Andreas Lieb

Background: Dopamine receptors (DRs) are G-protein-coupled receptors (GPCRs) found in the central nervous system (CNS). DRs are essential for mediating various downstream signaling cascades and play a critical role in regulating the dopaminergic nigrostriatal pathway, which is involved in motor control. Recently, mutations in DRD2 (WT), p.Ile212Phe (I212F), and p.Met345Arg (M345R) have been associated with hyperkinetic movement disorders and shown to alter heterotrimeric G-protein complex signaling and β-arrestin recruitment. Methods: To conduct a detailed investigation of the I212F and M345R functional phenotypes, we used the TRansdUcer PATHway (TRUPATH) assay to study heterotrimeric G-protein recruitment and the Parallel Receptorome Expression and Screening via Transcriptional Output (PRESTO-Tango) assay to evaluate transcriptional activation following arrestin translocation for β-arrestin recruitment. Results: In our study, we could confirm the reported mutant’s loss-of-function phenotype in β-arrestin 2 recruitment (reduced agonist potency and decreased maximal signaling efficacy in comparison to the WT). However, a detailed analysis of basal/constitutive activity also revealed a gain-of-function phenotype for mutant M345R. For a more comprehensive investigation of heterotrimeric G-protein complex signaling, we investigated the impact of WT mutants in combination with (i) a specifically suggested assay, and (ii) the most abundantly expressed heterotrimeric G-protein complex combinations in WT receptor-enriched regions. We were able to confirm the reported gain-of-function phenotype by Rodriguez-Contreras et al. and extend it by the use of the most abundant heterotrimeric G-protein subunits, Gα_oA and Gα_i1, β₁ and β₂, and γ₃ and γ₇, in mouse and human basal ganglia. Conclusions: Although our results indicate that the interaction of the two variants with the most highly expressed heterotrimeric G-protein complex subunit combinations also results in a gain-of-function phenotype, they also clearly demonstrate that the phenotype can be significantly altered, dependent on heterotrimeric G-protein complex expression.

We report an Osprey-based computational protocol to prospectively identify oncogenic mutations that act via disruption of molecular interactions. It is applicable to analyse both protein-protein and protein-DNA interfaces and it is validated on a dataset of clinically relevant mutations. In addition, it is used to predict previously uncharacterised patient mutations in CDK6 and p16 genes, which are experimentally confirmed to impair complex formation.

We report an Osprey-based computational protocol to prospectively identify oncogenic mutations that act via disruption of molecular interactions. It is applicable to analyze both protein-protein and protein-DNA interfaces and has been validated on a dataset of clinically relevant mutations. In addition, it was used to predict previously uncharacterized patient mutations in CDK6 and p16 genes, which were experimentally confirmed to impair complex formation.

Mutations in the prodynorphin gene (PDYN) are associated with the development of spinocerebellar ataxia type 23 (SCA23). Pathogenic missense mutations are localized predominantly in the PDYN region coding for the dynorphin A (DynA) neuropeptide and lead to persistently elevated mutant peptide levels with neurotoxic properties. The main DynA target in the central nervous system is the kappa opioid receptor (KOR), a member of the G-protein coupled receptor family, which can elicit signaling cascades mediated by G-protein dissociation as well as β-arrestin recruitment. To date, a thorough analysis of the functional profile for the pathogenic SCA23 DynA mutants at KOR is still missing. To elucidate the role of DynA mutants, we used a combination of assays to investigate the differential activation of G-protein subunits and β-arrestin. In addition, we applied molecular modelling techniques to provide a rationale for the underlying mechanism. Our results demonstrate that DynA mutations, associated with a severe ataxic phenotype, decrease potency of KOR activation, both for G-protein dissociation as well as β-arrestin recruitment. Molecular modelling suggests that this loss of function is due to disruption of critical interactions between DynA and the receptor. In conclusion, this study advances our understanding of KOR signal transduction upon DynA wild type or mutant peptide binding.

AMPA receptors (AMPARs) are tetrameric ligand-gated channels made up of combinations of GluA1-4 subunits encoded by GRIA1-4 genes. GluA2 has an especially important role because, following post-transcriptional editing at the Q607 site, it renders heteromultimeric AMPARs Ca²⁺-impermeable, with a linear relationship between current and trans-membrane voltage. Here, we report heterozygous de novo GRIA2 mutations in 28 unrelated patients with intellectual disability (ID) and neurodevelopmental abnormalities including autism spectrum disorder (ASD), Rett syndrome-like features, and seizures or developmental epileptic encephalopathy (DEE). In functional expression studies, mutations lead to a decrease in agonist-evoked current mediated by mutant subunits compared to wild-type channels. When GluA2 subunits are co-expressed with GluA1, most GRIA2 mutations cause a decreased current amplitude and some also affect voltage rectification. Our results show that de-novo variants in GRIA2 can cause neurodevelopmental disorders, complementing evidence that other genetic causes of ID, ASD and DEE also disrupt glutamatergic synaptic transmission.

Designer receptors exclusively activated by designer drugs (DREADDs) derived from muscarinic receptors not only are a powerful tool to test causality in basic neuroscience but also are potentially amenable to clinical translation. A major obstacle, however, is that the widely used agonist clozapine N-oxide undergoes conversion to clozapine, which penetrates the blood-brain barrier but has an unfavorable side effect profile. Perlapine has been reported to activate DREADDs at nanomolar concentrations but is not approved for use in humans by the Food and Drug Administration or the European Medicines Agency, limiting its translational potential. Here, we report that the atypical antipsychotic drug olanzapine, widely available in various formulations, is a potent agonist of the human M4 muscarinic receptor-based DREADD, facilitating clinical translation of chemogenetics to treat central nervous system diseases.