Natrium-Glukose Transporter (SGLT) gehören zur „solute carrier 5“ (SLC5) Familie, die sich durch einen sekundär aktiven, natriumabhängigen Transport von Zuckern und an-deren Molekülen nach intrazellulär auszeichnen. Die durch das Gen SLC5A4 kodierte Isoform SGLT3 transportiert dagegen keinen Zucker, sondern verhält sich als Glukosesensor, der nach Bindung seiner Liganden eine Membrandepolarisation induziert. In genomweiten Exomsequenzierungsstudien (whole exome sequencing, WES) mehrerer erweiterter Stammbäume mit hoher Prävalenz des Aufmerksamkeitsdefizit-/Hyperaktivitätssyndroms (ADHS) wurde im Vorfeld eine ATG-Tripletdeletion in SLC5A4 identifiziert, die zum Verlust einer Aminosäure (ΔM500) in SGLT3 führt und zumindest partiell mit dem klinischen Phänotyp kosegregiert. In der vorliegenden Arbeit wurde die zentralnervöse Expression von SGLT3 auf RNA- Ebene mittels Reverse-Transkriptase PCR sowie real-time PCR aus humanen Gesamt-RNAs nachgewiesen. Dabei konnte eine ubiquitäre Expression im Gehirn mit relativ erhöhter Expression unter anderem in Striatum und Hypothalamus, deren Dysfunktion in der Pathogenese des ADHS impliziert wurde, gezeigt werden. Da Mutationen in homologen Domänen der eng strukturverwandten Isoformen SGLT1 und SGLT2 sowohl intestinale als auch renale Funktionen schwer beeinträchtigen, wurden in dieser Arbeit funktionelle Charakteristika sowohl des wildtypischen als auch der ΔM500 und der benachbarten ΔI501 Deletionsvariante von SGLT3 mittels Zwei-Elektroden Spannungs- und Stromklemme in entsprechend cRNA-injizierten Xenopus laevis Oozyten untersucht. Der hochpotente SGLT3-spezifische Iminozuckeragonist 1-Desoxynojirimycin (DNJ) induzierte an SGLT3-exprimierenden Oozyten in sauren Bedingungen etwa dreifach größere Kationeneinströme als D-Glukose, was sowohl im Spannungsklemmen-, und anhand einer entsprechenden Membrandepolarisation im Stromklemmenmodus gezeigt wurde. Die mit der ΔM500 bzw. ΔI501 Variante injizierten Oozyten dagegen zeigten in den maximalen Aktivierungsbedingungen um 92% bzw. 96% (p<0,01) reduzierte Kationeneinströme, sodass diese als hochgradig schädliche „Loss of Function“ Mutationen in SGLT3 charakterisiert wurden. Dieser Befund wurde mittels bioinformatischer in-silico Effektvorhersage validiert. Um Konsequenzen der Sequenzalteration auf den Membraneinbau der Transporter zu untersuchen, wurden die mit einem gelb fluoreszierenden Farbstoff (YFP) markierten Transporter in Oozytenmembranen mittels Laser-Scanning Mikroskop nachgewiesen und die jeweiligen Mengen der Konstrukte anhand der Fluoreszenzintensitäten quantifiziert. Dabei zeigte sich eine um 53% bzw. 42% (p<0,01) reduzierte Menge der mutierten Konstrukte ΔM500 bzw. ΔI501 in der Membran, was zusätzliche schädliche Effekte der Mutationen auf das sogenannte Membrantargeting der Transporter belegt. Zusammenfassend demonstrieren die Ergebnisse dieser Arbeit, dass die ΔM500 Variante von SGLT3, welcher in ADHS-relevanten Hirnarealen exprimiert wird, dessen sub-stratinduzierte Natriumleitfähigkeit aufhebt und den Membraneinbau beeinträchtigen könnte, was in Wechselwirkung mit anderen genetischen ADHS Risikovarianten das Risiko für ADHS in Mutationsträgern beeinflussen kann.

Diese Arbeit widmet sich der Untersuchung einer Kopienzahlvariante (CNV) im Erbgut, die zu einer genomischen Duplikation des SLC2A3-Gens führt. Die Auswirkungen der SLC2A3- Duplikation wurden im Zellkulturmodell und durch bildgebende Verfahren untersucht. Für die SLC2A3-Duplikation konnte eine populationsspezifische Assoziation mit ADHS gezeigt werden (Merker et al. 2017). SLC2A3 kodiert für den neuronalen Glukosetransporter GLUT3, der u.a. Prozesse der Neurotransmitterfreisetzung und Synaptogenese vermittelt und daher wichtig für die Hirnreifung ist. Mögliche Endpunkte für Endophänotypen, die auf einem alterierten Glukosemetabolismus basieren, sind dysfunktionale Hungerregulationsmechanismen ebenso wie eine veränderte neurale Reaktivität gegenüber emotionalen Stimuli und Belohnungsreizen. In zwei peripheren Zellmodellen konnte gezeigt werden, dass die SLC2A3-Duplikation Gen-Dosis-abhängig zu einer Steigerung der basalen SLC2A3-mRNA Expression führt. Ein Expressionsunterschied auf Proteinebene konnte jedoch nicht gefunden werden. Metabolischer Zellstress durch Aushungern der Zellkulturen und eine niedrige Glukosekonzentration im Zellkulturmedium führten zu einer signifikanten Erhöhung des schon unter basalen Bedingungen vorhandenen SLC2A3-Expressionsunterschiedes zwischen Duplikations- und Kontrollzelllinien. Dies deutet darauf hin, dass die SLC2A3-Duplikation bei verminderter zellulärer Energiezufuhr zu einer Überkompensation der Glukoseaufnahme führt. In einer fMRT-Untersuchung wurden erwachsene ADHS-Patienten mit SLC2A3- Duplikation mit ADHS-Patienten und gesunden Kontrollen mit jeweils 2 Genkopien hinsichtlich ereigniskorrelierter neuraler Aktivität als Antwort auf emotionale Stimuli und Essensreize verglichen. Es konnte gezeigt werden, dass die SLC2A3-Duplikation zu einer veränderten Reaktivität gegenüber hochkalorischen Essensreizen führt, was sich in einem durch maschinelles Lernen identifizierten multivariaten neuralen Antwortmuster und einer relativen Unterschätzung des Kaloriengehaltes hochkalorischer Nahrung zeigt. Bei der univariaten Gesamthirn-Analyse der Bilddaten wurden keine signifikanten Gruppenunterschiede gefunden, was darauf hinweist, dass unter den gewählten Versuchsbedingungen keine fokal umschriebenen Gruppenunterschiede der Hirnaktivierung bestehen. Diese Arbeit zeigt, dass die SLC2A3-Duplikation zu einer Erhöhung der SLC2A3- Genexpression mit bisher unbekannten Auswirkungen auf nachgeschaltete Stoffwechselwege und zu einem komplex veränderten neuralen Antwortmuster führt, das durch einen linearen Zusammenhang nicht zu beschreiben ist. Weitere Untersuchungen auf Zellebene und eine Erweiterung der bildgebenden Verfahren könnten zu einer besseren Einordnung der SLC2A3- Duplikation bezüglich ihres Anteils an der endophänotypischen Varianz der ADHS führen.

Disruptions in brain serotonin (5-hydroxytryptamine, 5-HT) signaling pathways have been associated with etiology and pathogenesis of various neuropsychiatric disorders, but specific neural mechanisms of 5-HT function are yet to be fully elucidated. Tryptophan hydroxylase 2 (TPH2) is the rate-limiting enzyme for brain 5-HT synthesis. Therefore, in this study a tamoxifen (Tam)-inducible cre-mediated conditional gene (Tph2) knockout in adult mouse brain (Tph2icKO) has been established to decipher the specific role of brain 5-HT in the regulation of behavior in adulthood. Immunohistochemistry and high-performance liquid chromatography (HPLC) were used first to test the efficacy of Tam-inducible inactivation of Tph2 and consequential reduction of 5-HT in adult mouse brain. Tam treatment resulted in ≥90% reduction in the number of 5-HT immuno-reactive cells in the anterior raphe nuclei. HPLC revealed a significant reduction in concentration of 5-HT and its metabolite 5-hydroxyindole acetic acid (5-HIAA) in selected brain regions of Tph2icKO, indicating the effectiveness of the protocol used. Second, standard behavioral tests were used to assess whether reduced brain 5-HT concentrations could alter anxiety-, fear- and depressive-like behavior in mice. No altered anxiety- and depressive-like behaviors were observed in Tph2icKO compared to control mice (Tph2CON) in all indices measured, but Tph2icKO mice exhibited intense and sustained freezing during context-dependent fear memory retrieval. Tph2icKO mice also exhibited locomotor hyperactivity in the aversive environments, such as the open field, and consumed more food and fluid than Tph2CON mice. Lastly, the combined effect of maternal separation (MS) stress and adult brain 5-HT depletion on behavior was assessed in male and female mice. Here, MS stress, 5-HT depletion and their interaction elicited anxiety-like behavior in a sex-dependent manner. MS reduced exploratory behavior in both male and female mice. Reduced 5-HT enhanced anxiety in female, but not in male mice. Furthermore, expression of genes related to the 5-HT system and emotionality (Tph2, Htr1a, Htr2a, Maoa and Avpr1a) was assessed by performing a quantitative real-time PCR. In Tph2icKO mice there was a reduction in expression of Tph2 in the raphe nuclei of both male and female mice. Interaction between MS stress and 5-HT deficiency was detected showing increased Htr2a and Maoa expression in raphe and hippocampus respectively of female mice. In male mice, MS stress and 5-HT depletion interaction effects reduced Avpr1a expression in raphe, while the expression of Htr1a, Htr2a and Maoa was differentially altered by 5-HT depletion and MS in various brain regions.

Neurodevelopmental disorders, including attention-deficit/hyperactivity disorder (ADHD) and autism spectrum disorder (ASD) are disorders of mostly unknown etiopathogenesis, for which both genetic and environmental influences are expected to contribute to the phenotype observed in patients. Changes at all levels of brain function, from network connectivity between brain areas, over neuronal survival, synaptic connectivity and axonal growth, down to molecular changes and epigenetic modifications are suspected to play a key roles in these diseases, resulting in life-long behavioural changes. Genome-wide association as well as copy-number variation studies have linked cadherin-13 (CDH13) as a novel genetic risk factor to neuropsychiatric and neurodevelopmental disorders. CDH13 is highly expressed during embryonic brain development, as well as in the adult brain, where it is present in regions including the hippocampus, striatum and thalamus (among others) and is upregulated in response to chronic stress exposure. It is however unclear how CDH13 interacts with environmentally relevant cues, including stressful triggers, in the formation of long-lasting behavioural and molecular changes. It is currently unknown how the environment influences CDH13 and which long term changes in behaviour and gene expression are caused by their interaction. This work therefore investigates the interaction between CDH13 deficiency and neonatal maternal separation (MS) in mice with the aim to elucidate the function of CDH13 and its role in the response to early-life stress (ELS). For this purpose, mixed litters of wild-type (Cdh13+/+), heterozygous (Cdh13+/-) and homozygous knockout (Cdh13-/-) mice were maternally separated from postnatal day 1 (PN1) to postnatal day 14 (PN14) for 3 hours each day (180MS; PN1-PN14). In a first series of experiments, these mice were subjected to a battery of behavioural tests starting at 8 weeks of age in order to assess motor activity, memory functions as well as measures of anxiety. Subsequently, expression of RNA in various brain regions was measured using quantitativ real-time polymerase chain reaction (qRT-PCR). A second cohort of mice was exposed to the same MS procedure, but was not behaviourally tested, to assess molecular changes in hippocampus using RNA sequencing. Behavioural analysis revealed that MS had an overall anxiolytic-like effect, with mice after MS spending more time in the open arms of the elevated-plus-maze (EPM) and the light compartment in the light-dark box (LDB). As a notable exception, Cdh13-/- mice did not show an increase of time spent in the light compartment after MS compared to Cdh13+/+ and Cdh13+/- MS mice. During the Barnes-maze learning task, mice of most groups showed a similar ability in learning the location of the escape hole, both in terms of primary latency and primary errors. Cdh13-/- control (CTRL) mice however committed more primary errors than Cdh13-/- MS mice. In the contextual fear conditioning (cFC) test, Cdh13-/- mice showed more freezing responses during the extinction recall, indicating a reduced extinction of fear memory. In the step-down test, an impulsivity task, Cdh13-/- mice had a tendency to wait longer before stepping down from the platform, indicative of more hesitant behaviour. In the same animals, qRT-PCR of several brain areas revealed changes in the GABAergic and glutamatergic systems, while also highlighting changes in the gatekeeper enzyme Glykogensynthase-Kinase 3 (Gsk3a), both in relation to Cdh13 deficiency and MS. Results from the RNA sequencing study and subsequent gene-set enrichment analysis revealed changes in adhesion and developmental genes due to Cdh13 deficiency, while also highlighting a strong link between CDH13 and endoplasmatic reticulum function. In addition, some results suggest that MS increased pro-survival pathways, while a gene x environment analysis showed alterations in apoptotic pathways and migration, as well as immune factors and membrane metabolism. An analysis of the overlap between gene and environment, as well as their interaction, highlighted an effect on cell adhesion factors, underscoring their importance for adaptation to the environment. Overall, the stress model resulted in increased stress resilience in Cdh13+/+ and Cdh13+/- mice, a change absent in Cdh13-/- mice, suggesting a role of CDH13 during programming and adaptation to early-life experiences, that can results in long-lasting consequences on brain functions and associated behaviours. These changes were also visible in the RNA sequencing, where key pathways for cell-cell adhesion, neuronal survival and cell-stress adaptation were altered. In conclusion, these findings further highlight the role of CDH13 during brain development, while also shedding light on its function in the adaptation and response during (early life) environmental challenges.

Early life stress, including exposure to prenatal stress (PS), has been shown to affect the developing brain and induce severe effects on emotional health in later life, concomitant with an increased risk for psychopathology. However, some individuals are more vulnerable to early-life stress, while others adapt successfully, i.e. they are resilient and do not succumb to adversity. The molecular substrates promoting resilience in some individuals and vulnerability in other individuals are as yet poorly investigated. A polymorphism in the serotonin transporter gene (5­HTT/SLC6A4) has been suggested to play a modulatory role in mediating the effects of early-life adversity on psychopathology, thereby rendering carriers of the lower-expressing short (s)-allele more vulnerable to developmental adversity, while long (l)-allele carriers are relatively resilient. The molecular mechanisms underlying this gene x environment interaction (GxE) are not well understood, however, epigenetic mechanisms such as DNA methylation and histone modifications have been discussed to contribute as they are at the interface of environment and the genome. Moreover, developmental epigenetic programming has also been postulated to underlie differential vulnerability/resilience independent of genetic variation. The present work comprises two projects investigating the effects of prenatal maternal restraint stress in 5-HTT deficient mice. In the first study, we examined to which extent previously observed changes in behavior and hippocampal gene expression of female 5-Htt+/- prenatally stressed (PS) offspring were associated with changes in DNA methylation patterns. Additionally, we investigated the expression of genes involved in myelination in hippocampus and amygdala of those animals using RT-qPCR. The genome-wide hippocampal DNA methylation screening was performed using methylated-DNA immunoprecipitation (MeDIP) on Affymetrix GeneChip® Mouse Promoter 1.0R arrays. In order to correlate individual gene-specific DNA methylation, mRNA expression and behavior, we used hippocampal DNA from the same mice as assessed before. 5-Htt genotype, PS and their interaction differentially affected the DNA methylation signature of numerous genes, a part of which were also differentially expressed. More specifically, we identified a differentially methylated region in the Myelin basic protein (Mbp) gene, which was associated with Mbp expression in a 5-Htt-, PS- and 5-Htt x PS-dependent manner. Subsequent fine-mapping linked the methylation status of two specific CpG sites in this region to Mbp expression and anxiety-related behavior. We furthermore found that not only the expression of Mbp but of large gene set associated with myelination was affected by a 5-Htt x PS interaction in a brain-region specific manner. In conclusion, hippocampal DNA methylation patterns and expression profiles of female PS 5-Htt+/- mice suggest that distinct molecular mechanisms, some of which are associated with changes in gene promoter methylation, and processes associated with myelination contribute to the behavioral effects of the 5-Htt genotype, PS exposure, and their interaction. In the second study, we aimed at investing the molecular substrates underlying resilience to PS. For this purpose, we exposed 5-Htt+/+ dams to the same restraint stress paradigm and investigated the effects of PS on depression- and anxiety-like behavior and corticosterone (CORT) secretion at baseline and after acute restraint stress in female 5-Htt+/+ and 5-Htt+/- offspring. We found that PS affected the offspring’s social behavior in a negative manner. When specifically examining those PS animals, we grouped the PS offspring of each genotype into a social, resilient and an unsocial, vulnerable group. While anxiety-like behavior in the EPM was reduced in unsocial, but not social, PS 5-Htt+/+ animals when compared to controls, this pattern could not be found in animals of the other genotype, indicating that social anxiety and state anxiety in the EPM were independent of each other. We then assessed genome-wide hippocampal gene expression profiles using mRNA sequencing in order to identify pathways and gene ontology (GO) terms enriched due to 5-Htt genotype (G), PS exposure (E) and their interaction (GxE) as well as enriched in social, but not unsocial, PS offspring, and vice versa. Numerous genes were affected by 5-Htt genotype, PS and most of all a GxE-interaction. Enrichment analysis using enrichr identified that the genotype affected mitochondrial respiration, while GxE-interaction-affected processes associated primarily with myelination and chromatin remodeling. We furthermore found that 5-Htt+/- mice showed profound expression changes of numerous genes in a genomic region located 10 mio kb upstream of the 5 Htt locus on the same chromosome. When looking at social vs. unsocial mice, we found that a much higher number of genes was regulated in 5 Htt+/- animals than in 5-Htt+/+ animals, reflecting the impact of GxE-interaction. Double the number of genes was regulated in social PS vs. control mice when compared to unsocial PS vs. control in both genotypes, suggesting that the successful adaption to PS might have required more active processes from the social group than the reaction to PS from the unsocial group. This notion is supported by the up-regulation of mitochondrial respiration in social, but not in unsocial, PS 5-Htt+/- mice when compared to controls, as those animals might have been able to raise energy resources the unsocial group was not. Next to this, processes associated with myelination seemed to be down-regulated in social 5-Htt+/- mice, but not in unsocial animals, when compared to controls. Taken together, PS exposure affected sociability and anxiety-like behavior dependent on the 5-Htt genotype in female offspring. Processes associated with myelination and epigenetic mechanisms involved in chromatin remodeling seemed be affected in a GxE-dependent manner in the hippocampus of these offspring. Our transcriptome data furthermore suggest that mitochondrial respiration and, with this, energy metabolism might be altered in 5-Htt+/- offspring when compared to 5-Htt+/+ offspring. Moreover, myelination and mitochondrial respiration might contribute to resilience towards PS exposure in 5-Htt+/- offspring, possibly by affecting brain connectivity and energy capabilities.