ADHD-associated PARK2 copy number variants: A pilot study on gene expression and effects of supplementary deprivation in patient-derived cell lines.

Recent studies show an association of Parkin RBR E3 ubiquitin protein ligase (PARK2) copy number variations (CNVs) with attention deficit hyperactivity disorder (ADHD). The aim of our pilot study to investigate gene expression associated with PARK2 CNVs in human-derived cellular models. We investigated gene expression in fibroblasts, hiPSC and dopaminergic neurons (DNs) of ADHD PARK2 deletion and duplication carriers by qRT PCR compared with healthy and ADHD cell lines without PARK2 CNVs. The selected 10 genes of interest were associated with oxidative stress response (TP53, NQO1, and NFE2L2), ubiquitin pathway (UBE3A, UBB, UBC, and ATXN3) and with a function in mitochondrial quality control (PINK1, MFN2, and ATG5). Additionally, an exploratory RNA bulk sequencing analysis in DNs was conducted. Nutrient deprivation as a supplementary deprivation stress paradigm was used to enhance potential genotype effects. At baseline, in fibroblasts, hiPSC, and DNs, there was no significant difference in gene expression after correction for multiple testing. After nutrient deprivation in fibroblasts NAD(P)H-quinone-dehydrogenase 1 (NQO1) expression was significantly increased in PARK2 CNV carriers. In a multivariate analysis, ubiquitin C (UBC) was significantly upregulated in fibroblasts of PARK2 CNV carriers. RNA sequencing analysis of DNs showed the strongest significant differential regulation in Neurontin (NNAT) at baseline and after nutrient deprivation. Our preliminary results suggest differential gene expression in pathways associated with oxidative stress, ubiquitine-proteasome, immunity, inflammation, cell growth, and differentiation, excitation/inhibition modulation, and energy metabolism in PARK2 CNV carriers compared to wildtype healthy controls and ADHD patients.

Expression of the adult ADHD-associated gene ADGRL3 is dysregulated by risk variants and environmental risk factors.

OBJECTIVES: ADGRL3 is a well-replicated risk gene for adult ADHD, encoding the G protein-coupled receptor latrophilin-3 (LPHN3). However, LPHN3's potential role in pathogenesis is unclear. We aimed to determine whether ADGRL3 expression could be dysregulated by genetic risk variants and/or ADHD-associated environmental risk factors. METHODS: Eighteen adult ADHD patients and healthy controls were genotyped for rs734644, rs1397547, rs1397548, rs2271338, rs2305339, rs2345039 and rs6551665 ADGRL3 SNPs, and fibroblast cells were derived from skin punches. The environmental ADHD risk factors 'low birthweight' and 'maternal smoking' were modelled in fibroblast cell culture using starvation and nicotine exposure, respectively. Quantitative real-time PCR and western blotting were performed to quantify ADGRL3 gene and protein expression under control, starvation and nicotine-exposed conditions. RESULTS: Starvation was found to significantly decrease ADGRL3 expression, whereas nicotine exposure significantly increased ADGRL3 expression. rs1397547 significantly elevated ADGRL3 transcription and protein expression. rs6551665 and rs2345039 interacted with environment to modulate ADGRL3 transcription. ADGRL3 SNPs were significantly able to predict its transcription under both baseline and starvation conditions, and rs1397547 was identified as a significant independent predictor. CONCLUSIONS: ADGRL3 SNPs and environmental risk factors can regulate ADGRL3 expression, providing a potential functional mechanism by which LPHN3 may play a role in ADHD pathogenesis.

Energy Metabolism Disturbances in Cell Models of PARK2 CNV Carriers with ADHD.

The main goal of the present study was the identification of cellular phenotypes in attention-deficit-/hyperactivity disorder (ADHD) patient-derived cellular models from carriers of rare copy number variants (CNVs) in the PARK2 locus that have been previously associated with ADHD. Human-derived fibroblasts (HDF) were cultured and human-induced pluripotent stem cells (hiPSC) were reprogrammed and differentiated into dopaminergic neuronal cells (mDANs). A series of assays in baseline condition and in different stress paradigms (nutrient deprivation, carbonyl cyanide m-chlorophenyl hydrazine (CCCP)) focusing on mitochondrial function and energy metabolism (ATP production, basal oxygen consumption rates, reactive oxygen species (ROS) abundance) were performed and changes in mitochondrial network morphology evaluated. We found changes in PARK2 CNV deletion and duplication carriers with ADHD in PARK2 gene and protein expression, ATP production and basal oxygen consumption rates compared to healthy and ADHD wildtype control cell lines, partly differing between HDF and mDANs and to some extent enhanced in stress paradigms. The generation of ROS was not influenced by the genotype. Our preliminary work suggests an energy impairment in HDF and mDAN cells of PARK2 CNV deletion and duplication carriers with ADHD. The energy impairment could be associated with the role of PARK2 dysregulation in mitochondrial dynamics.

The role of pre-, peri-, and postnatal risk factors in bipolar disorder and adult ADHD.

Gene-environment-development interactions are suggested to play a crucial role in psychiatric disorders. However, it is not clear if there are specific risk gene interactions with particular pre-, peri-, and postnatal risk factors for distinct disorders, such as adult attention-deficit-/hyperactivity disorder (aADHD) and bipolar disorder (BD). In this pilot study, the first aim was to investigate retrospective self-reports of pre-, peri-, and postnatal complications and risk factors from 126 participants (aADHD, BD, and healthy controls) and their mothers. The second aim was to investigate possible interaction between the previously published common risk gene variants of ADHD in the ADGRL3 (=LPHN3) gene (rs2305339, rs1397548, rs734644, rs1397547, rs2271338, rs6551665, and rs2345039) and shared risk gene variants of aADHD and BD in the DGKH gene (DGKH rs994856/rs9525580/rs9525584 GAT haplotype) and pre-, peri-, and postnatal risk factors in comparison to a healthy control group. After correction for multiple comparison, the following pre-, peri-, and postnatal risk factors remained statistically significant (p ≤ 0.0036) between healthy controls and ADHD and BD patients as one group: unplanned pregnancies, psychosocial stress of the mother during pregnancy, mode of delivery, shared decision-making regarding medical procedures during the delivery, perinatal bonding, number of crybabies, and quality of mother-child and father-child relationship. There were no significant environment-gene interactions. In our preliminary data, similar risk factors were found to be significantly associated with both disorders in comparison to healthy controls. However, larger and longitudinal studies and standardized and validated instruments to get a better understanding of the interaction of pre-, peri-, and postnatal complications and mental health in the offspring are needed.

Generation of human induced pluripotent stem cell lines (hiPSC) from one bipolar disorder patient carrier of a DGKH risk haplotype and one non-risk-variant-carrier bipolar disorder patient.

Fibroblasts were isolated from skin biopsies from two patients with bipolar I disorder. One patient was a 26 year old female carrying a risk haplotype in the DGKH (diacylglycerol kinase eta) gene and the other was a non-carrier 27 year old male. Patient fibroblasts were reprogrammed into human induced pluripotent stem cells (hiPSCs) by using a Sendai virus vector. DGKH-risk haplotype and non-risk haplotype hiPSCs showed expression of pluripotency markers and were able to differentiate into cells of the three germ layers. These cell models are useful to investigate the role of risk gene variants in bipolar disorder. Resource table.

Region specific up-regulation of oxytocin receptors in the opioid oprm1 (-/-) mouse model of autism.

Autism spectrum disorders (ASDs) are characterized by impaired communication, social impairments, and restricted and repetitive behaviors and interests. Recently, altered motivation and reward processes have been suggested to participate in the physiopathology of ASDs, and µ-opioid receptors (MORs) have been investigated in relation to social reward due to their involvement in the neural circuitry of reward. Mice lacking a functional MOR gene (Oprm1 (-/-) mice) display abnormal social behavior and major autistic-like core symptoms, making them an animal model of autism. The oxytocin (OXT) system is a key regulator of social behavior and co-operates with the opioidergic system in the modulation of social behavior. To better understand the opioid-OXT interplay in the central nervous system, we first determined the expression of the oxytocin receptor (OXTR) in the brain of WT C57BL6/J mice by quantitative autoradiography; we then evaluated OXTR regional alterations in Oprm1 (-/-) mice. Moreover, we tested these mice in a paradigm of social behavior, the male-female social interaction test, and analyzed the effects of acute intranasal OXT treatment on their performance. In autoradiography, Oprm1 (-/-) mice selectively displayed increased OXTR expression in the Medial Anterior Olfactory Nucleus, the Central and Medial Amygdaloid nuclei, and the Nucleus Accumbens. Our behavioral results confirmed that Oprm1 (-/-) male mice displayed social impairments, as indicated by reduced ultrasonic calls, and that these were rescued by a single intranasal administration of OXT. Taken together, our results provide evidence of an interaction between OXT and opioids in socially relevant brain areas and in the modulation of social behavior. Moreover, they suggest that the oxytocinergic system may act as a compensative mechanism to bypass and/or restore alterations in circuits linked to impaired social behavior.