BMBF - Bundesministerium für Bildung und Forschung 01EK2101B: Biomarker Evaluation Supporting Clinical Translation in schizophrenia (BEST), Subproject 3: Functional analysis of PBMCs and iPSC-derived neurons for the validation of schizophrenia biomarkers . 09/2021-08/2024.
An objectively measured and evaluated biomarker used as an indicator for a pathogenic process or pharmacological response needs to achieve analytical and clinical validity. The latter depends on qualifying measures linking peripheral biomarkers with a biological process and clinical endpoints of schizophrenia. Thus, biomarker signatures identified in subprojects (SP) 1 and SP2 will be evaluated and compared to schizophrenia-specific mechanistic endpoints in patient-derived neurons. Schizophrenia is a neurodevelopmental disorder characterized by developmental deficits such as impaired neuronal differentiation, reduced synapse densities in selected brain areas and dysfunctional neuronal network activity. Since brain samples from patients are not accessible for mechanistic biomarker validation in vitro, we apply neurons differentiated from patient-derived induced pluripotent stem cells (iPSC) which faithfully represent the genetic background of individual patients. To this end, iPSC clones from patients with schizophrenia will be delivered by partners LMU and CIMH. Diagnostic and imaging data as well as blood samples of the corresponding patients are at hand and serve for sequencing blood transcriptomes and the establishment of multi-modal disease signatures in SP1 and SP2. For biomarker validation, those iPSC specimens will be selected out of the available ones which display the most similar disease signature as compared to signatures obtained of the large scale patient cohorts in SP1 and SP2.
DFG - Deutsche Forschungsgemeinschaft : Deciphering alcohol addiction-associated gene regulation changes on a single cell level. 01/2020-12/2022.
Changes in brain structure and function that results from chronic exposure to alcohol suggest that neuroadaptive alterations in gene regulation substantially contribute to addictive behavior. Transcriptional and epigenetic profiling of brain tissue from animal models and post-mortem human samples has identified multiple candidate genes to be dysregulated upon chronic alcohol exposure. However, a detailed assignment of those changes to specific cell types has not been performed due to technical limitations and lack of appropriate tissue. In this planned consortium, we will apply paralleled single cell sequencing to decipher transcriptional and epigenetic changes underlying alcohol addiction. We will perform single nuclei RNAsequencing (snRNA-seq) and snATAC-seq epigenetic profiling using postmortem tissue from a well-defined, high-quality brain bank of deceased alcohol addicts. We have previously established a novel snRNA-seq platform that allows isolating and sequencing of individual nuclei from snap-frozen brain samples obtained from patients with other neuropsychiatric diseases. In parallel we will use standardized human iPSC-derived forebrain organoids from controls and alcohol addicts to monitor alcohol-induced changes in gene regulation and gene expression in an isogenic (non-exposed vs. exposed; acute, chronic intermitting, acute withdrawal) forward approach. We expect that the proposed project will deliver the largest available database on alcohol addiction-associated gene regulation changes on a single cell level and help define critical contributors in the pathogenesis of alcohol addiction eventually eading to new therapeutic paradigms.
Koch P, Ladewig J. BMBF - Bundesministerium für Bildung und Forschung : SysMedSUDs - WP2: A human brain Organoid-based addiction circuitry to investigate molecular changes associated with SUDs. 11/2019-10/2022.
Ladewig J. DFG - Deutsche Forschungsgemeinschaft LA 2933/3-1 : MECPer-3D -Personalized MECP2 gene therapy using CRISPR/Cas9 technology coupled to AAVmediated delivery in 3D cell culture and KI mice. 01/2024-02/2021.
Rett syndrome is one of the most common causes of intellectual disability in girls, resulting in severe cognitive and physical disabilities. The classic form is caused by mutations in the transcriptional regulator MECP2. Effective therapies are not currently available and gene editing based on CRISPR/Cas9 combined with Homology Directed Repair appears an appealing option for the development of new therapeutic approaches. We already engineered a gene editing toolkit and demonstrated its ability to efficiently correct one of the most common MECP2 mutation, c.473C>T - (p.(T158M)), in patient cells. Based on these results, in this project we will further validate constructs for this mutation and develop and validate toolkits for three other MECP2 mutation hotspots, namely c.502C>T (p(R168X)), c.763C>T (p.(R255X)), c.916C>T (p.(R306C)). To characterize the potential of our approach in a relevant context and define its efficiency in a human 3D model, we will employ brain organoids differentiated from patient-derived induced pluripotent stem cells. Thanks to the ability of specific AAV serotypes to cross the Blood Brain Barrier (BBB) following intravenous injection, we will test our system in KI mice to validate efficacy and safety in vivo. Moreover, since available AAV serotypes have an imperfect brain tropism, with significant distribution to other organs, new serotypes will be developed and validated for their ability to cross the BBB in the mouse and their efficacy and specificity in human cells. These experiments will allow us to demonstrate the full potential of gene editing as a therapeutic option for Rett and for other neurodevelopmental disorders currently lacking an effective treatment.
Ladewig J. DFG - Deutsche Forschungsgemeinschaft LA 2933/2-1: Cerebral organoids to decipher molecular mechanisms perturbed in EML1 induced ribbonlike subcortical heterotopia . 08/2020-07/2020.
Ladewig J. BMBF - Bundesministerium für Bildung und Forschung 01EW1611: Neuron Verbund STEM-MCD: Stammzellen und Mechanismen die zu humanen kortikalen. 04/2019-05/2020.
Die Entwicklung des menschlichen Gehirns sowie seine Architektur sind durch eine immense Vergrößerung der Oberfläche bedingt durch eine komplexe Faltung in Gyri und Sulci charakterisiert. Kortikale Fehlbildungen können sehr schwerwiegend sein und zu geistiger Behinderung sowie Epilepsie führen. In Mausmodelle können bestimmte Aspekte der fehlerhaften Entwicklung nachvollzogen werden, jedoch fehlen im Maus Gehirn Zelltypen die für die menschliche Gehirnentwicklung entscheidend sind. Diese Zellen spielen eine wichtige Rolle während der Proliferation, der Vervielfältigung und der Organisation von Nervenzellen. Um diese Prozesse während der Entwicklung des menschlichen Gehirns und die Variabilität von entsprechenden kortikalen Fehlbildungen zu studieren, können Patienten abgeleitete reprogrammierte Zellen genutzt werden (iPSZ), welche gewisse Aspekte der menschliche Gehirnentwicklung in der Zellkulturschale wiederspiegeln können. Unter Verwendung von modernsten Mikroskopie-Technologien sollen Entwicklungsstörungen des menschlichen Gehirns visualisiert werden. Molekulare Mechanismen der Fehlbildungen sollen mit Hilfe von globaler Genexpression sowie neusten Technologien zur Hochdurchsatz-Sequenzierung von RNA aus einzelnen Zellen geklärt werden. Die Identifizierung von molekularen und zellulären Mechanismen wird uns gestatten auf gestörte subzelluläre Prozesse zu fokussieren und nach korrigierenden Strategien zu suchen.
Meyer-Lindenberg A, Rietschel M. DFG - Deutsche Forschungsgemeinschaft SFB 636: TP B03: Plasticity in prefrontal circuits in the human: Genetic variation, cellular. 01/2012-12/2015.
In the previous funding period, repetitive transcranial magnetic stimulation (rTMS) was shown to induce plasticity in prefrontal circuits for executive function and working memory, and genome-wide significant variants for psychosis were found to impact on these circuits, as well as on prefrontal regulation of the limbic system. In the present project, plasticity in prefrontal circuits will be further dissected by three experiments: (a) a genetic association study examining the effects of genome-wide significant common variants for psychosis on modulation of prefrontal circuit plasticity by rTMS, (b) an experiment in healthy controls relating prefrontal plasticity to a direct cellular assay of synaptic and glutamatergic function by using neuronally-differentiated induced pluripotent stem cells (iPS cells) together with multimodal imaging and rTMS, and (c) an experiment in healthy controls to directly modulate prefrontal circuit (connectivity) features and dependent cognition through real-time-fMRI based neurofeedback. The experiments are independent, but subjects for experiments (b) and (c) will be subsamples of the group investigated in (a) If successful, these experiments will identify genetic variants linking prefrontal plasticity and risk for psychosis, establish a cellular model for systems-level plasticity in humans and provide a first cognitive approach to induce plasticity in connected brain circuits relevant for mental disorders.
Schloss P. DFG - Deutsche Forschungsgemeinschaft SCHL 353/13-1: Serotonylierung neuronaler Proteine durch Transglutminasen - ein Mechanismus neuronaler Plastizität. 01/2013-12/2015.
Serotonin (5-hydroxytryptamine, 5-HT) was first discovered in the blood serum as a vasoconstrictor substance. Here, 5-HT is also covalently incorporated into distinct proteins involved in thrombus formation. This process is mediated by transglutaminases and has been termed “serotonylation”. In the central nervous system (CNS) 5-HT plays important roles in both embryonic development as a mediator of neurogenesis and in the mature brain as a neurotransmitter. Disturbances in the 5-HT system have also been indicated in several psychiatric disorders, however, it is questionable whether this is only due to 5-HT acting as a classical neurotransmitter. Taking lessons from the fate of 5-HT during thrombin formation in the blood it is conceivable that also in the CNS 5-HT can serve as a substrate for transglutaminases to form cross-linked matrices – a possibility which has not been investigated so far. The major goal of this proposal is to unravel new mechanisms how serotonin can interact with neural proteins to form multivalent cross-links and how such yet unknown processes may contribute to neuronal plasticity.