny of chloroplast lineages among nearly all monogenomic Triticeae taxa and polyploid wheat species aiming at a deeper understanding of the tribe's evolution. We used on- and off-target reads of a target-enrichment experiment followed by Illumina sequencing. RESULTS: The read data was used to assemble the plastid locus ndhF for 194 individuals and the whole chloroplast genome for 183 individuals, representing 53 Triticeae species and 15 genera. We conducted Bayesian and multispecies coalescent analyses to infer relationships and estimate divergence times of the taxa. We present the most comprehensive dated Triticeae chloroplast phylogeny and review previous hypotheses in the framework of our results. Monophyly of Triticeae chloroplasts could not be confirmed, as either Bromus or Psathyrostachys captured a chloroplast from a lineage closely related to a Bromus-Triticeae ancestor. The most recent common ancestor of Triticeae occurred approximately between ten and 19 million years ago. CONCLUSIONS: The comparison of the chloroplast phylogeny with available nuclear data in several cases revealed incongruences indicating past hybridizations. Recent events of chloroplast capture were detected as individuals grouped apart from con-specific accessions in otherwise monopyhletic groups.

Impulsive Decision Making in Young Adult Social Drinkers and Detoxified Alcohol-Dependent Patients: A Cross-Sectional and Longitudinal Study.

BACKGROUND: Impulsive decision making relates to problematic substance use. Specifically, altered delay discounting (DD) has been suggested as a behavioral marker for addiction, while other relevant facets of choice impulsivity such as probability discounting (PD) or loss aversion are clearly understudied. METHODS: Two studies were performed collecting behavioral data on choice impulsivity with a value-based decision-making battery providing estimates of DD, PD for gains and losses, and loss aversion. Study (1): In a sample of 198 male 18-year-old social drinkers, we analyzed impulsive choice behavior and its association with alcohol consumption and self-report measures of substance use-related personality traits on a cross-sectional level. Additionally, the predictive value of baseline choice behavior for the trajectories of alcohol consumption over a 12-month follow-up period was evaluated. Study (2): Behavioral data on choice impulsivity were collected for 114 detoxified patients with alcohol use disorder (AUD) and 98 control participants. We analyzed group differences at baseline and assessed the predictive value of choice impulsivity for relapse to heavy alcohol use in patients during a follow-up period of 48 weeks. RESULTS: Study (1): Only DD was associated with baseline alcohol use, but no measure of choice impulsivity predicted the drinking trajectories over the following 12 months. Study (2): Compared to the control group, AUD patients showed higher DD, lower risk aversion regarding probabilistic gains, lower risk seeking regarding probabilistic losses, and lower loss aversion facing mixed prospects. Further, shallow discounting of probabilistic losses at baseline was predictive for relapse in patients. CONCLUSIONS: All 4 domains of impulsive decision making were considerably altered in AUD patients though mostly not related to alcohol use in young adult social drinkers. This suggests that these facets of impulsive behavior may develop as consequences of chronic alcohol consumption. Furthermore, discounting of probabilistic losses might prove valuable in identifying patients vulnerable for relapse.

Pavlovian-to-instrumental transfer effects in the nucleus accumbens relate to relapse in alcohol dependence.

In detoxified alcohol-dependent patients, alcohol-related stimuli can promote relapse. However, to date, the mechanisms by which contextual stimuli promote relapse have not been elucidated in detail. One hypothesis is that such contextual stimuli directly stimulate the motivation to drink via associated brain regions like the ventral striatum and thus promote alcohol seeking, intake and relapse. Pavlovian-to-Instrumental-Transfer (PIT) may be one of those behavioral phenomena contributing to relapse, capturing how Pavlovian conditioned (contextual) cues determine instrumental behavior (e.g. alcohol seeking and intake). We used a PIT paradigm during functional magnetic resonance imaging to examine the effects of classically conditioned Pavlovian stimuli on instrumental choices in n = 31 detoxified patients diagnosed with alcohol dependence and n = 24 healthy controls matched for age and gender. Patients were followed up over a period of 3 months. We observed that (1) there was a significant behavioral PIT effect for all participants, which was significantly more pronounced in alcohol-dependent patients; (2) PIT was significantly associated with blood oxygen level-dependent (BOLD) signals in the nucleus accumbens (NAcc) in subsequent relapsers only; and (3) PIT-related NAcc activation was associated with, and predictive of, critical outcomes (amount of alcohol intake and relapse during a 3 months follow-up period) in alcohol-dependent patients. These observations show for the first time that PIT-related BOLD signals, as a measure of the influence of Pavlovian cues on instrumental behavior, predict alcohol intake and relapse in alcohol dependence.

Manipulation of the nuclear envelope-associated protein SLAP during mammalian brain development affects cortical lamination and exploratory behavior.

Here, we report the first characterization of the effects resulting from the manipulation of Soluble-Lamin Associated Protein (SLAP) expression during mammalian brain development. We found that SLAP localizes to the nuclear envelope and when overexpressed causes changes in nuclear morphology and lengthening of mitosis. SLAP overexpression in apical progenitors of the developing mouse brain altered asymmetric cell division, neurogenic commitment and neuronal migration ultimately resulting in unbalance in the proportion of upper, relative to deeper, neuronal layers. Several of these effects were also recapitulated upon Cas9-mediated knockdown. Ultimately, SLAP overexpression during development resulted in a reduction in subcortical projections of young mice and, notably, reduced their exploratory behavior. Our study shows the potential relevance of the previously uncharacterized nuclear envelope protein SLAP in neurodevelopmental disorders.

DCC mediated axon guidance of spinal interneurons is essential for normal locomotor central pattern generator function.

Coordinated limb rhythmic movements take place through organized signaling in local spinal cord neuronal networks. The establishment of these circuitries during development is dependent on the correct guidance of axons to their targets. It has previously been shown that the well-known axon guidance molecule netrin-1 is required for configuring the circuitry that provides left-right alternating coordination in fictive locomotion. The attraction of commissural axons to the midline in response to netrin-1 has been shown to involve the netrin-1 receptor DCC (deleted in Colorectal Cancer). However, the role of DCC for the establishment of CPG coordination has not yet been resolved. We show that mice carrying a null mutation of DCC displayed an uncoordinated left-right activity during fictive locomotion accompanied by a loss of interneuronal subpopulations originating from commissural progenitors. Thus, DCC plays a crucial role in the formation of spinal neuronal circuitry coordinating left-right activities. Together with the previously published results from netrin-1 deficient mice, the data presented in this study suggest a role for the most ventral originating V3 interneurons in synchronous activities over the midline. Further, it provides evidence that axon crossing in the spinal cord is more intricately controlled than in previously suggested models of DCC-netrin-1 interaction.

Supplement Treatment with NAC and Omega-3 Polyunsaturated Fatty Acids during Pregnancy Partially Prevents Schizophrenia-Related Outcomes in the Poly I:C Rat Model.

BACKGROUND: Heightened levels of inflammation and oxidative stress are thought to be involved in the pathophysiology of schizophrenia. We aimed to assess whether intake of anti-inflammatory and anti-oxidant drugs during pregnancy prevents later schizophrenia-related outcomes in a neurodevelopmental rat model of this disorder. METHODS: Pregnant Wistar rats were injected with polyriboinosinic-polyribocytidilic acid (Poly I:C) or saline and subsequently treated with either N-acetyl cysteine (NAC) or omega-3 polyunsaturated fatty acids (PUFAs) until delivery. Controls rats received no treatment. In the offspring, neuroinflammation and anti-oxidant enzyme activity were assessed on postnatal day (PND) 21, 33, 48, and 90. Behavioral testing was performed at PND 90, followed by post-mortem neurochemical assessment and ex vivo MRI. RESULTS: The supplement treatment led to a quicker restoration of the wellbeing of dams. In the adolescent Poly I:C offspring, the supplement treatment prevented an increase in microglial activity and partially prevented a deregulation in the anti-oxidant defense system. In the adult Poly I:C offspring, supplement treatment partially prevented dopamine deficits, which was paralleled by some changes in behavior. Exposure to omega-3 PUFAs prevented the enlargement of lateral ventricles. CONCLUSION: Intake of over-the-counter supplements may assist in especially targeting the inflammatory response related to schizophrenia pathophysiology, aiding in diminishing later disease severity in the offspring.

A multicentric consortium study demonstrates that dimethylarginine dimethylaminohydrolase 2 is not a dimethylarginine dimethylaminohydrolase.

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects against cardiovascular disease by metabolising the risk factor asymmetric dimethylarginine (ADMA). However, the question whether the second DDAH isoform, DDAH2, directly metabolises ADMA has remained unanswered. Consequently, it is still unclear if DDAH2 may be a potential target for ADMA-lowering therapies or if drug development efforts should focus on DDAH2's known physiological functions in mitochondrial fission, angiogenesis, vascular remodelling, insulin secretion, and immune responses. Here, an international consortium of research groups set out to address this question using in silico, in vitro, cell culture, and murine models. The findings uniformly demonstrate that DDAH2 is incapable of metabolising ADMA, thus resolving a 20-year controversy and providing a starting point for the investigation of alternative, ADMA-independent functions of DDAH2.