Attention-deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder with a complex genetic background, hampering identification of underlying genetic risk factors. We hypothesized ...that combining linkage analysis and whole-exome sequencing (WES) in multi-generation pedigrees with multiple affected individuals can point toward novel ADHD genes. Three families with multiple ADHD-affected members (N
= 70) and apparent dominant inheritance pattern were included in this study. Genotyping was performed in 37 family members, and WES was additionally carried out in 10 of those. Linkage analysis was performed using multi-point analysis in Superlink Online SNP 1.1. From prioritized linkage regions with a LOD score ≥ 2, a total of 24 genes harboring rare variants were selected. Those genes were taken forward and were jointly analyzed in gene-set analyses of exome-chip data using the MAGMA software in an independent sample of patients with persistent ADHD and healthy controls (N = 9365). The gene-set including all 24 genes together, and particularly the gene-set from one of the three families (12 genes), were significantly associated with persistent ADHD in this sample. Among the latter, gene-wide analysis for the AAED1 gene reached significance. A rare variant (rs151326868) within AAED1 segregated with ADHD in one of the families. The analytic strategy followed here is an effective approach for identifying novel ADHD risk genes. Additionally, this study suggests that both rare and more frequent variants in multiple genes act together in contributing to ADHD risk, even in individual multi-case families.
Fibroblasts isolated from a skin biopsy of a healthy 46-year-old female were infected with Sendai virus containing the Yamanaka factors to produce transgene-free human induced pluripotent stem cells ...(iPSCs). CRISPR/Cas9 was used to generate isogenic cell lines with a gene dose-dependent deficiency of CDH13, a risk gene associated with neurodevelopmental and psychiatric disorders. Thereby, a heterozygous CDH13 knockout (CDH13+/−) and a CDH13 null mutant (CDH13−/−) iPSC line was obtained. All three lines showed expression of pluripotency-associated markers, the ability to differentiate into cells of the three germ layers in vitro, and a normal female karyotype.
During early prenatal stages of brain development, serotonin (5-HT)-specific neurons migrate through somal translocation to form the raphe nuclei and subsequently begin to project to their target ...regions. The rostral cluster of cells, comprising the median and dorsal raphe (DR), innervates anterior regions of the brain, including the prefrontal cortex. Differential analysis of the mouse 5-HT system transcriptome identified enrichment of cell adhesion molecules in 5-HT neurons of the DR. One of these molecules, cadherin-13 (Cdh13) has been shown to play a role in cell migration, axon pathfinding, and synaptogenesis. This study aimed to investigate the contribution of Cdh13 to the development of the murine brain 5-HT system.
For detection of Cdh13 and components of the 5-HT system at different embryonic developmental stages of the mouse brain, we employed immunofluorescence protocols and imaging techniques, including epifluorescence, confocal and structured illumination microscopy. The consequence of
loss-of-function mutations on brain 5-HT system development was explored in a mouse model of Cdh13 deficiency.
Our data show that in murine embryonic brain Cdh13 is strongly expressed on 5-HT specific neurons of the DR and in radial glial cells (RGCs), which are critically involved in regulation of neuronal migration. We observed that 5-HT neurons are intertwined with these RGCs, suggesting that these neurons undergo RGC-guided migration. Cdh13 is present at points of intersection between these two cell types. Compared to wildtype controls, Cdh13-deficient mice display increased cell densities in the DR at embryonic stages E13.5, E17.5, and adulthood, and higher serotonergic innervation of the prefrontal cortex at E17.5.
Our findings provide evidence for a role of CDH13 in the development of the serotonergic system in early embryonic stages. Specifically, we indicate that Cdh13 deficiency affects the cell density of the developing DR and the posterior innervation of the prefrontal cortex (PFC), and therefore might be involved in the migration, axonal outgrowth and terminal target finding of DR 5-HT neurons. Dysregulation of CDH13 expression may thus contribute to alterations in this system of neurotransmission, impacting cognitive function, which is frequently impaired in neurodevelopmental disorders including attention-deficit/hyperactivity and autism spectrum disorders.
Fibroblasts were isolated from a skin biopsy of a clinically diagnosed 51-year-old female attention-deficit/hyperactivity disorder (ADHD) patient carrying a duplication of SLC2A3, a gene encoding ...neuronal glucose transporter-3 (GLUT3). Patient fibroblasts were infected with Sendai virus, a single-stranded RNA virus, to generate transgene-free human induced pluripotent stem cells (iPSCs). SLC2A3-D2-iPSCs showed expression of pluripotency-associated markers, were able to differentiate into cells of the three germ layers in vitro and had a normal female karyotype. This in vitro cellular model can be used to study the role of risk genes in the pathogenesis of ADHD, in a patient-specific manner.
SLC2A3 encodes the predominantly neuronal glucose transporter 3 (GLUT3), which facilitates diffusion of glucose across plasma membranes. The human brain depends on a steady glucose supply for ATP ...generation, which consequently fuels critical biochemical processes, such as axonal transport and neurotransmitter release. Besides its role in the central nervous system, GLUT3 is also expressed in nonneural organs, such as the heart and white blood cells, where it is equally involved in energy metabolism. In cancer cells, GLUT3 overexpression contributes to the Warburg effect by answering the cell's increased glycolytic demands. The SLC2A3 gene locus at chromosome 12p13.31 is unstable and prone to non‐allelic homologous recombination events, generating multiple copy number variants (CNVs) of SLC2A3 which account for alterations in SLC2A3 expression. Recent associations of SLC2A3 CNVs with different clinical phenotypes warrant investigation of the potential influence of these structural variants on pathomechanisms of neuropsychiatric, cardiovascular, and immune diseases. In this review, we accumulate and discuss the evidence how SLC2A3 gene dosage may exert diverse protective or detrimental effects depending on the pathological condition. Cellular states which lead to increased energetic demand, such as organ development, proliferation, and cellular degeneration, appear particularly susceptible to alterations in SLC2A3 copy number. We conclude that better understanding of the impact of SLC2A3 variation on disease etiology may potentially provide novel therapeutic approaches specifically targeting this GLUT.
This review summarizes the cellular and clinical implications of deletion and duplication variants of the SLC2A3 gene which encodes the glucose transporter 3. Disease associations of these copy number variants with neurodevelopmental and neurodegenerative disorders, congenital heart defects, and autoimmune diseases are discussed, focussing on how metabolic alterations may influence cellular development and degeneration.
Human induced pluripotent stem cells (hiPSCs) have revolutionized the generation of experimental disease models, but the development of protocols for the differentiation of functionally active ...neuronal subtypes with defined specification is still in its infancy. While dysfunction of the brain serotonin (5-HT) system has been implicated in the etiology of various neuropsychiatric disorders, investigation of functional human 5-HT specific neurons in vitro has been restricted by technical limitations. We describe an efficient generation of functionally active neurons from hiPSCs displaying 5-HT specification by modification of a previously reported protocol. Furthermore, 5-HT specific neurons were characterized using high-end fluorescence imaging including super-resolution microscopy in combination with electrophysiological techniques. Differentiated hiPSCs synthesize 5-HT, express specific markers, such as tryptophan hydroxylase 2 and 5-HT transporter, and exhibit an electrophysiological signature characteristic of serotonergic neurons, with spontaneous rhythmic activities, broad action potentials and large afterhyperpolarization potentials. 5-HT specific neurons form synapses reflected by the expression of pre- and postsynaptic proteins, such as Bassoon and Homer. The distribution pattern of Bassoon, a marker of the active zone along the soma and extensions of neurons, indicates functionality via volume transmission. Among the high percentage of 5-HT specific neurons (~ 42%), a subpopulation of CDH13 + cells presumably designates dorsal raphe neurons. hiPSC-derived 5-HT specific neuronal cell cultures reflect the heterogeneous nature of dorsal and median raphe nuclei and may facilitate examining the association of serotonergic neuron subpopulations with neuropsychiatric disorders.
Background
Attention‐deficit/hyperactivity disorder (ADHD) is a common, highly heritable neurodevelopmental disorder with profound cognitive, behavioral, and psychosocial impairments with persistence ...across the life cycle. Our initial genome‐wide screening approach for copy number variants (CNVs) in ADHD implicated a duplication of SLC2A3, encoding glucose transporter‐3 (GLUT3). GLUT3 plays a critical role in cerebral glucose metabolism, providing energy for the activity of neurons, which, in turn, moderates the excitatory–inhibitory balance impacting both brain development and activity‐dependent neural plasticity. We therefore aimed to provide additional genetic and functional evidence for GLUT3 dysfunction in ADHD.
Methods
Case–control association analyses of SLC2A3 single‐nucleotide polymorphisms (SNPs) and CNVs were conducted in several European cohorts of patients with childhood and adult ADHD (SNP, n = 1,886 vs. 1,988; CNV, n = 1,692 vs. 1,721). These studies were complemented by SLC2A3 expression analyses in peripheral cells, functional EEG recordings during neurocognitive tasks, and ratings of food energy content.
Results
Meta‐analysis of all cohorts detected an association of SNP rs12842 with ADHD. While CNV analysis detected a population‐specific enrichment of SLC2A3 duplications only in German ADHD patients, the CNV + rs12842 haplotype influenced ADHD risk in both the German and Spanish cohorts. Duplication carriers displayed elevated SLC2A3 mRNA expression in peripheral blood cells and altered event‐related potentials reflecting deficits in working memory and cognitive response control, both endophenotypic traits of ADHD, and an underestimation of energy units of high‐caloric food.
Conclusions
Taken together, our results indicate that both common and rare SLC2A3 variation impacting regulation of neuronal glucose utilization and energy homeostasis may result in neurocognitive deficits known to contribute to ADHD risk.
Neuropsychiatric disorders, such as attention-deficit/hyperactivity disorder (ADHD), represent a burden which deeply impair the patient’s life. Neurobiological research has therefore increasingly ...focused on the examination of brain neurotransmitter systems, such as the serotonin (5-HT) system, since a dysfunction has been repeatedly implicated in the pathology of these diseases. However, investigation of functional human neurons in vitro has been restricted by technical limitations for a long time until the discovery of human induced pluripotent stem cells (iPSCs) revolutionized the field of experimental disease models. Since the pathogenesis of neuropsychiatric disorders involves a complex genetic component, genome-wide association studies (GWAS) revealed numerous risk genes that are associated with an increased risk for ADHD. For instance, the novel ADHD candidate gene SLC2A3 which encodes the glucose transporter-3 (GLUT3), facilitates the transport of glucose across plasma membranes and is essential for the high energy demand of several cell types, such as stem cells and neurons. Specifically, copy number variants (CNVs) of SLC2A3 might therefore impact cerebral glucose metabolism as well as the assembly of synaptic proteins in human neurons which might contribute to the pathogenesis of ADHD.
We hypothesized that an altered SLC2A3 gene dosage in human neurons can exert diverse protective or detrimental effects on neurodevelopmental processes as well as the coping of glucometabolic stress events, such as hypo- and hyperglycaemic conditions. The generation of specific iPSC lines from ADHD patients and healthy probands served as basis to efficiently differentiate stem cells into 5-HT specific neurons. Using this neuronal culture, we were able to examine effects of SLC2A3 CNVs on the basal expression of SCL2A3 and GLUT3 in human neurons. Furthermore, the focus was on potentially altered coping of the cells with glucose deprivation and the treatment with specific high- and low glycaemic media.
High-resolution fluorescence imaging in combination with electrophysiological and molecular biological techniques showed that:
1) The generated human iPSCs are fully reprogrammed human stem cells showing typical characteristics of embryonic stem cell-like morphology, growth behaviour, the ability to differentiate into different cell types of the human body and the expression of pluripotency-specific markers.
2) The neuronal subtype derived from our stem cells display typical characteristics of 5-HT specific median and dorsal neurons and forms synapses reflected by the expression of pre- and postsynaptic proteins.
3) Even if SLC2A3 CNVs influence SLC2A3 and GLUT3 basal expression, no significant alterations in gene and protein expression caused by hyper- and hypoglycaemic conditions, nor in the assembly of proteins associated with synapse formation could be observed in human iPSC-derived neurons.
Neuropsychiatrische Erkrankungen, wie das Aufmerksamkeits-Defizit/Hyperaktivitäts-Syndrom (ADHS), stellen eine Belastung dar, die das Leben des Patienten schwerwiegend beeinträchtigen. Die neurobiologische Forschung hat sich deshalb zunehmend auf die Untersuchung der Neurotransmittersysteme des Gehirns, wie das serotonerge (5-HT) System fokussiert, da eine Dysfunktion wiederholt in Zusammenhang mit der Pathogenese solcher Erkrankungen gebracht wurde. Die in vitro-Untersuchung funktioneller humaner Neurone war jedoch lange Zeit durch technische Limitierungen eingeschränkt, bis die Entdeckung humaner induzierter pluripotenter Stammzellen (iPSCs) das Feld der experimentellen Krankheitsmodelle revolutionierte. Da die Pathogenese neuropsychiatrischer Erkrankungen eine komplexe genetische Komponente einschließt, haben genomweite Assoziationsstudien zahlreiche Risikogene aufgedeckt, die mit einem erhöhten Risiko für ADHS assoziiert werden. Das Kandidatengen SLC2A3, das den Glukosetransporter-3 (GLUT3) codiert, ermöglicht beispielsweise den Transport von Glukose über Plasmamembranen und ist somit essenziell für die hohe Energieanforderung verschiedenster Zelltypen, wie etwa Stammzellen und Neurone. Im Besonderen könnten die Kopienzahlvarianten (CNVs) des Gens SLC2A3 daher den cerebralen Glukosemetabolismus, sowie die Ansammlung synaptischer Proteine beeinflussen und so zur Pathogenese des ADHS beitragen.
Wir nahmen an, dass eine veränderte SLC2A3-Gendosis in humanen Neuronen diverse protektive oder schädliche Effekte auf Neuroentwicklungsprozesse, sowie den Umgang mit glukometabolischen Stress-Ereignissen, wie etwa hypo- und hyperglykämische Bedingungen haben könnte. Die Generierung spezieller iPSC-Linien von ADHS-Patienten und gesunden Probanden diente als Basis, um Stammzellen effizient in 5-HT spezifische Neurone zu differenzieren. Diese neuronale Kultur ermöglichte uns die Untersuchung der Effekte der SLC2A3 CNVs auf die Basalexpression von SLC2A3 und GLUT3 in humanen Neuronen. Des Weiteren war der Focus auf einen potenziell veränderten Umgang der Zellen mit Glukoseentzug und der Behandlung mit speziellen hoch- und niederglykämischen Medien.
Hochauflösende Fluoreszenzaufnahmen in Kombination mit elektrophysiologischen und molekularbiologischen Techniken zeigten, dass:
1) Die generierten human iPSCs vollständig reprogrammierte humane Stammzellen sind, die die typischen Merkmale der embryonalen Stammzell-Morphologie, des Wachstumsverhaltes, der Fähigkeit in verschiedenste Zelltypen des menschlichen Körpers zu differenzieren und der Expression Pluripotenz-assoziierter Marker aufweisen.
2) Der neuronale Subtyp, der aus unseren Stammzellen generiert wurde, die typischen Charakteristiken medianer und dorsaler 5-HT Neurone aufweist und Synapsen formt, verdeutlicht durch die Expression prä- und postsynaptischer Proteine.
3) Selbst wenn die SLC2A3 CNVs einen Einfluss auf die basale Gen- und Proteinexpression haben, zeigte sich jedoch in humanen iPSC-erzeugten Neuronen keine signifikante Veränderung der Gen- und Proteinexpression aufgrund hyper- und hypoglykämischer Konditionen, noch der Ansammlung der Proteine, die mit der Formation der Synapsen assoziiert sind.
Background
Attention‐deficit/hyperactivity disorder (
ADHD
) is a common, highly heritable neurodevelopmental disorder with profound cognitive, behavioral, and psychosocial impairments with ...persistence across the life cycle. Our initial genome‐wide screening approach for copy number variants (
CNV
s) in
ADHD
implicated a duplication of
SLC
2A3
, encoding glucose transporter‐3 (
GLUT
3).
GLUT
3 plays a critical role in cerebral glucose metabolism, providing energy for the activity of neurons, which, in turn, moderates the excitatory–inhibitory balance impacting both brain development and activity‐dependent neural plasticity. We therefore aimed to provide additional genetic and functional evidence for
GLUT
3 dysfunction in
ADHD
.
Methods
Case–control association analyses of
SLC
2A3
single‐nucleotide polymorphisms (
SNP
s) and
CNV
s were conducted in several European cohorts of patients with childhood and adult
ADHD
(
SNP
,
n
= 1,886 vs. 1,988;
CNV
,
n
= 1,692 vs. 1,721). These studies were complemented by
SLC
2A3
expression analyses in peripheral cells, functional
EEG
recordings during neurocognitive tasks, and ratings of food energy content.
Results
Meta‐analysis of all cohorts detected an association of
SNP
rs12842 with
ADHD
. While
CNV
analysis detected a population‐specific enrichment of
SLC
2A3
duplications only in German
ADHD
patients, the
CNV
+ rs12842 haplotype influenced
ADHD
risk in both the German and Spanish cohorts. Duplication carriers displayed elevated
SLC
2A3
m
RNA
expression in peripheral blood cells and altered event‐related potentials reflecting deficits in working memory and cognitive response control, both endophenotypic traits of
ADHD
, and an underestimation of energy units of high‐caloric food.
Conclusions
Taken together, our results indicate that both common and rare
SLC
2A3
variation impacting regulation of neuronal glucose utilization and energy homeostasis may result in neurocognitive deficits known to contribute to
ADHD
risk.
PDF
