@article{JanschZieglerForeroetal.2021, author = {Jansch, Charline and Ziegler, Georg C. and Forero, Andrea and Gredy, Sina and W{\"a}ldchen, Sina and Vitale, Maria Rosaria and Svirin, Evgeniy and Z{\"o}ller, Johanna E. M. and Waider, Jonas and G{\"u}nther, Katharina and Edenhofer, Frank and Sauer, Markus and Wischmeyer, Erhard and Lesch, Klaus-Peter}, title = {Serotonin-specific neurons differentiated from human iPSCs form distinct subtypes with synaptic protein assembly}, series = {Journal of Neural Transmission}, volume = {128}, journal = {Journal of Neural Transmission}, number = {2}, issn = {1435-1463}, doi = {10.1007/s00702-021-02303-5}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-268519}, pages = {225-241}, year = {2021}, abstract = {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.}, language = {en} } @phdthesis{Vitale2023, author = {Vitale, Maria Rosaria}, title = {Excitatory/inhibitory balance in iPSC-derived glutamatergic/GABAergic neuronal networks: differential Cadherin-13 genotype effects}, doi = {10.25972/OPUS-28789}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-287895}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {While the healthy brain works through balanced synaptic communication between glutamatergic and GABAergic neurons to coordinate excitation (E) and inhibition (I), disruption of E/I balance interferes with synaptic communication, information processing, and ultimately cognition. Multiple line of evidence indicates that E/I imbalance represents the pathophysiological basis of a wide spectrum of mental disorders. Genetic screening approaches have identified Cadherin-13 (CDH13). as a risk gene across neurodevelopmental and mental disorders. CDH13 regulates several cellular and synaptic processes in brain development and neuronal plasticity in adulthood. In addition to other functions, it is specifically localized at inhibitory synapses of parvalbumin- and somatostatin-expressing GABAergic neurons. In support of CDH13's function in moderating E/I balance, electrophysiological recordings of hippocampal slices in a CDH13-deficient mouse model revealed an increase in basal inhibitory but not excitatory synaptic transmission. Moreover, the search for genetic variants impacting functional expression of the CDH13 gene identified SNP (single nucleotide polymorphism)) rs2199430 in intron 1 to be associated with differential mRNA concentrations in human post-mortem brain across the three genotypes CDH13G/G, CDH13A/G and CDH13A/A . This work therefore aimed to further validate these findings in a complementary human model by using induced pluripotent stem cells (iPSCs). The application of human iPSCs in research has replaced the use of embryonic cells, resolving the ethical conflict of destructive usage of human embryos. Investigating CDH13's mode of action in inhibitory synapses was predicted to facilitate mechanistic insight into the effects of CDH13 gene variants on E/I network activity, which can then be targeted to reinstate balance. Genome-wide association studies have identified rare copy number variants (CNVs) resulting in a deletion (or duplication) of CDH13. To reduce genetic background variance, a set of isogenic iPSC lines with a gene dose-dependent deficiency of CDH13 (CDH13-/- and CDH13+/- ) was generated by using the Clustered Regulatory Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. These CRISPRed iPSCs carrying a single or two allele(s) with CDH13 inactivation facilitate investigation of CDH13 function in cellular processes, at inhibitory synapses and in neuronal network activity. In addition, iPSCs carrying allelic SNP rs2199430 variants were used to study the effects of common genetic variation of CDH13. These cell lines were differentiated into pure glutamatergic and GABAergic neurons and co-cultured to generate neuronal networks allowing its activity to be measured and correlated with electrophysiological signatures of differential CDH13 genotypes. The work towards assessment of neuronal network activity of the iPSC lines was subdivided into three major steps: first, generating rtTA/Ngn2 and rtTA/Ascl1-positive iPSCs via a lentivirus-mediated approach; second, differentiating pure glutamatergic and GABAergic neurons from the genetically transduced iPSCs and co-culturing of pure glutamatergic and GABAergic neurons in a pre-established ratio (65:35) by direct differentiation upon supplementation with doxycycline and forskolin on a microelectrode array (MEA) chip; and, finally, recording of neuronal network activity of iPSC lines after 49 days in vitro, followed by extraction and analyses of multiple MEA parameters. x Based on the MEA parameters, it was confirmed that complete CDH13 knockout as well as heterozygous deficiency influence E/I balance by increasing inhibition. It was further revealed that common SNP variation alters the signature of neuronal network activity. Specifically, CDH13 deficiency resulted in a significant reduction in network burst duration (NBD), reduced number of detected spikes within a network burst and reduction in network burst rate (NBR) compared to the control (CDH13G/G). CDH13A/G and CDH13A/A showed similarities with the CRISPRed CDH13-deficient networks by showing a significant reduction in the NBD and a reduced number of detected spikes within a network compared to CDH13G/G. Strikingly. there was a significant increase in the NBR of the CDH13A/G and CDH13A/A compared to CDH13G/G networks. CDH13A/G networks exhibited significant differences in both parameters. At the cellular level, this indicates that signalling pathways which determine the length and frequency of network bursts differ among allelic variants of SNP rs2199430, thus confirming functional relevance of this intronic SNP. In summary, CDH13-deficient isogenic iPSC lines were generated using CRISPR/Cas9, iPSCs were genetically transduced via a lentivirus approach, direct differentiation of glutamatergic/GABAergic neurons derived from transduced iPSCs were used to establish a scalable co-culture system, and network activity was recorded by MEA using pre-established parameters to extract and analyze activity information. The results indicate that iPSC-derived neuronal networks following CRISPR/Cas9-facilitated CDH13 inactivation, as well as networks with allelic SNP variants of CDH13, moderate E/I balance, thus advancing understanding of CDH13 function at inhibitory synapses and elucidating the effects of rare and common CDH13 gene variation.}, subject = {Induzierte pluripotente Stammzelle}, language = {en} } @article{VitaleZoellerJanschetal.2021, author = {Vitale, Maria Rosaria and Z{\"o}ller, Johanna Eva Maria and Jansch, Charline and Janz, Anna and Edenhofer, Frank and Klopocki, Eva and van den Hove, Daniel and Vanmierlo, Tim and Rivero, Olga and Kasri, Nael Nadif and Ziegler, Georg Christoph and Lesch, Klaus-Peter}, title = {Generation of induced pluripotent stem cell (iPSC) lines carrying a heterozygous (UKWMPi002-A-1) and null mutant knockout (UKWMPi002-A-2) of Cadherin 13 associated with neurodevelopmental disorders using CRISPR/Cas9}, series = {Stem Cell Research}, volume = {51}, journal = {Stem Cell Research}, doi = {10.1016/j.scr.2021.102169}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-260331}, year = {2021}, abstract = {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.}, language = {en} } @article{ZieglerEhlisWeberetal.2021, author = {Ziegler, Georg C. and Ehlis, Ann-Christine and Weber, Heike and Vitale, Maria Rosaria and Z{\"o}ller, Johanna E. M. and Ku, Hsing-Ping and Schiele, Miriam A. and K{\"u}rbitz, Laura I. and Romanos, Marcel and Pauli, Paul and Kalisch, Raffael and Zwanzger, Peter and Domschke, Katharina and Fallgatter, Andreas J. and Reif, Andreas and Lesch, Klaus-Peter}, title = {A Common CDH13 Variant is Associated with Low Agreeableness and Neural Responses to Working Memory Tasks in ADHD}, series = {Genes}, volume = {12}, journal = {Genes}, number = {9}, issn = {2073-4425}, doi = {10.3390/genes12091356}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-245220}, year = {2021}, abstract = {The cell—cell signaling gene CDH13 is associated with a wide spectrum of neuropsychiatric disorders, including attention-deficit/hyperactivity disorder (ADHD), autism, and major depression. CDH13 regulates axonal outgrowth and synapse formation, substantiating its relevance for neurodevelopmental processes. Several studies support the influence of CDH13 on personality traits, behavior, and executive functions. However, evidence for functional effects of common gene variation in the CDH13 gene in humans is sparse. Therefore, we tested for association of a functional intronic CDH13 SNP rs2199430 with ADHD in a sample of 998 adult patients and 884 healthy controls. The Big Five personality traits were assessed by the NEO-PI-R questionnaire. Assuming that altered neural correlates of working memory and cognitive response inhibition show genotype-dependent alterations, task performance and electroencephalographic event-related potentials were measured by n-back and continuous performance (Go/NoGo) tasks. The rs2199430 genotype was not associated with adult ADHD on the categorical diagnosis level. However, rs2199430 was significantly associated with agreeableness, with minor G allele homozygotes scoring lower than A allele carriers. Whereas task performance was not affected by genotype, a significant heterosis effect limited to the ADHD group was identified for the n-back task. Heterozygotes (AG) exhibited significantly higher N200 amplitudes during both the 1-back and 2-back condition in the central electrode position Cz. Consequently, the common genetic variation of CDH13 is associated with personality traits and impacts neural processing during working memory tasks. Thus, CDH13 might contribute to symptomatic core dysfunctions of social and cognitive impairment in ADHD.}, language = {en} } @article{ZieglerRadtkeVitaleetal.2021, author = {Ziegler, Georg C. and Radtke, Franziska and Vitale, Maria Rosaria and Preuße, Andr{\´e} and Klopocki, Eva and Herms, Stefan and Lesch, Klaus-Peter}, title = {Generation of multiple human iPSC lines from peripheral blood mononuclear cells of two SLC2A3 deletion and two SLC2A3 duplication carriers}, series = {Stem Cell Research}, volume = {56}, journal = {Stem Cell Research}, doi = {10.1016/j.scr.2021.102526}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-264696}, year = {2021}, abstract = {Copy number variants of SLC2A3, which encodes the glucose transporter GLUT3, are associated with several neuropsychiatric and cardiac diseases. Here, we report the successful reprogramming of peripheral blood mononuclear cells from two SLC2A3 duplication and two SLC2A3 deletion carriers and subsequent generation of two transgene-free iPSC clones per donor by Sendai viral transduction. All eight clones represent bona fide hiPSCs with high expression of pluripotency genes, ability to differentiate into cells of all three germ layers and normal karyotype. The generated cell lines will be helpful to enlighten the role of glucometabolic alterations in pathophysiological processes shared across organ boundaries.}, language = {en} }