Attention problems and decreased quality of life are frequently accompanied in Cerebral Palsy (CP), which can negatively affect rehabilitation of physical disability. However, the majority of affected children remain untreated in the aspects of attention or psychosocial factors. Equine-Assisted Activities and Therapies (EAAT) use horse as a therapeutic modality including grooming as well as mounted riding activities in which patients exercise and experience mounted stimulation. It is known to help improve attention in children with ADHD, so that it can be an exercise therapy that is expected to improvement of attention as well as rehabilitating effects in CP patients. EAA may be a promising strategy to address the unmet need for CP patients. This study aims to investigate the efficacy of EAA for children with CP, those with both CP and ADHD and confirm the comorbidity between CP and ADHD.Forty-six children with cerebral palsy participated in this study. For the exercise group, they participated in a 40-min session twice a week for a 16-week period, while the control group engaged in daily life without any special treatments. Each children individually were assessed on attention and psychological wellbeing at baseline and post-treatment. Comorbidity were identified based on the Diagnostic and Statistical Manual of Mental Disorder 5th edition (DSM-5) and confirmed by Korean Kiddie-Schedule for Affective Disorders and Schizophrenia Present and Lifetime Version (K-SADS-PL).p < .024). However, no significant improvement in children’s quality of life was observed after EAA program compared with control group. Among the total participants, fifteen children (31.91%) were diagnosed with ADHD. When conducting an additional analysis with the subsample of CP patients diagnosed with ADHD, the d’, commission error and perseveration showed a significant decrease only in the exercise group. Children with CP and ADHD reported an improvement in quality of life both in exercise and control group, but only in the exercise group social functioning exhibited a significant difference.Perseveration rated using the Conner’s Performance Test (CPT) showed a significant decrease only in the exercise group . Registered 26 July 2017.This trial was registered on ClinicalTrials.gov ( Cerebral palsy (CP) is a group of neurological disorders resulting from damage to the developing brain. It represents a heterogeneous group of motor impairments , and oftADHD is one of the most common neurodevelopmental disorders diagnosed in the field of child psychiatry, with a worldwide prevalence of approximately 3–10% . It is aVarious studies have demonstrated that executive function of the cognitive domain in children with neurodevelopmental disorders are not fully developed during childhood. As a result, those with CP and ADHD appear to continuously experience difficulties in the academic fields and life than children with CP but without ADHD. To start with, both populations experience difficulties with sustained attention. The reduced processing speed and performance interferes with complex cognitive tasks that require cognitive and logical skills . In factAs mentioned above, numerous studies have shown that the presence of concurrent CP and ADHD leads to the accumulation of negative experience, which may increase the risk of secondary psychological ailments , 15, 16.Recently, Equine-Assisted Activities and Therapies (EAAT) have received attention as it helps people suffering from a variety of psychological issues. Equine Assisted Activities and Therapies (EAAT) is a broad term that encompasses two main interventions; Equine Assisted Activities (EAA) and Equine Assisted Therapies (EAT). EAA includes instructions in riding skills, how to control the horse with verbal and nonverbal cues and horsemanship skills . This inAlthough the effectiveness of EAA on attention or psychosocial functioning has been investigated , 9, studThe study involved 47 CP outpatients aged 6 to 13 years who visited the Department of Rehabilitation within a university hospital located in Seoul from August 2017 to January 2019. The psychiatric diagnoses were based on the diagnostic criteria of the Diagnostic and Statistical Manual for Mental Disorders-5 (DSM-5) and confirmed the comorbidity by Kiddie Schedule for Affective Disorders and Schizophrenia Present and Lifetime Version (K-SADS-PL). The exclusion criteria were: patients with weight exceeding 35 kg, uncontrolled seizures; inability to perform one-step instruction due to visual and hearing impairment or intellectual disability; hip dislocation; scoliosis greater than 30°; less than 1-year history of musculoskeletal surgery; unrecovered fracture; botulinum toxin injection within 3 months; or riding that was inappropriate for other medical conditions.The participants were randomly assigned to the EAA exercise group or the control group. The participants were assigned with trial ID numbers, created by a researcher not otherwise involved in the study. Prior to the 16-week study program, both groups participated in a pre-comprehensive assessment within 2 weeks, including intelligence, attention and psychological evaluation using Korean-Wechsler Intelligence Scale for Children-Fourth Edition (K-WISC-IV), Conner’s Continuous Performance Test (CPT), Korean ADHD Rating Scale (K-ARS) and Pediatric Quality of Life Inventory (PedsQl), which were used in previous studies to examine therapeutic effects on ADHD or CP children , 23. 2-wThose included in the EAA exercise group participated in a 40-min class twice a week over the course of 16 weeks . A single instructor conducted a group program for three children, and each child was assisted by a horse leader and a side walker. The instructor holds national therapeutic riding instructor certification and previously had obtained Professional Association of Therapeutic Horsemanship (PATH) registered therapeutic riding instructor certification. The sessions focused on mounted activities which consisted of exercises to facilitate accurate posture, balance and practice basic riding skills. The control group did not receive intervention and continued with their usual daily activities.K-WISC-IV is a standardized measurement tool to assess intellectual function in children between ages 6 and 16 years and 11 months. The profile provides Full Scale Intelligence Quotient (FSIQ) for overall intellectual abilities and a total of five composite scores for specific cognitive abilities. K-WISC-IV showed high internal consistency and good test-retest correlation in previous studies .This visual vigilance computerized test was developed to measure attention problems and has been widely used as a supplementary diagnostic tool for ADHD. The participants are instructed to respond to the computer screen by pressing the left button for every picture presented except the soccer ball. Based on the response, d’ (the accuracy in discriminating non-targets from targets), omission error (the number of times that the subject has failed to respond to the target), commission error (the number of times that the subject provided an incorrect response to the non-target), preservation error , mean response time (measured between the presentation of the target stimulus and a correct response), standard deviation of reaction time (variability or consistency of attention), and standard deviation of variable response time are calculated to determine index scores of attention and impulsivity. In the current study, all subjects underwent a 3- to 4-min practice session before starting the main evaluation. The test required a total of 30 min and the examiner remained in the room during the administration.The scale is used to assess ADHD symptoms in children. The score is based on each 9 items that reflect inattentiveness and hyperactivity/impulsivity, with a total of 18 items. Higher scores indicate increased attention impairment and impulse inhibition abilities. The Korean version of ARS was validated with reasonable internal consistency of 0.77–0.89 .The scale measures children’s general quality of life. It is composed of 21 items based on 4 dimensions: physical, emotional, social and academic aspects. Higher scores indicate higher quality of life. In this study, scores for each subscale were used to evaluate the treatment effect.K-SADS-PL was introduced by Kaufman and his colleges (1997) to assess current and past episodes of psychiatric disorders listed in Diagnostic and Statistical Manual of Mental Disorders-IV (DSM-IV). This semi-structured diagnostic instrument has been reported to be valid for the screening and diagnosis of childhood and adolescent disorders. The Korean Version of K-SADS-PL was translated and validated by Kim and his colleagues (2004) who reported excellent reliability and validity. In the present study, the tool was used to confirm the diagnoses of any existing psychiatric diagnosis.t-test. Repeated measures of analysis of variance (repeated ANOVA) were used to compare the overall improvement in attention and quality of life before and after treatment in the exercise and control group. The secondary analysis for intra-group comparison among subjects who were diagnosed with ADHD was based on t-test. A p-value less than 0.05 was considered statistically significant and results were analyzed using SPSS ver. 24 .The demographic data of 46 participants  were compared via independent Twenty-three participants were assigned to the exercise group  and the control group . The average age of participants in the exercise and control group is 7.78 ± 1.68 years, 7.30 ± 1.61 years, respectively. The average FSIQ for the exercise and control group is 79.91 ± 19.86, 79.70 ± 16.03, respectively. There was no group difference in demographic characteristics of sex or age and intelligence. Participant characteristics are summarized in Table p < .001). Group differences existed , representing a difference in effectiveness between groups. The change in mean perseveration score showed a significant decrease pre- and post-treatment from 61.30 to 53.04 in the exercise group; 51.57 to 49.91 in the control group. The d’ (p < .001), commission (p < .001), and hit reaction time standard deviation (p < .05) decreased in a time-dependent manner, but the group difference was insignificant.Analyzed by repeated measures of ANOVA, the perseveration error score decreased in a time-dependent manner , emotional (p = .011), social (p = .000), and academic (p = .024) subdomains significantly increased in a time-dependent manner, but no group difference existed. The above results are presented in Table The effects on quality of life suggest that physical , commission , and perseveration score  showed a significant difference during the 16-week program. Further, omission, hit reaction time, and hit block change showed a gradual improvement but was not statistically significant. In the control group, d’, omission and hit reaction time showed a decrease but was not significant. In the parent’s ARS self-report, the scores showed a decrease in both groups but without any significance.A secondary analysis was conducted with 15 CP children who were diagnosed with ADHD. For attention, in the exercise group the change of d’  showed a significant increase after the intervention. Although the results were insignificant, the domains of physical and emotional functioning showed an increase whereas academic performance decreased. In the control group, physical, emotional, social functioning, and academic performance showed an increase, but was statistically insignificant. The above results are presented in Table In the case of quality of life, in the exercise group the change in social function (Previous studies that applied EAA to children with CP or ADHD evaluated the changes in CP and ADHD separately in attention, social problems, and motor function but did not consider symptomatic improvements collectively or the comorbidity of these disorders. Therefore, the primary purpose of the current study was to evaluate the therapeutic effect of 16-week EAA on attention and psychosocial functioning in children with CP. The secondary purpose was to confirm the comorbidity between CP and ADHD and examine the effectiveness of EAA for those who have both CP and ADHD.The effects of EAA program on attention and quality of life in CP children are as follows. Participants in the EAA exercise group showed an improvement in attention. Specifically, their ability to distinguish target from non-target stimulus was enhanced, suggesting that the ability for sustained attention and suppression of impulse to unrelated stimulus was improved by the intervention. Our results are consistent with previously studies reporting that sustained visual and auditory attention increased post-computerized EAA in children with ADHD . These fA secondary analysis of a subsample of CP children who were diagnosed with ADHD was performed. The overall function of the ADHD group performing exercise showed a greater improvement than the control group with ADHD. More specifically, most of the indices of attention showed an increase. The sub-domain of social functioning in quality of life showed an increase. Consistent with earlier studies, the social skills of disabled children improved significantly after an eight-week EAA program, whereas the control group revealed no significant differences . This reThe broader symptomatic improvement in attentional and psychosocial aspects of the CP and ADHD comorbidity groups may suggest that EAA is highly appropriate for patients with neurodevelopmental disorders. In fact, in the current study 31.91% of CP patients were diagnosed with ADHD. It is essential to scrutinize the ADHD symptoms of CP patients under clinical settings. As the coexistence of the disease is increased, the poor adaptability interferes with functional ability for activities of daily life, underscoring the importance of early intervention in these children.The mechanism of EAA in improving attention and social skills is uncertain, but there are several possible explanations. One of the possible hypotheses is that during the repetitive and rhythmic movements of the horse in EAA, every participant begins to anticipate every movement. This practice leads to modification and reorganization of multiple systems in the brain including the sensory, muscular and limbic systems , which eA positive effect on social maladaptation may occur via interactions with the horses. The behavioral response and social structure of horses is similar to that of humans, which provides an opportunity to simulate relationships in a non-threatening environment , 36. AlsThe strength of this study is that unlike previous studies, an objective measurement tool of CPT was used to evaluate attention levels. In most previous studies, the assessment of psychological symptoms in early childhood relied heavily on caregivers or teachers’ response obtained through self-reports . This apThe limitations are as follows. First, although participants were randomly assigned to each group the baseline CPT scores showed a significant difference. However, after the EAA exercise group showed a radical increase in attention improvement post-treatment compared to the control group, it could be interpreted as therapeutic effect. Second, this study was restricted to a small sample of outpatients in one particular hospital, and therefore the findings may not be generalized to other CP patients. Additional studies with larger samples are needed to replicate the findings and determine whether the results can be generalized across CP outpatients. Lastly, the current study did not examine the long-term therapeutic effect, so maintenance of the effect was not shown. Further studies that include a follow-up session after a certain period a time seems meaningful.In conclusion, to the best of our knowledge, this is the first study examining the clinical effects of EAA using the objective measurement in the group with both CP and ADHD for improving attention and social skills. For the CP population, the exercise group showed an enhanced attention, establishing the effectiveness of EAA. Furthermore, it seems to be more effective when CP and ADHD coexists, as both attention and social skills improved. Lastly, our study confirmed a high comorbidity of CP and ADHD. Based on these results, EAA can be considered as a non-pharmacological option to treat attentional problems for children with CP, and especially those with both CP and ADHD. Further epidemiological investigations in this area are certainly warranted for further understanding.


As we all know that stroke is still a leading cause of death and acquired disability. Etiological treatment and brain protection are equally important. This review aimed to summarize the advance of normobaric-hyperoxia (NBHO) on brain protection in the setting of experimental stroke and brain trauma. We analyzed the data from relevant studies published on PubMed Central (PMC) and EMBASE, about NBHO on brain protection in the setting of experimental ischemic and hemorrhagic strokes and brain trauma, which revealed that NBHO had important value on improving hypoxia and attenuating ischemia damage. The mechanisms of NBHO involved increasing the content of oxygen in brain tissues, restoring the function of mitochondria, enhancing the metabolism of neurons, alleviating blood-brain barrier (BBB) damage, weakening brain cell edema, reducing intracranial pressure, and improving cerebral blood flow, especially in the surrounding of injured area of the brain, to make the neurons in penumbral area alive. Compared to hyperbaric oxygen (HBO), NBHO is more safe and more easily to transform to clinical use, whereby, further studies about the safety and efficacy as well as the proper treatment protocol of NBHO on human may be still needed. The benefits of oxygen therapy in neuroprotection are unproven, with mechanisms of action still unclear , 2. ThisHypoxia is known to result in edema, apoptosis, and the death of nerve cells. Low oxygen environments cause leukocytes to infiltrate into hypoxic tissue, leading to tumor necrosis factor elevation, oxygen free radicals all resulting in an inflammatory reaction , 4. To dIn this review, we have analyzed data from relevant studies published in PubMed Central (PMC) and EMBASE, relating to normobaric-hyperoxia (NBHO) and brain protection in the setting of experimental ischemic and hemorrhagic strokes and brain trauma –8.Herein, we summarize the experimental data, and the mechanisms of NBHO in acute stroke to create a go-to resource for future study.The common oxygen therapies include hyperbaric oxygen (HBO) and NBHO. Hyperbaric oxygen (HBO) refers to administering high concentrations of oxygen in an environment where ambient pressure is greater than atmospheric pressure , 10. HBONBHO (high-flow oxygen or high-concentration oxygen) refers to the method of continuously administering oxygen to patients via mask or oxygen hood under normal atmospheric pressure. The commonly utilized oxygen concentration used is 40–100% , 12.Compared to HBO, the advantages of NBHO are convenience, cost and safety. NBHO can be performed anywhere, even in the acute stroke setting. NBHO can reduce the infarcted volume and prolong the time window of thrombolytic treatment in ischemic stroke . In addiResults from previous NBHO studies have been controversial. The role of NBHO in brain protection is mainly to improve brain metabolism and increase the tolerance of cells to damage , 14. TheOther studies have shown that NBHO does not improve the hypoxic area after cerebral infarction or hemorrhage. Some have concluded that too much oxygen use might increase oxygen free radicals and lung injury , 16.NBHO protocols from numerous studies were reviewed and no clear protocol noted. Differences in starting time, frequency, concentration of oxygen, and the cycles of NBHO treatment were noted. Systemic conditions can also affect the outcomes of NBHO, such as glucose and other nutritional supply deficiencies. Poor ventilation and anemia can cause difficulty with oxygenation and render this therapy inadequate . Figure Local neurons surrounding the hemorrhagic lesion are in a hypoperfused state, related to the hematoma compression-mediated arterial perfusion decrease and venous outflow insufficiency. This results in mitochondrial dysfunction in affected cells, causing further cascading damage, all of which can have detrimental outcomes on function –19.Previous studies have identified that blood pressure and body temperature control as well as that anti-oxygen free radical agent use could attenuate brain edema . HoweverAn animal study assessing NBHO therapy with oxygen delivered at a 90% concentration resulted in remarkable neuro-protection in rats with cerebral hemorrhage (ICH) . In thisResearch by Zhou et al. depicted the relationship between early NBHO therapy after ICH, blood-brain barrier damage and cerebral edema . This stA study by Norio Fujiwara et al. applied 100% of NBHO per-hour to 2 h in rats after ICH. Although the arterial oxygen concentration increased, NBHO did not affect any of the functional outcomes . The autBlood flow interruption and hypoxia are the main causes of cell death after ischemic stroke. Thrombolysis and mechanical thrombectomy are time limited therapies . The preA previous study revealed that early NBHO use attenuated BBB damage and improved the outcome of stroke treated with delayed rtPA. The authors concluded NBHO might be an effective adjunctive agent in extending the time window of rtPA thrombolysis . NHBO usResearch by Ding et al. demonstrated that NBHO started in the acute stage inhibited lactic acidosis in transient and permanent ischemic stroke . NBHO stMeanwhile, a study by Jean-Claude Baron suggested that NBHO alleviated brain damage in rats with transient cerebral ischemia by preventing neuronal death and microglial inflammation, as well as sensorimotor impairment .2 at 3 L/min. NBHO started at the 15th minute after MCAO and lasted for 90 min. We consider that the negative result of the NBHO was that the intervention time was too short and there was no continuous multi-day oxygen supply.Conversely, Elmira Pasban showed that early NBHO use did not reduce ischemic brain injuries and also did not produce protective effects against vasogenic cerebral edema formation or BBB disruption following acute ischemic stroke , 28. TheThe protective role of NBHO in acute cerebral infarction had also been studied in clinical cases. Different studies have concluded with various results. In cerebral infarction animal model, NBHO protected the BBB by improving cerebral perfusion and oxygen metabolism, inhibiting of MMP-9, AQP-4, and NHE1 expression and reducing of TJP degradation. This may be a plausible molecular mechanism of protection , 24, 26.In patients with cranio-cerebral trauma, secondary injury refers to cerebral ischemia  caused by hypoxia, hypotension, intracranial hypertension, and cerebral vasospasm. Among them, cerebral ischemia and hypoxia are the most common pathological changes in secondary injury and are important prognostic factors after brain trauma , 30. In Use of micro-dialysis technology to detect the efficacy of early NBHO therapy in patients with brain trauma has shown increased levels of glucose and decreased lactic acid in cerebrospinal fluid resulting in the decline of the ratios of lactic acid to glucose and lactic acid to pyruvate. This signifies NBHO could improve the redox state of the brain , 33. Mor2, altering the timing, or extending the duration of NBHO exposure along with varying the range of injury severity will be necessary to improve the understanding of the potential benefits or limitations of NBHO therapy after TBI.Conversely, Talley et al. demonstrated that NBHO increased the volume of brain damage in a rat model with a moderate traumatic brain injury (TBI), in which 0.5–3 h of 100% oxygen was administered after TBI , 36. TheNBHO use may have some adverse effects such as oxygen poisoning, lung injury and brain excito-toxicity.2 for 1 h) increased brain edema and worsened neurological function at 24 h following TBI  or NBHO  in acute stroke patients with chronic obstructive pulmonary disease. Ordinary masks and oxygen storage masks are not recommended. COy weight . High-riy weight .Current research is focused on improving oxygen supply and metabolism in brain tissue. There are few reports on the effects of NBHO on excitatory amino acids and intracellular calcium ion concentrations. Further monitoring of potential adverse effects related to NBHO will be necessary in the future.To sum up, standard NBHO appears to be a promising potential neurotherapeutic. The molecular mechanism of NBHO may inhibit the expression of HIF-1α, MMP-9, Caspase-3, and increase the expression of VEGF, ROS, etc., thereby increasing the utilization of oxygen molecules by cells .NBHO's easy use allows it to be performed anywhere with a mask or nasal catheter. No special requirements for the treatment environment are necessary. This therapy is inexpensive and can be administered in remote areas.2 could be controlled at 50–90% (All studies mentioned above revealed that the initial oxygenation time was as early as possible, especially within 2 h after stroke. The ideal protocol for this appears to be controlled at 1-2 h/time, 3–4 times/d for 1 week, FiOt 50–90% . FurtherRM: manuscript drafting and revision, and study concept and design. ZC: manuscript drafting and revision, study concept and design, collection, assembly, and interpretation of the study. YD and XJ: deeply edited the revised version and contributed critical revision.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Attention-Deficit/Hyperactivity Disorder (ADHD) is a common neurodevelopmental disorder with only symptomatic care available. Genome-wide association (GWA) studies can provide a starting point in the search for novel drug targets and possibilities of drug repurposing. Here, we explored the druggable genome in ADHD by utilising GWA studies on ADHD and its co-morbid conditions. First, we explored whether the genes targeted by current ADHD drugs show association with the disorder and/or its co-morbidities. Second, we aimed to identify genes and pathways involved in the biological processes underlying ADHD that can be targeted by pharmacological agents. These ADHD-associated druggable genes and pathways were also examined in co-morbidities of ADHD, as commonalities in their aetiology and management may lead to novel pharmacological insights. Strikingly, none of the genes encoding targets of first-line pharmacotherapeutics for ADHD were significantly associated with the disorder, suggesting that FDA-approved ADHD drugs may act through different mechanisms than those underlying ADHD. In the examined druggable genome, three loci on chromosomes 1, 4 and 12 revealed significant association with ADHD and contained nine druggable genes, five of which encode established drug targets for malignancies, autoimmune and neurodevelopmental disorders. To conclude, we present a framework to assess the druggable genome in a disorder, exemplified by ADHD. We highlight signal transduction and cell adhesion as potential novel avenues for ADHD treatment. Our findings add to knowledge on known ADHD drugs and present the exploration of druggable genome associated with ADHD, which may offer interventions at the aetiological level of the disorder. Attention deficit/hyperactivity disorder (ADHD) is a common and highly heritable childhood-onset neurodevelopmental disorder that often persists into adulthood , 2. The Current management of ADHD is based on either non-pharmacologic or pharmacologic treatments as well as the combination of the two. The non-pharmacologic treatments usually involve psychological and/or behavioural therapies, while the pharmacologic interventions include stimulant and/or non-stimulant drugs , 8. For An important feature of ADHD relevant to the search of new medications is the existence of co-morbid conditions , 13. As Randomised controlled studies have shown that pharmacotherapy reduces the ADHD symptom burden  and obseImprovements in the pharmacotherapeutic options for ADHD may require a fuller understanding of its underlying biological processes . As knowIn this study, we explored the druggable genome in ADHD by utilising the summary statistics from GWA studies on ADHD and its major co-morbid conditions. We aimed to (1) explore whether the genes targeted by current FDA-approved ADHD drugs show association with the disorder and/or its co-morbidities, (2) identify genes and pathways involved in the biological processes underlying ADHD, its co-morbidities and quality of life phenotypes that can be targeted by pharmacological compounds and (3) examine the identified druggable genes and pathways as potential options for novel drug development and repurposing.Figure The druggable genome was defined as described in Finan et al.  as a selThe identification of these druggable genes was based on the protein targets of known and experimental drugs, sequence similarities to those targets , drug–gene interactions, biotherapeutics and a number of databases documenting pharmacological molecules and their therapeutic targets.For the purpose of this study, we focused only on common conditions with well-documented evidence for association with ADHD based on large-scale genetic  and epidAdditional criteria were the availability of large  GWA studies and their summary statistics. Thus, where such data were not available, we used proxy phenotypes .We characterised the co-morbidities into the three main groups: (1) cardiometabolic, (2) immune-inflammatory-autoimmune (referred to as immune) and (3) neuropsychiatric.In addition to well-defined clinical diagnoses, ADHD has been reported to be associated with reduced quality of life, reduced educational attainment and sleep disturbances . TherefoWe relied on summary statistics derived from large-scale GWA studies. Where possible, we restricted our analyses to individuals of European descent only, meta-analysed sample size equal to or larger than 70% of the total sample, variants with minor allele frequency above or equal to 1% and of good imputation quality (INFO ≥ 0.8). For ADHD, summary statistics were acquired from the large-scale meta-analysis of 19,099 cases and 34,194 controls . For thep ≤ 0.001) were further analysed in the GWA data of its co-morbidities and quality of life phenotypes. We applied Bonferroni correction to account for multiple testing.Statistical analyses were divided into two main steps to address our first two aims: (1) examination of genes targeted by current FDA-approved ADHD drugs and (2) examination of the genes within the druggable genome and their pathways defined as known biological pathways containing at least one gene from the druggable genome. In step 1, we examined all genes in all curated GWA data. In step 2, we first examined ADHD and only genes and pathways that revealed suggestive association with it  were assigned to that gene (i.e. the default settings of MAGMA). The 1000 Genomes CEU population was used as the reference panel to correct for linkage disequilibrium (LD). We conducted gene-based analyses of all genes on autosomal chromosomes. Genes represented by a single parameter  in MAGMA were excluded. To evaluate each gene’s contribution to the examined gene-set, the association p-value of each gene was converted to a Z-value and used as an outcome variable for a regression model with gene-set membership as a predictor. Gene size, gene-sets’ gene density and LD were taken into account to adjust for possible confounding effects and prevent spurious association.The genes targeted by the current FDA-approved ADHD medications were defined by Gaspar and Breen . We examThe gene associations with ADHD, its co-morbidities and quality of life phenotypes were tested on two levels: (a) DNA variation and (b) gene expression. The first was tested in MAGMA as described above. The latter was tested in S-PrediXcan . In shorThe biological pathways were defined as determined by Gene Ontology (GO)  and the To address our third aim, we explored the pharmacology of genes (or their encoded proteins) pinpointed in our statistical analyses.http://dgidb.org/), and (4) small molecule compounds with reported molarity measurement for bioactivity.To identify the pharmacological agents, we developed a systematic pipeline utilising publically available databases, where the agents were assessed in four stages: (1) FDA-approved drugs, (2) drugs in clinical trials, (3) compounds reported in the Drug–Gene Interaction Database  and characterised the identified FDA-approved drugs and compounds in clinical trials using Drugbank (https://www.drugbank.ca/), CLUE Repurposing Hub (https://clue.io/repurposing-app), and DGIdb databases. The FDA approval and clinical trial status of the compounds were crosschecked using the publically available FDA labels and ClinicalTrials.gov  database. The paediatric approval status was investigated on Medscape (https://reference.medscape.com/) and in FDA labels. For FDA-approved compounds, the approved indication reported in FDA label was noted. For compounds in clinical trials, the indication was researched in ClinicalTrials.gov, applying the following filters: “not yet recruiting”, “recruiting”, “enroling by invitation”, “active, not recruiting” and “completed”, in order to select trials that are currently active. In addition, the mechanism of action on the specific gene of interest was noted from Drugbank.First, we evaluated each gene of interest in Uniprot (https://www.ebi.ac.uk/chembl/), downloading all compounds reported to interact with a gene of interest along with their reported “Target Associated Bioactivity” as their affinity or potency to the human gene product stated in molarity (referred to as “Bioactivity”). The compounds from ChEMBL were then prioritised from the lowest “Bioactivity” value to the highest and, in order to investigate the most relevant compounds, the top 50 were characterised further by being individually investigated in PubChem (https://pubchem.ncbi.nlm.nih.gov/) to confirm their target-associated bioactivity.Next, the genes were researched as targets in ChEMBL , bringing the Bonferroni-corrected significance threshold to p = 1.29E−04.In the co-morbidities and quality of life phenotypes, we examined 385 genes and three gene sets (two GO pathways and one set of FDA-approved ADHD genes) in the druggable genome. The Bonferroni-adjusted significance thresholds for these analyses was set to Associations stronger than the determined Bonferroni thresholds were considered significant.DRD2 and the frequency of alcohol consumption (p = 2.88E−08), followed by associations between the same gene (DRD2) and SCZ (p = 1.55E−07), CYP2D6 and SCZ (p = 1.81E−06), CHRM2 and major depressive disorder (p = 2.56E−06). In addition, SLC6A3 revealed significant association with sleep duration (p = 2.41E−05). All of these genes encode protein targets of atomoxetine. Furthermore, SLC6A3 is also targeted by MPH and AMP, while DRD2 is a secondary target of MPH and AMP.We identified 23 genes targeted by the FDA-approved ADHD drugs . Individually, none of these genes showed significant association with ADHD Table . The strExamining all the genes as a set revealed no significant association with neither ADHD nor its co-morbidities Table S.ST3GAL3 gene, p = 3.10E−12, Table For ADHD, four loci on chromosomes one, three, four and twelve showed significant association Table . The locITPR3 and rheumatoid arthritis . Among the three loci associated with ADHD, those on chromosomes one and four revealed druggable genes also significantly associated with SCZ, ulcerative colitis, autism spectrum disorder and the frequency of alcohol consumption  and LEPRE1 —showed significant association and 18 additional genes showed signs of suggestive association (p < 0.001)  Table S.HLA-DPB1 and rheumatoid arthritis  were also significantly associated with body mass index, rheumatoid arthritis and SCZ . Two GO pathways revealed nominal associations with ADHD  and significant association with any of the examined co-morbidities. Among the 17 druggable genes within those 14 loci, 13 interact with drugs that are in clinical trials or are FDA-approved, with the majority of indications being autoimmune disorders and/or malignancies , three are targeted by compounds in clinical trials and two of them are also FDA-approved. All of these compounds are nutraceuticals, with malignancies and immune dysfunctions among their indications Tables S and S10.Despite ADHD being a highly heritable disorder, it has been challenging to utilise genetic information in its treatment. Nonetheless, the more insight we gain into the molecular genetics of ADHD, the more options for its treatment may become available . In thisTo answer the first question, we examined the association between the genes encoding the immediate targets of the first-line ADHD pharmacotherapeutics and ADHD as well as its co-morbidities. We observed no significant association between these genes and ADHD, suggesting that these drugs may act through mechanisms different to those underlying ADHD. However, as the current GWA study on ADHD reveals only a small fraction of the biological processes underlying this condition , larger Overall, drugs that are FDA-approved or currently undergoing clinical trials to treat ADHD  target only a limited number of known pharmacological targets, essentially enhancing catecholamine signalling. This illustrates that all active ADHD drugs belong to a small pharmacological niche and we should aim to move beyond it. Hence, we examined whether any known druggable genes and pathways are associated with ADHD and/or its co-morbidities and quality of life phenotypes (second question), following by pharmacological characterisation of identified associations (third question). These analyses aimed to pinpoint novel avenues for ADHD drug development as well as repurposing. Because the de novo discovery and development of entirely new drugs targeting unique biology of a disorder is a tedious and expensive process with a low success rate, the possibility of repurposing already existing drugs towards new indications may be more effective . Here, wWithin the loci associated with ADHD, 5 druggable genes encode proteins interacting with drugs that are FDA-approved or are in clinical trials. The common indications of those pharmaceuticals are autoimmune disorders and malignancies, with some also being tested in clinical trials for treatment of neurodevelopmental disorders. Interestingly, autoimmune disorders and malignancies are also common indications for drugs interacting with genes associated with co-morbidities of ADHD, suggesting that these two fields of research could present novel paths for ADHD treatment.PTPRF is the gene with the most prominent association signal. This gene encodes a tyrosine phosphatase, a signalling molecule involved in a myriad of cellular processes, including cell adhesion, neuronal development and functioning [PTPRF has mainly been studied in the context of cancer. However, its involvement in hyperactivity [SLC6A9, a gene encoding a glycine transporter that is targeted by such compounds as bitopertin, sarcosine and glycine [The locus on chromosome one shows the strongest association with ADHD and also contains the most genes interacting with drugs that are FDA-approved or in clinical trials. Among them, ctioning , 42. PTPactivity  has also glycine . In ADHD glycine . Similar glycine .KCNH3 gene is also interacting with drugs that are FDA-approved or are in clinical trials. This gene encodes a voltage-dependent potassium channel, a selective inhibitor of which was recently described [KCNH3 in mice has been reported to enhance cognitive skills, including attention, further supporting a potential role of dalfampridine-like drugs in the treatment of ADHD [Outside the chromosome one locus, the escribed . It is aescribed  used to escribed , 49. Kno of ADHD .The aforementioned druggable genes also showed significant association with educational attainment, suggesting that drugs targeting them may have a possible impact on quality of life of ADHD patients.MANBA and LEPRE1, among which only LEPRE1 interacts with a number of compounds in clinical trials, such as nutraceutical ascorbate, succinic acid and L-proline. This gene encodes an enzyme needed for collagen synthesis and assembly, which has recently been proposed as a novel therapeutic vista for protection and regeneration of neurons [The analyses of correlation between ADHD and gene expression levels in brain pinpointed druggable genes  neurons . MoreoveIn pathway analyses, the GO pathway of negative regulation of protein binding (GO:0032091) showed the strongest, albeit non-significant, association with ADHD. This pathway encompasses any process that negatively affects any protein binding, such as actin binding (e.g. synaptic plasticity), microtubule binding, receptor binding and homodimerization activity of a protein. The latter processes affect a G-protein-coupled receptor signalling, tapping into the largest class of targets in current drug development , 53 and KCNJ13, encoding a druggable potassium channel targeted by dalfampridine, the same compound that also targets the KCNH3-protein discussed above.Among the examined co-morbidities, the neuropsychiatric (mostly SCZ) and immune groups revealed significant associations, with 12 genes interacting with compounds that are FDA-approved or in clinical trials. Interestingly, one of these genes is SEMA3F. This gene also showed significant association with educational attainment. SEMA3F encodes semaphoring-3F protein involved in cell signalling, affecting cell adhesion and migration and being explored mostly in cancer therapies [The gene that revealed significant associations with the largest number of co-morbidities is herapies , 55. Nonherapies .Our study has some limitations. As we examined associations observed in GWA studies, where it is difficult to obtain adequate sample sizes to detect associations of small effects, our findings are limited by their statistical power. Moreover, as we imposed a sample size limit of 20,000 individuals, some of the co-morbid conditions, where GWA studies of such size were not available, were replaced by proxy phenotypes.The current statistical methods allow us to identify chromosomal loci only. Further studies on the genes of interest as well as fine mapping are needed to unambiguously establish which gene(s) lies on the causal pathway to developing ADHD. This knowledge would allow for a higher resolution search for therapeutic targets, especially on chromosome one locus where the LD structure is particularly complicated.KCNH3 in brain tissues in reference transcriptome), preventing a comprehensive investigation of the transcriptome.The gene expression analyses have several limitations , includiAs we used publicly available databases, it was not possible to control their quality. Furthermore, the information provided in the utilised drug target databases may be incomplete.PTPRF, SEMA3F, KCNH3, KCNJ13) as potential novel avenues for ADHD treatment. Our findings add to the knowledge on known ADHD drugs and present an exploration of druggable genome associated with ADHD, which may offer intervention at the aetiological level of the disorder.To conclude, we present a framework for assessment of the druggable genome in a disorder, exemplified by ADHD. We present possibilities for drug repurposing  and highlight processes of signal transduction and cell adhesion (negative regulation of protein binding, Supplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6Supplementary Table 7Supplementary Table 8Supplementary Table 9Supplementary Table 10Supplemental Legends


These abnormalities are thought to depend on abnormal proprioceptive input coming from dystonic muscles. Since Botulinum toxin-A (BT-A) reduces proprioceptive input in the injected muscles, our study investigated the effects of BT-A on STDT tested at rest and during voluntary movement execution in patients with focal dystonia. (2) Methods: We enrolled 35 patients with focal dystonia: 14 patients with cervical dystonia (CD), 11 patients with blepharospasm (BSP), and 10 patients with focal hand dystonia (FHD); and 12 age-matched HS. STDT tested by delivering paired stimuli was measured in all subjects at rest and during index finger abductions. (3) Results: Patients with dystonia had higher STDT values at rest and during movement execution than HS. While BT-A did not modify STDT at rest, it reduced the abnormal values of STDT during movement in CD and FHD patients, but not in BSP patients. (4) Conclusions: BT-A improved abnormal sensorimotor integration in CD and FHD, most likely by decreasing the overflow of proprioceptive signaling from muscle dystonic activity to the thalamus. Temporal discrimination can be tested in humans with the somatosensory temporal discrimination threshold (STDT) technique, which assesses the interval needed to recognize a pair of stimuli as separate in time . ExperimBotulinum toxin A (BT-A), the gold standard therapy for focal dystonia ,10, imprThe aim of the present study was to investigate whether BT-A influences STDT modulation induced by movement execution in patients with dystonia. In order to do so, we tested STDT during voluntary movement execution in HS and in patients with focal dystonia before and 1 month after treatment with BT-A. Investigating how BT-A modifies STDT modulation in patients with focal dystonia could help clarify the role of proprioception and sensorimotor integration in the pathophysiology of different types of focal dystonia.p = 0.01). STDT values were similar in the three dystonia patient groups (p > 0.05).Before BT-A, STDT values tested at rest were higher in dystonia patients than in HS  and ‘ISI’, where ISI is considered the interstimulus interval between movement onset and the deliverance of electrical stimuli , and a significant ‘GROUP × ISI’ interaction . Between-group ANOVA comparing STDT changes during movement execution in patients after BT-A therapy and HS disclosed significant factors ‘GROUP’  and ‘ISI’  and a significant ‘GROUP × ISI’ interaction .Between-group ANOVA comparing STDT changes during movement execution in patients before BT-A and HS disclosed significant factors ‘GROUP’  and ‘ISI’  and a significant ‘BT-A x ISI’ interaction . Similarly, in FHD patients, analysis disclosed a significant factor ‘ISI’ , a significant ‘BT-A x ISI’ interaction , and a trend towards a significant factor ‘BT-A’ .Repeated measures ANOVA evaluating changes in STDT values during movement execution before and 1 month after BT-A injection in CD patients showed significant factors ‘BT-A’ , but no significant factor ‘BT-A’  or ‘BT-A x ISI’ interaction . Therefore, BT-A injection changed STDT modulation during index finger movement in CD and FHD patients, but not in BSP patients .Repeated-measures ANOVA evaluating the kinematic features of index finger movements showed no significant factors ‘BT-A’ or ‘ISI’, nor any significant interactions in the three patient groups .The duration of BT-A treatment did not differ in the three groups of patients .BT-A treatment improved disease severity scale scores of dystonic muscle spasms in the three patient groups (CD patients: p > 0.05).Pearson’s correlation coefficient did not disclose any significant relationship between clinical severity score changes and the extent of BT-A-induced STDT modulation changes  because we pseudorandomized the order of sessions before and after BT-A injections. We also randomized the different trials  in each participant. If the improvement in STDT modulation after BT-A was due to a learning effect, we should have observed the same extent of improvement in the three groups. Conversely, STDT modulation before and after BT-A did not significantly change in BSP patients. Finally, previous studies in healthy subjects investigating STDT gating during movement ,16 showeThe novel finding of the present study is that BT-A improves sensorimotor integration as tested by STDT modulation in CD and FHD patients. In HS we have previously demonstrated that STDT increases when tested during voluntary movement execution and that the increase is maximal at movement onset and returns to normal values at the end of the movement ,17. SimiBT-A-induced improvement in STDT modulation in CD and FHD patients did not correlate with motor symptom improvement as clinically tested using standardized clinical scales. It is known that clinical motor improvement induced by BT-A in dystonia is mostly dependent on its effect on extrafusal fibers , while SIn the present study, we observed that the kinematic parameters of index finger abduction were normal in patients affected by CD, BSP, and FHD. Previous studies reported conflicting results regarding movement performance in dystonia patients ,26,27,28In conclusion, we have provided evidence that BT-A improves abnormal sensorimotor integration in CD and FHD patients, as tested by STDT movement-induced modulation. We believe that the BT-A-induced improvement depends on BT-A’s indirect central effects, which modulate proprioceptive overflow from dystonic muscles to the thalamus. Concordantly, BT-A treatment did not influence STDT modulation during movement in BSP patients, who conceivably have negligible proprioceptive overflow related to the dystonic ocular muscle as compared with CD and FHD patients . Our stuThirty-five patients with different types of focal dystonia were enrolled, including 14 patients with CD, aged 54 ± 9 years ; 11 pati®, Merz] and after a BT-A wash-out period at least 3-4 months after the last BT-A injection (pre-BT-A). The order of the two sessions (pre-BT-A and post-BT-A) was pseudorandomized across individuals, except for three FHD patients who performed the pre-BT-A evaluation first. The timeframe of 3-4 months after the last BT-A treatment was established in each patient according to his/her own clinical history, previous response to BT-A, and the frequency of injections. To ensure the reliability of STDT testing, patients with a Mini-Mental State Examination (MMSE) score lower than 26 and those with clinical signs of peripheral neuropathy were excluded. The study was approved by the institutional review board Sapienza University of Rome Ethics Committee  and the experimental procedure was conducted in accordance with the Declaration of Helsinki. All participants gave their written informed consent.All patients underwent the experimental procedure and the clinical examination twice: 1 month after BT-A injection (post-BT-A) (incobotulinum toxin A) [XeominTo assess STDT values we applied the experimental protocol used in previous studies ,31,32,33Electromyographic (EMG) activity was recorded through a pair of surface (Ag/AgCl) cup electrodes placed over the first dorsal interosseous (FDI) muscle in a belly-tendon configuration. EMG signals were recorded and filtered with Digitimer D360  (bandwidth 20 Hz-1 kHz), then analyzed offline with a personal computer through a 1401-plus A/D laboratory interface . Data were stored on a laboratory computer for online visual display and further offline analysis . EMG activity from the FDI muscle during voluntary movement was measured by assessing the root mean square (RMS) amplitude. Electromyographic recordings were used to trigger the stimuli.The SMART analyzer motion system , equipped with three infrared cameras (sampling rate 120 Hz), was used to record index finger abductions. The arm was abducted at the shoulder by about 45–50˚, and the elbow joint was flexed at about 90˚. We used four optical markers to record finger abduction: one marker was placed over the distal phalanx of the index finger, another one was placed on the first metacarpophalangeal joint, and two were distributed over the first and fifth metacarpal, respectively. After a verbal “go” signal, subjects abducted the index finger, then returned the finger to the starting position. The movement was performed as rapidly and widely as possible. Marker displacements were reconstructed via a dedicated software program that ran an automatic algorithm to compute the range of motion (ROM), which represented the displacement of the index finger around its metacarpophalangeal joint expressed as the degree of the angle and mean velocity (degrees per second) .First, we measured STDT at rest  and then during index finger abduction on the volar surface of the right index finger in each subject. In patients with FHD we tested STDT on the affected hand. Patients were asked to abduct the index finger as widely and rapidly as possible. We first recorded 10 index finger abductions without delivering any paired stimuli before starting the experimental procedure. Experimental tasks consisted of index finger abductions, with paired stimuli for STDT triggered at 0, 100, or 200 ms after movement onset. Movement threshold was set at 100 μV of EMG activity (defined as “0 ms” for simplicity). To define the time course of movement-induced STDT changes, paired stimuli were delivered as soon as EMG signals reached an amplitude of 100 μV (defined as “0 ms” for simplicity), 100 and 200 ms after movement onset. STDT values were thus calculated for each time lapse . At each index finger abduction, the interstimulus interval for STDT testing was progressively increased in 10-ms intervals until the subject recognized the two stimuli as sequential (10). The order of sessions for each time lapse was pseudorandomized. All participants were blinded to the time-lapse condition.Separate repeated measures analyses of variance (rmANOVAs) were performed to evaluate STDT changes in the three patient groups before and after BT-A. Separate between-group rmANOVAs with the factor ‘ISI’ were performed to evaluate any differences in STDT modulation between patient groups before and after BT-A and HS.p value < 0.05 indicated statistical significance.We also investigated possible correlations between changes in clinical scale values (post-BT-A/pre-BT-A ratio) and percentage changes in STDT modulation after BT-A (Pearson’s correlation coefficient). A


Toxoplasma gondii (T. gondii) has a high worldwide prevalence and an underestimated impact on neuropsychiatric disorders. Previous studies related T. gondii to disorders associated with the dysfunctional dopaminergic system. However, an association between T. gondii infection and adult attention-deficit/hyperactivity disorder (ADHD) has not yet been studied. In a sex- and age-matched case–control study, we investigated the seropositivity, serointensity, and avidity of latent T. gondii infection in adult ADHD patients and examined the influence of those variables on the symptomatology of ADHD. Of 140 participants, 20.0% were seropositive for anti-T. gondii IgG and 0% for anti-T. gondii IgM. T. gondii seropositivity was associated with 2.8-fold increase in the odds of ADHD in a confounder-adjusted multivariable analysis. Age and consumption of raw/undercooked meat were confirmed as significant predictors of T. gondii seropositivity. Multiple linear regression analysis of self-rated ADHD-related symptom severity in all participants revealed a significant association with T. gondii seropositivity, elevated IgG titers (serointensity), and stronger anti-T. gondii IgG avidity. Overall symptom severity was increased in seropositive ADHD patients compared to seronegative subjects with ADHD. In particular, hyperactivity was significantly associated with serointensity. We conclude that there is a high rate of T. gondii seropositivity in adults with ADHD. Additionally, our results suggest a clinical impact of latent T. gondii infection on ADHD-related symptoms in a serointensity- and avidity-dependent manner. This condition is characterized by inattention, impulsivity, and/or hyperactivity2. The pathogenesis is considered multifactorial: several genetic and environmental risk factors have been shown to contribute to an increased susceptibility to ADHD, and a wide range of functional and structural brain anomalies, e.g., in the dopaminergic system have been found to be associated with the disorder3.Adult attention-deficit/hyperactivity disorder (ADHD) is a frequently diagnosed neurodevelopmental disorder with an average worldwide prevalence of 2.8%6 and adults9 suggest that infections are associated with an increased risk of mental disorders by a direct influence on the nervous system, immune activation, or inflammatory mediators10. However, studies focusing on the general relationship of infectious agents and ADHD are scarce13.Recent population-based registry studies in childrenToxoplasma gondii (T. gondii) is an obligate intracellular parasite with a high (30–50%) worldwide prevalence and growing importance in neuropsychiatric research15. The parasite shows high affinity for brain tissue, wide distribution in the brain and potential life-long persistence causing latent infections17. Studies in rodents and humans have already linked T. gondii to various neuropsychiatric diseases, especially those associated with a dysfunctional dopaminergic system, e.g., bipolar disorder type I, schizophrenia, and Parkinson’s disease19. However, the association between T. gondii infection and ADHD in adulthood has not yet been investigated.T. gondii is the domestic cat, which harbors the sexual parasitic cycle in the feline intestine and spreads infectious oocysts through feces20. The asexual reproduction of the parasite takes place in a broad spectrum of intermediate hosts21. While in the past, our ancestors might have been prey of larger cats21, humans currently represent dead-end hosts of T. gondii20. The main risk factors for infection in humans are consuming the parasite’s cysts through cyst-carrying undercooked meat, oocyst-contaminated soil, or contact with fecal material of infected cats22. In immunocompetent humans, infections are mostly regarded as clinically asymptomatic23. However, the results of animal and behavioral studies showed evidence that latent toxoplasmosis may lead to behavioral changes, the so-called manipulation hypothesis27. Several studies describe reduced fear displayed by rodents toward cats, which reportedly increases the parasite’s chance of being transmitted to its definite host17. Studies comparing infected and uninfected humans in terms of personality traits, behavior, and psychomotor performance as well as intelligence suggest the applicability of the manipulation hypothesis to humans29. In particular, the ability of T. gondii to influence and dysregulate dopamine metabolism as well as disease susceptibility genes of its host has come into focus in current research on different psychiatric, neurological, and somatic disorders32. The dopaminergic neurotransmission pathway is also strongly related to ADHD34. Previous studies discussed that behavioral changes of T. gondii-infected rodents might resemble clinical symptoms of patients with ADHD30, such as impaired learning capacity and memory36, rapid loss of concentration37, and elevated activity levels38. Furthermore, latent toxoplasmosis has been associated with lengthened reaction times38, deficits in motor performance39, reduced anxiety40, and increased novelty-seeking behavior41. In concordance, impaired concentration capacity, motor skills and motor control as well as lengthened reaction time42, increased novelty-seeking behavior41, and an increased risk of injury43 have also been described in patients with ADHD.Today, the most relevant definite host of T. gondii infection and adult ADHD compared to an age- and sex-matched healthy control sample. We further investigated the possible influences of the parasite on the symptomatology of ADHD in detail.In a case–control study, we investigated the association between latent 44. Furthermore, when linking T. gondii with psychiatric diseases, prior research revealed that the robustness of the antibody response  rather than seropositivity is an important factor that plays a crucial role in behavioral changes46. However, available data on T. gondii seropositivity in ADHD involve only children and do not provide information on serointensity48. In contrast, we included both the anti-T. gondii IgG avidity and the measurement of serointensity in this study.The antigen–antibody binding avidity of immunoglobulin G (IgG) increases over time and provides further information on the age of infectionT. gondii seropositivity in adult ADHD patients compared to healthy controls. The secondary hypotheses are as follows: we hypothesize that the presence of a latent T. gondii infection (seropositivity) and the concentrations of anti-T. gondii IgG (serointensity) contribute to the severity of ADHD symptomatology, measured by valid diagnostic instruments.Our main hypothesis is a higher T. gondii, one participant in the ADHD group (0.7%) had equivocal anti-T. gondii IgG titers (serointensity) of 3–6 (U/ml) and was thus excluded from analysis, resulting in a final study sample size of 140 participants.A total of 193 individuals were contacted for study participation; 41 were ineligible or not interested. Overall, 152 of 193 contacted participants were assessed for eligibility (78.8%). Of these 152, four patients did not want to stop medication for study participation, two participants refused to undergo blood sampling, three patients did not show up for blood sampling for unknown reasons, and two participants handed back incomplete questionnaires. As a consequence, 141 participants [71 subjects with ADHD (50.4%) and 70 healthy controls (49.6%)] were taken into account for analysis. Among 141 individuals screened for IgG and IgM antibodies against C-reactive protein (CRP) concentrations below 1 mg/dl were present in 69 (98.6%) individuals in the control group and in 67 patients (95.7%) in the ADHD group. Three participants in the ADHD group (4.3%) had CRP concentrations between 2 and 3 mg/dl. None of the included participants showed any changes in the differential blood count according to acute inflammation or infection. In total, 140 individuals without signs of acute infection were included in the study.The groups were matched for sex and age; thus, the groups were balanced. The sociodemographic characteristics, including medical and family history, of the study population are shown in Table p < 0.001), tended to have more direct soil contact (n = 33 (47.1%) vs. n = 23 (33.3%); φ = 0.15, p = 0.085), lived significantly more often in towns  and less often in cities , and had significantly lower educational status than controls . In concordance, ADHD patients were significantly less often students  and more frequently job seekers . In turn, the highest education status in controls was significantly more often university-entrance diploma, grade 12/13 , or university degree , see Table p = 0.366). The number of participants who were currently married or had ever been married was n = 25 (18.1%) in the ADHD group versus n = 18 (13%) in controls . Rates of raw/undercooked meat consumption habits were well balanced between groups (ADHD n = 36 (51.4%) vs. controls n = 35 (50.7%); φ = 0.01, p = 0.866). The distribution of body mass index (BMI) revealed that ADHD patients were significantly more often overweight or obese with a BMI above 25 . In contrast, controls had significantly more often spent several months abroad (n = 29 (42.6%) vs. ADHD n = 10 (14.9%); φ = 0.25, p = 0.002).With regard to known risk factors for seropositivity, ADHD patients more often revealed current contact with cats (n = 43 61.4%) versus controls n = 20 (28.6%); φ = 0.33, .4% versuT. gondii IgG, 0% for IgM. In the ADHD group, 27.1% (n = 19) were seropositive for anti-T. gondii IgG versus 12.9% (n = 9) in the control group. ADHD showed a significant association with anti-T. gondii seropositivity in the unavailable analysis (odds ratio (OR) 2.53; 95% confidence interval (CI) 1.05–6.06; p = 0.038). This was confirmed by confounder-adjusted multivariable analysis .Out of 140 individuals, 20% (n = 28) were seropositive for anti-T. gondii seropositivity, a multiple logistic regression was performed (see Table T. gondii seropositivity with regard to the entire study sample. Other less-fitting covariates were eliminated following the Akaike information criterion (AIC) model selection. The variable living abroad was excluded from analysis, as only four seropositive participants lived abroad for a longer period. Among them, only one participant had ADHD. These case counts were too low to allow further analysis.In order to examine an association between potential risk factors for p = 0.085), age , never been married , and raw/undercooked meat consumption  were predictive for seropositivity of T. gondii.Similarly, a separate regression analysis of risk factors for seropositivity in the ADHD group yielded a model in which male sex  .When linking Ordinary linear regression models revealed a positive association between serointensity and the hyperactivity subscales in subjects with and without ADHD Fig. .Figure 22) were found for hyperactivity. As a result, seropositivity demonstrated a positive association with hyperactivity in males as measured by the Conners' Adult ADHD Rating Scale (CAARS) hyperactivity subscale, while females revealed a weak negative association with a low R2 value .To clarify to what extent ADHD symptoms were influenced by serointensity, multiple linear regression models were estimated Tables  and 5. Is = − 0.543; p 0.003; Fig. T. gondii avidity on ADHD-related symptoms (Table p = 0.014), Supplementary Table The mean avidity index was m = 0.43 , with one individual having a small avidity index strength of < 0.20, three individuals having a medium avidity index strength and the majority (n = 24) showing a high avidity index strength of > 0.30. Avidity and serointensity showed a significant negative correlation , carried out in Egypt, reported a significant association of T. gondii seropositivity with neurodevelopmental disorders among fourteen subjects with ADHD13. This cited study is limited to the small sample size of ADHD patients, the lack of a control group, and the lack of inclusion of risk factors for seropositivity. Finally, the preceding studies did not investigate the impact of T. gondii serointensity49 or IgG avidity on the clinical course of ADHD.This case–control study is the first to show that seropositive individuals had more than 2.5 times the adult ADHD risk of seronegative individuals. Two previous studies from Iran conducted with children and adolescents found no significant differences in T. gondii in humans varies between 1 and 100% throughout different countries, depending on eating habits, hygiene standards, health behavior, age, culture, geographic and climatic factors53. For Germany, an age-related prevalence of IgG antibodies has recently been found, indicating a seroprevalence of 20% (95%-CI 17–23%) in the group of 18–29 years of age and 77% (95%-CI 73–81%) in the group of 70–79 years of age22. In concordance with the previous findings, our study, which was also conducted in Germany, revealed a seropositivity of 20% in a study population of 31.8 years on average (SD: 10.4).The seroprevalence of T. gondii in the ADHD group were investigated by integrating identified risk factors for seropositivity into our analyses. The main risk factors in humans are consuming the parasite’s cysts through cyst-carrying undercooked meat, oocyst-contaminated soil, or contact with fecal material of infected cats22. Moreover, a previous population-based investigation on T. gondii seroprevalence demonstrated that an older age, male sex, BMI > 25, low socioeconomic status, living in rural or moderately urbanized areas, and marital status were associated with T. gondii seropositivity54. In Germany, eating habits, particularly consuming raw meat, were found to be of high epidemiological relevance22. The higher seroprevalence observed in men has been discussed as a result of eating habits, as male Germans are known to eat approximately twice as much meat as females, which in turn contributes to an elevated risk of ingesting contaminated meat22. Our results are in line with these findings, as the presented data confirm male sex, age, marital status, and consumption of raw/undercooked meat as predictors for T. gondii seropositivity. Cat contact, BMI, municipality size, and low educational or professional status were not associated with seropositivity in our analysis. The risk for infection of cat owners has been controversial22. A previous study suggested an elevated risk for individuals living with three or more cats55. This specific question was not addressed in the questionnaire used in our study. Last, we did not evaluate lifetime cat contact or whether the individuals had contact with cat litter, which has been shown to be associated with an elevated risk of infection22.Associations between epidemiological aspects and the higher seropositivity of T. gondii seropositivity on the clinical course of adult ADHD and reveal specific influences of T. gondii serointensity and IgG avidity on the severity of ADHD symptoms for the first time. The influences were found in subjects with ADHD and controls, which hints at the fact that latent T. gondii infection might be able to trigger ADHD-related symptoms in individuals without ADHD. Above all, the present study illustrates an aggravation of symptoms in ADHD patients, especially in males, which was dependent on the serum levels of T. gondii antibodies and the strength of the antibody-binding avidity.To our knowledge, our results suggest an impact of 27. Indeed, our finding of increased hyperactivity in T. gondii-seropositive participants correspond with behavioral changes found in T. gondii-infected rodents, which also showed higher activity levels than uninfected controls58. The underlying mechanism by which T. gondii manipulates brain function is not yet fully understood. Experimental studies in mice revealed neurological and behavioral abnormalities following inflammation and loss of brain parenchyma due to T. gondii infection59. Moreover, several behavioral changes in rodents have been linked to changes in the dopaminergic pathway of various origins: local immune responses due to infection with the parasite  cause modifications of the turnover, efficiency, and levels of neuromodulators such as dopamine, serotonin, and glutamate62. Finally, T. gondii showed its capability to directly influence neurotransmitter levels by enhancing the release of dopamine from neurons in vitro and producing dopamine in vivo with its own genes encoding tyrosine hydroxylase, which represents a rate-limiting enzyme of dopamine biosynthesis60. In infected mice, T. gondii cysts were found throughout the brain with higher percentages in the amygdala, nucleus accumbens32, and hypothalamus63. The amygdala and the nucleus accumbens, as dopamine-containing brain regions, are well known to have important functions in the control of movements  and fear , respectively32. Altered dopamine levels induced by T. gondii are discussed to have functional consequences leading to behavioral changes and neurological dysfunction26. Previous studies found that the manipulation patterns vary among the three existing T. gondii strains due to different prevalence, virulence, and neuropathogenic potential64. Only infections with the type I strain showed abnormalities in the three neurotransmitter systems  and two neuropeptides (PROK2 and TAC1) compared to controls31. This may be one explanation for the heterogeneity of T. gondii-associated neurological changes31 and should therefore be taken into account in future studies on ADHD and T. gondii.Previous animal models and behavioral studies in humans have indicated that latent toxoplasmosis may lead to behavioral changesT. gondii has been found to interact with approximately 3000 host genes or proteins during its life cycle, including 18% of ADHD susceptibility genes in humans with a specific emphasis on the calcium signaling and neurotransmitter pathways 30. Moreover, overlapping susceptibility genes of ADHD and T. gondii in various metabolic pathways, e.g., phenylalanine, tyrosine, tyrosine, histidine, and unsaturated fatty acid synthesis, were found30. Furthermore, T. gondii was linked to increased expression of miR-132 in mice, which led to reduced expression of D1-like dopamine receptors , metabolizing enzyme monoamine oxidase A (MAOA), and several intracellular proteins linked to the transduction of dopamine-mediated signaling31. In ADHD, changed levels of peripheral miRNA can be found in both ADHD animal models and humans34. Furthermore, miRNAs, as posttranscriptional regulators, have been shown to modulate the gene expression of several genes linked to ADHD etiology, e.g., dopamine transporter (DAT1)67. As a consequence, changes in the dopaminergic pathway, which have been shown by both T. gondii and attention-deficit/hyperactivity disorder, might display the link to the clinical alterations in T. gondii-seropositive adults with ADHD seen in our study.Additionally, T. gondii on human behavior was found with older age and longer duration of infection, assessed via IgG avidity68. While in our study seropositivity was associated with stronger ADHD-related symptoms in the whole sample, avidity had a significant influence on the ADHD Index only in ADHD patients. This result suggests a stronger impact of a latent infection on the severity of ADHD symptomatology in ADHD-affected individuals which increases with the duration of the infection. Thus, it can be assumed that a longer infection period enables the parasite to be more likely to influence the host’s neuromodulators, immunity, or hormonal system74. Two previous studies discussed the impact of the timing of infection with T. gondii on seropositivity among children with ADHD48. Although both studies were conducted in the same country, the seropositivity found in children and adolescents was not in line with available data on the prevalence of T. gondii in their country, suggesting greater parasite exposure in later life48. It has been remarked that the maturation of the IgG response varies noticeably between individuals75. Although it remained unclear whether a linearity of the avidity-maturation curve over time can be assumed, the antibody-binding avidity increases over time and therefore provides additional information about the age of an infection75. High avidity index strength was measured in 86% of participants in our study , while associations with dysthymia as well as mild and moderate depression have been found77. However, our data did not reveal an influence of T. gondii seropositivity or serointensity on depressive symptoms.Our study was conducted at a time when the current meta-analysis found no significant association between 78. Our study suggests that T. gondii should be regarded as one of these environmental risk factors for ADHD in adulthood that may contribute to symptom severity. Our data hints at the fact that the pathomechanism of a latent T. gondii infection, in addition to an affected dopaminergic system in ADHD patients, may lead to an aggravation of ADHD symptoms, especially contributing to increased hyperactivity. Despite the high T. gondii seropositivity found, the presented results should be judged with care, as our data are based on a case–control study and are not population-based. Discussions on causative or influencing effects should consider additional factors, which are generally difficult to assess, e.g., individual immunity and resistance of the host, timing and severity of infection, epigenetics and confounding medical and environmental factors30. Additional studies are required to clarify the underlying neurobiological mechanisms of our clinical findings. Moreover, the impact of T. gondii on the course of ADHD symptomatology in the long term should be taken into account in future research with regard to prevention, diagnostic proceedings, and therapeutic implications. Further longitudinal studies are needed to investigate possible differences in the pharmacological response or treatment outcome of T. gondii seropositive versus seronegative ADHD patients. Different psychopharmacological medications like the antipsychotic dopamine receptor agonist haloperidol, the mood stabilizer valproic acid, or the selective dopamine reuptake inhibitor vanoxerine were found to inhibit replication in vitro or inhibit the behavioral effects of T. gondii in rodents80. With regard to these experimental findings, potential interactions of methylphenidate and T. gondii require exploration. The norepinephrine-dopamine reuptake inhibitor methylphenidate is the recommended first-line treatment for ADHD81. However, the response to this medication as well as long-term efficacy can vary among individuals83. Studies are warranted to clarify whether the effect of methylphenidate might be reduced or even augmented through a co-occurring latent T. gondii infection. On the one hand, methylphenidate should in turn be investigated for its ability to influence parasite replication, and on the other hand, it is worthwhile to explore whether T. gondii seropositive patients with ADHD profit from dopamine-enhancing medication.Current discussions point out that environmental risk factors may alter the subsequent course of ADHDT. gondii infection and the symptom severity of ADHD patients. Our findings suggest that T. gondii might display a disregarded environmental influencing factor of ADHD in adulthood.Our results provide support for the hypothesis of an association between latent T. gondii or who are particularly familiar with the subject . Recruitment was performed between May 2016 and November 2017. Laboratory analyses were performed at the Institute for Laboratory Diagnostics and Microbiology at the Klinikum Oldenburg. Anti-T. gondii avidity was analyzed at the Medical Laboratory in Bremen. The study received ethics committee approval from the local ethics committee . All methods were performed in accordance with the relevant guidelines and regulations. Written informed consent was obtained from all included subjects before study participation.In this clinic-based case–control study, patients were consecutively recruited from the University Hospital of Psychiatry and Psychotherapy at the Carl von Ossietzky University of Oldenburg, European Medical School Oldenburg in Germany. The hospital was running specialized psychiatric and psychotherapeutic ADHD inpatient and outpatient units. Medical staff as well as research facilities were broadly experienced in the assessment and treatment of ADHD in adulthood. The controls were recruited via announcements at the website of the same university. In addition, the mention of toxoplasmosis on the website was omitted to avoid overrepresentation of groups of people who may have a particular interest in an antibody test against T. gondii seropositivity with an OR of 2.5 between adult ADHD patients and healthy controls, a minimum power of 80%, and a significance level of 5% in the logistic regression, 140 participants were needed.To detect a higher We included sex- and age-matched participants, if at least 18 years old, who spoke and understood German and lacked clinically significant abnormalities detected on physical examination or blood samples. Patients were required to fulfill the criteria for ADHD according to DSM-IV with a chronic course of ADHD symptoms from childhood to adulthood. Only patients with the combined subtype of ADHD were included.T. gondii , treatment with stimulants or ADHD-specific medication that cannot be terminated three days prior to blood sampling and questionnaires, pregnancy or breast-feeding, and severe abnormality known or detected on routine blood testing .Exclusion criteria for patients and controls were acute severe inflammation or infection, detected via CRP; excluded ≥ 5 mg/dl) and differential blood count, unwillingness or incapability to adhere to the study protocol, comorbid neuropsychiatric disorders known to be associated with T. gondii is assumed.Moreover, controls were excluded if they had any psychiatric disorder except tobacco dependency or specific phobia, as no problematic relationship between these disorders and 84 and the Wender-Utah-Rating-Scale for the retrospective assessment of ADHD in childhood (WURS-k85).All participants took part in the full diagnostic process and received 10 Euros as monetary compensation. In the diagnostic process, we used diagnostic interviews as well as self-rating scales in German. The diagnosis (or exclusion) of ADHD in adulthood and other psychiatric disorders were established by psychiatric expert assessment and validated using instruments such as the ADHD Self-Rating Scale 86), the self-report inventory for the assessment of depression (Beck Depression Inventory (BDI-II87), revised version 1996), and a self-rating form to assess autistic symptoms [Autismus Spektrum Quotienten (AQ88)] were used.To assess further disorders, the structured clinical interviews for DSM-IV , hyperactivity (CAARS HY), impulsivity/emotional instability (CAARS I/EL), and problems with self-concept (CAARS SKP)). Three further subscales corresponding to the DSM-IV were also included (inattention/memory problems according to DSM-IV (UA-DSM), ADHD symptoms according to DSM , and hyperactivity/impulsivity according to DSM (DSM-HY/I)).Current ADHD symptoms were assessed via the CAARS-S:LAll applied instruments were tested for reliability and validity . Serological assays were performed on the automated BEP 2000 system , and T. gondii antibody titers were categorized as negative (< 6 U/ml) or positive (> 6 U/ml), according to the instructions of the manufacturer. In seropositive respondents, a subanalysis of the serointensity was performed. Concentrations of anti-T. gondii IgG were obtained in units per milliliter (U/ml). We further tested the avidity of T. gondii antibodies by using the fully automated chemiluminescence analyzer LIAISON XL  Italy). The avidity index allows specimen classification as low , moderate , or high . In addition, the CRP concentrations were determined using an immunoturbidimetric assay on a Cobas 6000 analyzer system . Differential blood counts were performed using an Advia 2120-System . Blood serum aliquots of each sample were stored at − 80 °C for testing of Toxoplasma IgG avidity, which was conducted after completion of all blood sample collections.All blood samples were tested for T. gondii IgG seropositivity between groups were compared by Pearson’s chi-squared test.A descriptive analysis was performed for all variables included in the analyses. Frequencies are reported for categorical variables. For continuous variables, the mean and standard deviation were calculated. Risk factors for anti-T. gondii seropositivity as the dependent variable to assess the crude and confounder-adjusted OR for the association between T. gondii seropositivity and ADHD. Covariates were taken into account as confounders based on a priori hypotheses considering covariates associated with anti-T. gondii seropositivity. The fully adjusted model included sex, age, education status, marital status, professional status, current cat contact, soil contact by gardening without gloves, and raw/undercooked meat consumption. The results are presented as ORs with 95% CIs.In the main analysis, a logistic regression model was used with T. gondii IgG seropositivity, a logistic regression model was developed. Multivariable regression analyses were performed by using backward variable elimination based on the AIC. Risk factors were included as covariates with T. gondii seropositivity as the dependent variable. The following covariates including the reference category (ref) were set: age (in years), sex , BMI , education status (ref ‘university-entrance diploma’), municipality size (ref ‘city’), marital status (ref ‘currently or previously married’), professional status, (ref ‘job seeker), current cat contact (ref ‘no’), soil contact by gardening without gloves (ref ‘no’), raw/undercooked meat consumption (ref ‘no’), and living abroad (ref ‘no’). A second model was developed for the analysis of ADHD patients only.In order to investigate associations between explanatory variables of infection and anti-22. At the same time, ADHD patients are known to suffer from a higher BMI than controls93. As the seroprevalence varies throughout different countries between 1 and 100%, depending on eating habits, hygiene standards, and health behavior53, we assessed whether the participants had already spent (or lived) several months abroad during their lifespan, defined as more than 4 weeks. The results are presented as ORs with 95% CIs.Age was self-reported. Marital status was dichotomized as ‘never married’ and ‘currently or previously married’ due to the small sample size of divorced and widowed participants. Professional status was categorized as ‘job seeker’, ‘student’, ‘employee or pensioner’, and ‘self-employed’. The BMI was included as studies indicate that a BMI > 25 might be a risk factor for seropositivity91 questionnaires. For each linear regression model, a separate stepwise backward variable elimination based on the AIC was performed starting with all predictors in a full model. Based on the AIC, one term was removed from the model in each step until the minimum value of the AIC was reached. As a result, the ‘final models’ represent the models of the best fit with the lowest AIC value of each data set94.In secondary analyses, the association between IgG and ADHD symptom severity was estimated. This association was explored in scatter plots with bivariate linear regression models of the ADHD group and sex. Moreover, linear regression models were constructed for the metric responses of the CAARS-S:LCovariates that entered in the variable selection process were age (in years), sex , IgG (U/ml), ADHD (ref ‘no’), borderline personality disorder (BPD) as the most frequent Axis II disorder in this sample (ref ‘no’), Axis II other than BPD (ref ‘no’), anxiety disorder (ref ‘no’), affective disorders (ref ‘no’), lifetime substance abuse (ref ‘no’), eating disorder (ref ‘no’), antidepressant medication (ref ‘no’), antipsychotic medication (ref ‘no’), ADHD medication (ref ‘no’), and sedatives (ref ‘no’). In addition, ordinary linear regression models were estimated with IgG as a single covariate to offer minimal models for comparison.T. gondii seropositivity (ref ‘no’) or the avidity (measured as share of affinity). For sensitivity, these models were estimated for the entire study and for the ADHD group. Linear regression analysis results were reported as regression coefficients with 95% CIs. Statistical analyses were conducted with SPSS Version 25 and R 3.5.0.In addition, exploratory analyses, separate linear regression models were constructed for the ADHD Index with either the Supplementary information.