Protocol for a Wnt reporter assay to measure its activity in human neural stem cells derived from induced pluripotent stem cells.

The canonical Wnt signaling is an essential pathway that regulates cellular proliferation, maturation, and differentiation during neurodevelopment and maintenance of adult tissue homeostasis. This pathway has been implicated with the pathophysiology of neuropsychiatric disorders and was associated with cognitive processes, such as learning and memory. However, the molecular investigation of the Wnt signaling in functional human neural cell lines might be challenging since brain biopsies are not possible and animal models may not represent the polygenic profile of some neurological and neurodevelopmental disorders. In this context, using induced pluripotent stem cells (iPSCs) has become a powerful tool to model disorders that affect the Central Nervous System (CNS) in vitro, by maintaining patients' genetic backgrounds. In this method paper, we report the development of a virus-free Wnt reporter assay in neural stem cells (NSCs) derived from human iPSCs from two healthy individuals, by using a vector containing a reporter gene (luc2P) under the control of a TCF/LEF (T-cell factor/lymphoid enhancer factor) responsive element. Dose-response curve analysis from this luciferase-based method might be useful when testing the activity of the Wnt signaling pathway after agonists (e.g. Wnt3a) or antagonists (e.g. DKK1) administration, comparing activity between cases and controls in distinct disorders. Using such a reporter assay method may help to elucidate whether neurological or neurodevelopmental mental disorders show alterations in this pathway, and testing whether targeted treatment may reverse these. Therefore, our established assay aims to help researchers on the functional and molecular investigation of the Wnt pathway in patient-specific cell types comprising several neuropsychiatric disorders.

Limited usefulness of neurocognitive functioning indices as predictive markers for treatment response to methylphenidate or neurofeedback@home in children and adolescents with ADHD.

Introduction: Earlier studies exploring the value of executive functioning (EF) indices for assessing treatment effectiveness and predicting treatment response in attention-deficit/hyperactivity disorder (ADHD) mainly focused on pharmacological treatment options and revealed rather heterogeneous results. Envisioning the long-term goal of personalized treatment selection and intervention planning, this study comparing methylphenidate treatment (MPH) and a home-based neurofeedback intervention (NF@Home) aimed to expand previous findings by assessing objective as well as subjectively reported EF indices and by analyzing their value as treatment and predictive markers. Methods: Children and adolescents (n = 146 in the per protocol sample) aged 7-13 years with a formal diagnosis of an inattentive or combined presentation of ADHD were examined. We explored the EF performance profile using the Conners Continuous Performance Task (CPT) and the BRIEF self-report questionnaire within our prospective, multicenter, randomized, reference drug-controlled NEWROFEED study with sites in five European countries (France, Spain, Switzerland, Germany, and Belgium). As primary outcome for treatment response, the clinician-rated ADHD Rating Scale-IV was used. Patients participating in this non-inferiority trial were randomized to either NF@home (34-40 sessions of TBR or SMR NF depending on the pre-assessed individual alpha peak frequency) or MPH treatment (ratio: 3:2). Within a mixed-effects model framework, analyses of change were calculated to explore the predictive value of neurocognitive indices for ADHD symptom-related treatment response. Results: For a variety of neurocognitive indices, we found a significant pre-post change during treatment, mainly in the MPH group. However, the results of the current study reveal a rather limited prognostic value of neurocognitive indices for treatment response to either NF@Home or MPH treatment. Some significant effects emerged for parent-ratings only. Discussion: Current findings indicate a potential value of self-report (BRIEF global score) and some objectively measured neurocognitive indices (CPT commission errors and hit reaction time variability) as treatment markers (of change) for MPH. However, we found a rather limited prognostic value with regard to predicting treatment response not (yet) allowing recommendation for clinical use. Baseline symptom severity was revealed as the most relevant predictor, replicating robust findings from previous studies.

Impact of the COVID-19 pandemic on mental health and family situation of clinically referred children and adolescents in Switzerland: results of a survey among mental health care professionals after 1 year of COVID-19.

The aim of this study was to assess the impact of the COVID-19 pandemic on mental well-being of clinically referred children and adolescents and on their families from the perspective of mental health care professionals in Switzerland during the first year of the pandemic. Psychiatrists and psychologists for children and adolescents participated in an anonymous survey conducted online in April/May 2021. The survey was completed by 454 mental health care professionals, most of them working in outpatient clinics for child and adolescent psychiatry or in independent practices. Most participants indicated an important increase of referrals for depression (86.8% of respondents), anxiety disorders (81.5%), crisis interventions (76.2%), psychosomatic disorders (66.1%), suicidality (63.8%), and behavioral addictions, e.g., excessive gaming (64.6%). In contrast, referrals or treatment demands for disorders such as autism spectrum disorder or psychosis showed no substantial change or a slight decrease, respectively. According to 69% of respondents, patients experienced the highest psychological burden in January/February/March 2021. Family problems very frequently reported by mental health professionals were parents' worries about loneliness/isolation of the child (49%), child's education and academic future (33%), increased media use due to missing options of recreational activities (37.6%), as well as multiple stresses of mothers (36.3%). To conclude, the pandemic has substantially changed the pattern of disorders and the number of clinical referrals of children and adolescents with mental health problems, which has serious consequences for the treatment supply in Switzerland.

Problematic use of digital media in children and adolescents with a diagnosis of attention-deficit/hyperactivity disorder compared to controls. A meta-analysis.

Objective: Problematic use of digital media and problematic use of the internet (PUI) in particular are growing problems in the general population. Moreover, studies have shown links between PUI and symptoms of attention-deficit/hyperactivity disorder (ADHD). This meta-analysis investigated whether children and adolescents with ADHD are more often affected by PUI compared to control groups. Method: Multiple databases (EBSCOhost, Pubmed) were reviewed. Studies were eligible if individuals (aged 6-18 years) were diagnosed with ADHD, assessed on PUI-related measures, and compared to non-clinical or/and clinical controls without a diagnosis of ADHD. Out of 3,859 identified studies, 14 studies assessing 2,488 participants met all inclusion criteria. Four meta-analyses examining time-based and scale-based measures, different informants and non-clinical vs. clinical controls using random-effects models were performed. Funnel plots were used to investigate publication bias. Results: The analyses revealed significantly more severe PUI in individuals with ADHD compared to controls, both when PUI was assessed via rating scale (scaled-based) and via units for time (time-based measures). Different informants (self- vs. parent-rating) had no impact on results. Differences in PUI between groups with ADHD and non-clinical controls were significant, whereas differences between ADHD and clinical controls were not. Due to the high heterogeneity observed and the small sample sizes, these latter findings should be interpreted cautiously. Conclusion: Children and adolescents with ADHD show more severe PUI compared to non-clinical controls without ADHD. However, the small number of studies does not allow for a systematic comparison between ADHD and groups with other psychopathologies.

The Impact of the COVID-19 Pandemic on Mental Health Care of Children and Adolescents in Switzerland: Results of a Survey among Mental Health Care Professionals after One Year of COVID-19.

BACKGROUND: To assess the impact of the COVID-19 pandemic on treatment demand and supply in children and adolescents with mental disorders during the first year of the pandemic from the perspective of child and adolescent psychiatrists and psychologists in Switzerland. METHODS: The survey was conducted anonymously, in German or French and online in April/May 2021. Mental health professionals working in child and adolescent psychiatry, psychotherapy services or independent practices were contacted by email. RESULTS: N = 454 professionals completed the survey (176 child and adolescent psychiatrists and 276 psychologists). After an initial period of decreased demand during the lockdown in spring 2020, requests for treatment increased, considerably exceeding the demand pre-pandemic and reaching a peak in January/February/March 2021. The vast majority of professionals (78.2%) estimated that there was currently too little supply during the pandemic, which differed from the evaluation of the pre-pandemic situation (37%). A total of 65% of participants indicated that waiting time until the initiation of treatment increased during the pandemic, 41% reported their current workload to be somewhat higher and 44.5% much higher. CONCLUSIONS: For the first pandemic year, youth mental health professionals reported a large increase in the treatment demand and waiting time and a worrisome overload of treatment services.

Media use and emotional distress under COVID-19 lockdown in a clinical sample referred for internalizing disorders: A Swiss adolescents' perspective.

The COVID-19 outbreak has profoundly affected adolescents' life. Adolescents with pre-existing psychiatric disorders have been at particular risk of increased mental health problems and problematic media use. 178 patients, aged 12-18 years, referred before the COVID-19 outbreak to child and adolescent psychiatry, participated in an anonymous online survey on the impact of the lockdown on media use and mental well-being. The survey was conducted approximately one month after the first easing of restrictions following a six-week lockdown in Switzerland. Based on self-report, half of the patients had been diagnosed with internalizing disorders (ID; depression or anxiety disorder) and the other half with other disorders (non-ID, e.g. ADHD, autistic spectrum disorder). Patients with ID reported higher emotional distress during the lockdown, and a larger number of patients with ID indicated a deterioration of pre-existing symptoms compared to non-ID patients. Although more patients with ID than with non-ID indicated spending a large amount of time on social media, social media time per day in hours was not significantly higher in ID. Patients with ID indicated a higher impact of media use on well-being and mood in everyday life during the lockdown. Social media time was higher in worsened than in improved non-ID patients, while the opposite was found in ID patients, indicating a possible protective effect of media use at least for some ID patients. The results confirm positive as well as negative associations between mental health, emotional well-being and media use for adolescents with ID during the lockdown.

Personalized at-home neurofeedback compared to long-acting methylphenidate in children with ADHD: NEWROFEED, a European randomized noninferiority trial.

BACKGROUND: Neurofeedback is considered a promising intervention for the treatment of attention-deficit hyperactivity disorder (ADHD). NEWROFEED is a prospective, multicentre, randomized (3:2), reference drug-controlled trial in children with ADHD aged between 7 and 13 years. The main objective of NEWROFEED was to demonstrate the noninferiority of personalized at-home neurofeedback (NF) training versus methylphenidate in the treatment of children with ADHD. METHODS: The NF group (n = 111) underwent eight visits and two treatment phases of 16 to 20 at-home sessions with down-training of the theta/beta ratio (TBR) for children with high TBR and enhancing the sensorimotor rhythm (SMR) for the others. The control group (n = 67) received optimally titrated long-acting methylphenidate. The primary endpoint was the change between baseline and endpoint in the Clinician ADHD-RS-IV total score in the per-protocol population (90 NF/59 controls). TRIAL REGISTRATION: US National Institute of Health, ClinicalTrials.gov #NCT02778360. RESULTS: Our study failed to demonstrate noninferiority of NF versus methylphenidate (mean between-group difference 8.09 90% CI [8.09; 10.56]). However, both treatment groups showed significant pre-post improvements in core ADHD symptoms and in a broader range of problems. Reduction in the Clinician ADHD-RS-IV total score between baseline and final visit (D90) was 26.7% (SMD = 0.89) in the NF and 46.9% (SMD = 2.03) in the control group. NF effects increased whereas those of methylphenidate were stable between intermediate and final visit. CONCLUSIONS: Based on clinicians' reports, the effects of at-home NF were inferior to those of methylphenidate as a stand-alone treatment.

Problematic use of the internet during the COVID-19 pandemic: Good practices and mental health recommendations.

With the onset of the COVID-19 pandemic and the accelerated spread of the SARS-CoV-2 virus came jurisdictional limitations on mobility of citizens and distinct alterations in their daily routines. Confined to their homes, many people increased their overall internet use, with problematic use of the internet (PUI) becoming a potential reason for increased mental health concerns. Our narrative review summarizes information on the extent of PUI during the pandemic, by focusing on three types: online gaming, gambling and pornography viewing. We conclude by providing guidance for mental health professionals and those affected by PUI (with an outline of immediate research priorities and best therapeutic approaches), as well as for the general public (with an overview of safe and preventative practices).

Media use before, during and after COVID-19 lockdown according to parents in a clinically referred sample in child and adolescent psychiatry: Results of an online survey in Switzerland.

BACKGROUND: To investigate the consequences of COVID-19 lockdown on screen media use in children and adolescents with mental health problems, an online survey was conducted on leisure media use before, during and after the lockdown of spring 2020. METHOD: Parents of patients (10-18 yrs) referred to child and adolescent psychiatry participated in an anonymous online survey, approximately six weeks after the first easing of lockdown measures. Parents rated the amount, the content and the psychological impact of their children's media use before, during and after the lockdown. RESULTS: N = 477 parents completed the survey. Patients showed a significant increase in media time during the lockdown (including devices such as mobile, tablet/PC, video game console, TV, and activities such as gaming, social media) and a moderate increase in the negative impact of media use on everyday life. After the lockdown, total media time returned to pre-COVID-19 levels in most patients, but remained slightly higher in males. A worsening of the main psychopathological problem during lockdown was related to elevated media time in children (10-13 yrs), but not in adolescents (14-18 yrs). CONCLUSION: According to parents' retrospective ratings, the increase in screen media time was reversible, and seems to reflect an expected coping strategy during lockdown. However, male patients did not completely return to pre-COVID-19 gaming time, and a small number continued to display excessive gaming.

Impact of the COVID-19 lockdown on screen media use in patients referred for ADHD to child and adolescent psychiatry: an introduction to problematic use of the internet in ADHD and results of a survey.

The COVID-19 outbreak and lockdown have been associated with multiple consequences for mental health, including an excessive and potentially harmful increase in screen media use. The specific consequences for children, adolescents and young adults with ADHD are still unknown. In the first part of this study, a short review of problematic use of the internet (PUI) in ADHD is presented, showing that patients with ADHD are at risk for different aspects of PUI, such as excessive gaming or problematic social media use. In the second part, we report original data of an online survey on screen media use before, during and after the lockdown completed by parents of children and adolescents clinically referred for ADHD. Parents rated children's/adolescents' media-related behavior and media time on a new screening questionnaire for PUI. Each item was rated three times, referring to the observed behavior before, during and 1-2 months after the lockdown. N = 126 parents of patients referred for ADHD aged 10-18 years participated in the study. Total media time increased by 46% during the lockdown and did not completely return to pre-Corona levels afterwards. Patients with difficulties concentrating, high irritability or deterioration of ADHD problems under lockdown spent more time with screen media than those with milder or no such problems. While the effects of the lockdown on screen media use and its negative impact on everyday life appear to be largely reversible, a small proportion of patients with ADHD apparently continue to show increased media use.

Generation of integration-free induced pluripotent stem cell lines from four pediatric ADHD patients.

Human induced pluripotent stem cell (iPSC) lines have been derived from four male patients with childhood attention-deficit hyperactivity disorder (ADHD). Children and adolescents between the ages 6 and 18 suffering from ADHD were recruited for this work. Isolated keratinocytes or peripheral blood mononuclear cells from the participants were reprogrammed into iPSCs using non-integrating Sendai virus to deliver the reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc.

Generation of integration-free induced pluripotent stem cells from healthy individuals.

Ten human induced pluripotent stem cell (iPSC) lines have been derived from five healthy controls matched to a study including Attention-Deficit Hyperactivity Disorder patients (ADHD). Both female and male children and adolescents aged 6-18 years were recruited. Isolated keratinocyte cells from the participants were reprogrammed into iPSCs using non-integrating Sendai virus to deliver the reprogramming factors Oct3/4, Sox2, Klf4 and cMyc.

The stress-Wnt-signaling axis: a hypothesis for attention-deficit hyperactivity disorder and therapy approaches.

Attention-deficit hyperactivity disorder (ADHD) is one of the most common psychiatric neurodevelopmental disorders in children and adolescents. Although ADHD has been studied for nearly a century, the cause and pathophysiology of ADHD is yet largely unknown. However, findings from previous studies have resulted in the formation of a new hypothesis: Apart from the well-known multifactorial etiology of ADHD, recent evidence suggests that the interaction between genetic and environmental factors and especially Wnt- and mTOR-signaling pathways might have an important role in the pathophysiology of ADHD. The Wnt-signaling pathway is known to orchestrate cellular proliferation, polarity, and differentiation, and the mTOR pathway is involved in several significant processes of neurodevelopment and synaptic plasticity. As a result, dysregulations of these pathways in a time-dependent manner could lead to neurodevelopmental delays, resulting in ADHD phenotype. This review presents further evidence supporting our hypothesis by combining results from studies on ADHD and Wnt- or mTOR-signaling and the influence of genetics, methylphenidate treatment, Omega-3 supplementation, and stress.

High-resolution chromosomal microarray analysis for copy-number variations in high-functioning autism reveals large aberration typical for intellectual disability.

Copy-number variants (CNVs), in particular rare, small and large ones (< 1% frequency) and those encompassing brain-related genes, have been shown to be associated with neurodevelopmental disorders like autism spectrum disorders (ASDs), attention deficit hyperactivity disorder (ADHD), and intellectual disability (ID). However, the vast majority of CNV findings lack specificity with respect to autistic or developmental-delay phenotypes. Therefore, the aim of the study was to investigate the size and frequency of CNVs in high-functioning ASD (HFA) without ID compared with a random population sample and with published findings in ASD and ID. To investigate the role of CNVs for the "core symptoms" of high-functioning autism, we included in the present exploratory study only patients with HFA without ID. The aim was to test whether HFA have similar large rare (> 1 Mb) CNVs as reported in ASD and ID. We performed high-resolution chromosomal microarray analysis in 108 children and adolescents with HFA without ID. There was no significant difference in the overall number of rare CNVs compared to 124 random population samples. However, patients with HFA carried significantly more frequently CNVs containing brain-related genes. Surprisingly, six HFA patients carried very large CNVs known to be typically present in ID. Our findings provide new evidence that not only small, but also large CNVs affecting several key genes contribute to the genetic etiology/risk of HFA without affecting their intellectual ability.

Association study and a systematic meta-analysis of the VNTR polymorphism in the 3'-UTR of dopamine transporter gene and attention-deficit hyperactivity disorder.

Attention-deficit hyperactivity disorder (ADHD) has been postulated to associate with dopaminergic dysfunction, including the dopamine transporter (DAT1). Several meta-analyses showed small but significant association between the 10-repeat allele in the DAT1 gene in 3'-untranslated region variant number tandem repeat polymorphism and child and adolescent ADHD, whereas in adult ADHD the 9-repeat allele was suggested to confer as risk allele. Interestingly, recent evidence indicated that the long-allele variants (10 repeats and longer) might confer to lower expression of the transporter in comparison to the short-allele. Therefore, we assessed here the association in samples consisting of families with child and adolescent ADHD as well as a case-control sample, using either the 10- versus 9-repeat or the long- versus short-allele approach. Following, we conducted a systematic review and meta-analysis, including family and case-control studies, using the two aforementioned approaches as well as stratifying to age and ethnicity. The first approach (10-repeat) resulted in nominal significant association in child and adolescent ADHD (OR 1.1050 p = 0.0128), that became significant stratifying to European population (OR 1.1301 p = 0.0085). The second approach (long-allele) resulted in significant association with the whole ADHD population (OR 1.1046 p = 0.0048), followed by significant association for child and adolescent ADHD (OR 1.1602 p = 0.0006) and in Caucasian and in European child and adolescent ADHD (OR 1.1310 p = 0.0114; OR 1.1661 p = 0.0061; respectively). We were not able to confirm the association reported in adults using both approaches. In conclusion, we found further indication for a possible DAT1 gene involvement; however, further studies should be conducted with stringent phenotyping to reduce heterogeneity, a limitation observed in most included studies.

The involvement of the canonical Wnt-signaling receptor LRP5 and LRP6 gene variants with ADHD and sexual dimorphism: Association study and meta-analysis.

Wnt-signaling is one of the most abundant pathways involved in processes such as cell-proliferation, -polarity, and -differentiation. Altered Wnt-signaling has been linked with several neurodevelopmental disorders including attention-deficit/hyperactivity disorder (ADHD) as well as with cognitive functions, learning and memory. Particularly, lipoprotein receptor-related protein 5 (LRP5) or LRP6 coreceptors, responsible in the activation of the canonical Wnt-pathway, were associated with cognitive alterations in psychiatric disorders. Following the hypothesis of Wnt involvement in ADHD, we investigated the association of genetic variations in LRP5 and LRP6 genes with three independent child and adolescent ADHD (cADHD) samples (total 2,917 participants), followed by a meta-analysis including previously published data. As ADHD is more prevalent in males, we stratified the analysis according to sex and compared the results with the recent ADHD Psychiatric Genomic Consortium (PGC) GWAS. Meta-analyzing our data including previously published cADHD studies, association of LRP5 intronic rs4988319 and rs3736228 (Ala1330Val) with cADHD was observed among girls (OR = 1.80 with 95% CI = 1.07-3.02, p = .0259; and OR = 2.08 with 95% CI = 1.01-4.46, p = .0026, respectively), whereas in boys association between LRP6 rs2302685 (Val1062Ile) and cADHD was present (OR = 1.66, CI = 1.20-2.31, p = .0024). In the PGC-ADHD dataset (using pooled data of cADHD and adults) tendency of associations were observed only among females with OR = 1.09 (1.02-1.17) for LRP5 rs3736228 and OR = 1.18 (1.09-1.25) for LRP6 rs2302685. Together, our findings suggest a potential sex-specific link of cADHD with LRP5 and LRP6 gene variants, which could contribute to the differences in brain maturation alterations in ADHD affected boys and girls, and suggest possible therapy targets.

Improved Generation of Induced Pluripotent Stem Cells From Hair Derived Keratinocytes - A Tool to Study Neurodevelopmental Disorders as ADHD.

In the last decade, there is an increasing application of induced pluripotent stem cells (iPSCs) for disease modeling. The iPSC technology enables the study of patient-specific neuronal cell lines in vitro to evaluate dysfunction at the cellular level and identify the responsible genetic factors. This approach might be particularly valuable for filling the gap of knowledge at the cellular and molecular levels underlying the pathophysiology of various neurodevelopmental and/or psychiatric disorders, such as attention-deficit hyperactivity disorder (ADHD). However, the invasiveness of skin biopsy or blood withdrawal might represent a major impediment in such protected population. Using hair derived keratinocytes as starting somatic cells circumvents this problem as sample collections can be performed non-invasively. Here we describe an improved, convenient, standardized and effective method to culture and reprogram hair derived keratinocytes from three healthy controls and one ADHD patient into iPSCs, which in turn will be used to generate differentiated neuronal cells. All the cell types were maintained in highly defined, serum-free conditions and showed expression of the respective key marker genes, assessed by both immunocytochemistry and qRT-PCR. The described in vitro personalized neuronal model has its advantage in modeling neurodevelopmental trajectories since it can recapitulate key processes of brain development at the cellular and molecular level and is intended to be used as for example studying ADHD etiopathology.

CNTNAP2 gene in high functioning autism: no association according to family and meta-analysis approaches.

The Contactin Associated Protein-like 2 (CNTNAP2) gene has been discussed to be associated with different symptoms of autism spectrum disorders (ASDs) and other neurodevelopmental disorders. We aimed to elucidate the genetic association of CNTNAP2 within high functioning ASD (HFA), focusing on autism specific symptoms and reducing intelligence related factors. Furthermore, we compared our findings conducting a meta-analysis in patients with ASD and HFA only. A case-control association study was performed for HFA (HFA, n = 105; controls, n = 133). Moreover, we performed a family-based association study (DFAM) analysis (HFA, n = 44; siblings, n = 57). Individuals were genotyped for the two most frequently reported single nucleotide polymorphisms (SNPs) in the CNTNAP2 gene (rs2710102, rs7794745). Furthermore, a meta-analysis using the MIX2 software integrated our results with previously published data. A significant association for the carriers of the T-allele of the rs7794745 with HFA was found in the case-control sample [OR = 1.547; (95 % CI 1.056-2.266); p = 0.025]. No association could be found by DFAM with any of the CNTNAP2 SNPs with HFA. The meta-analysis of both SNPs did not show a significant association with either ASD or with HFA. Overall, including case-control, sibs, and meta-analysis, we could not detect any significant association with the CNTNAP2 gene and HFA. Our results point in the direction that CNTNAP2 may not play a major role in HFA, but rather seems to have a significance in neurodevelopmental disorders or in individuals displaying intellectual delays.