Cilia play critical roles in cell signal transduction and organ development. Defects in cilia function result in a variety of genetic disorders. Cep290 is an evolutionarily conserved ciliopathy protein that bridges the ciliary membrane and axoneme at the basal body (BB) and plays critical roles in the initiation of ciliogenesis and TZ assembly. How Cep290 is maintained at BB and whether axonemal and ciliary membrane localized cues converge to determine the localization of Cep290 remain unknown. Here, we report that the Cep131-Cep162 module near the axoneme and the Cby-Fam92 module close to the membrane synergistically control the BB localization of Cep290 and the subsequent initiation of ciliogenesis in Drosophila. Concurrent deletion of any protein of the Cep131-Cep162 module and of the Cby-Fam92 module leads to a complete loss of Cep290 from BB and blocks ciliogenesis at its initiation stage. Our results reveal that the first step of ciliogenesis strictly depends on cooperative and retroactive interactions between Cep131-Cep162, Cby-Fam92 and Cep290, which may contribute to the complex pathogenesis of Cep290-related ciliopathies.

The evolution and development of the head have long captivated researchers due to the crucial role of the head as the gateway for sensory stimuli and the intricate structural complexity of the head. Although significant progress has been made in understanding head development in various vertebrate species, our knowledge of early human head ontogeny remains limited. Here, we used advanced whole-mount immunostaining and 3D imaging techniques to generate a comprehensive 3D cellular atlas of human head embryogenesis. We present detailed developmental series of diverse head tissues and cell types, including muscles, vasculature, cartilage, peripheral nerves, and exocrine glands. These datasets, accessible through a dedicated web interface, provide insights into human embryogenesis. We offer perspectives on the branching morphogenesis of human exocrine glands and unknown features of the development of neurovascular and skeletomuscular structures. These insights into human embryology have important implications for understanding craniofacial defects and neurological disorders and advancing diagnostic and therapeutic strategies.

Human limbs emerge during the fourth post-conception week as mesenchymal buds, which develop into fully formed limbs over the subsequent months1. This process is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common2. Decades of work with model organisms have defined the fundamental mechanisms underlying vertebrate limb development, but an in-depth characterization of this process in humans has yet to be performed. Here we detail human embryonic limb development across space and time using single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells from a few multipotent progenitors to myriad differentiated cell states, including several novel cell populations. We uncover two waves of human muscle development, each characterized by different cell states regulated by separate gene expression programmes, and identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity. Through assembly of multiple anatomically continuous spatial transcriptomic samples using VisiumStitcher, we map cells across a sagittal section of a whole fetal hindlimb. We reveal a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncover transcriptionally and spatially distinct populations of the mesenchyme in the autopod. Finally, we perform single-cell RNA sequencing on mouse embryonic limbs to facilitate cross-species developmental comparison, finding substantial homology between the two species.

Objective: Small-cell lung cancer (SCLC) is the malignancy most frequently associated with paraneoplastic neurological syndromes (PNS) and can trigger different antibody responses against intracellular (Hu) or neuronal surface (GABAB R) antigens. Our aim was to clarify if the genomic and transcriptomic features of SCLC are different in patients with anti-GABAB R or anti-Hu PNS compared with SCLC without PNS.
Methods: A total of 76 SCLC tumor samples were collected: 34 anti-Hu, 14 anti-GABAB R, and 28 SCLC without PNS. The study consisted of four steps: (i) pathological confirmation; (ii) Next Generation Sequencing using a panel of 98 genes, including those encoding the autoantibodies targets ELAVL1-4, GABBR1-2, KCTD16; (iii) genome-wide Copy Number Variation (CNV); (iv) whole-transcriptome RNA sequencing.
Results: CNV analysis revealed that patients with anti-GABAB R PNS have commonly a gain in chromosome 5q, which contains KCTD16, while anti-Hu and control patients often harbor a loss. No significantly different number of mutations regarding any onconeural genes was observed. Conversely, transcriptomic profile of SCLC was different and the differentially expressed genes permitted to effectively cluster the samples into three groups, reflecting the antibody-based classification, with an overexpression of KCTD16 specific to anti-GABAB R PNS. Pathway analysis revealed that tumors of patients with anti-GABAB R encephalitis were enriched in B cell signatures, as opposed to those of patients with anti-Hu in which T cell and IFN-γ-related signatures were overexpressed.
Interpretation: SCLC genetic and transcriptomic features differentiate anti-GABAB R, anti-Hu, and non-PNS tumors. The role of KCTD16 appears to be pivotal in the tumor immune tolerance breakdown of anti-GABAB R PNS. This article is protected by copyright. All rights reserved.

Background
Among KCNH2 missense loss of function (LOF) variants, homozygosity –at any position in the Kv11.1/hERG channel – is very rare and generally leads to intrauterine death, while heterozygous variants in the pore are responsible for severe Type 2 long-QT syndrome (LQTS). We report a novel homozygous p.Gly603Ser missense variant in the pore of Kv11.1/hERG (KCNH2 c.1807G > A) discovered in the context of a severe LQTS.
Methods
We carried out a phenotypic family study combined with a functional analysis of mutated and wild-type (WT) Kv11.1 by two-electrode voltage-clamp using the Xenopus laevis oocyte heterologous expression system.
Results
The variant resulted in a severe LQTS phenotype (very prolonged corrected QT interval, T-wave alternans, multiple Torsades de pointes) with a delayed clinical expression in later childhood in the homozygous state, and in a Type 2 LQTS phenotype in the heterozygous state. Expression of KCNH2 p.Gly603Ser cRNA alone elicited detectable current in Xenopus oocytes. Inactivation kinetics and voltage dependence of activation were not significantly affected by the variant. The macroscopic slope conductance of the variant was three-fold less compared to the WT (18.5 ± 9.01 vs 54.7 ± 17.2 μS, p < 0.001).
Conclusions
We characterized the novel p.Gly603Ser KCNH2 missense LOF variant in the pore region of Kv11.1/hERG leading to a severe but viable LQTS in the homozygous state and an attenuated Type 2 LQTS in heterozygous carriers. To our knowledge we provide the first description of a homozygous variant in the pore-forming region of Kv11.1 with a functional impact but a delayed clinical expression.

Co-occurring anti-tripartite motif-containing protein 9 and 67 autoantibodies (TRIM9/67-IgG) have been reported in only a very few cases of paraneoplastic cerebellar syndrome. The value of these biomarkers and the most sensitive methods of TRIM9/67-IgG detection are not known. We performed a retrospective, multi-center study to evaluate the cerebrospinal fluid (CSF) and serum of candidate TRIM9/67-IgG cases by tissue-based immunofluorescence (TBIF), peptide phage display immunoprecipitation sequencing (PhIP-Seq), overexpression cell-based assay (CBA), and immunoblot. Cases in whom TRIM9/67-IgG was detected by at least two assays were considered TRIM9/67-IgG positive. Among these cases (N=13), CBA was the most sensitive (100%) and revealed that all cases had TRIM9 and TRIM67 autoantibodies. Of TRIM9/67-IgG cases with available clinical history, a subacute cerebellar syndrome was the most common presentation (N = 7/10), followed by encephalitis (N = 3/10). Of these ten, 70% had comorbid cancer (7/10), 85% of whom (N = 6/7) had confirmed metastatic disease. All evaluable cancer biopsies expressed TRIM9 protein (N = 5/5), whose expression was elevated in the cancerous regions of the tissue in 4 of 5 cases. TRIM9/67-IgG are rare but likely high-risk paraneoplastic biomarkers for which CBA appears to be the most sensitive diagnostic assay. This article is protected by copyright. All rights reserved.

The nematode Caenorhabditis elegans is a research model organism particularly suited to the mechanistic understanding of synapse genesis in the nervous system. Armed with powerful genetics, knowledge of complete connectomics, and modern genomics, studies using C. elegans have unveiled multiple key regulators in the formation of a functional synapse. Importantly, many signaling networks display remarkable conservation throughout animals, underscoring the contributions of C. elegans research to advance the understanding of our brain. In this chapter, we will review up-to-date information of the contribution of C. elegans to the understanding of chemical synapses, from structure to molecules and to synaptic remodeling.

The molecular code that controls synapse formation and maintenance in vivo has remained quite sparse. Here, we identify that the secreted protein Adamtsl3 functions as critical hippocampal synapse organizer acting through the transmembrane receptor DCC (deleted in colorectal cancer). Traditionally, DCC function has been associated with glutamatergic synaptogenesis and plasticity in response to Netrin-1 signaling. We demonstrate that early post-natal deletion of Adamtsl3 in neurons impairs DCC protein expression, causing reduced density of both glutamatergic and GABAergic synapses. Adult deletion of Adamtsl3 in either GABAergic or glutamatergic neurons does not interfere with DCC-Netrin-1 function at glutamatergic synapses but controls DCC signaling at GABAergic synapses. The Adamtsl3-DCC signaling unit is further essential for activity-dependent adaptations at GABAergic synapses, involving DCC phosphorylation and Src kinase activation. These findings might be particularly relevant for schizophrenia because genetic variants in Adamtsl3 and DCC have been independently linked with schizophrenia in patients.

Evolutionary transitions from oviparity to viviparity are frequent across diverse taxa. Some species also display intraspecific variation in parity mode, or they exhibit an intermediate mode by laying eggs containing embryos at variable, often advanced stages of development. How such natural quantitative variation in egg retention arises through differences in genetics, behaviour, and physiology – and how this variation ultimately connects to variation in specific fitness components – is not well-understood. Here, we study this problem by characterizing intraspecific variation in constitutive retention of fertilized eggs of the nematode Caenorhabditis elegans. Analysing a panel of ∼300 wild strains, we find highly variable intra-uterine retention of fertilized eggs, with a fraction of strains showing either strongly reduced or increased egg retention with partial viviparity. We provide evidence for multiple evolutionary origins of such phenotypic extremes and we identify candidate loci explaining this natural variation. Characterizing a subset of wild strains, we confirm that natural variation in egg-laying behaviour contributes to observed differences in egg retention. Using multiple neuromodulatory agents and controlled CRISPR-Cas9-mediated genetic manipulation of endogenous serotonin levels in 10 wild strains, we then show that this behavioural variation arises through an evolutionarily divergent neuromodulatory architecture of the egg-laying circuitry. Intraspecific variation in C. elegans neural circuit activity therefore connects with variation in reproductive strategy, including transitions from oviparity to partial viviparity. In a second objective, we asked why natural variation in C. elegans egg retention might be maintained. Examining potential fitness costs and benefits of this natural variation, we show that strong egg retention reduces maternal fertility and survival, mostly due to detrimental larval hatching in utero. On the other hand, such genotypes with strong egg retention can benefit from improved offspring protection against environmental insults and by gaining a competitive advantage as offspring exhibit a shortened extra-uterine developmental time to reproductive maturity. Observed natural variation in C. elegans egg-laying behaviour may therefore reflect modifications of a trade-off between alternative fitness components expressed across generations. Our study uncovers underappreciated natural diversity in the C. elegans egg-laying circuit and provides insights into the fitness consequences of this behavioural variation. We propose that intraspecific variation in nematode egg-laying behaviour can serve as an ideal system to pinpoint the molecular changes underlying evolutionary transitions between invertebrate ovi- and viviparity.

Metastatic melanoma patients carrying a BRAFV600 mutation can be treated with a combination of BRAF and MEK inhibitors (BRAFi/MEKi), but innate and acquired resistance invariably occurs. Predicting patient response to targeted therapies is crucial to guide clinical decision. We describe here the development of a highly efficient patient-derived xenograft model adapted to patient melanoma biopsies, using the avian embryo as a host (AVI-PDX). In this in vivo paradigm, we depict a fast and reproducible tumor engraftment of patient samples within the embryonic skin, preserving key molecular and phenotypic features. We show that sensitivity and resistance to BRAFi/MEKi can be reliably modeled in these AVI-PDX , as well as synergies with other drugs. We further provide proof-of-concept that the AVI-PDX models the diversity of responses of melanoma patients to BRAFi/MEKi, within days, hence positioning it as a valuable tool for the design of personalized medicine assays and for the evaluation of novel combination strategies.

The plasma membrane of excitable cells is highly structured and molecular scaffolds recruit proteins to specific membrane compartments. Here, we show that potassium channels and proteins belonging to the dystrophin-associated protein complex define multiple types of planar-polarized membrane compartments at the surface of C. elegans muscle cells. Surprisingly, conserved planar cell polarity proteins are not required for this process. However, we implicate a Wnt signaling module involving the Wnt ligand EGL-20, the Wnt receptor CAM-1, and the intracellular effector DSH-1/disheveled in the formation of this cell polarity pattern. Moreover, using time-resolved and tissue-specific protein degradation, we demonstrate that muscle cell polarity is a dynamic state, requiring continued presence of DSH-1 throughout post-embryonic life. Our results reveal the intricate, highly reproducible, and entirely unsuspected complexity of the worm's sarcolemma. This novel case of planar cell polarity in a tractable genetic model organism may provide valuable insight into the molecular and cellular mechanisms that regulate cellular organization, allowing specific functions to be compartmentalized within distinct plasma membrane domains.

Behaviors consist of distinct states and state transitions. How a neural system continuously modulates each state, which underlies smooth state transitions, is not well understood. C. elegans spontaneously switches between two mutually exclusive motor states, forward and backward movements, a behavior long thought to reflect the reciprocal antagonism between interneurons that separately gate the forward and backward motor circuits. We report here that during spontaneous locomotion, these interneurons are not functionally equivalent, and are not strictly reciprocally inhibitory. Instead, AVA, the premotor interneuron thought to exclusively gate the backward motor circuit, functions as a master regulator for both motor states. AVA's transient activation inhibits the forward circuit while initiating backward movement. In parallel, AVA maintains a constitutive and excitatory input to the forward circuit to promote the exit from backward movement and to maintain forward movement. This excitation results from AVA's sustained depolarized membrane potential, which makes its chemical synapse tonically active. A tonically active master neuron breaks the symmetry between the forward and backward motor circuit and offers a powerful circuit solution for smooth transitions when animals switch between two mutually exclusive motor states.

Cilia are highly conserved organelles critical for animal development and perception. Dysfunction of cilia has been linked to a wide spectrum of human genetic diseases, termed ciliopathies.1,2 Transition fibers (TFs) are striking ciliary base structures essential for cilia assembly. Vertebrates' TFs that originate from centriole distal appendages (DAs) mediate basal body docking to ciliary vesicles to initiate ciliogenesis and regulate the entry of ciliary proteins for axoneme assembly via intraflagellar transport (IFT) machinery.3 Although no distal appendages can be observed on Drosophila centrioles,4,5 three key TF proteins, FBF1, CEP164, and CEP89, have obvious homologs in Drosophila. We aimed to compare their functions with their mammalian counterparts in Drosophila ciliogenesis. Here, we show that all three proteins are localized like TF proteins at the ciliary base in both sensory neurons and spermatocytes, the only two types of ciliated cells in flies. Fbf1 and Cep89 are essential for the formation of IFT-dependent neuronal cilia, but Cep164 is dispensable for ciliogenesis in flies. Strikingly, none are required for basal body docking and transition zone (TZ) assembly in IFT-dependent neuronal cilia or IFT-independent spermatocyte cilia. Furthermore, we demonstrate that Unc is essential to recruit all three TF proteins and establish a hierarchical order, with Cep89 acting on Fbf1. Collectively, our results not only demonstrate that TF proteins are required for IFT-dependent ciliogenesis in Drosophila, in agreement with an evolutionarily conserved function of these proteins in regulating ciliary protein entry, but also that the basal body docking function of TFs has diverged during evolution.

Quantifying animal behavior during microscopy is crucial to associate optically recorded neural activity with behavioural outputs and states. Here I describe an imaging and tracking system for head-restrained larval zebrafish compatible with functional microscopy. This system is based on the Raspberry Pi computer, Pi NoIR camera, and open-source software for the real-time tail segmentation and skeletonization of the zebrafish tail at over 100hz. This allows for precise and long-term analyses of swimming behaviour, that can be related to functional signals recorded in individual neurons. This system offers a simple but performant solution for quantifying the behavior of head-restrained larval zebrafish, which can be built for 340€.

Studying chemosensory processing desires precise chemical cue presentation, behavioral response monitoring, and large-scale neuronal activity recording. Here we present Fish-on-Chips, a set of optofluidic tools for highly-controlled chemical delivery while simultaneously imaging behavioral outputs and whole-brain neuronal activities at cellular resolution in larval zebrafish. These include a fluidics-based swimming arena and an integrated microfluidics-light sheet fluorescence microscopy (µfluidics-LSFM) system, both of which utilize laminar fluid flows to achieve spatiotemporally precise chemical cue presentation. To demonstrate the strengths of the platform, we used the navigation arena to reveal binasal input-dependent behavioral strategies that larval zebrafish adopt to evade cadaverine, a death-associated odor. The µfluidics-LSFM system enables sequential presentation of odor stimuli to individual or both nasal cavities separated by only ~100 µm. This allowed us to uncover brainwide neural representations of cadaverine sensing and binasal input summation in the vertebrate model. Fish-on-Chips is readily generalizable and will empower the investigation of neural coding in the chemical senses.

Habituation allows animals to learn to ignore persistent but inconsequential stimuli. Despite being the most basic form of learning, a consensus model on the underlying mechanisms has yet to emerge. To probe relevant mechanisms we took advantage of a visual habituation paradigm in larval zebrafish, where larvae reduce their reactions to abrupt global dimming (a dark flash). We used Ca2+ imaging during repeated dark flashes and identified 12 functional classes of neurons that differ based on their rate of adaptation, stimulus response shape, and anatomical location. While most classes of neurons depressed their responses to repeated stimuli, we identified populations that did not adapt, or that potentiated their response. These neurons were distributed across brain areas, consistent with a distributed learning process. Using a small molecule-screening approach, we confirmed that habituation manifests from multiple distinct molecular mechanisms, and we have implicated molecular pathways in habituation, including: Melatonin, Estrogen and GABA signaling. However, by combining anatomical analyses and pharmacological manipulations with Ca2+ imaging, we failed to identify a simple relationship between pharmacology, altered activity patterns, and habituation behaviour. Collectively, our work indicates that habituation occurs via a complex and distributed plasticity processes that cannot be captured by a simple model. Therefore, untangling the mechanisms of habituation will likely require dedicated approaches aimed at sub-component mechanisms underlying this multidimensional learning process.

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 new study identifies a conserved regulatory mechanism for cilia assembly in the closest unicellular relatives of animals, suggesting that this mechanism was already present in a common unicellular ancestor and was repurposed during the transition to multicellularity.

During corticogenesis, dynamic regulation of apical adhesion is fundamental to generate correct numbers and cell identities. While radial glial cells (RGCs) maintain basal and apical anchors, basal progenitors and neurons detach and settle at distal positions from the apical border. Whether diffusible signals delivered from the cerebrospinal fluid (CSF) contribute to the regulation of apical adhesion dynamics remains fully unknown. Secreted class 3 Semaphorins (Semas) trigger cell responses via Plexin-Neuropilin (Nrp) membrane receptor complexes. Here, we report that unconventional Sema3-Nrp preformed complexes are delivered by the CSF from sources including the choroid plexus to Plexin-expressing RGCs via their apical endfeet. Through analysis of mutant mouse models and various ex vivo assays mimicking ventricular delivery to RGCs, we found that two different complexes, Sema3B/Nrp2 and Sema3F/Nrp1, exert dual effects on apical endfeet dynamics, nuclei positioning, and RGC progeny. This reveals unexpected balance of CSF-delivered guidance molecules during cortical development.

Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here, we reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding and, together with mutant proteins, show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell guidance, where finely balanced Unc5-GPC3 interactions regulate cell migration.

Embryonic malignant transformation is concomitant to organogenesis, often affecting multipotent and migratory progenitors. While lineage relationships between malignant cells and their physiological counterparts are extensively investigated, the contribution of exogenous embryonic signals is not fully known. Neuroblastoma (NB) is a childhood malignancy of the peripheral nervous system arising from the embryonic trunk neural crest (NC) and characterized by heterogeneous and interconvertible tumor cell identities. Here, using experimental models mimicking the embryonic context coupled to proteomic and transcriptomic analyses, we show that signals released by embryonic sympathetic ganglia, including Olfactomedin-1, induce NB cells to shift from a noradrenergic to mesenchymal identity, and to activate a gene program promoting NB metastatic onset and dissemination. From this gene program, we extract a core signature specifically shared by metastatic cancers with NC origin. This reveals non-cell autonomous embryonic contributions regulating the plasticity of NB identities and setting pro-dissemination gene programs common to NC-derived cancers.

The Wnt/β-catenin signaling pathway is highly conserved throughout evolution, playing crucial roles in several developmental and pathological processes. Wnt ligands can act at a considerable distance from their sources and it is therefore necessary to examine not only the Wnt-producing but also the Wnt-receiving cells and tissues to fully appreciate the many functions of this pathway. To monitor Wnt activity, multiple tools have been designed which consist of multimerized Wnt signaling response elements (TCF/LEF binding sites) driving the expression of fluorescent reporter proteins (e.g. GFP, RFP) or of LacZ. The high stability of those reporters leads to a considerable accumulation in cells activating the pathway, thereby making them easily detectable. However, this makes them unsuitable to follow temporal changes of the pathway's activity during dynamic biological events. Even though fluorescent transcriptional reporters can be destabilized to shorten their half-lives, this dramatically reduces signal intensities, particularly when applied in vivo. To alleviate these issues, we developed two transgenic quail lines in which high copy number (12× or 16×) of the TCF/LEF binding sites drive the expression of destabilized GFP variants. Translational enhancer sequences derived from viral mRNAs were used to increase signal intensity and specificity. This resulted in transgenic lines efficient for the characterization of TCF/β-catenin transcriptional dynamic activities during embryogenesis, including using in vivo imaging. Our analyses demonstrate the use of this transcriptional reporter to unveil novel aspects of Wnt signaling, thus opening new routes of investigation into the role of this pathway during amniote embryonic development.

Background and objectives: Paraneoplastic cerebellar degeneration (PCD) with anti-Yo antibodies is a cancer-related autoimmune disease directed against neural antigens expressed by tumor cells. A putative trigger of the immune tolerance breakdown is genetic alteration of Yo antigens. We aimed to identify the tumors' genetic and immune specificities involved in Yo-PCD pathogenesis.
Methods: Using clinicopathologic data, immunofluorescence (IF) imaging, and whole-transcriptome analysis, 22 breast cancers (BCs) associated with Yo-PCD were characterized in terms of oncologic characteristics, genetic alteration of Yo antigens, differential gene expression profiles, and morphofunctional specificities of their in situ antitumor immunity by comparing them with matched control BCs.
Results: Yo-PCD BCs were invasive carcinoma of no special type, which early metastasized to lymph nodes. They overexpressed human epidermal growth factor receptor 2 (HER2) but were hormone receptor negative. All Yo-PCD BCs carried at least 1 genetic alteration (variation or gain in copy number) on CDR2L, encoding the main Yo antigen that was found aberrantly overexpressed in Yo-PCD BCs. Analysis of the differentially expressed genes found 615 upregulated and 54 downregulated genes in Yo-PCD BCs compared with HER2-driven control BCs without PCD. Ontology enrichment analysis found significantly upregulated adaptive immune response pathways in Yo-PCD BCs. IF imaging confirmed an intense immune infiltration with an overwhelming predominance of immunoglobulin G-plasma cells.
Discussion: These data confirm the role of genetic alterations of Yo antigens in triggering the immune tolerance breakdown but also outline a specific biomolecular profile in Yo-PCD BCs, suggesting a cancer-specific pathogenesis.

Multiple advances have been made to increase the efficiency of CRISPR/Cas9 editing using the model genetic organism Caenorhabditis elegans (C. elegans). Here we report on the use of co-CRISPR 'marker' genes: worms in which co-CRISPR events have occurred have overt, visible phenotypes which facilitates the selection of worms that harbour CRISPR events in the target gene. Mutation in the co-CRISPR gene is then removed by outcrossing to wild type but this can be challenging if the CRISPR and co-CRISPR gene are hard to segregate. However, segregating away the co-CRISPR modified gene can be less challenging if the worms selected appear wild type and are selected from a jackpot brood. These are broods in which a high proportion of the progeny of a single injected worm display the co-CRISPR phenotype suggesting high CRISPR efficiency. This can deliver worms that harbour the desired mutation in the target gene locus without the co-CRISPR mutation. We have successfully generated a discrete mutation in the C. elegans nlg-1 gene using this method. However, in the process of sequencing to authenticate editing in the nlg-1 gene we discovered genomic rearrangements that arise at the co-CRISPR gene unc-58 that by visual observation were phenotypically silent but nonetheless resulted in a significant reduction in motility scored by thrashing behaviour. This highlights that careful consideration of the hidden consequences of co-CRISPR mediated genetic changes should be taken before downstream analysis of gene function. Given this, we suggest sequencing of co-CRISPR genes following CRISPR procedures that utilise phenotypic selection as part of the pipeline.

Background and objectives: To report the clinical, biological, and imaging features and clinical course of a French cohort of patients with glial fibrillary acidic protein (GFAP) autoantibodies. Methods: We retrospectively included all patients who tested positive for GFAP antibodies in the CSF by immunohistochemistry and confirmed by cell-based assay using cells expressing human GFAPα since 2017 from 2 French referral centers. Results: We identified 46 patients with GFAP antibodies. Median age at onset was 43 years, and 65% were men. Infectious prodromal symptoms were found in 82%. Other autoimmune diseases were found in 22% of patients, and coexisting neural autoantibodies in 11%. Tumors were present in 24%, and T-cell dysfunction in 23%. The most frequent presentation was subacute meningoencephalitis (85%), with cerebellar dysfunction in 57% of cases. Other clinical presentations included myelitis (30%) and visual (35%) and peripheral nervous system involvement (24%). MRI showed perivascular radial enhancement in 32%, periventricular T2 hyperintensity in 41%, brainstem involvement in 31%, leptomeningeal enhancement in 26%, and reversible splenial lesions in 4 cases. A total of 33 of 40 patients had a monophasic course, associated with a good outcome at last follow-up (Rankin Score ≤2: 89%), despite a severe clinical presentation. Adult and pediatric features are similar. Thirty-two patients were treated with immunotherapy. A total of 11/22 patients showed negative conversion of GFAP antibodies. Discussion: GFAP autoimmunity is mainly associated with acute/subacute meningoencephalomyelitis with prodromal symptoms, for which tumors and T-cell dysfunction are frequent triggers. The majority of patients followed a monophasic course with a good outcome.

Glioblastomas (GBMs), the most frequent brain tumours, are highly invasive and their prognosis is still poor despite the use of combination treatment. MG624 is a 4-oxystilbene derivative that is active on α7- and α9-containing neuronal nicotinic acetylcholine receptor (nAChR) subtypes. Hybridisation of MG624 with a non-nicotinic resveratrol-derived pro-oxidant mitocan has led to two novel compounds (StN-4 and StN-8) that are more potent than MG624 in reducing the viability of GBM cells, but less potent in reducing the viability of mouse astrocytes. Functional analysis of their activity on α7 receptors showed that StN-4 is a silent agonist, whereas StN-8 is a full antagonist, and neither alters intracellular [Ca2+] levels when acutely applied to U87MG cells. After 72 h of exposure, both compounds decreased U87MG cell proliferation, and pAKT and oxphos ATP levels, but only StN-4 led to a significant accumulation of cells in phase G1/G0 and increased apoptosis. One hour of exposure to either compound also decreased the mitochondrial and cytoplasmic ATP production of U87MG cells, and this was not paralleled by any increase in the production of reactive oxygen species. Knocking down the α9 subunit (which is expressed at relatively high levels in U87MG cells) decreased the potency of the effects of both compounds on cell viability, but cell proliferation, ATP production, pAKT levels were unaffected by the presence of the noncell-permeable α7/α9-selective antagonist αBungarotoxin. These last findings suggest that the anti-tumoral effects of StN-4 and StN-8 on GBM cells are not only due to their action on nAChRs, but also to other non-nicotinic mechanisms.

The appearance of synapses was a crucial step in the creation of the variety of nervous systems that are found in the animal kingdom. With increased complexity of the organisms came a greater number of synaptic proteins. In this review we describe synaptic proteins that contain the structural domains CUB, CCP, or TSP-1. These domains are found in invertebrates and vertebrates, and CUB and CCP domains were initially described in proteins belonging to the complement system of innate immunity. Interestingly, they are found in synapses of the nematode C. elegans, which does not have a complement system, suggesting an ancient function. Comparison of the roles of CUB-, CCP-, and TSP-1 containing synaptic proteins in various species shows that in more complex nervous systems, these structural domains are combined with other domains and that there is partial conservation of their function. These three domains are thus basic building blocks of the synaptic architecture. Further studies of structural domains characteristic of synaptic proteins in invertebrates such as C. elegans and comparison of their role in mammals will help identify other conserved synaptic molecular building blocks. Furthermore, this type of functional comparison across species will also identify structural domains added during evolution in correlation with increased complexity, shedding light on mechanisms underlying cognition and brain diseases.

Embryonic malignant transformation is concomitant to organogenesis, often affecting multipotent and migratory progenitors. While lineage relationships between malignant cells and their physiological counterparts are extensively investigated, the contribution of exogenous embryonic signals is not fully known. Neuroblastoma (NB) is a childhood malignancy of the peripheral nervous system arising from the embryonic trunk neural crest (NC) and characterized by heterogeneous and interconvertible tumor cell identities. Here, using experimental models mimicking the embryonic context coupled to proteomic and transcriptomic analyses, we show that signals released by embryonic sympathetic ganglia, including Olfactomedin-1, induce NB cells to shift from a noradrenergic to mesenchymal identity, and to activate a gene program promoting NB metastatic onset and dissemination. From this gene program, we extract a core signature specifically shared by metastatic cancers with NC origin. This reveals non-cell autonomous embryonic contributions regulating the plasticity of NB identities and setting pro-dissemination gene programs common to NC-derived cancers.

Background: Arthrogryposis multiplex congenita (AMC) is characterised by congenital joint contractures in two or more body areas. AMC exhibits wide phenotypic and genetic heterogeneity. Our goals were to improve the genetic diagnosis rates of AMC, to evaluate the added value of whole exome sequencing (WES) compared with targeted exome sequencing (TES) and to identify new genes in 315 unrelated undiagnosed AMC families.
Methods: Several genomic approaches were used including genetic mapping of disease loci in multiplex or consanguineous families, TES then WES. Sanger sequencing was performed to identify or validate variants.
Results: We achieved disease gene identification in 52.7% of AMC index patients including nine recently identified genes (CNTNAP1, MAGEL2, ADGRG6, ADCY6, GLDN, LGI4, LMOD3, UNC50 and SCN1A). Moreover, we identified pathogenic variants in ASXL3 and STAC3 expanding the phenotypes associated with these genes. The most frequent cause of AMC was a primary involvement of skeletal muscle (40%) followed by brain (22%). The most frequent mode of inheritance is autosomal recessive (66.3% of patients). In sporadic patients born to non-consanguineous parents (n=60), de novo dominant autosomal or X linked variants were observed in 30 of them (50%).
Conclusion: New genes recently identified in AMC represent 21% of causing genes in our cohort. A high proportion of de novo variants were observed indicating that this mechanism plays a prominent part in this developmental disease. Our data showed the added value of WES when compared with TES due to the larger clinical spectrum of some disease genes than initially described and the identification of novel genes.

The Wnt/β-catenin signaling pathway is highly conserved throughout evolution, playing crucial roles in several developmental and pathological processes. Wnt ligands can act at a considerable distance from their sources and it is therefore necessary to examine not only the Wnt-producing but also the Wnt-receiving cells and tissues to fully appreciate the many functions of this pathway. To monitor Wnt activity, multiple tools have been designed which consist of multimerized Wnt signaling response elements (TCF/LEF binding sites) driving the expression of fluorescent reporter proteins (e.g. GFP, RFP) or of LacZ. The high stability of those reporters leads to a considerable accumulation in cells activating the pathway, thereby making them easily detectable. However, this makes them unsuitable to follow temporal changes of the pathway's activity during dynamic biological events. Even though fluorescent transcriptional reporters can be destabilized to shorten their half-lives, this dramatically reduces signal intensities, particularly when applied in vivo. To alleviate these issues, we developed two transgenic quail lines in which high copy number (12× or 16×) of the TCF/LEF binding sites drive the expression of destabilized GFP variants. Translational enhancer sequences derived from viral mRNAs were used to increase signal intensity and specificity. This resulted in transgenic lines efficient for the characterization of TCF/β-catenin transcriptional dynamic activities during embryogenesis, including using in vivo imaging. Our analyses demonstrate the use of this transcriptional reporter to unveil novel aspects of Wnt signaling, thus opening new routes of investigation into the role of this pathway during amniote embryonic development.

Biophysical properties of ligand-gated receptors can be profoundly modified by auxiliary subunits or by the lipid microenvironment of the membrane. Hence, it is sometimes challenging to relate the properties of receptors reconstituted in heterologous expression systems to those of their native counterparts. Here we show that the properties of Caenorhabditis elegans levamisole-sensitive acetylcholine receptors (L-AChRs), the ionotropic acetylcholine receptors targeted by the cholinergic anthelmintic levamisole at neuromuscular junctions, can be profoundly modified by their clustering machinery. We uncovered that L-AChRs exhibit a strong outward rectification in vivo, which was not previously described in heterologous systems. This unusual feature for an ionotropic AChR is abolished by disrupting the interaction of the receptors with the extracellular complex required for their synaptic clustering. When recorded at -60 mV, levamisole-induced currents are similar in the wild type and in L-AChR-clustering-defective mutants, while they are halved in these mutants at more depolarized physiological membrane potentials. Consequently, levamisole causes a strong muscle depolarization in the wild type, which leads to complete inactivation of the voltage-gated calcium channels and to an irreversible flaccid paralysis. In mutants defective for L-AChR clustering, the levamisole-induced depolarization is weaker, allowing voltage-gated calcium channels to remain partially active, which eventually leads to adaptation and survival of the worms. This explains why historical screens for C. elegans mutants resistant to levamisole identified the components of the L-AChR clustering machinery, in addition to proteins required for receptor biosynthesis or efficacy. This work further emphasizes the importance of pursuing ligand-gated channel characterization in their native environment.

During aging, preservation of locomotion is generally considered an indicator of sustained good health, in elderlies and in animal models. In Caenorhabditis elegans, mutants of the insulin-IGF-1 receptor DAF2/IIRc represent a paradigm of healthy aging, as their increased lifespan is accompanied by a delay in age-related loss of motility. Here, we investigated the DAF-2/IIRc-dependent relationship between longevity and motility using an auxin-inducible degron to trigger tissue-specific degradation of endogenous DAF-2/IIRc. As previously reported, inactivation of DAF-2/IIRc in neurons or intestine was sufficient to extend the lifespan of worms, whereas depletion in epidermis, germline, or muscle was not. However, neither intestinal nor neuronal depletion of DAF-2/IIRc prevented the age-related loss of motility. In 1-day-old adults, DAF-2/IIRc depletion in neurons reduced motility in a DAF-16/FOXO dependent manner, while muscle depletion had no effect. By contrast, DAF-2 depletion in the muscle of middle-age animals improved their motility independently of DAF-16/FOXO but required UNC-120/SRF. Yet, neuronal or muscle DAF-2/IIRc depletion both preserved the mitochondria network in aging muscle. Overall, these results show that the motility pattern of daf-2 mutants is determined by the sequential and opposing impact of neurons and muscle tissues and can be dissociated from the regulation of the lifespan. This work also provides the characterization of a versatile tool to analyze the tissue-specific contribution of insulin-like signaling in integrated phenotypes at the whole organism level.

Animals have a remarkable ability to use local cues to orient in space in the absence of a panoramic fixed reference frame. Here we use the mechanosensory lateral line in larval zebrafish to understand rheotaxis, an innate oriented swimming evoked by water currents. We generated a comprehensive light-microscopy cell-resolution projectome of lateralis afferent neurons (LANs) and used clustering techniques for morphological classification. We find surprising structural constancy among LANs. Laser-mediated microlesions indicate that precise topographic mapping of lateral-line receptors is not essential for rheotaxis. Recording neuronal-activity during controlled mechanical stimulation of neuromasts reveals unequal representation of water-flow direction in the hindbrain. We explored potential circuit architectures constrained by anatomical and functional data to suggest a parsimonious model under which the integration of lateralized signals transmitted by direction-selective LANs underlies the encoding of water-flow direction in the brain. These data provide a new framework to understand how animals use local mechanical cues to orient in space.

Delayed emergence from anesthesia was previously reported in a case study of a child with Glycine Encephalopathy. To investigate the neural basis of this delayed emergence, we developed a zebrafish glial glycine transporter (glyt1 − / −) mutant model. We compared locomotor behaviors; dose–response curves for tricaine, ketamine, and 2,6-diisopropylphenol (propofol); time to emergence from these anesthetics; and time to emergence from propofol after craniotomy in glyt1−/− mutants and their siblings. To identify differentially active brain regions in glyt1−/− mutants, we used pERK immunohistochemistry as a proxy for brain-wide neuronal activity. We show that glyt1−/− mutants initiated normal bouts of movement less frequently indicating lethargy-like behaviors. Despite similar anesthesia dose–response curves, glyt1−/− mutants took over twice as long as their siblings to emerge from ketamine or propofol, mimicking findings from the human case study. Reducing glycine levels rescued timely emergence in glyt1−/− mutants, pointing to a causal role for elevated glycine. Brain-wide pERK staining showed elevated activity in hypnotic brain regions in glyt1−/− mutants under baseline conditions and a delay in sensorimotor integration during emergence from anesthesia. Our study links elevated activity in preoptic brain regions and reduced sensorimotor integration to lethargy-like behaviors and delayed emergence from propofol in glyt1−/− mutants.

Skeletal muscle regenerates through the activation of resident stem cells. Termed satellite cells, these normally quiescent cells are induced to proliferate by wound-derived signals1. Identifying the source and nature of these cues has been hampered by an inability to visualize the complex cell interactions that occur within the wound. Here we use muscle injury models in zebrafish to systematically capture the interactions between satellite cells and the innate immune system after injury, in real time, throughout the repair process. This analysis revealed that a specific subset of macrophages 'dwell' within the injury, establishing a transient but obligate niche for stem cell proliferation. Single-cell profiling identified proliferative signals that are secreted by dwelling macrophages, which include the cytokine nicotinamide phosphoribosyltransferase (Nampt, which is also known as visfatin or PBEF in humans). Nampt secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (Ccr5), which is expressed on muscle stem cells. This analysis shows that in addition to their ability to modulate the immune response, specific macrophage populations also provide a transient stem-cell-activating niche, directly supplying proliferation-inducing cues that govern the repair process that is mediated by muscle stem cells. This study demonstrates that macrophage-derived niche signals for muscle stem cells, such as NAMPT, can be applied as new therapeutic modalities for skeletal muscle injury and disease.

Heparan sulfate (HS) proteoglycans contribute to the structural organization of various neurochemical synapses. Depending on the system, their role involves either the core protein or the glycosaminoglycan chains. These linear sugar chains are extensively modified by HS modification enzymes, resulting in highly diverse molecules. Specific modifications of glycosaminoglycan chains may thus contribute to a sugar code involved in synapse specificity. Caenorhabditis elegans is particularly useful to address this question because of the low level of genomic redundancy of these enzymes, as opposed to mammals. Here, we systematically mutated the genes encoding HS modification enzymes in C. elegans and analyzed their impact on excitatory and inhibitory neuromuscular junctions (NMJs). Using single chain antibodies that recognize different HS modification patterns, we show in vivo that these two HS epitopes are carried by the SDN-1 core protein, the unique C. elegans syndecan ortholog, at NMJs. Intriguingly, these antibodies differentially bind to excitatory and inhibitory synapses, implying unique HS modification patterns at different NMJs. Moreover, while most enzymes are individually dispensable for proper organization of NMJs, we show that 3-O-sulfation of SDN-1 is required to maintain wild-type levels of the extracellular matrix protein MADD-4/Punctin, a central synaptic organizer that defines the identity of excitatory and inhibitory synaptic domains at the plasma membrane of muscle cells.

The extracellular matrix has emerged as an active component of chemical synapses regulating synaptic formation, maintenance, and homeostasis. The heparan sulfate proteoglycan (HSPG) syndecans are known to regulate cellular and axonal migration in the brain. They are also enriched at synapses, but their synaptic functions remain more elusive. Here, we show that SDN-1, the sole orthologue of syndecan in C. elegans, is absolutely required for the synaptic clustering of homomeric α7-like acetylcholine receptors (AChRs) and regulates the synaptic content of heteromeric AChRs. SDN-1 is concentrated at neuromuscular junctions (NMJs) by the neurally secreted synaptic organizer Ce-Punctin/MADD-4, which also activates the transmembrane netrin receptor DCC. Those cooperatively recruit the FARP and CASK orthologues that localize α7-like-AChRs at cholinergic NMJs through physical interactions. Therefore, SDN-1 stands at the core of the cholinergic synapse organization by bridging the extracellular synaptic determinants to the intracellular synaptic scaffold that controls the postsynaptic receptor content.

Neurobeachin (NBEA) was initially identified as a candidate gene for autism. Recently, variants in NBEA have been associated with neurodevelopmental delay and childhood epilepsy. Here, we report on a novel NBEA missense variant (c.5899G > A, p.Gly1967Arg) in the Domain of Unknown Function 1088 (DUF1088) identified in a child enrolled in the Undiagnosed Diseases Network (UDN), who presented with neurodevelopmental delay and seizures. Modeling of this variant in the Caenorhabditis elegans NBEA ortholog, sel-2, indicated that the variant was damaging to in vivo function as evidenced by altered cell fate determination and trafficking of potassium channels in neurons. The variant effect was indistinguishable from that of the reference null mutation suggesting that the variant is a strong hypomorph or a complete loss-of-function. Our experimental data provide strong support for the molecular diagnosis and pathogenicity of the NBEA p.Gly1967Arg variant and the importance of the DUF1088 for NBEA function.

Genetic assimilation-the evolutionary process by which an environmentally induced phenotype is made constitutive-represents a fundamental concept in evolutionary biology. Thought to reflect adaptive phenotypic plasticity, matricidal hatching in nematodes is triggered by maternal nutrient deprivation to allow for protection or resource provisioning of offspring. Here, we report natural Caenorhabditis elegans populations harboring genetic variants expressing a derived state of near-constitutive matricidal hatching. These variants exhibit a single amino acid change (V530L) in KCNL-1, a small-conductance calcium-activated potassium channel subunit. This gain-of-function mutation causes matricidal hatching by strongly reducing the sensitivity to environmental stimuli triggering egg-laying. We show that reestablishing the canonical KCNL-1 protein in matricidal isolates is sufficient to restore canonical egg-laying. While highly deleterious in constant food environments, KCNL-1 V530L is maintained under fluctuating resource availability. A single point mutation can therefore underlie the genetic assimilation-by either genetic drift or selection-of an ancestrally plastic trait.

Objective: Recurrent seizures of autoimmune origin (AEp) are one of the most frequent causes of recurrent seizures or suspected epilepsy of unknown cause. The aim of this study was to identify specific phenotypes corresponding to AEp.
Methods: We retrospectively reviewed features of patients with recurrent seizures of unknown cause and investigated for suspected AEp (January 2015-May 2018). Patients were separated in: (1) AEpAb+: AEp with positive autoantibodies; (2) AEpAb-: suspected AEp (inflammatory central nervous system (CNS) profile) without autoantibodies; (3) NAEp: epilepsy without CNS inflammation.
Results: Eighty-nine epileptic patients underwent a CSF antibody detection. From the remaining 57 epileptic patients (32 excluded for a differential diagnosis), 61.4% were considered as AEp. 21% were AEpAb+ (4 NMDAR, 2 GABAbR, 3 GAD-Ab, 2 LGi1, 1 CASPR2), 40.4% AEpAb-, and 38.6% NAE. AE (AEpAb+ and AEpAb-) was significantly associated with antibody prevalence in epilepsy (APE) score ≥ 4 (80%), encephalitic phase (71.4%), psychiatric involvement (64.7%), cognitive impairment (50%), and status epilepticus (41.2%). Within the group of 29 patients without encephalitic phase and with chronic epilepsy (NEPp), 34.5% were defined as AEp. 10.4% were AEpAb+ (2 GAD, 1 CASPR2) and 24.1% were AEpAb-. NEP AEp was associated with non-cerebral autoimmune disorders, short epileptic disease duration, and cognitive impairment.
Conclusions: Autoimmune cause (AEp) should be assessed in patient suffering from recurrent seizures of unknown cause. Acute encephalitis is clearly the main AEp phenotype. AEp was also defined in more than one-third of chronic epilepsy patients (NEP) of unknown cause. Then, AEp may be combined with other autoimmune comorbidities, a shorter evolution of recurrent seizures, and cognitive impairment.

The purpose of this study is to propose and validate a preclinical in vivo magnetic resonance imaging (MRI) tool to monitor neuroinflammation following ischemic stroke, based on injection of a novel multimodal nanoprobe, NanoGd, specifically designed for internalization by phagocytic cells. First, it is verified that NanoGd is efficiently internalized by microglia in vitro. In vivo MRI coupled with intravenous injection of NanoGd in a permanent middle cerebral artery occlusion mouse model results in hypointense signals in the ischemic lesion. In these mice, longitudinal two-photon intravital microscopy shows NanoGd internalization by activated CX3CR1-GFP/+ cells. Ex vivo analysis, including phase contrast imaging with synchrotron X-ray, histochemistry, and transmission electron microscopy corroborate NanoGd accumulation within the ischemic lesion and uptake by immune phagocytic cells. Taken together, these results confirm the potential of NanoGd-enhanced MRI as an imaging biomarker of neuroinflammation at the subacute stage of ischemic stroke. As far as it is known, this work is the first to decipher the working mechanism of MR signals induced by a nanoparticle passively targeted at phagocytic cells by performing intravital microscopy back-to-back with MRI. Furthermore, using a gadolinium-based rather than an iron-based contrast agent raises future perspectives for the development of molecular imaging with emerging computed tomography technologies.

The vascular endothelial growth factor (VEGF) pathway regulates key processes in synapse function, which are disrupted in early stages of Alzheimer's disease (AD) by toxic-soluble amyloid-beta oligomers (Aβo). Here, we show that VEGF accumulates in and around Aβ plaques in postmortem brains of patients with AD and in APP/PS1 mice, an AD mouse model. We uncover specific binding domains involved in direct interaction between Aβo and VEGF and reveal that this interaction jeopardizes VEGFR2 activation in neurons. Notably, we demonstrate that VEGF gain of function rescues basal synaptic transmission, long-term potentiation (LTP), and dendritic spine alterations, and blocks long-term depression (LTD) facilitation triggered by Aβo. We further decipher underlying mechanisms and find that VEGF inhibits the caspase-3-calcineurin pathway responsible for postsynaptic glutamate receptor loss due to Aβo. These findings provide evidence for alterations of the VEGF pathway in AD models and suggest that restoring VEGF action on neurons may rescue synaptic dysfunction in AD.

Limbic encephalitis with antibodies against adenylate kinase 5 (AK5) has been difficult to characterize because of its rarity. In this study, we identified 10 new cases and reviewed 16 previously reported patients, investigating clinical features, IgG subclasses, human leucocyte antigen and CSF proteomic profiles. Patients with anti-AK5 limbic encephalitis were mostly male (20/26, 76.9%) with a median age of 66 years (range 48-94). The predominant symptom was severe episodic amnesia in all patients, and this was frequently associated with depression (17/25, 68.0%). Weight loss, asthenia and anorexia were also highly characteristic, being present in 11/25 (44.0%) patients. Although epilepsy was always lacking at disease onset, seizures developed later in a subset of patients (4/25, 16.0%). All patients presented CSF abnormalities, such as pleocytosis (18/25, 72.0%), oligoclonal bands (18/25, 72.0%) and increased Tau (11/14, 78.6%). Temporal lobe hyperintensities were almost always present at disease onset (23/26, 88.5%), evolving nearly invariably towards severe atrophy in subsequent MRIs (17/19, 89.5%). This finding was in line with a poor response to immunotherapy, with only 5/25 (20.0%) patients responding. IgG1 was the predominant subclass, being the most frequently detected and the one with the highest titres in nine CSF-serum paired samples. A temporal biopsy from one of our new cases showed massive lymphocytic infiltrates dominated by both CD4+ and CT8+ T cells, intense granzyme B expression and abundant macrophages/microglia. Human leucocyte antigen (HLA) analysis in 11 patients showed a striking association with HLA-B*08:01 [7/11, 63.6%; odds ratio (OR) = 13.4, 95% confidence interval (CI): 3.8-47.4], C*07:01 (8/11, 72.7%; OR = 11.0, 95% CI: 2.9-42.5), DRB1*03:01 (8/11, 72.7%; OR = 14.4, 95% CI: 3.7-55.7), DQB1*02:01 (8/11, 72.7%; OR = 13.5, 95% CI: 3.5-52.0) and DQA1*05:01 (8/11, 72.7%; OR = 14.4, 95% CI: 3.7-55.7) alleles, which formed the extended haplotype B8-C7-DR3-DQ2 in 6/11 (54.5%) patients (OR = 16.5, 95% CI: 4.8-57.1). Finally, we compared the CSF proteomic profile of five anti-AK5 patients with that of 40 control subjects and 10 cases with other more common non-paraneoplastic limbic encephalitis (five with antibodies against leucine-rich glioma inactivated 1 and five against contactin-associated protein-like 2), as well as 10 cases with paraneoplastic neurological syndromes (five with antibodies against Yo and five against Ma2). These comparisons revealed 31 and seven significantly upregulated proteins in anti-AK5 limbic encephalitis, respectively mapping to apoptosis pathways and innate/adaptive immune responses. These findings suggest that the clinical manifestations of anti-AK5 limbic encephalitis result from a distinct T cell-mediated pathogenesis, with major cytotoxicity-induced apoptosis leading to a prompt and aggressive neuronal loss, likely explaining the poor prognosis and response to immunotherapy.

Lack of preclinical patient-derived xenograft cancer models in which to conduct large-scale molecular studies seriously impairs the development of effective personalized therapies. We report here an in vivo concept consisting of implanting human tumor cells in targeted tissues of an avian embryo, delivering therapeutics, evaluating their efficacy by measuring tumors using light sheet confocal microscopy, and conducting large-scale RNA-seq analysis to characterize therapeutic-induced changes in gene expression. The model was established to recapitulate triple-negative breast cancer (TNBC) and validated using TNBC standards of care and an investigational therapeutic agent.

athogenic variants in L1CAM, the gene encoding the L1 cell adhesion molecule, are responsible for a wide clinical spectrum including X-linked hydrocephalus with stenosis of the Sylvius aqueduct, MASA syndrome (mental retardation, aphasia, shuffling gait, adducted thumbs), and a form of spastic paraplegia (SPG1). A moderate phenotype with mild intellectual disability (ID) and X-linked partial corpus callosum agenesis (CCA) has only been related to L1CAM in one family. We report here a second family, including 5 patients with mild to moderate ID and partial CCA without signs usually associated with L1CAM pathogenic variations (such as hydrocephalus, pyramidal syndrome, thumb adductus, aphasia). We identified a previously unreported c.3226A > C transversion leading to a p.Thr1076Pro amino acid substitution in the fifth fibronectin type III domain (FnIII) of the protein which co-segregates with the phenotype within the family. We performed in vitro assays to assess the pathogenic status of this variation. First, the expression of the novel p.Thr1076Pro mutant in COS7 cells resulted in endoplasmic reticulum (ER) retention and reduced L1CAM cell surface expression, which is expected to affect both L1CAM-mediated cell-cell adhesion and neurite growth. Second, immunoblotting techniques showed that the immature form of the L1CAM protein was increased, indicating that this variation led to a lack of maturation of the protein. ID associated with CCA is not a common clinical presentation of L1CAM pathogenic variants. Genome-wide analyses will identify such variations and it is important to acknowledge this atypical phenotype.

Fusion of nascent myoblasts to pre-existing myofibres is critical for skeletal muscle growth and repair. The vast majority of molecules known to regulate myoblast fusion are necessary in this process. Here, we uncover, through high-throughput in vitro assays and in vivo studies in the chicken embryo, that TGFβ (SMAD2/3-dependent) signalling acts specifically and uniquely as a molecular brake on muscle fusion. While constitutive activation of the pathway arrests fusion, its inhibition leads to a striking over-fusion phenotype. This dynamic control of TGFβ signalling in the embryonic muscle relies on a receptor complementation mechanism, prompted by the merging of myoblasts with myofibres, each carrying one component of the heterodimer receptor complex. The competence of myofibres to fuse is likely restored through endocytic degradation of activated receptors. Altogether, this study shows that muscle fusion relies on TGFβ signalling to regulate its pace.

Nesprin-1 is a large scaffold protein connecting nuclei to the actin cytoskeleton via its KASH and Calponin Homology domains, respectively. Nesprin-1 disconnection from nuclei results in altered muscle function and myonuclei mispositioning. Furthermore, Nesprin-1 mutations are associated with muscular pathologies such as Emery Dreifuss muscular dystrophy and arthrogryposis. Nesprin-1 was thus proposed to mainly contribute to muscle function by controlling nuclei position. However, Nesprin-1's localisation at sarcomere's Z-discs, its involvement in organelles' subcellular localization, as well as the description of numerous isoforms presenting different combinations of Calponin Homology (CH) and KASH domains, suggest that the contribution of Nesprin-1 to muscle functions is more complex. Here, we investigate the roles of Nesprin-1/Msp300 isoforms in muscle function and subcellular organisation using Drosophila larvae as a model. Subsets of Msp300 isoform were down-regulated by muscle-specific RNAi expression and muscle global function and morphology were assessed. We show that nuclei anchoring in mature muscle and global muscle function are disconnected functions associated with different Msp300 isoforms. Our work further uncovers a new and unsuspected role of Msp300 in myofibril registration and nuclei peripheral displacement supported by Msp300 CH containing isoforms, a function performed by Desmin in mammals.

The common co-occurrence of autoimmune systemic diseases in patients with neurological disorders and antibodies against glutamic acid decarboxylase 65 (GAD65) suggests a shared genetic predisposition to these disorders. However, the nature and frequency of familial aggregation of autoimmune diseases, which might also support this hypothesis, have been poorly investigated. Herein, an exploratory, interview-based study was conducted with the aim of describing the autoimmune diseases displayed by the relatives of GAD65 neurological patients, their frequency, kinship, and potential patterns of inheritance. Patients were enrolled only if they had GAD65 antibodies in the cerebrospinal fluid and typical clinical phenotypes associated with such antibodies (stiff-person syndrome, cerebellar ataxia, limbic encephalitis, or temporal lobe epilepsy). A total of 65 patients were included in the study, and 44/65 (67.7%) reported family history of autoimmunity, including first-degree relatives in 36/65 (55.4%); the sibling recurrence risk (λS) was 5.5, reinforcing the hypothesis of an underlying strong genetic predisposition. Most pedigrees with familial autoimmunity (38/44, 86.4%) showed multiple autoimmune diseases, all but 2 of them with diabetes mellitus or autoimmune thyroid disease, therefore resembling autoimmune polyendocrine syndromes. Inheritance patterns were diverse, possibly autosomal dominant in 17/44 (38.6%) pedigrees or autosomal recessive in 5/44 (11.4%), and un-defined or complex in 24/44 (54.5%). However, a total of 21/65 (32.3%) patients had no identified family history of autoimmunity. In conclusion, these results suggest a variable and heterogeneous genetic predisposition to GAD65 neurological disorders, possibly involving multiple loci and modes of inheritance with different contribution in each family.

In this study, we report the clinical features of Kelch-like protein 11 antibody-associated paraneoplastic neurological syndrome, design and validate a clinical score to facilitate the identification of patients that should be tested for Kelch-like protein 11 antibodies, and examine in detail the nature of the immune response in both the brain and the tumour samples for a better characterization of the immunopathogenesis of this condition. The presence of Kelch-like protein 11 antibodies was retrospectively assessed in patients referred to the French Reference Center for paraneoplastic neurological syndrome and autoimmune encephalitis with (i) antibody-negative paraneoplastic neurological syndrome [limbic encephalitis (n = 105), cerebellar degeneration (n = 33)] and (ii) antibody-positive paraneoplastic neurological syndrome [Ma2-Ab encephalitis (n = 34), antibodies targeting N-methyl-D-aspartate receptor encephalitis with teratoma (n = 49)]. Additionally, since 1 January 2020, patients were prospectively screened for Kelch-like protein 11 antibodies as new usual clinical practice. Overall, Kelch-like protein 11 antibodies were detected in 11 patients [11/11, 100% were male; their median (range) age was 44 (35–79) years], 9 of them from the antibody-negative paraneoplastic neurological syndrome cohort, 1 from the antibody-positive (Ma2-Ab) cohort and 1 additional prospectively detected patient. All patients manifested a cerebellar syndrome, either isolated (4/11, 36%) or part of a multi-system neurological disorder (7/11, 64%). Additional core syndromes were limbic encephalitis (5/11, 45%) and myelitis (2/11, 18%). Severe weight loss (7/11, 64%) and hearing loss/tinnitus (5/11, 45%) were common. Rarer neurologic manifestations included hypersomnia and seizures (2/11, 18%). Two patients presented phenotypes resembling primary neurodegenerative disorders (progressive supranuclear palsy and flail arm syndrome, respectively). An associated cancer was found in 9/11 (82%) patients; it was most commonly (7/9, 78%) a spontaneously regressed (‘burned-out’) testicular germ cell tumour. A newly designed clinical score (MATCH score: male, ataxia, testicular cancer, hearing alterations) with a cut-off ≥4 successfully identified patients with Kelch-like protein 11 antibodies (sensitivity 78%, specificity 99%). Pathological findings (three testicular tumours, three lymph node metastases of testicular tumours, one brain biopsy) showed the presence of a T-cell inflammation with resulting anti-tumour immunity in the testis and one chronic, exhausted immune response—demonstrated by immune checkpoint expression—in the metastases and the brain. In conclusion, these findings suggest that Kelch-like protein 11 antibody paraneoplastic neurological syndrome is a homogeneous clinical syndrome and its detection can be facilitated using the MATCH score. The pathogenesis is probably T-cell mediated, but the stages of inflammation are different in the testis, metastases and the brain.

Objective To identify and characterize autoantibodies (Abs) as novel biomarkers for an autoimmune context in patients with central and peripheral neurologic diseases. Methods Two distinct approaches (immunoprecipitation/mass spectrometry–based proteomics and protein microarrays) and patients' sera and CSF were used. The specificity of the identified target was confirmed by cell-based assay (CBA) in 856 control samples. Results Using the 2 methods as well as sera and CSF of patients with central and peripheral neurologic involvement, we identified Abs against the family of Argonaute proteins (mainly AGO1 and AGO2), which were already reported in systemic autoimmunity. AGO-Abs were mostly of immunoglobulin G 1 subclass and conformation dependent. Using CBA, AGO-Abs were detected in 21 patients with a high suspicion of autoimmune neurologic diseases (71.4% were women; median age 57 years) and only in 4/856 (0.5%) controls analyzed by CBA (1 diagnosed with small-cell lung cancer and the other 3 with Sjögren syndrome). Among the 21 neurologic patients identified, the main clinical presentations were sensory neuronopathy (8/21, 38.1%) and limbic encephalitis (6/21, 28.6%). Fourteen patients (66.7%) had autoimmune comorbidities and/or co-occurring Abs, whereas AGO-Abs were the only autoimmune biomarker for the remaining 7/21 (33.3%). Thirteen (61.9%) patients were treated with immunotherapy; 8/13 (61.5%) improved, and 3/13 (23.1%) remained stable, suggesting an efficacy of these treatments. Conclusions AGO-Abs might be potential biomarkers of autoimmunity in patients with central and peripheral nonparaneoplastic neurologic diseases. In 7 patients, AGO-Abs were the only biomarkers; thus, their identification may be useful to suspect the autoimmune character of the neurologic disorder. Classification of Evidence This study provides Class III evidence that AGO-Abs are more frequent in patients with autoimmune neurologic diseases than controls.

Objective Antibodies against leucine-rich glioma-inactivated 1 (LGI1-Abs) characterize a limbic encephalitis (LE) strongly associated with HLA-DRB1*07:01, although some patients lack LGI1-Abs in CSF or do not carry this allele. Whether they represent a different subtype of disease or have different prognoses is unclear. Methods Retrospective analysis of clinical features, IgG isotypes, and outcome according to LGI1-Ab CSF positivity and DRB1*07:01 in a cohort of anti-LGI1 LE patients. Results Patients with LGI1-Abs detected in both CSF and serum (105/134, 78%) were compared with those who were CSF negative (29/134, 22%). Both groups had similar clinical features and serum levels, but CSF-positive patients had shorter diagnostic delay, more frequently hyponatremia, inflammatory CSF, and abnormal MRI (p < 0.05). Human leukocyte antigen (HLA) genotyping was performed in 72/134 (54%) patients and 63/72 (88%) carried DRB1*07:01. Noncarriers (9/72, 12%) were younger, more commonly women, and had less frequently psychiatric and frontal symptoms (p < 0.05). No difference in IgG isotypes according to CSF positivity or HLA was found (p > 0.05). HLA and IgG isotypes were not associated with poor outcome (mRS >2 at last follow-up) in univariate analyses; CSF positivity was only identified as a poor outcome predictor in the multivariate analysis including the complete follow-up, whereas age and female sex also remained when just the first year was considered. Conclusions LE without CSF LGI1-Abs is clinically indistinguishable and likely reflects just a lesser LGI1-Ab production. HLA association is sex and age biased and presents clinical particularities, suggesting subtle differences in the immune response. Long-term outcome depends mostly on demographic characteristics and the intensity of the intrathecal synthesis.

Habituation is an adaptive learning process that enables animals to adjust innate behaviors to changes in their environment. Despite its well-documented implications for a wide diversity of behaviors, the molecular and cellular basis of habituation learning is not well understood. Using whole-genome sequencing of zebrafish mutants isolated in an unbiased genetic screen, we identified the palmitoyltransferase Huntingtin interacting protein 14 (Hip14) as a critical regulator of habituation learning. We demonstrate that Hip14 regulates depression of sensory inputs onto an identified hindbrain neuron and provide evidence that Hip14 palmitoylates the Shaker-like K+ voltage-gated channel subunit (Kv1.1), thereby regulating Kv1.1 subcellular localization. Furthermore, we show that, like for Hip14, loss of Kv1.1 leads to habituation deficits and that Hip14 is dispensable in development and instead acts acutely to promote habituation. Combined, these results uncover a previously unappreciated role for acute posttranslational palmitoylation at defined circuit components to regulate learning.

Increasing evidence indicates that guidance molecules used during development for cellular and axonal navigation also play roles in synapse maturation and homeostasis. In C. elegans the netrin receptor UNC-40/DCC controls the growth of dendritic-like muscle cell extensions towards motoneurons and is required to recruit type A GABA receptors (GABAARs) at inhibitory neuromuscular junctions. Here we show that activation of UNC-40 assembles an intracellular synaptic scaffold by physically interacting with FRM-3, a FERM protein orthologous to FARP1/2. FRM-3 then recruits LIN-2, the ortholog of CASK, that binds the synaptic adhesion molecule NLG-1/Neuroligin and physically connects GABAARs to prepositioned NLG-1 clusters. These processes are orchestrated by the synaptic organizer CePunctin/MADD-4, which controls the localization of GABAARs by positioning NLG-1/neuroligin at synapses and regulates the synaptic content of GABAARs through the UNC-40-dependent intracellular scaffold. Since DCC is detected at GABA synapses in mammals, DCC might also tune inhibitory neurotransmission in the mammalian brain.

Spinal commissural axon navigation across the midline in the floor plate requires repulsive forces from local Slit repellents. The long-held view is that Slits push growth cones forward and prevent them from turning back once they became sensitized to these cues after midline crossing. We analyzed with fluorescent reporters Slits distribution and FP glia morphology. We observed clusters of Slit-N and Slit-C fragments decorating a complex architecture of glial basal process ramifications. We found that PC2 proprotein convertase activity contributes to this pattern of ligands. Next, we studied Slit-C acting via PlexinA1 receptor shared with another FP repellent, the Semaphorin3B, through generation of a mouse model baring PlexinA1Y1815F mutation abrogating SlitC but not Sema3B responsiveness, manipulations in the chicken embryo, and ex vivo live imaging. This revealed a guidance mechanism by which SlitC constantly limits growth cone exploration, imposing ordered and forward-directed progression through aligned corridors formed by FP basal ramifications.

Cilia assembly is under strict transcriptional control during animal development. In vertebrates, a hierarchy of transcription factors (TFs) are involved in controlling the specification, differentiation and function of multiciliated epithelia. RFX TFs play key functions in the control of ciliogenesis in animals. Whereas only one RFX factor regulates ciliogenesis in C. elegans, several distinct RFX factors have been implicated in this process in vertebrates. However, a clear understanding of the specific and redundant functions of different RFX factors in ciliated cells remains lacking. Using RNA-seq and ChIP-seq approaches we identified genes regulated directly and indirectly by RFX1, RFX2 and RFX3 in mouse ependymal cells. We show that these three TFs have both redundant and specific functions in ependymal cells. Whereas RFX1, RFX2 and RFX3 occupy many shared genomic loci, only RFX2 and RFX3 play a prominent and redundant function in the control of motile ciliogenesis in mice. Our results provide a valuable list of candidate ciliary genes. They also reveal stunning differences between compensatory processes operating in vivo and ex vivo.

Vesicular release is one of the release mechanisms of various signaling molecules. In neurons, the molecular machinery involved in vesicular release has been designed through evolution to trigger fast and synchronous release of neurotransmitters. Similar machinery with a slower kinetic and a slightly different molecular assembly allows astrocytes to release various transmitters such as adenosine triphosphate (ATP), glutamate, and D-serine. Astrocytes are important modulators of neurotransmission through gliotransmitter release. We recently demonstrated that microglia, another type of glia, release ATP to modulate synaptic transmission using astrocytes as intermediate. We now report that microglia regulate astrocytic gliotransmission through the regulation of SNARE proteins in astrocytes. Indeed, we found that gliotransmission triggered by P2Y1 agonist is impaired in slices from transgenic mice devoid of microglia. Using total internal reflection fluorescence imaging, we found that the vesicular release of gliotransmitter by astrocytes was different in cultures lacking microglia compared to vesicular release in astrocytes cocultured with microglia. Quantification of the kinetic of vesicular release indicates that the overall release appears to be faster in pure astrocyte cultures with more vesicles close to the membrane when compared to astrocytes cocultured with microglia. Finally, biochemical investigation of SNARE protein expression indicates an upregulation of VAMP2 in absence of microglia. Altogether, these results indicate that microglia seems to be involved in the regulation of an astrocytic phenotype compatible with proper gliotransmission. The mechanisms described in this study could be of importance for central nervous system diseases where microglia are activated.

Due to its amenability to manipulations, to live observation and its striking similarities to mammals, the chicken embryo has been one of the major animal models in biomedical research. Although it is technically possible to genome-edit the chicken, its long generation time (6 months to sexual maturity) makes it an impractical lab model and has prevented it widespread use in research. The Japanese quail (Coturnix coturnix japonica) is an attractive alternative, very similar to the chicken, but with the decisive asset of a much shorter generation time (1.5 months). In recent years, transgenic quail lines have been described. Most of them were generated using replication-deficient lentiviruses, a technique that presents diverse limitations. Here, we introduce a novel technology to perform transgenesis in quail, based on the in vivo transfection of plasmids in circulating Primordial Germ Cells (PGCs). This technique is simple, efficient and allows using the infinite variety of genome engineering approaches developed in other models. Furthermore, we present a website centralizing quail genomic and technological information to facilitate the design of genome-editing strategies, showcase the past and future transgenic quail lines and foster collaborative work within the avian community.

Accurate perception of guidance cues is crucial for cell and axon migration. During initial navigation in the spinal cord, commissural axons are kept insensitive to midline repellents. Upon midline crossing in the floor plate, they switch on responsiveness to Slit and Semaphorin repulsive signals and are thus propelled away and prevented from crossing back. Whether and how the different midline repellents control specific aspects of this navigation remain to be elucidated. We set up a paradigm for live-imaging and super-resolution analysis of PlexinA1, Neuropilin-2, and Robo1/2 receptor dynamics during commissural growth cone navigation in chick and mouse embryos. We uncovered a remarkable program of sensitization to midline cues achieved by unique spatiotemporal sequences of receptor allocation at the growth-cone surface that orchestrates receptor-specific growth-cone behavior changes. This reveals post-translational mechanisms whereby coincident guidance signals are temporally resolved to allow the generation of specific guidance responses.

In the developing organism, complex molecular programs orchestrate the generation of cells in adequate numbers, drive them to migrate along the correct pathways towards appropriate territories, eliminate superfluous cells, and induce terminal differentiation of survivors into the appropriate cell-types. Despite strict controls constraining developmental processes, malignancies can emerge in still immature organisms. This is the case of neuroblastoma (NB), a highly heterogeneous disease, predominantly affecting children before the age of 5 years. Highly metastatic forms represent half of the cases and are diagnosed when disseminated foci are detectable. NB arise from a transient population of embryonic cells, the neural crest (NC), and especially NC committed to the establishment of the sympatho-adrenal tissues. The NC is generated at the dorsal edge of the neural tube (NT) of the vertebrate embryo, under the action of NC specifier gene programs. NC cells (NCCs) undergo an epithelial to mesenchymal transition, and engage on a remarkable journey in the developing embryo, contributing to a plethora of cell-types and tissues. Various NCC sub-populations and derived lineages adopt specific migratory behaviors, moving individually as well as collectively, exploiting the different embryonic substrates they encounter along their path. Here we discuss how the specific features of NCC in development are re-iterated during NB metastatic behaviors.

Septins, discovered almost half a century ago in yeast, have prominent contributions in a broad range of morphological and functional processes from yeast to human. Septins now emerge as key players of neurodevelopment and more specifically of the mechanisms driving the complex morphological differentiation and compartmentalization of neurons that are fundamental to their function. We review here recent advances in Septin-mediated processes of neuron differentiation, which enlighten similarities and differences between neuron and yeast polarity programs.

The navigation of commissural axons in the developing spinal cord has attracted multiple studies over the years. Many important concepts emerged from these studies which have enlighten the general mechanisms of axon guidance. The navigation of commissural axons is regulated by a series of cellular territories which provides the diverse guidance information necessary to ensure the successive steps of their pathfinding towards, across, and away from the ventral midline. In this review, we discuss how repulsive forces, by propelling, channelling, and confining commissural axon navigation, bring key contributions to the formation of this neuronal projection.

Cilia and flagella are conserved eukaryotic organelles essential for cellular signaling and motility. Cilia dysfunctions cause life-threatening ciliopathies, many of which are due to defects in the transition zone (TZ), a complex structure of the ciliary base. Therefore, understanding TZ assembly, which relies on ordered interactions of multiprotein modules, is of critical importance. Here, we show that Drosophila Dzip1 and Fam92 form a functional module which constrains the conserved core TZ protein, Cep290, to the ciliary base. We identify cell type specific roles of this functional module in two different tissues. While it is required for TZ assembly in all Drosophila ciliated cells, it also regulates basal-body growth and docking to the plasma membrane during spermatogenesis. We therefore demonstrate a novel regulatory role for Dzip1 and Fam92 in mediating membrane/basal-body interactions and show that these interactions exhibit cell type specific functions in basal-body maturation and TZ organization.

Producing mature spermatozoa is essential for sexual reproduction in metazoans. Spermiogenesis involves dramatic cell morphological changes going from sperm tail elongation and nuclear reshaping to cell membrane remodeling during sperm individualization and release. The sperm manchette plays a critical scaffolding function during nuclear remodeling by linking the nuclear lamina to the cytoskeleton. Here, we describe the role of an uncharacterized protein in Drosophila, salto/CG13164, involved in nuclear shaping and spermatid individualization. Salto has dynamic localization during spermatid differentiation, being progressively relocated from the sperm-nuclear dense body, which is equivalent to the mammalian sperm manchette, to the centriolar adjunct and acrosomal cap during spermiogenesis. salto-null male flies are sterile and exhibit complete spermatid individualization defects. salto-deficient spermatids show coiled spermatid nuclei at late maturation stages and stalled individualization complexes. Our work sheds light on a novel component involved in cytoskeleton-based cell-morphological changes during spermiogenesis.

Acrocallosal syndrome (ACLS) is a rare genetic disorder characterized by agenesis or hypoplasia of corpus callosum (CC), polydactyly, craniofacial dysmorphism and severe intellectual deficiency. We previously identified KIF7, a key ciliary component of the Sonic hedgehog (SHH) pathway, as being a causative gene for this syndrome, thus including ACLS in the group of ciliopathies. In both humans and mice, KIF7 depletion leads to abnormal GLI3 processing and over-activation of SHH target genes. To understand the pathological mechanisms involved in CC defects in this syndrome, we took advantage of a previously described Kif7-/- mouse model to demonstrate that in addition to polydactyly and neural tube closure defects, these mice present CC agenesis with characteristic Probst bundles, thus recapitulating major ACLS features. We show that CC agenesis in these mice is associated with specific patterning defects of the cortical septum boundary leading to altered distribution of guidepost cells required to guide the callosal axons through the midline. Furthermore, by crossing Kif7-/- mice with Gli3Δ699 mice exclusively producing the repressive isoform of GLI3 (GLI3R), we demonstrate that decreased GLI3R signaling is fully responsible for the ACLS features in these mice, as all phenotypes are rescued by increasing GLI3R activity. Moreover, we show that increased FGF8 signaling is responsible in part for CC defects associated to KIF7 depletion, as modulating FGF8 signaling rescued CC formation anteriorly in Kif7-/- mice. Taken together our data demonstrate that ACLS features rely on defective GLI3R and FGF8 signaling.

Mutations that modulate the activity of ion channels are essential tools to understand the biophysical determinants that control their gating. Here, we reveal the conserved role played by a single amino acid position (TM2.6) located in the second transmembrane domain of two-pore domain potassium (K2P) channels. Mutations of TM2.6 to aspartate or asparagine increase channel activity for all vertebrate K2P channels. Using two-electrode voltage-clamp and single-channel recording techniques, we find that mutation of TM2.6 promotes channel gating via the selectivity filter gate and increases single channel open probability. Furthermore, channel gating can be progressively tuned by using different amino acid substitutions. Finally, we show that the role of TM2.6 was conserved during evolution by rationally designing gain-of-function mutations in four Caenorhabditis elegans K2P channels using CRISPR/Cas9 gene editing. This study thus describes a simple and powerful strategy to systematically manipulate the activity of an entire family of potassium channels.

Autoantibodies against CASPR2 (contactin-associated protein-like 2) have been linked to autoimmune limbic encephalitis that manifests with memory disorders and temporal lobe seizures. According to the growing number of data supporting a role for CASPR2 in neuronal excitability, CASPR2 forms a molecular complex with transient axonal glycoprotein-1 (TAG-1) and shaker-type voltage-gated potassium channels (Kv1.1 and Kv1.2) in compartments critical for neuronal activity and is required for Kv1 proper positioning. Whereas the perturbation of these functions could explain the symptoms observed in patients, the pathogenic role of anti-CASPR2 antibodies has been poorly studied. In the present study, we find that patient autoantibodies alter Caspr2 distribution at the cell membrane promoting cluster formation. We confirm in a HEK cellular model that the anti-CASPR2 antibodies impede CASPR2/TAG-1 interaction and we identify the domains of CASPR2 and TAG-1 taking part in this interaction. Moreover, introduction of CASPR2 into HEK cells induces a marked increase of the level of Kv1.2 surface expression and in cultures of hippocampal neurons Caspr2-positive inhibitory neurons appear to specifically express high levels of Kv1.2. Importantly, in both cellular models, anti-CASPR2 patient autoAb increase Kv1.2 expression. These results provide new insights into the pathogenic role of autoAb in the disease.

Encephalitis with anti-NMDAR antibodies (NMDAR-E) is a severe autoimmune neurological disorder, defined by a clinical presentation of encephalitis and the presence of IgG targeting the GluN1 subunit of NMDA receptors in the CSF. An underlying ovarian teratoma is commonly associated with this autoimmune disease suggesting a role of the tumor in immunopathogenesis. In this study, we characterized the salient histopathological features of 27 ovarian teratomas associated with NMDAR-E (3 immature and 24 mature teratomas) and 40 controls without associated encephalitis. All but one NMDAR-E-associated teratomas contained a nervous tissue component, while less than 40% of control teratomas did (p < 0.001). GluN1 expression by teratomatous nervous tissue seemed to be more often glial in NMDAR-E teratomas than in control teratomas (73% vs. 29%, p < 0.05). Strikingly, 3 out of 24 NMDAR-E-associated mature teratomas contained neuroglial tissue exhibiting histopathological features of central nervous system neuroglial tumor, while such glioma-like features are exceptionally described in the literature on ovarian teratomas. Moreover, NMDAR-E associated teratomas differed from sporadic ovarian teratomas by consistent and prominent infiltration of the nervous tissue component by immune cells, comprised of T- and B-cells and mature dendritic cells organized in tertiary lymphoid structures, with IgG and IgA deposits and plasma cells in close contact to the neuroglial tissue.These data demonstrate an association between massive infiltration of NMDAR-E-associated teratomas by immune cells and particular glial features of its neuroglial component, suggesting that this glial tissue might be involved in triggering or sustaining the anti-tumor response associated with the auto-immune neurological disease

Purpose: Identifying autoantigens of serological autoantibodies requires expensive methods, such as protein microarrays or IP+MS. Thus, sera are commonly pre-screened for interesting immunopatterns via immunocytochemistry/immunohistochemistry. However, distinguishing immunopatterns can be difficult and intracellular antigens are less accessible. Therefore, a simple and cheap immunoblot screening able to distinguish immunopatterns and to detect refractory proteins is presented.
Experimental design: Five steps of immunoblotting-based autoantigen screening are revised: (1) choice of protein source, (2) protein extraction, (3) protein separation, (4) protein transfer, (5) antigen detection. Thereafter, 52 patients' sera with chronic inflammatory demyelinating polyneuropathy (CIDP) and 45 controls were screened.
Results: The protein source impacts the detected antigen set. Steps 2-4 can be adapted for refractory proteins. Furthermore, longitudinal cutting of protein lanes saves ≥75% of time and material and allows for exact comparison of band patterns. As the latter are individually specific and temporarily constant, we call them 'immunological fingerprints'. In a proof-of-principle, a 155 kDa immunoband was detected with two anti-neurofascin-155-positive CIDP sera and two further immunobands (120/220 kDa) specific to a subgroup of 3-6 of 52 CIDP patients.
Conclusions and clinical relevance: Adapted immunoblotting is a cheap and simple method for accurate serum screening including refractory and intracellular antigens.

Genomic studies have identified hundreds of candidate genes near loci associated with risk for schizophrenia. To define candidates and their functions, we mutated zebrafish orthologs of 132 human schizophrenia-associated genes. We created a phenotype atlas consisting of whole-brain activity maps, brain structural differences, and profiles of behavioral abnormalities. Phenotypes were diverse but specific, including altered forebrain development and decreased prepulse inhibition. Exploration of these datasets identified promising candidates in more than 10 gene-rich regions, including the magnesium transporter cnnm2 and the translational repressor gigyf2, and revealed shared anatomical sites of activity differences, including the pallium, hypothalamus, and tectum. Single-cell RNA sequencing uncovered an essential role for the understudied transcription factor znf536 in the development of forebrain neurons implicated in social behavior and stress. This phenotypic landscape of schizophrenia-associated genes prioritizes more than 30 candidates for further study and provides hypotheses to bridge the divide between genetic association and biological mechanism.

Animals have evolved specialized neural circuits to defend themselves from pain- and injury-causing stimuli. Using a combination of optical, behavioral and genetic approaches in the larval zebrafish, we describe a novel role for hypothalamic oxytocin (OXT) neurons in the processing of noxious stimuli. In vivo imaging revealed that a large and distributed fraction of zebrafish OXT neurons respond strongly to noxious inputs, including the activation of damage-sensing TRPA1 receptors. OXT population activity reflects the sensorimotor transformation of the noxious stimulus, with some neurons encoding sensory information and others correlating more strongly with large-angle swims. Notably, OXT neuron activation is sufficient to generate this defensive behavior via the recruitment of brainstem premotor targets, whereas ablation of OXT neurons or loss of the peptide attenuates behavioral responses to TRPA1 activation. These data highlight a crucial role for OXT neurons in the generation of appropriate defensive responses to noxious input.

Habituation is a simple form of learning where animals learn to reduce their responses to repeated innocuous stimuli [1]. Habituation is thought to occur via at least two temporally and molecularly distinct mechanisms, which lead to short-term memories that last for seconds to minutes and long-term memories that last for hours or longer [1, 2]. Here, we focus on long-term habituation, which, due to the extended time course, necessitates stable alterations to circuit properties [2-4]. In its simplest form, long-term habituation could result from a plasticity event at a single point in a circuit, and many studies have focused on identifying the site and underlying mechanism of plasticity [5-10]. However, it is possible that these individual sites are only one of many points in the circuit where plasticity is occurring. Indeed, studies of short-term habituation in C. elegans indicate that in this paradigm, multiple genetically separable mechanisms operate to adapt specific aspects of behavior [11-13]. Here, we use a visual assay in which larval zebrafish habituate their response to sudden reductions in illumination (dark flashes) [14, 15]. Through behavioral analyses, we find that multiple components of the dark-flash response habituate independently of one another using different molecular mechanisms. This is consistent with a modular model in which habituation originates from multiple independent processes, each adapting specific components of behavior. This may allow animals to more specifically or flexibly habituate based on stimulus context or internal states.

The assembly of neurotransmitter receptors in the endoplasmic reticulum limits the number of receptors delivered to the plasma membrane, ultimately controlling neurotransmitter sensitivity and synaptic transfer function. In a forward genetic screen conducted in the nematode C. elegans, we identified crld-1 as a gene required for the synaptic expression of ionotropic acetylcholine receptors (AChR). We demonstrated that the CRLD-1A isoform is a membrane-associated ER-resident protein disulfide isomerase (PDI). It physically interacts with AChRs and promotes the assembly of AChR subunits in the ER. Mutations of Creld1, the human ortholog of crld-1a, are responsible for developmental cardiac defects. We showed that Creld1 knockdown in mouse muscle cells decreased surface expression of AChRs and that expression of mouse Creld1 in C. elegans rescued crld-1a mutant phenotypes. Altogether these results identify a novel and evolutionarily-conserved maturational enhancer of AChR biogenesis, which controls the abundance of functional receptors at the cell surface.

Aging is commonly defined as the loss of global homeostasis, which results from progressive alteration of all organs function. This model is currently challenged by recent data showing that interventions that extend lifespan do not always increase the overall fitness of the organism. These data suggest the existence of tissue-specific factors that regulate the pace of aging in a cell-autonomous manner. Here, we investigated aging of Caenorhabditis elegans striated muscles at the subcellular and the physiological level. Our data show that muscle aging is characterized by a dramatic decrease in the expression of genes encoding proteins required for muscle contraction, followed by a change in mitochondria morphology, and an increase in autophagosome number. Myofilaments, however, remain unaffected during aging. We demonstrated that the conserved transcription factor UNC-120/SRF regulates muscle aging biomarkers. Interestingly, the role of UNC-120/SRF in the control of muscle aging can be dissociated from its broader effect on lifespan. In daf-2/insulin/IGF1 receptor mutants, which exhibit a delayed appearance of muscle aging biomarkers and are long-lived, disruption of unc-120 accelerates muscle aging but does not suppress the lifespan phenotype of daf-2 mutant. Conversely, unc-120 overexpression delays muscle aging but does not increase lifespan. Overall, we demonstrate that UNC-120/SRF controls the pace of muscle aging in a cell-autonomous manner downstream of the insulin/IGF1 receptor.

Accumulating evidence has showed that anti-CASPR2 autoantibodies occur in a long list of neurological immune disorders including limbic encephalitis (LE). Belonging to the well-known neurexin superfamily, CASPR2 has been suggested to be a central node in the molecular networks controlling neurodevelopment. Distinct from other subfamilies in the neurexin superfamily, the CASPR subfamily features a unique discoidin (Disc) domain. As revealed by our and others' recent studies, CASPR2 Disc domain bears a major epitope for autoantibodies. However, structural information on CASPR2 recognition by autoantibodies has been lacking. Here, we report the crystal structure of human CASPR2 Disc domain at a high resolution of 1.31 Å, which is the first atomic-resolution structure of the CASPR subfamily members. The Disc domain adopts a total β structure and folds into a distorted jellyroll-like barrel with a conserved disulfide-bond interlocking its N- and C-termini. Defined by four loops and located in one end of the barrel, the 'loop-tip surface' is totally polar and easily available for protein docking. Based on structure-guided epitope prediction, we generated nine mutants and evaluated their binding to autoantibodies of cerebrospinal fluid from twelve patients with limbic encephalitis. The quadruple mutant G69N/A71S/S77N/D78R impaired CASPR2 binding to autoantibodies from eleven LE patients, which indicates that the loop L1 in the Disc domain bears hot spots for autoantibody interaction. Structural mapping of autoepitopes within human CASPR2 Disc domain sheds light on how autoantibodies could sequester CASPR2 ectodomain and antagonize its functionalities in the pathogenic processes.

Transcriptional regulation of proteins involved in neuronal polarity is a key process that underlies the ability of neurons to transfer information in the central nervous system. The Collapsin Response Mediator Protein (CRMP) family is best known for its role in neurite outgrowth regulation conducting to neuronal polarity and axonal guidance, including CRMP5 that drives dendrite differentiation. Although CRMP5 is able to control dendritic development, the regulation of its expression remains poorly understood. Here we identify a Sox5 consensus binding sequence in the putative promoter sequence upstream of the CRMP5 gene. By luciferase assays we show that Sox5 increases CRMP5 promoter activity, but not if the putative Sox5 binding site is mutated. We demonstrate that Sox5 can physically bind to the CRMP5 promoter DNA in gel mobility shift and chromatin immunoprecipitation assays. Using a combination of real-time RT-PCR and quantitative immunocytochemistry, we provide further evidence for a Sox5-dependent upregulation of CRMP5 transcription and protein expression in N1E115 cells: a commonly used cell line model for neuronal differentiation. Furthermore, we report that increasing Sox5 levels in this neuronal cell line inhibits neurite outgrowth. This inhibition requires CRMP5 because CRMP5 knockdown prevents the Sox5-dependent effect. We confirm the physiological relevance of the Sox5-CRMP5 pathway in the regulation of neurite outgrowth using mouse primary hippocampal neurons. These findings identify Sox5 as a critical modulator of neurite outgrowth through the selective activation of CRMP5 expression.

Paraneoplastic cerebellar degenerations with anti-Yo antibodies (Yo-PCD) are rare syndromes caused by an auto-immune response against neuronal antigens (Ags) expressed by tumor cells. However, the mechanisms responsible for such immune tolerance breakdown are unknown. We characterized 26 ovarian carcinomas associated with Yo-PCD for their tumor immune contexture and genetic status of the 2 onconeural Yo-Ags, CDR2 and CDR2L. Yo-PCD tumors differed from the 116 control tumors by more abundant T and B cells infiltration occasionally organized in tertiary lymphoid structures harboring CDR2L protein deposits. Immune cells are mainly in the vicinity of apoptotic tumor cells, revealing tumor immune attack. Moreover, contrary to un-selected ovarian carcinomas, 65% of our Yo-PCD tumors presented at least one somatic mutation in Yo-Ags, with a predominance of missense mutations. Recurrent gains of the CDR2L gene with tumor protein overexpression were also present in 59% of Yo-PCD patients. Overall, each Yo-PCD ovarian carcinomas carried at least one genetic alteration of Yo-Ags. These data demonstrate an association between massive infiltration of Yo-PCD tumors by activated immune effector cells and recurrent gains and/or mutations in autoantigen-encoding genes, suggesting that genetic alterations in tumor cells trigger immune tolerance breakdown and initiation of the auto-immune disease.

Background: Paraneoplastic neurological syndromes are rare conditions where an autoimmune reaction against the nervous system appears in patients suffering from a tumour, but not linked to the spreading of the tumour. A break in the immune tolerance is thought to be the trigger.
Methods: The transcriptomic profile of 12 ovarian tumours (OT) from patients suffering from paraneoplastic cerebellar degeneration (PCD) linked to anti-Yo antibodies (anti-Yo PCD OT) was compared with 733 ovarian tumours (OT control) from different public databases using linear model analysis.
Results: A prominent significant transcriptomic over-representation of CD8+ and Treg cells was found in anti-Yo PCD OT, as compared to the OT control. However, the overall degree of immune cell infiltration was similar, according to the ESTIMATE immune score. We also found an under-representation of M2 macrophages in anti-Yo PCD OT. Furthermore, the differentially expressed genes were enriched for AIRE-related genes, a well-known transcription factor associated with a broad range of autoimmune diseases. Finally, we found that the differentially expressed genes were correlated to the transcriptomic profiling of the cerebellar structures.
Conclusions: Our data pinpointed the enrichment of acquired immune response, particularly high density of CD8+ lymphocytes, and high-level expression of CDR-related antigens in anti-Yo PCD OT.

To describe autoantibodies (Abs) against tripartite motif-containing (TRIM) protein 9 and 67 in two patients with paraneoplastic cerebellar degeneration (PCD) associated with lung adenocarcinoma. Abs were characterized using immunohistochemistry, Western blotting, cultures of murine cortical, and hippocampal neurons, immunoprecipitation, mass spectrometry, knockout mice for Trim9 and 67, and cell-based assay. Control samples included sera from 63 patients with small cell lung cancer without any paraneoplastic neurological syndrome, 36 patients with lung adenocarcinoma and PNS, CSF from 100 patients with autoimmune encephalitis, and CSF from 165 patients with neurodegenerative diseases. We found Abs targeting TRIM9 and TRIM67 at high concentration in the serum and the cerebrospinal fluid (CSF) of a 78-year-old woman and a 65-year-old man. Both developed subacute severe cerebellar ataxia. Brain magnetic resonance imaging found no abnormality and no cerebellar atrophy. Both had CSF inflammation with mild pleiocytosis and a few oligoclonal bands. We identified a pulmonary adenocarcinoma, confirming the paraneoplastic neurological syndrome in both patients. They received immunomodulatory and cancer treatments without improvement of cerebellar ataxia, even though both were in remission of their cancer (for more than 10 years in one patient). Anti-TRIM9 and anti-TRIM67 Abs were specific to these two patients. All control serum and CSF samples tested were negative for anti-TRIM9 and 67. Anti-TRIM9 and anti-TRIM67 Abs appeared to be specific biomarkers of PCD and should be added to the panel of antigens tested when this is suspected.

Simultaneous recordings of large populations of neurons in behaving animals allow detailed observation of high-dimensional, complex brain activity. However, experimental approaches often focus on singular behavioral paradigms or brain areas. Here, we recorded whole-brain neuronal activity of larval zebrafish presented with a battery of visual stimuli while recording fictive motor output. We identified neurons tuned to each stimulus type and motor output and discovered groups of neurons in the anterior hindbrain that respond to different stimuli eliciting similar behavioral responses. These convergent sensorimotor representations were only weakly correlated to instantaneous motor activity, suggesting that they critically inform, but do not directly generate, behavioral choices. To catalog brain-wide activity beyond explicit sensorimotor processing, we developed an unsupervised clustering technique that organizes neurons into functional groups. These analyses enabled a broad overview of the functional organization of the brain and revealed numerous brain nuclei whose neurons exhibit concerted activity patterns.

CRISPR/Cas9 genome engineering strategies allow the directed modification of the Caenorhabditis elegans genome to introduce point mutations, generate knock-out mutants, and insert coding sequences for epitope or fluorescent tags. Three practical aspects, however, complicate such experiments. First, the efficiency and specificity of single-guide RNAs (sgRNA) cannot be reliably predicted. Second, the detection of animals carrying genome edits can be challenging in the absence of clearly visible or selectable phenotypes. Third, the sgRNA target site must be inactivated after editing to avoid further double-strand break events. We describe here a strategy that addresses these complications by transplanting the protospacer of a highly efficient sgRNA into a gene of interest to render it amenable to genome engineering. This sgRNA targeting the dpy-10 gene generates genome edits at comparatively high frequency. We demonstrate that the transplanted protospacer is cleaved at the same time as the dpy-10 gene. Our strategy generates scarless genome edits because it no longer requires the introduction of mutations in endogenous sgRNA target sites. Modified progeny can be easily identified in the F1 generation, which drastically reduces the number of animals to be tested by PCR or phenotypic analysis. Using this strategy, we reliably generated precise deletion mutants, transcriptional reporters, and translational fusions with epitope tags and fluorescent reporter genes. In particular, we report here the first use of the new red fluorescent protein mScarlet in a multicellular organism. wrmScarlet, a C. elegans-optimized version, dramatically surpassed TagRFP-T by showing an eightfold increase in fluorescence in a direct comparison.

Neuroblastoma (NB) is a childhood cancer arising from sympatho-adrenal neural crest cells. Disseminated forms have high frequency of multiple tumoral foci whose etiology remains unknown; NB embryonic origin limits investigations in patients and current models. We developed an avian embryonic model driving human NB tumorigenesis in tissues homologous to patients. We found that aggressive NBs display a metastatic mode, secondary dissemination via peripheral nerves and aorta. Through tumor transcriptional profiling, we found that NB dissemination is induced by the shutdown of a pro-cohesion autocrine signal, SEMA3C, which constrains the tumoral mass. Lowering SEMA3C levels shifts the balance toward detachment, triggering NB cells to collectively evade the tumor. Together with patient cohort analysis, this identifies a microenvironment-driven pro-metastatic switch for NB.

Transmission of polarity established early during cell lineage history is emerging as a key process guiding cell differentiation. Highly polarized neurons provide a fascinating model to study inheritance of polarity over cell generations and across morphological transitions. Neural crest cells (NCCs) migrate to the dorsal root ganglia to generate neurons directly or after cell divisions in situ. Using live imaging of vertebrate embryo slices, we found that bipolar NCC progenitors lose their polarity, retracting their processes to round for division, but generate neurons with bipolar morphology by emitting processes from the same locations as the progenitor. Monitoring the dynamics of Septins, which play key roles in yeast polarity, indicates that Septin 7 tags process sites for re-initiation of process growth following mitosis. Interfering with Septins blocks this mechanism. Thus, Septins store polarity features during mitotic rounding so that daughters can reconstitute the initial progenitor polarity.

The diaphragm muscle is essential for breathing in mammals. Its asymmetric elevation during contraction correlates with morphological features suggestive of inherent left-right (L/R) asymmetry. Whether this asymmetry is due to L versus R differences in the muscle or in the phrenic nerve activity is unknown. Here, we have combined the analysis of genetically modified mouse models with transcriptomic analysis to show that both the diaphragm muscle and phrenic nerves have asymmetries, which can be established independently of each other during early embryogenesis in pathway instructed by Nodal, a morphogen that also conveys asymmetry in other organs. We further found that phrenic motoneurons receive an early L/R genetic imprint, with L versus R differences both in Slit/Robo signaling and MMP2 activity and in the contribution of both pathways to establish phrenic nerve asymmetry. Our study therefore demonstrates L-R imprinting of spinal motoneurons and describes how L/R modulation of axon guidance signaling helps to match neural circuit formation to organ asymmetry.

The diaphragm muscle is essential for breathing in mammals. Its asymmetric elevation during contraction correlates with morphological features suggestive of inherent left-right (L/R) asymmetry. Whether this asymmetry is due to L versus R differences in the muscle or in the phrenic nerve activity is unknown. Here, we have combined the analysis of genetically modified mouse models with transcriptomic analysis to show that both the diaphragm muscle and phrenic nerves have asymmetries, which can be established independently of each other during early embryogenesis in pathway instructed by Nodal, a morphogen that also conveys asymmetry in other organs. We further found that phrenic motoneurons receive an early L/R genetic imprint, with L versus R differences both in Slit/Robo signaling and MMP2 activity and in the contribution of both pathways to establish phrenic nerve asymmetry. Our study therefore demonstrates L-R imprinting of spinal motoneurons and describes how L/R modulation of axon guidance signaling helps to match neural circuit formation to organ asymmetry.

Objective: To report 10 patients with limbic encephalitis (LE) and adenylate kinase 5 autoantibodies (AK5-Abs).
Methods: We conducted a retrospective study in a cohort of 50 patients with LE with uncharacterized autoantibodies and identified a specific target using immunohistochemistry, Western blotting, immunoprecipitation, mass spectrometry, and cell-based assay.
Results: AK5 (a known autoantigen of LE) was identified as the target of antibodies in the CSFs and sera of 10 patients with LE (median age 64 years; range 57-80), which was characterized by subacute anterograde amnesia without seizure and sometimes preceded by a prodromal phase of asthenia or mood disturbances. Anterograde amnesia can be isolated, but some patients also complained of prosopagnosia, paroxysmal anxiety, or abnormal behavior. No associated cancer was observed. All 10 patients had bilateral hippocampal hypersignal on a brain MRI. CSF analysis generally showed a mild pleiocytosis with elevated immunoglobulin G index and oligoclonal bands, as well as high levels of tau protein with normal concentration of Aβ42 and phospho-tau, suggesting a process of neuronal death. Except for one patient, clinical response to immunotherapy was unfavorable, with persistence of severe anterograde amnesia. Two patients evolved to severe cognitive decline. Hippocampal atrophy was observed on control brain MRI. Using in vitro tests on hippocampal neurons, we did not identify clues suggesting a direct pathogenic role of AK5-Abs.
Conclusions: AK5-Abs should be systematically considered in aged patients with subacute anterograde amnesia. Recognition of this disorder is important to develop new treatment strategies to prevent irreversible limbic damage.

The identification of circulating autoantibodies against neuronal receptors in neuropsychiatric disorders has fostered new conceptual and clinical frameworks. However, detection reliability, putative presence in different diseases and in health have raised questions about potential pathogenic mechanism mediated by autoantibodies. Using a combination of single molecule-based imaging approaches, we here ascertain the presence of circulating autoantibodies against glutamate NMDA receptor (NMDAR-Ab) in about 20% of psychotic patients diagnosed with schizophrenia and very few healthy subjects. NMDAR-Ab from patients and healthy subjects do not compete for binding on native receptor. Strikingly, NMDAR-Ab from patients, but not from healthy subjects, specifically alter the surface dynamics and nanoscale organization of synaptic NMDAR and its anchoring partner the EphrinB2 receptor in heterologous cells, cultured neurons and in mouse brain. Functionally, only patients' NMDAR-Ab prevent long-term potentiation at glutamatergic synapses, while leaving NMDAR-mediated calcium influx intact. We unveil that NMDAR-Ab from psychotic patients alter NMDAR synaptic transmission, supporting a pathogenically relevant role.

Objective: To report paroxysmal episodes of cerebellar ataxia in a patient with anti-contactin-associated protein-like 2 (CASPR2) antibody-related autoimmune encephalitis and to search for similar paroxysmal ataxia in a cohort of patients with anti-CASPR2 antibody-associated autoimmune encephalitis.
Methods: We report a patient with paroxysmal episodes of cerebellar ataxia observed during autoimmune encephalitis with anti-CASPR2 antibodies. In addition, clinical analysis was performed in a retrospective cohort of 37 patients with anti-CASPR2 antibodies to search for transient episodes of ataxia. Paroxysmal symptoms were further specified from the referral physicians, the patients, or their relatives.
Results: A 61-year-old man with limbic encephalitis and anti-CASPR2 antibodies developed stereotyped paroxysmal episodes of cerebellar ataxia, including gait imbalance, dysarthria, and dysmetria, 1 month after the onset of the encephalitis. The ataxic episodes were specifically triggered by orthostatism and emotions. Both limbic symptoms and transient ataxic episodes resolved after treatment with steroids and IV cyclophosphamide. Among 37 other patients with anti-CASPR2 antibodies, we identified 5 additional cases with similar paroxysmal ataxic episodes that included gait imbalance (5 cases), slurred speech (3 cases), limb dysmetria (3 cases), and nystagmus (1 case). All had concomitant limbic encephalitis. Paroxysmal ataxia was not observed in patients with neuromyotonia or Morvan syndrome. Triggering factors (orthostatism or anger) were reported in 4 patients. Episodes resolved with immunomodulatory treatments in 4 patients and spontaneously in 1 case.
Conclusions: Paroxysmal cerebellar ataxia must be added to the spectrum of the anti-CASPR2 antibody syndrome.

Circulating autoantibodies against glutamatergic N-methyl-D-aspartate receptor (NMDAR) have been reported in a proportion of patients with psychotic disorders, raising hopes for more appropriate treatment for these antibody-positive patients. However, the prevalence of circulating autoantibodies against glutamatergic NMDAR in psychotic disorders remains controversial, with detection prevalence rates and immunoglobulin classes varying considerably between studies, perhaps because of different detection methods. Here, we compared the results of serum assays for a large cohort of patients with first-episode psychosis using classical cell-based assays in three labs and a single molecule-based imaging method. Most assays and single molecule imaging in live hippocampal neurons revealed the presence of circulating autoantibodies against glutamatergic NMDAR in approximately 5% of patients with first-episode psychosis. However, some heterogeneity between cell-based assays was clearly observed, highlighting the urgent need for new sensitive methods to detect the presence of low-titer autoantibodies against glutamatergic NMDAR in seropositive patients who cannot be clinically identified from seronegative ones.

Expansion microscopy (ExM) allows scalable imaging of preserved 3D biological specimens with nanoscale resolution on fast diffraction-limited microscopes. Here, we explore the utility of ExM in the larval and embryonic zebrafish, an important model organism for the study of neuroscience and development. Regarding neuroscience, we found that ExM enabled the tracing of fine processes of radial glia, which are not resolvable with diffraction-limited microscopy. ExM further resolved putative synaptic connections, as well as molecular differences between densely packed synapses. Finally, ExM could resolve subsynaptic protein organization, such as ring-like structures composed of glycine receptors. Regarding development, we used ExM to characterize the shapes of nuclear invaginations and channels, and to visualize cytoskeletal proteins nearby. We detected nuclear invagination channels at late prophase and telophase, potentially suggesting roles for such channels in cell division. Thus, ExM of the larval and embryonic zebrafish may enable systematic studies of how molecular components are configured in multiple contexts of interest to neuroscience and developmental biology.

High-resolution serial-section electron microscopy (ssEM) makes it possible to investigate the dense meshwork of axons, dendrites, and synapses that form neuronal circuits. However, the imaging scale required to comprehensively reconstruct these structures is more than ten orders of magnitude smaller than the spatial extents occupied by networks of interconnected neurons, some of which span nearly the entire brain. Difficulties in generating and handling data for large volumes at nanoscale resolution have thus restricted vertebrate studies to fragments of circuits. These efforts were recently transformed by advances in computing, sample handling, and imaging techniques, but high-resolution examination of entire brains remains a challenge. Here, we present ssEM data for the complete brain of a larval zebrafish (Danio rerio) at 5.5 days post-fertilization. Our approach utilizes multiple rounds of targeted imaging at different scales to reduce acquisition time and data management requirements. The resulting dataset can be analysed to reconstruct neuronal processes, permitting us to survey all myelinated axons (the projectome). These reconstructions enable precise investigations of neuronal morphology, which reveal remarkable bilateral symmetry in myelinated reticulospinal and lateral line afferent axons. We further set the stage for whole-brain structure-function comparisons by co-registering functional reference atlases and in vivo two-photon fluorescence microscopy data from the same specimen. All obtained images and reconstructions are provided as an open-access resource.

Diffuse extrasynaptic neurotransmitter receptors constitute an abundant pool of receptors that can be recruited to modulate synaptic strength. Whether the diffuse distribution of receptors in extrasynaptic membranes is a default state or is actively controlled remains essentially unknown. Here we show that RSU-1 (Ras Suppressor-1) is required for the proper distribution of extrasynaptic acetylcholine receptors (AChRs) in Caenorhabditis elegans muscle cells. RSU-1 is an evolutionary conserved cytoplasmic protein that contains multiple leucine-rich repeats (LRRs) and interacts with integrin-dependent adhesion complexes. In rsu-1 mutants, neuromuscular junctions differentiate as in the wild type, but AChRs assemble into ectopic clusters that progressively enlarge during development. As a consequence, the synaptic content of AChRs is reduced. Our study provides the first evidence that an RSU-1-dependent active mechanism maintains extrasynaptic receptors dispersed and indirectly regulates synapse maturation.

Positioning type A GABA receptors (GABA(A)Rs) in front of GABA release sites sets the strength of inhibitory synapses. The evolutionarily conserved Ce-Punctin/MADD-4 is an anterograde synaptic organizer that specifies GABAergic versus cholinergic identity of postsynaptic domains at the C. elegans neuromuscular junctions (NMJs). Here we show that the Ce-Punctin secreted by GABAergic motor neurons controls the clustering of GABA(A)Rs through the synaptic adhesion molecule neuroligin (NLG-1) and the netrin receptor UNC-40/DCC. The short isoform of Ce-Punctin binds and clusters NLG-1 postsynaptically at GABAergic NMJs. NLG-1 disruption causes a strong reduction of GABA(A)R content at GABAergic synapses. Ce-Punctin also binds and localizes UNC-40 receptors in the postsynaptic membrane of NMJs, which promotes the recruitment of GABA(A)Rs by NLG-1. Since the mammalian orthologs of these genes are expressed in the central nervous system and their mutations are implicated in neuropsychiatric diseases, this molecular pathway might have been evolutionarily conserved.

The ciliary transition zone (TZ) is a complex structure found at the cilia base. Defects in TZ assembly are associated with human ciliopathies. In most eukaryotes, three protein complexes (CEP290, NPHP, and MKS) cooperate to build the TZ. We show that in Drosophila melanogaster, mild TZ defects are observed in the absence of MKS components. In contrast, Cby and Azi1 cooperate to build the TZ by acting upstream of Cep290 and MKS components. Without Cby and Azi1, centrioles fail to form the TZ, precluding sensory cilia assembly, and no ciliary membrane cap associated with sperm ciliogenesis is made. This ciliary cap is critical to recruit the tubulin-depolymerizing kinesin Klp59D, required for regulation of axonemal growth. Our results show that Drosophila TZ assembly in sensory neurons and male germ cells involves cooperative actions of Cby and Dila. They further reveal that temporal control of membrane cap assembly by TZ components and microtubule elongation by kinesin-13 is required for axoneme formation in male germ cells.

The fruit fly, Drosophila melanogaster, is one of the most extensively studied organisms in biological research and has centrioles/basal bodies and cilia that can be modelled to investigate their functions in animals generally. Centrioles are nine-fold symmetrical microtubule-based cylindrical structures required to form centrosomes and also to nucleate the formation of cilia and flagella. When they function to template cilia, centrioles transition into basal bodies. The fruit fly has various types of basal bodies and cilia, which are needed for sensory neuron and sperm function. Genetics, cell biology and behaviour studies in the fruit fly have unveiled new basal body components and revealed different modes of assembly and functions of basal bodies that are conserved in many other organisms, including human, green algae and plasmodium. Here we describe the various basal bodies of Drosophila, what is known about their composition, structure and function.

In the hematopoietic system, Syk family tyrosine kinases are essential components of immunoreceptor ITAM-based signaling. While there is increasing data indicating the involvement of immunoreceptors in neural functions, the contribution of Syk kinases remains obscure. Previously, we identified phosphorylated forms of Syk kinases in specialized populations of migrating neurons or projecting axons. Moreover, we identified ephrin/Eph as guidance molecules utilizing the ITAM-bearing CD3zeta (Cd247) and associated Syk kinases for the growth cone collapse response induced in vitro Here, we show that in the developing spinal cord, Syk is phosphorylated in navigating commissural axons. By analyzing axon trajectories in open-book preparations of Syk(-/-); Zap70(-/-) mouse embryos, we show that Syk kinases are dispensable for attraction towards the midline but confer growth cone responsiveness to repulsive signals that expel commissural axons from the midline. Known to serve a repulsive function at the midline, ephrin B3/EphB2 are obvious candidates for driving the Syk-dependent repulsive response. Indeed, Syk kinases were found to be required for ephrin B3-induced growth cone collapse in cultured commissural neurons. In fragments of commissural neuron-enriched tissues, Syk is in a constitutively phosphorylated state and ephrin B3 decreased its level of phosphorylation. Direct pharmacological inhibition of Syk kinase activity was sufficient to induce growth cone collapse. In conclusion, Syk kinases act as a molecular switch of growth cone adhesive and repulsive responses.

Importance: Autoantibodies against contactin-associated protein-like 2 (CASPR2) are observed in several neurological syndromes, including neuromyotonia (NMT), Morvan syndrome (MoS), and limbic encephalitis.
Objective: To characterize the clinical and biological presentations of patients with anti-CASPR2 antibodies in the cerebrospinal fluid (CSF).
Design, setting, and participants: We conducted a retrospective cohort analysis of 18 patients who had anti-CASPR2 antibodies in their CSF between March 2009 and November 2015 at the Centre National de Référence pour les Syndromes Neurologiques Paranéoplasiques in Lyon, France. Additionally, we analyzed 15 patients who were diagnosed as having NMT or MoS as a comparative group.
Main outcomes and measures: Clinical presentations, anti-CASPR2 antibodies specificities, brain magnetic resonance imaging, and CSF analyses, cancer prevalence, and evolution.
Results: In this cohort of 18 patients with anti-CASPR2 antibodies in their CSF, 17 (94.4%) were male and had a median (range) age of 64.5 (53-75) years; in the second group, 9 of 15 patients (60.0%) with NMT or MoS were male and had a median (range) age of 51 years (1 month to 75 years). Only 3 patients (16.7%) in this cohort had a previous or concomitant history of cancer (prostate, hematological, or thyroid), whereas 9 patients (60.0%) in the second group had a malignant thymoma. Symptoms of limbic encephalitis were observed in all patients, including temporal lobe seizures in 16 patients (88.9%) and memory disorders in 17 patients (94.4%) from the cohort. Extralimbic signs were also evident in 12 of 18 patients (66.7%), including cerebellar ataxia in 6 patients (33.3%). Only 2 patients (11.1%) from the cohort were diagnosed as having NMT. Brain magnetic resonance imaging displayed T2-weighted temporolimbic abnormalities in 14 of 15 patients (93.3%) in the second group. Cerebrospinal fluid analysis was abnormal in 9 of 12 patients (75.0%). For 16 of 18 patients (88.9%), follow-up was performed for at least a 6-month period (median [range], 34 [11-114] months). Of these, 15 (93.8%) improved and 6 (37.5%) relapsed. In all patients in this cohort, IgG4 autoantibodies were detected in the CSF. Anti-CASPR2 antibodies in the CSF targeted the laminin G1 and discoidin domains of CASPR2 in all patients. Importantly, anti-CASPR2 antibodies were detected in the serum but not in the CSF of all patients with NMT or MoS.
Conclusions and relevance: In this cohort study, anti-CASPR2 antibodies in the CSF are associated with a subtype of autoimmune encephalitis with prominent limbic involvement and seizures that is rarely associated with cancer. Conversely, patients with NMT and MoS have anti-CASPR2 antibodies only in the serum but not in the CSF and frequently present with a malignant thymoma. The anti-CASPR2 antibodies found in these patients targeted the discoidin and laminin G1 domains of CASPR2 and always included IgG4 autoantibodies.

Vascular endothelial growth factor (VEGF) is known to be required for the action of antidepressant therapies but its impact on brain synaptic function is poorly characterized. Using a combination of electrophysiological, single-molecule imaging and conditional transgenic approaches, we identified the molecular basis of the VEGF effect on synaptic transmission and plasticity. VEGF increases the postsynaptic responses mediated by the N-methyl-D-aspartate type of glutamate receptors (GluNRs) in hippocampal neurons. This is concurrent with the formation of new synapses and with the synaptic recruitment of GluNR expressing the GluN2B subunit (GluNR-2B). VEGF induces a rapid redistribution of GluNR-2B at synaptic sites by increasing the surface dynamics of these receptors within the membrane. Consistently, silencing the expression of the VEGF receptor 2 (VEGFR2) in neural cells impairs hippocampal-dependent synaptic plasticity and consolidation of emotional memory. These findings demonstrated the direct implication of VEGF signaling in neurons via VEGFR2 in proper synaptic function. They highlight the potential of VEGF as a key regulator of GluNR synaptic function and suggest a role for VEGF in new therapeutic approaches targeting GluNR in depression.

Encephalitis associated with antibodies against leucine-rich glioma-inactivated 1 (LGI1) protein is increasingly recognized as an auto-immune disorder associated with characteristic tonic-dystonic seizures. The cortical or subcortical origin of these motor events is not clear. Some patients also present with different epileptic seizures and with cognitive impairment. The frequency of these features and their timing during the natural history of this encephalitis have not been fully described. We therefore reviewed data from 34 patients harbouring antibodies against LGI1 protein (21-81 years, median age 64) referred to the French Reference Centre for Neurological Paraneoplastic Syndrome. Three types of evidence suggested tonic-dystonic seizures were of cortical origin: (i) a slow, unilateral, frontal electroencephalographic wave, of duration ∼580 ms and amplitude ∼71 µV, preceded the contralateral tonic-dystonic seizures in simultaneous electroencephalographic and myographic records from seven of seven patients tested; (ii) 18-Fluorodeoxyglucose imaging revealed a strong hypermetabolism in primary motor cortex, controlateral to the affected limb, during encephalitis for five patients tested, as compared with data from the same patients after remission or from 16 control subjects; and (iii) features of polymyographic records of tonic-dystonic seizure events pointed to a cortical origin. Myoclonic patterns with brief, rhythmic bursts were present in three of five patients tested and a premyoclonic potential was identified in the cortex of one patient. Initially during encephalitis, 11 of 34 patients exhibited tonic-dystonic seizures (32%). Distinct epileptic syndromes were evident in 13 patients (38%). They were typically simple, focal seizures from the temporal lobe, consisting of vegetative symptoms or fear. At later stages, 22 of 32 patients displayed tonic-dystonic seizures (68%) and 29 patients presented frequent seizures (91%) including status epilepticus. Cognitive impairment, either anterograde amnesia or confusion was evident in 30 of 34 patients (88%). Brain imaging was normal in patients with isolated tonic-dystonic seizures; in patients with limbic symptoms it revealed initially a hippocampal hyperintensity in 8 of 19 patients (42%) and 17 of 24 patients (70%) at later stages. Our data suggest that the major signs of LGI1-antibody encephalitis can be linked to involvement of motor cortex and hippocampus. They occur in parallel with striatum involvement. One of these cortical targets is involved, often unilaterally at disease onset. As the encephalitis progresses, in the absence of immunomodulatory treatment, the second cortical target is affected and effects become bilateral. Progression to the second cortical target occurs with a variable delay of days to several months.

How cells in the embryo coordinate epithelial plasticity with cell fate decision in a fast changing cellular environment is largely unknown. In chick embryos, skeletal muscle formation is initiated by migrating Delta1-expressing neural crest cells that trigger NOTCH signaling and myogenesis in selected epithelial somite progenitor cells, which rapidly translocate into the nascent muscle to differentiate. Here, we uncovered at the heart of this response a signaling module encompassing NOTCH, GSK-3β, SNAI1 and β-catenin. Independent of its transcriptional function, NOTCH profoundly inhibits GSK-3β activity. As a result SNAI1 is stabilized, triggering an epithelial to mesenchymal transition. This allows the recruitment of β-catenin from the membrane, which acts as a transcriptional co-factor to activate myogenesis, independently of WNT ligand. Our results intimately associate the initiation of myogenesis to a change in cell adhesion and may reveal a general principle for coupling cell fate changes to EMT in many developmental and pathological processes.

In the absence of salient sensory cues to guide behavior, animals must still execute sequences of motor actions in order to forage and explore. How such successive motor actions are coordinated to form global locomotion trajectories is unknown. We mapped the structure of larval zebrafish swim trajectories in homogeneous environments and found that trajectories were characterized by alternating sequences of repeated turns to the left and to the right. Using whole-brain light-sheet imaging, we identified activity relating to the behavior in specific neural populations that we termed the anterior rhombencephalic turning region (ARTR). ARTR perturbations biased swim direction and reduced the dependence of turn direction on turn history, indicating that the ARTR is part of a network generating the temporal correlations in turn direction. We also find suggestive evidence for ARTR mutual inhibition and ARTR projections to premotor neurons. Finally, simulations suggest the observed turn sequences may underlie efficient exploration of local environments.