Fiona Marshall: Welcome note and a personal note on GABAB receptor drug discovery

The 1990s was a time of great excitement in receptor pharmacology and drug discovery with the sequencing of the human genome enabling the cloning of known and novel receptors.  The G protein-coupled receptor (GPCR) superfamily was already a rich source of drug targets and stimulated a race to clone all the members of the family.  The GABA_B receptor was described as the ‘last wallflower at the party’ – remaining elusive from the cloners embrace.  In this talk I will describe the work done in parallel led by Benny Bettler and his team at Novartis and the Receptor systems team at GlaxoWellcome that eventually led to the cloning of the GABA_B receptor and the first demonstration of heterodimer formation in the GPCR family.

Klemens Kaupmann: The cloning of GABA-B receptors

I will present an overview of the molecular cloning of GABA_B receptors. Following the pharmacological discovery of GABA_B receptors in 1980 by Norman Bowery and colleagues, a team of chemists at Ciba-Geigy succeeded in developing selective, high-affinity radioligands for the GABA_B site. These ligands facilitated the isolation of the first GABA_B receptor cDNAs by expression cloning, and the elucidation of the heterodimeric nature of native GABA_B receptors.

Hans Bräuner: My journey from GABAB receptors to GPRC6A

My collaboration with Prof. Bettler started with a 6-month sabbatical at Novartis in 2001. During the visit I generated the two GABA_B1a and GABA_B1a splice-variant knockout mice lines, which were subsequently used by Bettler’s group to delineate the function of the two splice-variants. In collaboration we subsequently generated a knock-out mouse of the orphan GPRC6A receptor, which my group similarly used to investigate its elusive physiological functions.

Bernd Fakler: GABAB receptors - from interactome of a native GPCR to complexome profiling

Molecular appearance and architecture of native GABA_B receptors was uncovered by 'functional proteomics' combining affinity-isolation of the target protein(s) from brain membranes with quantitative mass spectrometry and functional analyses. Accordingly, GABA_B receptors are protein complexes assembled from two seven-segment subunits, GABA_B1 and GABA_B2, and a series of interaction partners including the heteromeric G-proteins, members of the KCTD (KCTDs 8, 12, 12b, 16), and 14-3-3 (B, E, F, G) family of proteins, single-span proteins (AJAP1, PIANP), ion channels (Cav2.2, HCN) and the amyloid protein complex (A4/APP, APLP2, ITM2B/C). I will briefly review these results obtained by AP-MS-based interactome analyses and their significance - and introduce a novel technology, high-resolution complexome profiling, demonstrating that the complex building of native GABA_B receptors is not an exception, but rather the rule for native membrane proteins in the mammalian brain.

Quing R. Fan: Molecular Mechanisms of Dimeric GPCR Signaling – Cryo-EM Approaches

Our research focuses on the molecular mechanisms by which G protein–coupled receptors (GPCRs) transduce extracellular signals across the cell membrane. We focus on the GABA_B receptor, a receptor first cloned and characterized by Dr. Bernhard Bettler. The GABA_B receptor is an obligatory heterodimer that mediates slow inhibitory neurotransmission in the central nervous system. Using cryogenic electron microscopy (cryo-EM), we have determined structures of the human GABA_B receptor in multiple function states to understand how it recognizes ligands and undergoes conformational transitions to become activated.

We also study other dimeric class C GPCRs, such as the calcium-sensing receptor (CaSR), to uncover both shared and divergent principles of signal transduction. By defining the molecular basis of receptor activation and G protein coupling in this receptor family, our work supports the development of targeted therapeutics for neurological and metabolic diseases.

Ryuichi Shigemoto: Asymmetrical modulation of fear expression via GABAB receptors in the mouse medial habenula

The medial habenula (MHb) regulates emotional responses to aversive events, and its pathway to the interpeducular nucleus (IPN) exhibits peculiar potentiation to activation of GABA_B receptors, opposite to other synaptic connections in the brain. Studies in zebrafish but not in mice have identified remarkable morphological and behavioral left-right asymmetries in the MHb-IPN pathway, but the lateralization of synaptic properties remains unclear. Here, we discovered that the left MHb-originating synapses exhibit lower release probability and higher GABA_BR-mediated potentiation than the right MHb-originating synapses in mice. Furthermore, the left but not the right MHb is involved in GABA_BR-dependent modulation of cue-dependent fear memory, suggesting that the lateralized MHb-IPN pathways represent a principle in emotional regulation across species.

Jean-Philippe Pin: Nanobody immunotherapy rescues behavioral deficits in NMDA hypofunction schizophrenia models

Metabotropic glutamate receptors have been considered as excellent targets for therapeutic intervention for many brain diseases since their discovery, 40 years ago. Despite years of research and multiple clinical trials, no drug reached the market so far. Here we show that nanobodies can be an alternative to small molecules, being specific of mGlu receptors of a specific subunit combination. We show that a nanobody activating mGlu2 homodimers selectively can rescue symptoms resulting from NMDA receptor hypofunction (Schizophrenia and GRIN models), up to one week after a single peripheral injection. This demonstrates nanobodies can be used to treat brain disorders.

Kristian Strømgaard: Targeting protein-protein interactions of receptor complexes with therapeutic peptides

We are interested in protein-protein interactions (PPIs) between integral membrane protein receptors and their intracellular protein partners, so-called ‘receptor complexes’ and examine how modulation of PPIs of such receptor complexes can provide novel biological insight and new therapeutics. We have developed peptide-based modulators of several receptor complexes, in particular those including ionotropic glutamate receptors and GABA_B receptors and explored the therapeutic relevance.

Marius Hoener: Angelman syndrome patient-derived neuron screen leads to clinical antisense oligonucleotide rugonersen targeting UBE3A-ATS with long lasting effect in monkey

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by loss of the neuronal ubiquitin E3 ligase UBE3A, with no approved treatment. Restoring UBE3A protein by silencing the paternally expressed long non-coding antisense transcript (UBE3A-ATS) offers a potentially disease-modifying strategy. To overcome the challenge of targeting UBE3A-ATS—selectively expressed in mature neurons and poorly conserved across species—we screened locked-nucleic acid (LNA)-modified antisense oligonucleotides in AS patient-derived neurons. This led to the identification of rugonersen (RO7248824), which potently reduces UBE3A-ATS and robustly increases UBE3A protein in neurons from neurotypical individuals, AS patients, and cynomolgus monkeys. In vivo studies in AS mice and monkeys showed that near-complete UBE3A-ATS knockdown was required for significant protein upregulation. Repeated lumbar intrathecal dosing in monkeys produced sustained UBE3A protein restoration in key brain regions without adverse effects. Rugonersen’s favorable pharmacology and durable activity supports its ongoing clinical development for AS.

Bernhard Bettler Title: De novo disease-causing mutations in the GABAB receptor complex

Whole-exome sequencing has uncovered hundreds of variants in the genes encoding the GBR subunits GABBR1 and GABBR2, many of which are linked to neurological and psychiatric disorders. Functional characterization of such variants in recombinant assay systems has revealed both gain-of-function (GOF) and loss-of-function (LOF) mutations, which surprisingly exhibit overlapping clinical symptoms. Moreover, variants in GBR-associated proteins have also been linked to human disease, with some shown to selectively impair presynaptic GBR functions in mouse models. I will discuss genetic evidence linking GBR dysfunction to human disease and emphasize the critical role of functional analyses of genetic variants in enhancing diagnostic precision and guiding therapeutic strategies.