Calcineurin-Dependent Homeostatic Response of C. elegans Muscle Cells upon Prolonged Activation of Acetylcholine Receptors.

Pharmacological adaptation is a common phenomenon observed during prolonged drug exposure and often leads to drug resistance. Understanding the cellular events involved in adaptation could provide new strategies to circumvent this resistance issue. We used the nematode Caenorhabditis elegans to analyze the adaptation to levamisole, an ionotropic acetylcholine receptor agonist, used for decades to treat nematode parasitic infections. Genetic screens in C. elegans identified "adapting mutants" that initially paralyze upon exposure to levamisole as the wild type (WT), but recover locomotion after a few hours whereas WT remain paralyzed. Here, we show that levamisole induces a sustained increase in cytosolic calcium concentration in the muscle cells of adapting mutants, lasting several hours and preceding a decrease in levamisole-sensitive acetylcholine receptors (L-AChR) at the muscle plasma membrane. This decrease correlated with a drop in calcium concentration, a relaxation of the animal's body and a resumption of locomotion. The decrease in calcium and L-AChR content depends on calcineurin activation in muscle cells. We also showed that levamisole adaptation triggers homeostatic mechanisms in muscle cells including mitochondria remodeling, lysosomal tubulation and an increase in autophagic activity. Levamisole adaptation thus provides a new experimental paradigm for studying how cells cope with calcium stress.

A genotype-to-phenotype approach suggests under-reporting of single nucleotide variants in nephrocystin-1 (NPHP1) related disease (UK 100,000 Genomes Project).

Autosomal recessive whole gene deletions of nephrocystin-1 (NPHP1) result in abnormal structure and function of the primary cilia. These deletions can result in a tubulointerstitial kidney disease known as nephronophthisis and retinal (Senior-Løken syndrome) and neurological (Joubert syndrome) diseases. Nephronophthisis is a common cause of end-stage kidney disease (ESKD) in children and up to 1% of adult onset ESKD. Single nucleotide variants (SNVs) and small insertions and deletions (Indels) have been less well characterised. We used a gene pathogenicity scoring system (GenePy) and a genotype-to-phenotype approach on individuals recruited to the UK Genomics England (GEL) 100,000 Genomes Project (100kGP) (n = 78,050). This approach identified all participants with NPHP1-related diseases reported by NHS Genomics Medical Centres and an additional eight participants. Extreme NPHP1 gene scores, often underpinned by clear recessive inheritance, were observed in patients from diverse recruitment categories, including cancer, suggesting the possibility of a more widespread disease than previously appreciated. In total, ten participants had homozygous CNV deletions with eight homozygous or compound heterozygous with SNVs. Our data also reveals strong in-silico evidence that approximately 44% of NPHP1 related disease may be due to SNVs with AlphaFold structural modelling evidence for a significant impact on protein structure. This study suggests historical under-reporting of SNVS in NPHP1 related diseases compared with CNVs.

Repurposing small molecules for nephronophthisis and related renal ciliopathies.

Nephronophthisis is an autosomal recessive tubulointerstitial nephropathy, belonging to the ciliopathy disorders, characterized by fibrosis and/or cysts. It is the most common genetic cause of kidney failure in children and young adults. Clinically and genetically heterogeneous, it is caused by variants in ciliary genes, resulting in either an isolated kidney disease or syndromic forms in association with other manifestations of ciliopathy disorders. No curative treatment is currently available. Over the past 2 decades, advances in understanding disease mechanisms have identified several dysregulated signaling pathways, some shared with other cystic kidney diseases. Notably, molecules previously developed to target these pathways have shown promising beneficial effects in orthologous mouse models. In addition to these knowledge-based repurposing approaches, unbiased "in cellulo" phenotypic screens of "repurposing" libraries identified small molecules able to rescue the ciliogenesis defects observed in nephronophthisis conditions. Those compounds appeared to act on relevant pathways and, when tested, showed beneficial nephronophthisis-associated kidney and/or extrarenal defects in mice. In this review, we have summarized those studies that highlight the drug repurposing strategies in the context of a rare disorders, such as nephronophthisis-related ciliopathies, with broad genetic heterogeneity and systemic manifestations but with shared disease mechanisms.

Agonists of prostaglandin E2 receptors as potential first in class treatment for nephronophthisis and related ciliopathies.

Nephronophthisis (NPH) is an autosomal recessive tubulointerstitial nephropathy belonging to the ciliopathy disorders and known as the most common cause of hereditary end-stage renal disease in children. Yet, no curative treatment is available. The major gene, NPHP1, encodes a protein playing key functions at the primary cilium and cellular junctions. Using a medium-throughput drug-screen in NPHP1 knockdown cells, we identified 51 Food and Drug Administration-approved compounds by their ability to alleviate the cellular phenotypes associated with the loss of NPHP1; 11 compounds were further selected for their physicochemical properties. Among those compounds, prostaglandin E1 (PGE1) rescued ciliogenesis defects in immortalized patient NPHP1 urine-derived renal tubular cells, and improved ciliary and kidney phenotypes in our NPH zebrafish and Nphp1 knockout mouse models. Furthermore, Taprenepag, a nonprostanoid prostaglandin E2 receptor agonist, alleviated the severe retinopathy observed in Nphp1−/− mice. Finally, comparative transcriptomics allowed identification of key signaling pathways downstream PGE1, including cell cycle progression, extracellular matrix, adhesion, or actin cytoskeleton organization. In conclusion, using in vitro and in vivo models, we showed that prostaglandin E2 receptor agonists can ameliorate several of the pleotropic phenotypes caused by the absence of NPHP1; this opens their potential as a first therapeutic option for juvenile NPH-associated ciliopathies.

Patient-Patient Similarity-Based Screening of a Clinical Data Warehouse to Support Ciliopathy Diagnosis.

A timely diagnosis is a key challenge for many rare diseases. As an expanding group of rare and severe monogenic disorders with a broad spectrum of clinical manifestations, ciliopathies, notably renal ciliopathies, suffer from important underdiagnosis issues. Our objective is to develop an approach for screening large-scale clinical data warehouses and detecting patients with similar clinical manifestations to those from diagnosed ciliopathy patients. We expect that the top-ranked similar patients will benefit from genetic testing for an early diagnosis. The dependence and relatedness between phenotypes were taken into account in our similarity model through medical concept embedding. The relevance of each phenotype to each patient was also considered by adjusted aggregation of phenotype similarity into patient similarity. A ranking model based on the best-subtype-average similarity was proposed to address the phenotypic overlapping and heterogeneity of ciliopathies. Our results showed that using less than one-tenth of learning sources, our language and center specific embedding provided comparable or better performances than other existing medical concept embeddings. Combined with the best-subtype-average ranking model, our patient-patient similarity-based screening approach was demonstrated effective in two large scale unbalanced datasets containing approximately 10,000 and 60,000 controls with kidney manifestations in the clinical data warehouse (about 2 and 0.4% of prevalence, respectively). Our approach will offer the opportunity to identify candidate patients who could go through genetic testing for ciliopathy. Earlier diagnosis, before irreversible end-stage kidney disease, will enable these patients to benefit from appropriate follow-up and novel treatments that could alleviate kidney dysfunction.

Author Correction: High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics.

Mining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes1. We present a method for high-throughput, single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays2, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100-1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450-900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility.

Single-cell deep phenotyping of IgG-secreting cells for high-resolution immune monitoring.

Studies of the dynamics of the antibody-mediated immune response have been hampered by the absence of quantitative, high-throughput systems to analyze individual antibody-secreting cells. Here we describe a simple microfluidic system, DropMap, in which single cells are compartmentalized in tens of thousands of 40-pL droplets and analyzed in two-dimensional droplet arrays using a fluorescence relocation-based immunoassay. Using DropMap, we characterized antibody-secreting cells in mice immunized with tetanus toxoid (TT) over a 7-week protocol, simultaneously analyzing the secretion rate and affinity of IgG from over 0.5 million individual cells enriched from spleen and bone marrow. Immunization resulted in dramatic increases in the range of both single-cell secretion rates and affinities, which spanned at maximum 3 and 4 logs, respectively. We observed differences over time in dynamics of secretion rate and affinity within and between anatomical compartments. This system will not only enable immune monitoring and optimization of immunization and vaccination protocols but also potentiate antibody screening.

Maternal LSD1/KDM1A is an essential regulator of chromatin and transcription landscapes during zygotic genome activation.

Upon fertilization, the highly specialised sperm and oocyte genomes are remodelled to confer totipotency. The mechanisms of the dramatic reprogramming events that occur have remained unknown, and presumed roles of histone modifying enzymes are just starting to be elucidated. Here, we explore the function of the oocyte-inherited pool of a histone H3K4 and K9 demethylase, LSD1/KDM1A during early mouse development. KDM1A deficiency results in developmental arrest by the two-cell stage, accompanied by dramatic and stepwise alterations in H3K9 and H3K4 methylation patterns. At the transcriptional level, the switch of the maternal-to-zygotic transition fails to be induced properly and LINE-1 retrotransposons are not properly silenced. We propose that KDM1A plays critical roles in establishing the correct epigenetic landscape of the zygote upon fertilization, in preserving genome integrity and in initiating new patterns of genome expression that drive early mouse development.

Proteolytic processing of the extracellular scaffolding protein LEV-9 is required for clustering acetylcholine receptors.

Correct positioning of neurotransmitter-gated receptors at postsynapses is essential for synaptic transmission. At Caenorhabditis elegans neuromuscular junctions, clustering of levamisole-sensitive acetylcholine receptors (L-AChRs) requires the muscle-secreted scaffolding protein LEV-9, a multidomain factor containing complement control protein (CCP) modules. Here we show that LEV-9 needs to be cleaved at its C terminus to exert its function. LEV-9 cleavage is not required for trafficking nor secretion but directly controls scaffolding activity. The cleavage site is evolutionarily conserved, and post-translational cleavage ensures the structural and functional decoupling between different isoforms encoded by the lev-9 gene. Data mining indicates that most human CCP-containing factors are likely cleaved C-terminally from CCP tandems, suggesting that not only domain architectures but also cleavage location can be conserved in distant architecturally related proteins.

cAMP protein kinase phosphorylates the Mos1 transposase and regulates its activity: evidences from mass spectrometry and biochemical analyses.

Genomic plasticity mediated by transposable elements can have a dramatic impact on genome integrity. To minimize its genotoxic effects, it is tightly regulated either by intrinsic mechanisms (linked to the element itself) or by host-mediated mechanisms. Using mass spectrometry, we show here for the first time that MOS1, the transposase driving the mobility of the mariner Mos1 element, is phosphorylated. We also show that the transposition activity of MOS1 is downregulated by protein kinase AMP cyclic-dependent phosphorylation at S170, which renders the transposase unable to promote Mos1 transposition. One step in the transposition cycle, the assembly of the paired-end complex, is specifically inhibited. At the cellular level, we provide evidence that phosphorylation at S170 prevents the active transport of the transposase into the nucleus. Our data suggest that protein kinase AMP cyclic-dependent phosphorylation may play a double role in the early stages of genome invasion by mariner elements.

Positive modulation of a Cys-loop acetylcholine receptor by an auxiliary transmembrane subunit.

Auxiliary subunits regulate the trafficking, localization or gating kinetics of voltage- and ligand-gated ion channels by associating tightly and specifically with pore-forming subunits. However, no auxiliary subunits have been identified for members of the Cys-loop receptor superfamily. Here we identify MOLO-1, a positive regulator of levamisole-sensitive acetylcholine receptors (L-AChRs) at the Caenorhabditis elegans neuromuscular junction. MOLO-1 is a one-pass transmembrane protein that contains a single extracellular globular domain-the TPM domain, found in bacteria, plants and invertebrates, including nonvertebrate chordates. Loss of MOLO-1 impairs locomotion and renders worms resistant to the anthelmintic drug levamisole. In molo-1 mutants, L-AChR-dependent synaptic transmission is reduced by half, while the number and localization of receptors at synapses remain unchanged. In a heterologous expression system, MOLO-1 physically interacts with L-AChRs and directly enhances channel gating without affecting unitary conductance. The identification of MOLO-1 expands the mechanisms for generating functional and pharmacological diversity in the Cys-loop superfamily.

Macoilin, a conserved nervous system-specific ER membrane protein that regulates neuronal excitability.

Genome sequence comparisons have highlighted many novel gene families that are conserved across animal phyla but whose biological function is unknown. Here, we functionally characterize a member of one such family, the macoilins. Macoilins are characterized by several highly conserved predicted transmembrane domains towards the N-terminus and by coiled-coil regions C-terminally. They are found throughout Eumetazoa but not in other organisms. Mutants for the single Caenorhabditis elegans macoilin, maco-1, exhibit a constellation of behavioral phenotypes, including defects in aggregation, O2 responses, and swimming. MACO-1 protein is expressed broadly and specifically in the nervous system and localizes to the rough endoplasmic reticulum; it is excluded from dendrites and axons. Apart from subtle synapse defects, nervous system development appears wild-type in maco-1 mutants. However, maco-1 animals are resistant to the cholinesterase inhibitor aldicarb and sensitive to levamisole, suggesting pre-synaptic defects. Using in vivo imaging, we show that macoilin is required to evoke Ca²(+) transients, at least in some neurons: in maco-1 mutants the O2-sensing neuron PQR is unable to generate a Ca²(+) response to a rise in O2. By genetically disrupting neurotransmission, we show that pre-synaptic input is not necessary for PQR to respond to O2, indicating that the response is mediated by cell-intrinsic sensory transduction and amplification. Disrupting the sodium leak channels NCA-1/NCA-2, or the N-,P/Q,R-type voltage-gated Ca²(+) channels, also fails to disrupt Ca²(+) responses in the PQR cell body to O2 stimuli. By contrast, mutations in egl-19, which encodes the only Caenorhabditis elegans L-type voltage-gated Ca²(+) channel α1 subunit, recapitulate the Ca²(+) response defect we see in maco-1 mutants, although we do not see defects in localization of EGL-19. Together, our data suggest that macoilin acts in the ER to regulate assembly or traffic of ion channels or ion channel regulators.

Phosphotriesterase variants with high methylphosphonatase activity and strong negative trade-off against phosphotriesters.

The most lethal organophosphorus nerve agents (NA), like sarin, soman, agent-VX and Russian-VX, share a methylphosphonate moiety. Pseudomonas diminuta phosphotriesterase (PTE) catalyses the hydrolysis of methylphosphonate NA analogues with a catalytic efficiency orders of magnitude lower than that towards the pesticide paraoxon. With a view to obtaining PTE variants that more readily accept methylphosphonate NA, ~75,000 PTE variants of the substrate-binding residues Gly-60, Ile-106, Leu-303 and Ser-308 were screened with fluorogenic analogues of the NA Russian-VX and cyclosarin. Seven new PTE variants were isolated, purified and their k(cat)/K(M) determined against five phosphotriesters and five methylphosphonate analogues of sarin, cyclosarin, soman, agent-VX and Russian-VX. The novel PTE variants exhibited as much as a 10-fold increase in activity towards the methylphosphonate compounds--many reaching a k(cat)/K(M) of 106 M⁻¹ s⁻¹--and as much as a 29,000-fold decrease in their phosphotriesterase activity. The mutations found in two of the variants, SS0.5 (G60V/I106L/S308G) and SS4.5 (G60V/I106A/S308G), were modelled into a high-resolution structure of PTE-wild type and docked with analogues of cyclosarin and Russian-VX using Autodock 4.2. The kinetic data and docking simulations suggest that the increase in activity towards the methylphosphonates and the loss of function against the phosphotriesters were due to an alteration of the shape and hydrophobicity of the binding pocket that hinders the productive binding of non-chiral racemic phosphotriesters, yet allows the binding of the highly asymmetric methylphosphonates.

Highest paraoxonase turnover rate found in a bacterial phosphotriesterase variant.

The bacterial phosphotriesterase (PTE) catalyses the hydrolysis of the man-made pesticide paraoxon with a diffusion-limited efficiency. Here we describe the selection and characterisation of PTE variant SS0.2 that possesses the highest paraoxonase turnover number so far described (k(cat) = 31,000 s⁻¹). The PTE-SS0.2 was selected from a library of binding-site mutants using a novel screening method that combines partial lysis of bacterial colonies and fluorogenic probes.

Analogues with fluorescent leaving groups for screening and selection of enzymes that efficiently hydrolyze organophosphorus nerve agents.

Enzymes that efficiently hydrolyze highly toxic organophosphorus nerve agents could potentially be used as medical countermeasures. As sufficiently active enzymes are currently unknown, we synthesized twelve fluorogenic analogues of organophosphorus nerve agents with the 3-chloro-7-oxy-4-methylcoumarin leaving group as probes for high-throughput enzyme screening. This set included analogues of the pesticides paraoxon, parathion, and dimefox, and the nerve agents DFP, tabun, sarin, cyclosarin, soman, VX, and Russian-VX. Data from inhibition of acetylcholinesterase, in vivo toxicity tests of a representative analogue (cyclosarin), and kinetic studies with phosphotriesterase (PTE) from Pseudomonas diminuta, and a mammalian serum paraoxonase (PON1), confirmed that the analogues mimic the parent nerve agents effectively. They are suitable tools for high-throughput screens for the directed evolution of efficient nerve agent organophosphatases.