Clinical and Molecular Profiling in GNAO1 Permits Phenotype-Genotype Correlation.

BACKGROUND: Defects in GNAO1, the gene encoding the major neuronal G-protein Gαo, are related to neurodevelopmental disorders, epilepsy, and movement disorders. Nevertheless, there is a poor understanding of how molecular mechanisms explain the different phenotypes. OBJECTIVES: We aimed to analyze the clinical phenotype and the molecular characterization of GNAO1-related disorders. METHODS: Patients were recruited in collaboration with the Spanish GNAO1 Association. For patient phenotyping, direct clinical evaluation, analysis of homemade-videos, and an online questionnaire completed by families were analyzed. We studied Gαo cellular expression, the interactions of the partner proteins, and binding to guanosine triphosphate (GTP) and G-protein-coupled receptors (GPCRs). RESULTS: Eighteen patients with GNAO1 genetic defects had a complex neurodevelopmental disorder, epilepsy, central hypotonia, and movement disorders. Eleven patients showed neurological deterioration, recurrent hyperkinetic crisis with partial recovery, and secondary complications leading to death in three cases. Deep brain stimulation improved hyperkinetic crisis, but had inconsistent benefits in dystonia. The molecular defects caused by pathogenic Gαo were aberrant GTP binding and hydrolysis activities, an inability to interact with cellular binding partners, and reduced coupling to GPCRs. Decreased localization of Gαo in the plasma membrane was correlated with the phenotype of "developmental and epileptic encephalopathy 17." We observed a genotype-phenotype correlation, pathogenic variants in position 203 were related to developmental and epileptic encephalopathy, whereas those in position 209 were related to neurodevelopmental disorder with involuntary movements. Milder phenotypes were associated with other molecular defects such as del.16q12.2q21 and I344del. CONCLUSION: We highlight the complexity of the motor phenotype, which is characterized by fluctuations throughout the day, and hyperkinetic crisis with a distinct post-hyperkinetic crisis state. We confirm a molecular-based genotype-phenotype correlation for specific variants. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Neomorphic Gαo mutations gain interaction with Ric8 proteins in GNAO1 encephalopathies.

GNAO1 mutated in pediatric encephalopathies encodes the major neuronal G-protein Gαo. Of >80 pathogenic mutations, most are single amino acid substitutions spreading across Gαo sequence. We perform extensive characterization of Gαo mutants showing abnormal GTP uptake and hydrolysis, and deficiencies to bind Gßγ and RGS19. Plasma membrane localization of Gαo is decreased for a subset of mutations that leads to epilepsy; dominant interactions with GPCRs also emerge for the more severe mutants. Pathogenic mutants massively gain interaction with Ric8A and, surprisingly, Ric8B proteins, delocalizing them from cytoplasm to Golgi. Of these two mandatory Gα-subunit chaperones, Ric8A is normally responsible for the Gαi/o, Gαq, and Gα12/13 subfamilies, and Ric8B solely for Gαs/olf. Ric8A/B mediate the disease dominance when engaging in neomorphic interactions with pathogenic Gαo through disbalancing the neuronal G protein signaling networks. As the strength of Gαo-Ric8B interactions correlates with disease severity, our study further identifies an efficient biomarker and predictor for clinical manifestations in GNAO1 encephalopathies. Our work discovers the neomorphic molecular mechanism of mutations underlying pediatric encephalopathies and offers insights to other maladies caused by G protein misfunctioning and further genetic diseases.

GNAO1 Mutations Affecting the N-Terminal α-Helix of Gαo Lead to Parkinsonism.

BACKGROUND: Patients carrying pathogenic variants in GNAO1 present a phenotypic spectrum ranging from severe early-onset epileptic encephalopathy and developmental delay to mild adolescent/adult-onset dystonia. Genotype-phenotype correlation and molecular mechanisms underlying the disease remain understudied. METHODS: We analyzed the clinical course of a child carrying the novel GNAO1 mutation c.38T>C;p.Leu13Pro, and structural, biochemical, and cellular properties of the corresponding mutant Gαo-GNAO1-encoded protein-alongside the related mutation c.68T>C;p.Leu23Pro. RESULTS: The main clinical feature was parkinsonism with bradykinesia and rigidity, unlike the hyperkinetic movement disorder commonly associated with GNAO1 mutations. The Leu ➔ Pro substitutions have no impact on enzymatic activity or overall folding of Gαo but uniquely destabilize the N-terminal α-helix, blocking formation of the heterotrimeric G-protein and disabling activation by G-protein-coupled receptors. CONCLUSIONS: Our study defines a parkinsonism phenotype within the spectrum of GNAO1 disorders and suggests a genotype-phenotype correlation by GNAO1 mutations targeting the N-terminal α-helix of Gαo. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Clinical Cases and the Molecular Profiling of a Novel Childhood Encephalopathy-Causing GNAO1 Mutation P170R.

De novo mutations in GNAO1, the gene encoding the major neuronal G protein Gαo, cause a spectrum of pediatric encephalopathies with seizures, motor dysfunction, and developmental delay. Of the >80 distinct missense pathogenic variants, many appear to uniformly destabilize the guanine nucleotide handling of the mutant protein, speeding up GTP uptake and deactivating GTP hydrolysis. Zinc supplementation emerges as a promising treatment option for this disease, as Zn2+ ions reactivate the GTP hydrolysis on the mutant Gαo and restore cellular interactions for some of the mutants studied earlier. The molecular etiology of GNAO1 encephalopathies needs further elucidation as a prerequisite for the development of efficient therapeutic approaches. In this work, we combine clinical and medical genetics analysis of a novel GNAO1 mutation with an in-depth molecular dissection of the resultant protein variant. We identify two unrelated patients from Norway and France with a previously unknown mutation in GNAO1, c.509C>G that results in the production of the Pro170Arg mutant Gαo, leading to severe developmental and epileptic encephalopathy. Molecular investigations of Pro170Arg identify this mutant as a unique representative of the pathogenic variants. Its 100-fold-accelerated GTP uptake is not accompanied by a loss in GTP hydrolysis; Zn2+ ions induce a previously unseen effect on the mutant, forcing it to lose the bound GTP. Our work combining clinical and molecular analyses discovers a novel, biochemically distinct pathogenic missense variant of GNAO1 laying the ground for personalized treatment development.

In-depth molecular profiling of an intronic GNAO1 mutant as the basis for personalized high-throughput drug screening.

BACKGROUND: The GNAO1 gene, encoding the major neuronal G protein Gαo, is mutated in a subset of pediatric encephalopathies. Most such mutations consist of missense variants. METHODS: In this study, we present a precision medicine workflow combining next-generation sequencing (NGS) diagnostics, molecular etiology analysis, and personalized drug discovery. FINDINGS: We describe a patient carrying a de novo intronic mutation (NM_020988.3:c.724-8G>A), leading to epilepsy-negative encephalopathy with motor dysfunction from the second decade. Our data show that this mutation creates a novel splice acceptor site that in turn causes an in-frame insertion of two amino acid residues, Pro-Gln, within the regulatory switch III region of Gαo. This insertion misconfigures the switch III loop and creates novel interactions with the catalytic switch II region, resulting in increased GTP uptake, defective GTP hydrolysis, and aberrant interactions with effector proteins. In contrast, intracellular localization, Gßγ interactions, and G protein-coupled receptor (GPCR) coupling of the Gαo[insPQ] mutant protein remain unchanged. CONCLUSIONS: This in-depth analysis characterizes the heterozygous c.724-8G>A mutation as partially dominant negative, providing clues to the molecular etiology of this specific pathology. Further, this analysis allows us to establish and validate a high-throughput screening platform aiming at identifying molecules that could correct the aberrant biochemical functions of the mutant Gαo. FUNDING: This work was supported by the Joint Seed Money Funding scheme between the University of Geneva and the Hebrew University of Jerusalem.

Restoration of the GTPase activity and cellular interactions of Gαo mutants by Zn2+ in GNAO1 encephalopathy models.

De novo point mutations in GNAO1, gene encoding the major neuronal G protein Gαo, have recently emerged in patients with pediatric encephalopathy having motor, developmental, and epileptic dysfunctions. Half of clinical cases affect codons Gly203, Arg209, or Glu246; we show that these mutations accelerate GTP uptake and inactivate GTP hydrolysis through displacement Gln205 critical for GTP hydrolysis, resulting in constitutive GTP binding by Gαo. However, the mutants fail to adopt the activated conformation and display aberrant interactions with signaling partners. Through high-throughput screening of approved drugs, we identify zinc pyrithione and Zn2+ as agents restoring active conformation, GTPase activity, and cellular interactions of the encephalopathy mutants, with negligible effects on wild-type Gαo. We describe a Drosophila model of GNAO1 encephalopathy where dietary zinc restores the motor function and longevity of the mutant flies. Zinc supplements are approved for diverse human neurological conditions. Our work provides insights into the molecular etiology of GNAO1 encephalopathy and defines a potential therapy for the patients.

Local and substrate-specific S-palmitoylation determines subcellular localization of Gαo.

Peripheral membrane proteins (PMPs) associate with cellular membranes through post-translational modifications like S-palmitoylation. The Golgi apparatus is generally viewed as the transitory station where palmitoyl acyltransferases (PATs) modify PMPs, which are then transported to their ultimate destinations such as the plasma membrane (PM). However, little substrate specificity among the many PATs has been determined. Here we describe the inherent partitioning of Gαo - α-subunit of heterotrimeric Go proteins - to PM and Golgi, independent from Golgi-to-PM transport. A minimal code within Gαo N-terminus governs its compartmentalization and re-coding produces G protein versions with shifted localization. We establish the S-palmitoylation at the outer nuclear membrane assay ("SwissKASH") to probe substrate specificity of PATs in intact cells. With this assay, we show that PATs localizing to different membrane compartments display remarkable substrate selectivity, which is the basis for PMP compartmentalization. Our findings uncover a mechanism governing protein localization and establish the basis for innovative drug discovery.

Gαi2-induced conductin/axin2 condensates inhibit Wnt/ß-catenin signaling and suppress cancer growth.

Conductin/axin2 is a scaffold protein negatively regulating the pro-proliferative Wnt/ß-catenin signaling pathway. Accumulation of scaffold proteins in condensates frequently increases their activity, but whether condensation contributes to Wnt pathway inhibition by conductin remains unclear. Here, we show that the Gαi2 subunit of trimeric G-proteins induces conductin condensation by targeting a polymerization-inhibiting aggregon in its RGS domain, thereby promoting conductin-mediated ß-catenin degradation. Consistently, transient Gαi2 expression inhibited, whereas knockdown activated Wnt signaling via conductin. Colorectal cancers appear to evade Gαi2-induced Wnt pathway suppression by decreased Gαi2 expression and inactivating mutations, associated with shorter patient survival. Notably, the Gαi2-activating drug guanabenz inhibited Wnt signaling via conductin, consequently reducing colorectal cancer growth in vitro and in mouse models. In summary, we demonstrate Wnt pathway inhibition via Gαi2-triggered conductin condensation, suggesting a tumor suppressor function for Gαi2 in colorectal cancer, and pointing to the FDA-approved drug guanabenz for targeted cancer therapy.

Pediatric Encephalopathy: Clinical, Biochemical and Cellular Insights into the Role of Gln52 of GNAO1 and GNAI1 for the Dominant Disease.

Heterotrimeric G proteins are immediate transducers of G protein-coupled receptors-the biggest receptor family in metazoans-and play innumerate functions in health and disease. A set of de novo point mutations in GNAO1 and GNAI1, the genes encoding the α-subunits (Gαo and Gαi1, respectively) of the heterotrimeric G proteins, have been described to cause pediatric encephalopathies represented by epileptic seizures, movement disorders, developmental delay, intellectual disability, and signs of neurodegeneration. Among such mutations, the Gln52Pro substitutions have been previously identified in GNAO1 and GNAI1. Here, we describe the case of an infant with another mutation in the same site, Gln52Arg. The patient manifested epileptic and movement disorders and a developmental delay, at the onset of 1.5 weeks after birth. We have analyzed biochemical and cellular properties of the three types of dominant pathogenic mutants in the Gln52 position described so far: Gαo[Gln52Pro], Gαi1[Gln52Pro], and the novel Gαo[Gln52Arg]. At the biochemical level, the three mutant proteins are deficient in binding and hydrolyzing GTP, which is the fundamental function of the healthy G proteins. At the cellular level, the mutants are defective in the interaction with partner proteins recognizing either the GDP-loaded or the GTP-loaded forms of Gαo. Further, of the two intracellular sites of Gαo localization, plasma membrane and Golgi, the former is strongly reduced for the mutant proteins. We conclude that the point mutations at Gln52 inactivate the Gαo and Gαi1 proteins leading to aberrant intracellular localization and partner protein interactions. These features likely lie at the core of the molecular etiology of pediatric encephalopathies associated with the codon 52 mutations in GNAO1/GNAI1.

HumanaFly: high-throughput transgenesis and expression of breast cancer transcripts in Drosophila eye discovers the RPS12-Wingless signaling axis.

Drosophila melanogaster has been a model for multiple human disease conditions, including cancer. Among Drosophila tissues, the eye development is particularly sensitive to perturbations of the embryonic signaling pathways, whose improper activation in humans underlies various forms of cancer. We have launched the HumanaFly project, whereas human genes expressed in breast cancer patients are screened for their ability to aberrate development of the Drosophila eye, hoping to thus identify novel oncogenes. Here we report identification of a breast cancer transgene, which upon expression in Drosophila produces eye malformation similar to the famous Glazed phenotype discovered by Thomas Morgan and decades later dissected to originate from mis-expression of Wingless (Wg). Wg is the ortholog of human Wnt proteins serving as ligands to initiate the developmental/oncogenic Wnt signaling pathway. Through genetic experiments we identified that this transgene interacted with the Wg production machinery, rather than with Wg signal transduction. In Drosophila imaginal discs, we directly show that the transgene promoted long-range diffusion of Wg, affecting expression of the Wg target genes. The transgene emerged to encode RPS12-a protein of the small ribosomal subunit overexpressed in several cancer types and known to also possess extra-ribosomal functions. Our work identifies RPS12 as an unexpected regulator of secretion and activity of Wnts. As Wnt signaling is particularly important in the context of breast cancer initiation and progression, RPS12 might be implicated in tumorigenesis in this and other Wnt-dependent cancers. Continuation of our HumanaFly project may bring further discoveries on oncogenic mechanisms.

Humanization of Drosophila Gαo to Model GNAO1 Paediatric Encephalopathies.

Several hundred genes have been identified to contribute to epilepsy-the disease affecting 65 million people worldwide. One of these genes is GNAO1 encoding Gαo, the major neuronal α-subunit of heterotrimeric G proteins. An avalanche of dominant de novo mutations in GNAO1 have been recently described in paediatric epileptic patients, suffering, in addition to epilepsy, from motor dysfunction and developmental delay. Although occurring in amino acids conserved from humans to Drosophila, these mutations and their functional consequences have only been poorly analysed at the biochemical or neuronal levels. Adequate animal models to study the molecular aetiology of GNAO1 encephalopathies have also so far been lacking. As the first step towards modeling the disease in Drosophila, we here describe the humanization of the Gαo locus in the fruit fly. A two-step CRISPR/Cas9-mediated replacement was conducted, first substituting the coding exons 2-3 of Gαo with respective human GNAO1 sequences. At the next step, the remaining exons 4-7 were similarly replaced, keeping intact the gene Cyp49a1 embedded in between, as well as the non-coding exons, exon 1 and the surrounding regulatory sequences. The resulting flies, homozygous for the humanized GNAO1 loci, are viable and fertile without any visible phenotypes; their body weight, locomotion, and longevity are also normal. Human Gαo-specific antibodies confirm the endogenous-level expression of the humanized Gαo, which fully replaces the Drosophila functions. The genetic model we established will make it easy to incorporate encephalopathic GNAO1 mutations and will permit intensive investigations into the molecular aetiology of the human disease through the powerful toolkit of Drosophila genetics.

High capacity in G protein-coupled receptor signaling.

G protein-coupled receptors (GPCRs) constitute a large family of receptors that activate intracellular signaling pathways upon detecting specific extracellular ligands. While many aspects of GPCR signaling have been uncovered through decades of studies, some fundamental properties, like its channel capacity-a measure of how much information a given transmission system can reliably transduce-are still debated. Previous studies concluded that GPCRs in individual cells could transmit around one bit of information about the concentration of the ligands, allowing only for a reliable on or off response. Using muscarinic receptor-induced calcium response measured in individual cells upon repeated stimulation, we show that GPCR signaling systems possess a significantly higher capacity. We estimate the channel capacity of this system to be above two, implying that at least four concentration levels of the agonist can be distinguished reliably. These findings shed light on the basic principles of GPCR signaling.

Golgi-Resident Gαo Promotes Protrusive Membrane Dynamics.

To form protrusions like neurites, cells must coordinate their induction and growth. The first requires cytoskeletal rearrangements at the plasma membrane (PM), the second requires directed material delivery from cell's insides. We find that the Gαo-subunit of heterotrimeric G proteins localizes dually to PM and Golgi across phyla and cell types. The PM pool of Gαo induces, and the Golgi pool feeds, the growing protrusions by stimulated trafficking. Golgi-residing KDELR binds and activates monomeric Gαo, atypically for G protein-coupled receptors that normally act on heterotrimeric G proteins. Through multidimensional screenings identifying > 250 Gαo interactors, we pinpoint several basic cellular activities, including vesicular trafficking, as being regulated by Gαo. We further find small Golgi-residing GTPases Rab1 and Rab3 as direct effectors of Gαo. This KDELR → Gαo → Rab1/3 signaling axis is conserved from insects to mammals and controls material delivery from Golgi to PM in various cells and tissues.

Reggie-1/Flotillin-2 regulates integrin trafficking and focal adhesion turnover via Rab11a.

Reggies/flotillins are implicated in trafficking of membrane proteins to their target sites and in the regulation of the Rab11a-dependent targeted recycling of E-cadherin to adherens junctions (AJs). Here we demonstrate a function of reggies in focal adhesion (FA) formation and α5- and ß1-integrin recycling to FAs. Downregulation of reggie-1 in HeLa and A431 cells by siRNA and shRNA increased the number of FAs, impaired their distribution and modified FA turnover. This was coupled to enhanced focal adhesion kinase (FAK) and Rac1 signaling and gain in plasma membrane motility. Wild type and constitutively-active (CA) Rab11a rescued the phenotype (normal number of FAs) whereas dominant-negative (DN) Rab11a mimicked the loss-of-reggie phenotype in control cells. That reggie-1 affects integrin trafficking emerged from the faster loss of internalized antibody-labeled ß1-integrin in reggie-deficient cells. Moreover, live imaging using TIRF microscopy revealed vesicles containing reggie-1 and α5- or ß1-integrin, trafficking close to the substrate-near membrane and making kiss-and-run contacts with FAs. Thus, reggie-1 in interaction with Rab11a controls Rac1 and FAK activation and coordinates the targeted recycling of α5- and ß1-integrins to FAs to regulate FA formation and membrane dynamics.

Heterotrimeric Go protein links Wnt-Frizzled signaling with ankyrins to regulate the neuronal microtubule cytoskeleton.

Drosophila neuromuscular junctions (NMJs) represent a powerful model system with which to study glutamatergic synapse formation and remodeling. Several proteins have been implicated in these processes, including components of canonical Wingless (Drosophila Wnt1) signaling and the giant isoforms of the membrane-cytoskeleton linker Ankyrin 2, but possible interconnections and cooperation between these proteins were unknown. Here, we demonstrate that the heterotrimeric G protein Go functions as a transducer of Wingless-Frizzled 2 signaling in the synapse. We identify Ankyrin 2 as a target of Go signaling required for NMJ formation. Moreover, the Go-ankyrin interaction is conserved in the mammalian neurite outgrowth pathway. Without ankyrins, a major switch in the Go-induced neuronal cytoskeleton program is observed, from microtubule-dependent neurite outgrowth to actin-dependent lamellopodial induction. These findings describe a novel mechanism regulating the microtubule cytoskeleton in the nervous system. Our work in Drosophila and mammalian cells suggests that this mechanism might be generally applicable in nervous system development and function.

Double suppression of the Gα protein activity by RGS proteins.

Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis on G protein α subunits, restricting their activity downstream from G protein-coupled receptors. Here we identify Drosophila Double hit (Dhit) as a dual RGS regulator of Gαo. In addition to the conventional GTPase-activating action, Dhit possesses the guanine nucleotide dissociation inhibitor (GDI) activity, slowing the rate of GTP uptake by Gαo; both activities are mediated by the same RGS domain. These findings are recapitulated using homologous mammalian Gαo/i proteins and RGS19. Crystal structure and mutagenesis studies provide clues into the molecular mechanism for this unprecedented GDI activity. Physiologically, we confirm this activity in Drosophila asymmetric cell divisions and HEK293T cells. We show that the oncogenic Gαo mutant found in breast cancer escapes this GDI regulation. Our studies identify Dhit and its homologs as double-action regulators, inhibiting Gαo/i proteins both through suppression of their activation and acceleration of their inactivation through the single RGS domain.

Conserved roles of the prion protein domains on subcellular localization and cell-cell adhesion.

Analyses of cultured cells and transgenic mice expressing prion protein (PrP) deletion mutants have revealed that some properties of PrP -such as its ability to misfold, aggregate and trigger neurotoxicity- are controlled by discrete molecular determinants within its protein domains. Although the contributions of these determinants to PrP biosynthesis and turnover are relatively well characterized, it is still unclear how they modulate cellular functions of PrP. To address this question, we used two defined activities of PrP as functional readouts: 1) the recruitment of PrP to cell-cell contacts in Drosophila S2 and human MCF-7 epithelial cells, and 2) the induction of PrP embryonic loss- and gain-of-function phenotypes in zebrafish. Our results show that homologous mutations in mouse and zebrafish PrPs similarly affect their subcellular localization patterns as well as their in vitro and in vivo activities. Among PrP's essential features, the N-terminal leader peptide was sufficient to drive targeting of our constructs to cell contact sites, whereas lack of GPI-anchoring and N-glycosylation rendered them inactive by blocking their cell surface expression. Importantly, our data suggest that the ability of PrP to homophilically trans-interact and elicit intracellular signaling is primarily encoded in its globular domain, and modulated by its repetitive domain. Thus, while the latter induces the local accumulation of PrPs at discrete punctae along cell contacts, the former counteracts this effect by promoting the continuous distribution of PrP. In early zebrafish embryos, deletion of either domain significantly impaired PrP's ability to modulate E-cadherin cell adhesion. Altogether, these experiments relate structural features of PrP to its subcellular distribution and in vivo activity. Furthermore, they show that despite their large evolutionary history, the roles of PrP domains and posttranslational modifications are conserved between mouse and zebrafish.

Reggies/flotillins interact with Rab11a and SNX4 at the tubulovesicular recycling compartment and function in transferrin receptor and E-cadherin trafficking.

The lipid raft proteins reggie-1 and -2 (flotillins) are implicated in membrane protein trafficking but exactly how has been elusive. We find that reggie-1 and -2 associate with the Rab11a, SNX4, and EHD1-decorated tubulovesicular recycling compartment in HeLa cells and that reggie-1 directly interacts with Rab11a and SNX4. Short hairpin RNA-mediated down-regulation of reggie-1 (and -2) in HeLa cells reduces association of Rab11a with tubular structures and impairs recycling of the transferrin-transferrin receptor (TfR) complex to the plasma membrane. Overexpression of constitutively active Rab11a rescues TfR recycling in reggie-deficient HeLa cells. Similarly, in a Ca(2+) switch assay in reggie-depleted A431 cells, internalized E-cadherin is not efficiently recycled to the plasma membrane upon Ca(2+) repletion. E-cadherin recycling is rescued, however, by overexpression of constitutively active Rab11a or SNX4 in reggie-deficient A431 cells. This suggests that the function of reggie-1 in sorting and recycling occurs in association with Rab11a and SNX4. Of interest, impaired recycling in reggie-deficient cells leads to de novo E-cadherin biosynthesis and cell contact reformation, showing that cells have ways to compensate the loss of reggies. Together our results identify reggie-1 as a regulator of the Rab11a/SNX4-controlled sorting and recycling pathway, which is, like reggies, evolutionarily conserved.