Дисертації з теми "IPSC-derived neurons"

Parkinson's disease (PD) is characterised by the loss of dopaminergic neurons in the Substantia Nigra pars compacta in the midbrain and the presence of intracellular aggregates, known as Lewy bodies (LBs), in the surviving neurons. The aetiology of PD is unknown but a causative role for α-Synuclein (SNCA) has been proposed. Although the function of αSyn is not well understood, a number of pathological mechanisms associated with αSyn toxicity have been proposed. In this study, nine induced pluripotent stem cells (iPSCs) lines from healthy individuals and PD patients carrying the A53T SNCA mutation or a triplication of SNCA were differentiated to dopaminergic neurons (iDAn). All iPSC lines differentiated with similar efficiency to iDAn, indicating that they could be used for phenotypic analysis. Quantification of αSyn expression showed increased αSyn intracellular staining and the novel detection of increased αSyn oligomerization in PD iDAn. Analysis of mitochondrial respiration found a decrease in basal respiration, maximal respiration, ATP production and spare capacity in PD iDAn, but not in undifferentiated iPSCs, indicating the cell-type specificity of these defects. Decreased phosphorylation of dynamin-1-like protein at Ser616 (DRP1^Ser616) and increased levels of Peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) in A53T SNCA iDAn suggest a new pathological mechanism linking αSyn to the imbalance in mitochondria homeostasis. Markers of endoplasmic reticulum (ER) stress were found to be up-regulated, along with increased β- Glucocerebrosidase (GBA) activity, perturbation of autophagy and decreased expression of fatty acids binding protein 7 (FAPB7) in PD iDAn. Lastly, lentiviral vectors for RNAi-mediated knockdown of αSyn were developed and these reduced αSyn protein levels in iDAn, resulting in increased expression of FABP7. These results describe a novel functional link between αSyn and FABP7. This work demonstrates that iDAn are a promising and relevant in vitro cell model for studying cellular dysfunctions in PD pathology, and the phenotypic analysis of A53T SNCA and SNCA triplication iDAn enabled the detection of novel pathological mechanisms associated with PD.

Parkinson's disease (PD) is a common neurodegenerative disorder, characterised by preferential loss of ventral midbrain dopaminergic (vmDA) neurons in the substantia nigra pars compacta (SNc). The majority of PD cases have unknown aetiology; however, between 5-10% arise due to known genetic mutations, the most common of which are found in the LRRK2 gene. LRRK2 is expressed in neurons and glia in the human brain; therefore, cell-autonomous and/or non-cell autonomous effects may participate in LRRK2-mutation-mediated degeneration of vmDA neurons. This study set out to understand the effects of LRRK2 mutations on human vmDA neurons and midbrain astrocytes, and to shed new light on the mechanisms of PD pathogenesis. To achieve this goal, differentiation protocols were generated to produce vmDA neurons and midbrain patterned (MP) astrocytes from induced pluripotent stem cells (iPSCs). iPSCs from patients carrying the LRRK2-G2019S mutation were differentiated into both cell types, and hypothesis-driven analysis of cellular functions including autophagy, mitochondrial respiration, glycolysis, and cellular migration was conducted; however, no disease phenotypes were observed. Following this, proteomics and transcriptomics techniques were then used to analyse the effects of the LRRK2-G2019S mutation in an unbiased manner. In the iPSC-derived MPastrocyte cultures, this technique highlighted stochastic X-chromosome reactivation events that led to difficulties in interpreting the resulting data; however, in the iPSC-derived vmDA-neuron cultures, LRRK2-G2019S-mediated inhibition of endocytosis and axon guidance was identified. These findings were found to be consistent in iPSC-derived vmDA-neuron cultures carrying the LRRK2-R1441C mutation, suggesting that these two mutations exert their pathogenic effects through similar mechanisms. Finally, phosphoproteomics analysis of iPSC-derived vmDA-neuron cultures was conducted to identify bone fide LRRK2-kinase substrates. Eleven potential LRRK2- kinase substrates were identified, nine of which have been previously shown to participate in endocytic vesicle trafficking, neurite outgrowth, and synaptic function. The findings of this study suggest that LRRK2 has neuron-specific functions, and that its mutations contribute to neurodegeneration in a cell-autonomous manner.

Rett syndrome is a severe neurodevelopmental disorder. The condition affects approximately one in every 10.000 females and is only rarely seen in males. Causative mutations in the transcriptional regulator MeCP2 have been identified in more than 95% of classic Rett patients; mutations in CDKL5 are responsible for the early onset seizures Rett variant and mutations in FOXG1 gene lead to the congenital Rett variant. To shed light on molecular mechanisms underlying Rett syndrome onset and progression in disease-relevant cells, we took advantage of the breakthrough genetic reprogramming technology and we investigated changes in iPSC-derived neurons from patients with different MECP2 and FOXG1 mutations and in the brain of Foxg1+/- mice. In total brains from Foxg1+/ − mutants we noticed a statistically significant overexpression of a group of neuropeptides expressed in the basal ganglia, cortex, hippocampus and hypothalamus: Oxytocin (Oxt), Arginine vasopressin (Avp) and Neuronatin (Nnat).Moreover, in iPSC-derived neuronal precursors and neurons mutated in FOXG1 and in Foxg1+/− mouse embryonic brain (E11.5) compared to wild type controls we found an increase in the expression of GluD1 and inhibitory synaptic markers, such as GAD67 and GABA AR-α1 and a decreased expression of excitatory synaptic markers, such as VGLUT1, GluA1, GluN1 and PSD-95, suggesting an excitation/inhibition imbalance in the developing brain of the congenital RTT variant. Furthermore, we investigated transcriptome changes in neurons differentiated from MECP2 mutated iPSC-derived neurons and we noticed a prominent GABAergic circuit disruption and a perturbation of cytoskeleton dynamics. In particular, in MECP2-mutated neurons we identified a significant decrease of acetylated α-tubulin which can be reverted by treatment with a selective inhibitor of HDAC6, the main α-tubulin deacetylase. Taken togheter, these findings contribute to shed light on Rett pathogenic mechanisms and provide hints for the definition of new therapeutic strategies for Rett syndrome.

The advent of human induced pluripotent stem cell (iPSC) technology has allowed for unprecedented investigation into the pathophysiology of human neurological and psychiatric diseases. Use of human iPSC-derived neural cells to study disease is complicated by the genetic heterogeneity of cell lines and diversity of differentiation protocols. Here, I address issues surrounding neuropsychiatric disease modeling with human iPSCs. Dozens of published protocols exist to differentiate iPSCs into forebrain neuronal cultures. Among the factors that distinguish these methods are: use of small molecules, monolayer vs. aggregate culture, choice of plating substrates, method of NPC isolation, and glial co-culture. Each of these factors is evaluated here, creating a resource that directly compares a variety of differentiation procedures. The most efficient and reproducible method was an embryoid aggregate differentiation protocol, including aggregate plating onto a Matrigel substrate, enzymatic neural rosette selection, and neuronal dissociation and plating onto Matrigel. This optimized protocol is used to model a schizophrenia-relevant mutation in human neural cells. Genetic and clinical association studies have identified disrupted-in-schizophrenia 1 (DISC1) as a strong candidate risk gene for major mental illness. DISC1 was initially associated with mental illness upon the discovery that its coding sequence is interrupted by a balanced chr(1;11) translocation in a Scottish family, in which the translocation cosegregates with psychiatric disorders. I investigate the functional and biochemical consequences of DISC1 interruption in human neurons using TALENs or CRISPR-Cas9 to introduce DISC1 frameshift mutations into iPSCs. I show that disease-relevant DISC1 targeting results in decreased DISC1 protein expression by nonsense-mediated decay, increases baseline Wnt signaling in neural progenitor cells, and causes a shift in neural cell fate. DISC1-dependent Wnt signaling and cell fate changes can be reversed by antagonizing the Wnt pathway during a critical window in neural progenitor development. These experiments suggest that DISC1-disruption increases Wnt signaling, which alters the balance and identity of neural progenitors, thereby subtly modifying cell fate. These studies evaluate the use of multiple differentiation procedures in neural disease modeling, shed light on the roles of DISC1 during human brain development, and further our understanding of the pathogenesis of major mental illness.
Medical Sciences

Parkinson's disease (PD) is characterised by the selective loss of dopaminergic neurons of the substantia nigra pars compacta. Patients suffer from a progressive motor disorder, defined by the presence of rigidity, resting tremor and bradykinesia. Current treatment options, relieve symptoms for a limited period, but are not curative, as the underlying molecular causes of neurodegeneration are unknown. Several causative PD mutations have been identified and could provide insight into the defective molecular pathways in PD. Multiplication or missense mutation of the SNCA gene leads to autosomal dominant PD. Alpha-synuclein, encoded by the SNCA gene, is a defining component of proteinaceous deposits found in surviving neurons in PD and a central protein in PD aetiology. Induced pluripotent stem cells (iPSCs) self-renew indefinitely and generate all germ layer lineages. Human iPSCs derived from an individual with a genetic variant known to cause disease, provide a platform to investigate the molecular basis of disease. However, genetic variation between iPSC lines can lead to functional disparities, masking or accentuating disease-specific phenotypes. Genome engineering facilitates the generation of iPSCs which differ exclusively at the locus of interest, providing a genetically stable cellular model. iPSCs from an individual with a SNCA missense mutation, G51D, and an unaffected relative were characterised, demonstrating ex vivo pluripotency was established and dopaminergic neurons could be derived. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system was exploited to introduce the G51D mutation into control iPSCs. Changes in dopamine turnover and protein metabolism were detected after differentiation of CRISPR-Cas9 generated iPSCs, now harbouring the heterozygote G51D mutation. A CRISPR-Cas9 G51D homozygote iPSC clone was generated and a reduction in the number of dopaminergic neurons produced observed. This study demonstrates human iPSCs can be used to detect phenotypic differences in specialised cells, despite the latency of PD, and before overt neurodegeneration.

IPSC-derived neurons and astrocytes: a novel patient-specific model to study the pre-degenerative molecular alteration in Parkinson's Disease
IPSC-derived neurons and astrocytes: a novel patient-specific model to study the pre-degenerative molecular alteration in Parkinson's Disease

Parkinson's disease (PD) primarily manifests as loss of motor control through the degeneration of nigrostriatal dopaminergic neurons. The microtubule-associated protein tau (MAPT) locus is highly genetically associated with PD, wherein the H1 haplotype confers disease risk and the H2 haplotype is protective. As this haplotype variation does not alter the amino acid sequence, disease risk may be conferred by altered gene expression, either of total MAPT or of specific isoforms, of which there are six in adult human brain. To investigate haplotype-specific control of MAPT expression in the neurons that die in PD, induced pluripotent stem cells (iPSCs) from H1/H2 heterozygous individuals were differentiated into dopaminergic neuronal cultures that expressed all six mature isoforms of MAPT after six months' maturation. A reporter construct using the human tyrosine hydroxylase locus was also generated to identify human dopaminergic neurons in mixed cultures. Haplotype-specific differences in the inclusion of exon 3 and total MAPT were observed in iPSC-derived dopaminergic neuronal cultures and a novel variant in MAPT intron 10 increased the inclusion of exon 10 by two-fold. RNA interference tools were generated to knockdown total MAPT or specific isoforms, wherein knockdown of the 4-repeat isoform of tau protein increased the velocity of axonal transport in iPSC-derived neurons. MAPT knockdown also reduced p62 levels, suggesting an impact of tau on macroautophagy, likely through altered axonal transport. These results demonstrate how variation at a disease susceptibility locus can alter gene expression, thereby impacting on neuronal function.

Parkinson’s disease (PD) is the second most common age-related neurodegenerative condition. It is neuropathologically characterised by the presence of Lewy pathology and the degeneration of the midbrain dopaminergic neurons from the substantia nigra pars compacta. Lewy pathology mainly consists of filamentous aggregated alpha-synuclein and familial forms of PD can be caused by genetic alternations in the SNCA gene encoding alpha-synuclein. Alpha-synuclein is primarily localised to neuronal presynaptic terminals and has been implicated in the maintenance of synaptic function. Studies have proposed that it regulates the docking, fusion, clustering and trafficking of neurotransmitter-loaded presynaptic vesicles. Nowadays, it is possible to model PD in vitro by obtaining adult somatic cells from patients, reprogramming them into induced pluripotent stem cells (iPSCs), and differentiating iPSCs into neurons. For this project, iPSCs derived from two PD patients, one harbouring the A53T SNCA mutation, the other a SNCA triplication, and three healthy individuals, were employed. In the initial stage, I optimised a neuronal differentiation protocol originally developed for human embryonic stem cells to produce neurons belonging to two distinct brain regions affected in PD, the forebrain and midbrain, from the available human iPSC lines. Next, I assessed the maturation of the generated neurons over time using protein expression and electrophysiological techniques. Finally, I examined PD-related phenotypes such as alpha-synuclein aggregation and release, susceptibility to cell death, and the redistribution of presynaptic proteins. All the iPSC lines used gave rise to forebrain and midbrain neuronal cultures. Maturation was similar across lines, as no consistent differences were observed in the changes of the expression of 4 repeat tau isoforms, presynaptic protein levels or electrophysiological properties over time. However, the emergence of astrocytes varied between cultures derived from distinct iPSC lines. No robust differences in alpha-synuclein release and susceptibility to cell death were detected between patient- and control-derived neurons. Apart from the presence of larger alpha-synuclein-positive puncta in patient-derived neurons, no other signs of alpha-synuclein aggregation were detected. Despite this, midbrain patient-derived neurons with a SNCA triplication exhibited a significant redistribution of presynaptic protein VAMP-2/synaptobrevin-2, which interacts with alpha-synuclein, relative to controls.

The expansion of the hexanucleotide repeat sequence GGGGCC (>30 repeats) in the first intron of C9ORF72 gene is the main genetic cause of two neurodegenerative diseases: amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The 5’ promoter of C9ORF72 gene has been found hypermethylated in 30% of C9ORF72 positive (C9+) ALS/FTLD patients and never in unexpanded patients and healthy controls. Promoter methylation seems to have a neuroprotective role from RNA toxicity and RAN translated dipeptide repeats (DPRs). The aim of this study has been to characterize C9+ ALS patient-derived induced pluripotent stem cells (iPSC) and differentiated motor neurons (iPSC-MN) correlating epigenetic marks of gene promoter and of the GC-rich repeat expansion (HRE) to C9ORF72-related pathological features (gene expression, RNA foci and DPRs). We initially studied 3 different C9+ iPSC lines which were cultured for up to 40 passages in vitro and, at each timepoint (10th, 20th, 30th and 40th passage), DNA was extracted for genetic and epigenetic characterization, RNA was obtained for Q-PCR analyses and slides were fixed for C9ORF72 RNA foci count. C9+ iPSCs were also differentiated into motor neurons for three times and iPSC-MNs were harvested for the same molecular characterization as for iPSCs. We observed a down-regulation of the two HRE-harboring mRNA isoforms (V1 and V3) both in iPSCs and iPSC-MNs when the promoter was methylated, while RNA foci number showed no correlation with methylation state. Moreover, we found that the epigenetic pattern of promoter methylation could change after C9+ iPSC reprogramming and through differentiation into iPSC-MNs. When we extended our analysis to a cohort of 8 different C9+ iPSC lines, we observed that both epigenetic marks and HRE length may influence RNA foci formation. Our study reports for the first time in C9+ iPSC and iPSC-MNs that promoter methylation can be considered a possible therapeutic target and corroborates that patient-derived cells represent a suitable model for further studies on C9ORF72-related neurodegeneration.

Heterozygous mutations in the glucocerebrosidase (GBA) gene represent the most common risk factor for Parkinson’s disease (PD), a disease in which midbrain dopaminergic neurons are preferentially vulnerable. However, the mechanisms underlying this association are still unknown, mostly due to the lack of an appropriate model of study. In this thesis, we aimed at elucidating the role of heterozygous GBA mutations in PD using a specific human induced pluripotent stem cell (hiPSC)-based model of disease. First we developed a protocol for the efficient differentiation of hiPSCs into dopaminergic cultures, and extensively characterized the derived dopaminergic neurons which expressed multiple midbrain relevant markers and produced dopamine. Next we screened a clinical cohort of PD patients to identify carriers of GBA mutations of interest. Using for the first time hiPSCs generated from PD patients heterozygous for a GBA mutation (together with idiopathic cases and control individuals) we were able to efficiently derive dopaminergic cultures and identify relevant disease mechanisms. Upon differentiation into dopaminergic neuronal cultures, we observed retention of mutant glucocerebrosidase (GCase) protein in the endoplasmic reticulum (ER) with no change in protein levels, leading to upregulation of ER stress machinery and resulting in increased autophagic demand. At the lysosomal level, we found a reduction of GCase activity in dopaminergic neuronal cultures, and the enlargement of the lysosomal compartment in identified dopaminergic neurons suggesting a decreased capacity for protein clearance. Together, these perturbations of cellular homeostasis resulted in increased release of α-synuclein and could likely represent critical early cellular phenotypes of Parkinson's disease and explain the high risk of heterozygous GBA mutations for PD.

Parkinson’s disease (PD) is an incurable neurodegenerative disorder, mainly characterized by a progressive loss of midbrain dopaminergic (DA) neurons, located in the substantia nigra pars compacta (SNpc), and frequently accompanied by the formation of insoluble cytosolic protein aggregates in the remaining surviving neurons, known as Lewy bodies. The progressive denervation of DA terminals that project to the basal ganglia striatum causes a lack of DA uptaking, and consequently a progressive manifestation of debilitating motor deficits, that leads to premature invalidity and death. To date, only symptomatic therapies can compensate efficiently the locomotor deficiencies over a period of 5 years, however they do not cure or halt disease progression. A lack of suitable animal and cellular models might explain the fragmentary knowledge of the pathogenic mechanisms leading to PD, and highlights the urgent need for developing reliable experimental models that can recapitulate the key features of this disorder. The utilization of induced pluripotent stem cells (iPSC) offers an unprecedented opportunity to model human diseases, since they can be generated from patients and differentiated into disease-relevant cell types, such as neurons. Recently, human iPSC-based models of PD have been described. iPSC-derived neurons from patients with familial and sporadic PD recapitulate human disease phenotypes in vitro such as abnormal α-synuclein accumulation, and alterations in the autophagy machinery. Here, we investigated the neuroprotective effect of several compounds, including the neurotrophic factor GDNF, in culture of iPSC-derived midbrain dopaminergic neurons from patients with a LRKK2 mutation or sporadic patients. Interestingly, we found that six weeks after GDNF treatment, the level of dendritic arborisation from DA neurons derived from ID-PD and LRRK2-PD-iPSC increased to normal levels found in Ctrl-iPSC derived DA neurons. The neuroprotective effects were associated to a decrease in the number of apoptotic cells, and to a GFR1α/RET downstream activation of cell survival pathways, as well as reduction of autophagosome vesicles, compared to the untreated PD-iPSC derived DA neurons. Additionally, we found that GDNF is, in part, a requirement for a properly systemic neuronal survival in this model, since levels of GDNF were found twice decreased within long-term DAn cultures derived from both ID-PD and LRRK2-PD, when compared to CTL. In conclusion, our data demonstrate that iPSC-derived neuronal cells are valuable models for measuring responses to neuroprotective therapies and they may help to identify potential new drugs, thus furthering the development of treatments for PD. The differential secretion of survival factors within the culture also highlights the importance of using this technology for studying the contribution of other neural cell types in the onset of PD.
La malaltia de Parkinson (MP) és un desordre neurodegeneratiu incurable, principalment caracteritzat per la pèrdua progressiva de neurones dopaminèrgiques (DA) del mesencèfal, localitzades a la substancia nigra pars compacta (SNpc), i freqüentment acompanyat per la formació d’agregats proteics citosòlics insolubles en les neurones supervivents residuals, coneguts com a cossos de Lewy. La progressiva denervació dels terminals dopaminèrgics que projecten als ganglis basals i l’estriat causa una manca d’alliberament i ús de dopamina, provocant com a conseqüència una progressiva manifestació de dèficits motors, que acaben conduïnt a la invalidesa i mort prematura. Actualment, la MP només es pot tractar amb teràpies simptomàtiques que compensen eficientment les deficiències locomotores durant un període al voltant dels 5 anys. No obstant, les teràpies existents no permeten curar ni aturar la progressió de la malaltia. La manca de models animals i cel·lulars adequats podrien explicar el coneixement fragmentari dels mecanismes patològics que condueixen a la MP, i posa en relleu la necessitat urgent de desenvolupar models experimentals fiables que puguin recapitular les principals característiques d’aquest trastorn. L’ús de cèl·lules mare amb pluripotència induïda (iPSC) ofereix una oportunitat sense precedents per a modelar malalties humanes, ja que poden ser generades a partir de pacients i diferenciades en tipus cel·lulars rellevants de la malaltia. Recentment, s’han descrit models basats en iPSC humanes per a l’estudi de la MP. Neurones derivades de iPSC provinents de pacients amb MP familiar i esporàdica recapitulen fenotips humans específics de la malaltia in vitro, com ara l’acumulació anormal d’α-sinucleïna, i alteracions en la maquinària de l’autofàgia. En aquesta tesi doctoral s’ha investigat l’efecte neuroprotectiu de diferents compostos, incloent el factor neurotròfic GDNF, en el cultiu in vitro de neurones DA derivades de iPSC de pacients amb la mutació familiar G2019S al gen lrrk2 (LRRK2-PD), i pacients esporàdics (ID-PD). Curiosament, s’ha trobat que sis setmanes després del tractament amb el GDNF, el nivell d’arborització dendrítica de les neurones DA derivades de LRRK2-PD i ID-PD-iPSC augmentà fins als nivells normals trobats en les neurones DA derivades d’individus control (Ctrl-iPSC). En comparació amb els cultius neuronals DA derivats de PD-iPSC sense el tractament, els efectes neuroprotectius estaven associats a una disminució en el número de neurones apoptòtiques, i a l’activació de vies de supervivència cel·lular, riu avall del complex receptor del GDNF GFR1α/RET, així com també la reducció de vesícules autofàgiques. A més, s’ha trobat que el GDNF és, en part, un requisit per a una correcta supervivència neuronal sistèmica en aquest model. Concretament, els nivells de GDNF a llarg temps de cultiu es troben dos vegades disminuïts tant en els cultius DA de ID-PD com els de LRRK2-PD, en comparació amb els nivells de les Ctrl. En conclusió, els nostres resultats demostren que les cèl·lules neuronals derivades de iPSC són models valuosos per a mesurar respostes a teràpies neuroprotectives, i per tant poden ajudar a identificar nous fàrmacs potencials, fomentant així el desenvolupament de tractaments per a la MP. La secreció diferencial de factors de supervivència dins del cultiu també destaca l’importància de l’ús d’aquesta tecnologia per a l’estudi de la contribució d’altres tipus neurals en la patogènesis de la MP.

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter involved in early developmental processes such as cell proliferation, migration, and differentiation. Recent research in humans showed that the brain 5-HT system and CDH13 are interlinked in the genetics of neurodevelopmental disorders including attention- deficit/hyperactivity disorder and autism spectrum disorder (Lesch et al., 2008; Neale et al., 2008; Neale, Medland, Ripke, Anney, et al., 2010; Neale, Medland, Ripke, Asherson, et al., 2010; Sanders et al., 2011; Sanders et al., 2015; Zhou et al., 2008). This study introduces Cadherin-13 (CDH13), a cell adhesion protein, as a contributor to the development and function of the 5-HT system. Our experiments show that the absence of CDH13 increases the density of 5-HT neurons in the developing dorsal raphe (DR) and increases the 5-HT innervation of the prefrontal cortex in mouse embryonic stages. CDH13 is also observed in radial glial cells, an important progenitor cell type linked to neuronal migration. A three-dimensional reconstruction carried out with super-resolution microscopy, identifies 5-HT neurons intertwined with radial glial cells, and CDH13 clusters at contact points between these cells. This indicates a potential contribution of CDH13 to the migration of DR 5-HT neurons. As CDH13 is strongly expressed in 5-HT neurons, we asked whether the selective deletion of CDH13 from these cells is sufficient to generate the alterations observed in the Cdh13 constitutive knockout mouse line. In 5-HT conditional Cdh13 knockout mice (Cdh13 cKO) an increase in DR 5-HT neurons in the embryonic and adult brains is observed, as well as 5-HT hyperinnervation of cortical regions. Therefore, illustrating that the lack of CDH13 from 5-HT neurons alone impacts DR formation and serotonergic innervation. Behavioral testing conducted on Cdh13 cKO mice showed delayed learning in visuospatial learning and memory processing, as well as, changes in sociability parameters. To find out how CDH13 localizes in human 5-HT neurons, CDH13 was visualized in neurons that derived from human induced pluripotent stem cells (iPSC). Super-resolution microscopy confirmed CDH13 expression in a subgroup of induced human neurons positive for typical hallmarks of 5-HT neurons, such as expression of Tph2, the neuron-specific tryptophan hydroxylase, and synaptic structures. In summary, the work included in this thesis presents a detailed analysis of CDH13 expression and localization in the 5-HT system and shows that deletion of CDH13 from 5-HT neurons affects specific higher-order functions of the brain
Serotonin (5-Hydroxytryptamin, 5-HT) ist ein Neurotransmitter, der in frühe Entwicklungsprozesse involviert ist, wie beispielsweise Zellproliferation, Migration und Differenzierung. Aktuelle Forschungsergebnisse im Menschen zeigten eine Verbindung zwischen dem 5-HT System des Gehirns und CDH13 in der Genetik neurologischer Entwicklungsstörungen, wie die Aufmerksamkeitsdefizit-/Hyperaktivitätsstörung und die Autismus-Spektrum-Störung (Lesch et al., 2008; Neale, Medland, Ripke, Anney, et al., 2010; Neale, Medland, Ripke, Asherson, et al., 2010; Sanders et al., 2011; Sanders et al., 2015; Zhou et al., 2008). Diese Studie präsentiert Cadherin-13 (CDH13), ein Zelladhäsionsprotein, als einen Gegenspieler in der Entwicklung und Funktion des 5-HT Systems. Unsere Experimente zeigen, dass die Abwesenheit von CDH13 die Dichte der 5-HT Neuronen in dem sich entwickelnden dorsalen Raphe (DR) sowie die 5-HT Innervation des Präfrontalen Kortex in den embryonalen Stadien der Maus steigert. CDH13 wird auch in Radialen Gliazellen beobachtet, ein wichtiger Vorläuferzelltyp, der mit neuronaler Migration in Verbindung gebracht wurde. Eine 3-dimensionale Rekonstruktion, durchgeführt mit Superresolutions-Mikroskopie, identifiziert 5-HT Neuronen verflochten mit Radialen Gliazellen und CDH13 in den Kontaktpunkten zwischen diesen Zellen. Dies verdeutlicht eine potenzielle Rolle von CDH13 bei der Migration der DR 5-HT Neuronen. Da CDH13 eine starke Expression in den 5-HT Neuronen aufweist, fragten wir uns, ob die selektive Deletion von CDH13 in den Zellen ausreichend sei, um die in der konstitutiven Cdh13 Knockout-Mauslinie beobachteten Veränderung zu erzeugen. In 5-HT konditionalen Cdh13 Knockout-Mäusen (Cdh13 cKO) wurde eine Erhöhung der Anzahl der DR 5-HT Neuronen im embryonalen und adulten Gehirn sowie eine 5-HT Überinnervation der kortikalen Regionen beobachtet. Dies veranschaulicht, dass bereits ein Mangel an CDH13 in 5-HT Neuronen die DR-Ausbildung und serotonerge Innervation beeinflusst. Verhaltensversuche, die an Cdh13 cKO-Mäusen durchgeführt wurden, zeigten verspätetes Lernen im visuell-räumlichen Spektrum und der Gedächtnisverarbeitung sowie Veränderungen der Soziabilitätsparameter. Um herauszufinden, wie CDH13 in humanen 5-HT Neuronen lokalisiert ist, wurde CDH13 in aus humanen pluripotenten Stammzellen (iPSC) erzeugten Neuronen visualisiert. Superresolutions-Mikroskopie bestätigte eine CDH13 Expression in einer Untergruppe induzierter humaner Neuronen, die typische Merkmale von 5-HT Neuronen, wie etwa die Expression der Neuronen-spezifischen Tryptophan-Hydroxylase Tph2 und synaptische Strukturen, aufweisen. Zusammengefasst präsentiert diese Doktorarbeit eine detaillierte Analyse der CDH13 Expression und Lokalisation im 5-HT System und zeigt, dass eine Deletion von CDH13 in 5-HT Neuronen spezifische höhergradige Funktionen des Gehirns beeinflusst

Caffeine is widely and massively consumed on daily basis in the form of coffee, tea or energy drinks. This very popular, psychoactive drug is sought after due to its ability to increase energy and alertness, enhance physical and cognitive performance as well as to improve our ability of focusing. Previous studies have investigated the ability of caffeine to inhibit the activation of adenosine receptors in low doses, amongst other pharmacological effects as well as its potential in several neurodegenerative disease-modeling studies. However, not much is known about the effects that caffeine exerts on gene expression of neuronal cells. In this study we aim to understand if and how physiological concentrations of caffeine affect gene expression in human induced pluripotent stem cell (iPSC)-derived neuronal cells. Moreover, we identify active enhancers in neuronal cells and investigate the extent to which enhancers might be involved in the regulation of neuronal responses to caffeine. Using Cap Analysis of Gene Expression (CAGE) RNA expression profiling, we obtained a comprehensive data set of transcription start sites and enhancer activity of neuronal cells exposed to various caffeine concentrations (0, 3 and 10 μM). We identified a set of genes that appears to be involved in the mediation of caffeine response. Synaptic activity is upregulated after 1 hour of 3 μM caffeine treatment as well as dopaminergic neurotransmission. Immune system processes as well as axon guidance events are downregulated after 3 hours of 10 μM caffeine exposure. These insights provide concrete hypotheses of physiological processes and associated genes for guiding further functional validation experiments with the potential to give valuable insights into the effects of caffeine to the human brain.