Klinik für Psychiatrie und Psychotherapie
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WG Cell Signalling

Head: PD Dr. rer. nat. Michael Wehr

Schizophrenia is a severe mental disorder affecting about 1% of the worldwide population and is considered to be a neurodevelopmental disorder with a high polygenic risk profile in the individual patient. The disease is characterised by positive symptoms (e.g., delusions, hallucinations, disorganised speech), negative symptoms (e.g., lack of motivation, social withdrawal, flat affect) and cognitive deficits (e.g., poor working memory, deficits in attention, processing speed and function). In our lab, we use molecular, biochemical and cell biology techniques and mouse models to investigate how molecular mechanisms are altered by schizophrenia candidate risk genes. In particular, we analyse the impact of selected schizophrenia risk genes on changes in signalling pathway activities and synaptic plasticity. In addition, we establish cell-based assays to perform drug repurposing screens (i.e., the repurposing of approved drugs for new disease indications) to identify new treatment options for schizophrenia. 

Details

Recent research on the aetiology of schizophrenia indicates that a large number of signalling pathways are altered as a result of multiple gene defects; these defects most likely occur during neurodevelopment. Schizophrenia may thus also be termed a ‘signalling disease’. In addition to signals that control activities of pharmacologically relevant receptors (e.g., the dopamine D2 receptor, a crucial target for treating positive symptoms), cellular signalling cascades also play an important role in neurons by coordinating the maturation of these cells and by promoting concomitant synaptic plasticity. For example, signalling pathways were identified that send signals from the synapse to the nucleus (e.g., mediated via calcium or MAP kinase signalling) and thus cause changes in transcriptional control mechanisms. Therefore, a better understanding of how individual risk genes and associated signalling activities contribute to the aetiology of schizophrenia may accelerate the development of new drugs.

Applications of the split TEV system in drug development
Figure 1. Applications of the split TEV system in drug development. The split TEV system has been used for a variety of applications, including the monitoring of various protein-protein interactions (PPI), high-throughput (HTP) screening, dose-response analyses and target selectivity studies. The lower inset illustrates the use of the split TEV system in multiparametric assays that use RNA barcode reporters and next-generation sequencing (NGS) as the final readout. R, receptor; bc, molecular barcode. (From Wehr and Rossner, 2016.)

We developed genetically encoded sensors that are applied in cell-based assays to profile actions of drug candidates on disease-relevant targets, such as G protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) (Wehr et al., 2016; Galinski et al., 2018; Wintgens et al., 2019). For example, we profiled the modulatory effects of neuroleptics on multiple GPCRs in a multiplexed assay using barcodes as readout (barcodes are short strings of nucleotides), and we validated known and identified new compound effects in this study (Galinski et al., 2018).

The genetically encoded sensors used in these assays are based on the complementation-based protein-protein interaction technique ‘split TEV’, which has also been used to study dynamic protein-protein interactions in living cells and to screen for genetic and pharmacological modulators of signalling pathways (Wehr et al., 2006, 2008, 2016, and 2017) (Figure 1). In addition, we combined the split TEV technique with an RNAi screening approach in Drosophila to identify modulators of Hippo signalling, a ubiquitously expressed pathway governing various aspects of cell fate and cell polarity, and differentiation of neurons (Wehr et al., 2013).

Outline of the drug discovery process for target-based and pathway-centric phenotypic campaigns. 
Figure 2. (A) Outline of the drug discovery process for target-based and pathway-centric phenotypic campaigns. (B) Patient cells from various sources (e.g., blood) can be reprogramed to human induced pluripotent stem cells (hiPSCs) and differentiated into 2D and 3D cellular disease models. Assays are performed in arrayed or pooled formats. (C) Multiparametric cell-based assays are used to profile chemical or genetic perturbations on target selectivity and pathway activities using molecular barcodes (BC). Next-generation sequencing (NGS) techniques allow a highly parallelized assay readout. Abbreviations: HT, high-throughput; ID, identification; MoA, mechanism of action. (modified from Herholt et al., 2020).

Furthermore, we developed multiplexed pathway-centric phenotypic assays that can be applied in disease-relevant cellular systems to assess both drug actions and genetic perturbations (Herholt et al., 2018). When barcoded pathway assays are combined with barcoded target-based assays, this dual approach is expected to better profile actions of drug candidates on target selectivity, potential off-targets, and pathway activities, thus promoting drug discovery and personalized medicine for complex diseases (Herholt et al., 2020) (Figure 2).

NRG1 mouse model. 
Figure  3. Spironolactone antagonises NRG1-ERBB4 signalling and ameliorates schizophrenia-relevant endophenotypes in a NRG1 mouse model. Using a split TEV-based co-culture assay that monitors ERBB4 receptor activity (measured by the NRG1 ligand-dependent association between activated ERBB4 and its adapter PIK3R1; inset top left) we screened the compound library of the NIH Clinical Collection, which contains 729 approved drugs, for modulators that decrease ERBB4 activities. Spironolactone was recovered as the top candidate and validated with orthogonal assays (e.g. biochemical and electrophysiological validation, behavioural profiling; inset top right). Mechanistically, NRG1 mice treated with spironolactone are believed to show an improvement in the excitation/inhibition balance.

Increased neuregulin-1 (NRG1)‐ERBB4 signalling is associated with schizophrenia, and corresponding mouse models display endophenotypes of the disease. Recently, we performed a drug repositioning screen using a cell-based split TEV assay to screen for modulators of the Nrg1-ERBB4 signalling pathway (Wehr et al., 2017). Spironolactone was identified in the screen as a substance that could inhibit NRG1‐ERBB4 signalling; in subsequent pre-clinical studies it was found to improve schizophrenia‐relevant phenotypes in NRG1‐transgenic mice (Figure 3). Based on these promising pre-clinical results and the fact that spironolactone is a licensed and thus safe drug, colleagues initiated a three-arm clinical trial of spironolactone as an add-on medication in schizophrenia (Hasan et al., 2020). In summary, the outcome of this approach indicates that combining cell-based assays with drug repositioning screenings can shorten drug discovery in comparison with standard approaches and may identify promising therapeutic options for psychiatric diseases.