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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.
The transport of glucose across the cell plasma membrane is vital to most mammalian cells. The glucose transporter (GLUT; also called SLC2A) family of transmembrane solute carriers is responsible for this function in vivo. GLUT proteins encompass 14 different isoforms in humans with different cell type-specific expression patterns and activities. Central to glucose utilization and delivery in the brain is the neuronally expressed GLUT3. Recent research has shown an involvement of GLUT3 genetic variation or altered expression in several different brain disorders, including Huntington’s and Alzheimer’s diseases. Furthermore, GLUT3 was identified as a potential risk gene for multiple psychiatric disorders. To study the role of GLUT3 in brain function and disease a more detailed knowledge of its expression in model organisms is needed. Zebrafish (Danio rerio) has in recent years gained popularity as a model organism for brain research and is now well-established for modeling psychiatric disorders. Here, we have analyzed the sequence of GLUT3 orthologs and identified two paralogous genes in the zebrafish, slc2a3a and slc2a3b. Interestingly, the Glut3b protein sequence contains a unique stretch of amino acids, which may be important for functional regulation. The slc2a3a transcript is detectable in the central nervous system including distinct cellular populations in telencephalon, diencephalon, mesencephalon and rhombencephalon at embryonic and larval stages. Conversely, the slc2a3b transcript shows a rather diffuse expression pattern at different embryonic stages and brain regions. Expression of slc2a3a is maintained in the adult brain and is found in the telencephalon, diencephalon, mesencephalon, cerebellum and medulla oblongata. The slc2a3b transcripts are present in overlapping as well as distinct regions compared to slc2a3a. Double in situ hybridizations were used to demonstrate that slc2a3a is expressed by some GABAergic neurons at embryonic stages. This detailed description of zebrafish slc2a3a and slc2a3b expression at developmental and adult stages paves the way for further investigations of normal GLUT3 function and its role in brain 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.
Converging evidence suggests a role of serotonin (5-hydroxytryptamine, 5-HT) and tryptophan hydroxylase 2 (TPH2), the rate-limiting enzyme of 5-HT synthesis in the brain, in modulating long-term, neurobiological effects of early-life adversity. Here, we aimed at further elucidating the molecular mechanisms underlying this interaction, and its consequences for socio-emotional behaviors, with a focus on anxiety and social interaction. In this study, adult, male Tph2 null mutant (Tph2\(^{-/-}\)) and heterozygous (Tph2\(^{+/-}\)) mice, and their wildtype littermates (Tph2\(^{+/+}\)) were exposed to neonatal, maternal separation (MS) and screened for behavioral changes, followed by genome-wide RNA expression and DNA methylation profiling. In Tph2\(^{-/-}\) mice, brain 5-HT deficiency profoundly affected socio-emotional behaviors, i.e., decreased avoidance of the aversive open arms in the elevated plus-maze (EPM) as well as decreased prosocial and increased rule breaking behavior in the resident-intruder test when compared to their wildtype littermates. Tph2\(^{+/-}\) mice showed an ambiguous profile with context-dependent, behavioral responses. In the EPM they showed similar avoidance of the open arm but decreased prosocial and increased rule breaking behavior in the resident-intruder test when compared to their wildtype littermates. Notably, MS effects on behavior were subtle and depended on the Tph2 genotype, in particular increasing the observed avoidance of EPM open arms in wildtype and Tph2\(^{+/-}\) mice when compared to their Tph2\(^{-/-}\) littermates. On the genomic level, the interaction of Tph2 genotype with MS differentially affected the expression of numerous genes, of which a subset showed an overlap with DNA methylation profiles at corresponding loci. Remarkably, changes in methylation nearby and expression of the gene encoding cholecystokinin, which were inversely correlated to each other, were associated with variations in anxiety-related phenotypes. In conclusion, next to various behavioral alterations, we identified gene expression and DNA methylation profiles to be associated with TPH2 inactivation and its interaction with MS, suggesting a gene-by-environment interaction-dependent, modulatory function of brain 5-HT availability.
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.
Aggressiveness is a behavioral trait that has the potential to be harmful to individuals and society. With an estimated heritability of about 40%, genetics is important in its development. We performed an exploratory genome-wide association (GWA) analysis of childhood aggressiveness in attention deficit hyperactivity disorder (ADHD) to gain insight into the underlying biological processes associated with this trait. Our primary sample consisted of 1,060 adult ADHD patients (aADHD). To further explore the genetic architecture of childhood aggressiveness, we performed enrichment analyses of suggestive genome-wide associations observed in aADHD among GWA signals of dimensions of oppositionality (defiant/vindictive and irritable dimensions) in childhood ADHD (cADHD). No single polymorphism reached genome-wide significance (P<5.00E-08). The strongest signal in aADHD was observed at rs10826548, within a long noncoding RNA gene (beta = -1.66, standard error (SE) = 0.34, P = 1.07E-06), closely followed by rs35974940 in the neurotrimin gene (beta = 3.23, SE = 0.67, P = 1.26E-06). The top GWA SNPs observed in aADHD showed significant enrichment of signals from both the defiant/vindictive dimension (Fisher's P-value = 2.28E-06) and the irritable dimension in cADHD (Fisher's P-value = 0.0061). In sum, our results identify a number of biologically interesting markers possibly underlying childhood aggressiveness and provide targets for further genetic exploration of aggressiveness across psychiatric disorders.
Sodium-glucose transporters (SGLT) belong to the solute carrier 5 family, which is characterized by sodium dependent transport of sugars and other solutes. In contrast, the human SGLT3 (hSGLT3) isoform, encoded by SLC5A4, acts as a glucose sensor that does not transport sugar but induces membrane depolarization by Na\(^{+}\) currents upon ligand binding. Whole-exome sequencing (WES) of several extended pedigrees with high density of attention-deficit/hyperactivity disorder (ADHD) identified a triplet ATG deletion in SLC5A4 leading to a single amino acid loss (ΔM500) in the hSGLT3 protein imperfectly co-segregating with the clinical phenotype of ADHD. Since mutations in homologous domains of hSGLT1 and hSGLT2 were found to affect intestinal and renal function, respectively, we analyzed the functional properties of hSGLT3[wt] and [ΔM500] by voltage clamp and current clamp recordings from cRNA-injected Xenopus laevis oocytes.
The cation conductance of hSGLT3[wt] was activated by application of glucose or the specific agonist 1-desoxynojirimycin (DNJ) as revealed by inward currents in the voltage clamp configuration and cell depolarization in the current clamp mode. Almost no currents and changes in membrane potential were observed when glucose or DNJ were applied to hSGLT3[ΔM500]-injected oocytes, demonstrating a loss of function by this amino acid deletion in hSGLT3. To monitor membrane targeting of wt and mutant hSGLT3, fusion constructs with YFP were generated, heterologously expressed in Xenopus laevis oocytes and analyzed for membrane fluorescence by confocal microscopy. In comparison to hSGLT3[wt] the fluorescent signal of mutant [ΔM500] was reduced by 43% indicating that the mutant phenotype might mainly result from inaccurate membrane targeting. As revealed by homology modeling, residue M500 is located in TM11 suggesting that in addition to the core structure (TM1-TM10) of the transporter, the surrounding TMs are equally crucial for transport/sensor function.
In conclusion, our findings indicate that the deletion [ΔM500] in hSGLT3 inhibits membrane targeting and thus largely disrupts glucose-induced sodium conductance, which may, in interaction with other ADHD risk-related gene variants, influence the risk for ADHD in deletion carriers.
Nonalcoholic fatty liver disease, induced by a Western diet (WD), evokes central and peripheral inflammation that is accompanied by altered emotionality. These changes can be associated with abnormalities in social behaviour, hippocampus-dependent cognitive functions, and metabolism. Female C57BL/6J mice were fed with a regular chow or with a WD containing 0.2% of cholesterol and 21% of saturated fat for three weeks. WD-treated mice exhibited increased social avoidance, crawl-over and digging behaviours, decreased body-body contacts, and hyperlocomotion. The WD-fed group also displayed deficits in hippocampal-dependent performance such as contextual memory in a fear conditioning and pellet displacement paradigms. A reduction in glucose tolerance and elevated levels of serum cholesterol and leptin were also associated with the WD. The peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1a) mRNA, a marker of mitochondrial activity, was decreased in the prefrontal cortex, hippocampus, hypothalamus, and dorsal raphe, suggesting suppressed brain mitochondrial functions, but not in the liver. This is the first report to show that a WD can profoundly suppress social interactions and induce dominant-like behaviours in naïve adult mice. The spectrum of behaviours that were found to be induced are reminiscent of symptoms associated with autism, and, if paralleled in humans, suggest that a WD might exacerbate autism spectrum disorder.