Characterization of Type I and Type II nNOS-Expressing Interneurons in the Barrel Cortex of Mouse

In the neocortex, neuronal nitric oxide (NO) synthase (nNOS) is essentially expressed in two classes of GABAergic neurons: type I neurons displaying high levels of expression and type II neurons displaying weaker expression. Using immunocytochemistry in mice expressing GFP under the control of the glutamic acid decarboxylase 67k (GAD67) promoter, we studied the distribution of type I and type II neurons in the barrel cortex and their expression of parvalbumin (PV), somatostatin (SOM), and vasoactive intestinal peptide (VIP). We found that type I neurons were predominantly located in deeper layers and expressed SOM (91.5%) while type II neurons were concentrated in layer II/III and VI and expressed PV (17.7%), SOM (18.7%), and VIP (10.2%). We then characterized neurons expressing nNOS mRNA (n = 42 cells) ex vivo, using whole-cell recordings coupled to single-cell reverse transcription-PCR and biocytin labeling. Unsupervised cluster analysis of this sample disclosed four classes. One cluster (n = 7) corresponded to large, deep layer neurons, displaying a high expression of SOM (85.7%) and was thus very likely to correspond to type I neurons. The three other clusters were identified as putative type II cells and corresponded to neurogliaform-like interneurons (n = 19), deep layer neurons expressing PV or SOM (n = 9), and neurons expressing VIP (n = 7). Finally, we performed nNOS immunohistochemistry on mouse lines in which GFP labeling revealed the expression of two specific developmental genes (Lhx6 and 5-HT3A). We found that type I neurons expressed Lhx6 but never 5-HT3A, indicating that they originate in the medial ganglionic eminence (MGE). Type II neurons expressed Lhx6 (63%) and 5-HT3A (34.4%) supporting their derivation either from the MGE or from the caudal ganglionic eminence (CGE) and the entopeduncular and dorsal preoptic areas. Together, our results in the barrel cortex of mouse support the view that type I neurons form a specific class of SOM-expressing neurons while type II neurons comprise at least three classes

Kinetochore component function in C. elegans oocytes revealed by 4D tracking of holocentric chromosomes

During cell division, chromosome congression to the spindle center, their orientation along the spindle long axis and alignment at the metaphase plate depend on interactions between spindle microtubules and kinetochores, and are pre-requisite for chromosome bi-orientation and accurate segregation. How these successive phases are controlled during oocyte meiosis remains elusive. Here we provide 4D live imaging during the first meiotic division in C. elegans oocytes with wild-type or disrupted kinetochore protein function. We show that, unlike in monocentric organisms, holocentric chromosome bi-orientation is not strictly required for accurate chromosome segregation. Instead, we propose a model in which initial kinetochore-localized BHC module (comprised of BUB-1Bub1, HCP-1/2CENP-F and CLS-2CLASP)-dependent pushing acts redundantly with Ndc80 complex-mediated pulling for accurate chromosome segregation in meiosis. In absence of both mechanisms, homologous chromosomes tend to co-segregate in anaphase, especially when initially mis-oriented. Our results highlight how different kinetochore components cooperate to promote accurate holocentric chromosome segregation in oocytes of C. elegans.This work was supported by CNRS and University ParisCité, by NIHR01GM117407 and R01GM130764 (J.C.C.), and by grants from the European Research Council ERC-CoG ChromoSOMe 819179 and from the Agence Nationale de la Recherche ANR-19-CE13-0015 (J.D.).Peer ReviewedPostprint (published version

Functional role of ɛ-tubulin in the assembly of the centriolar microtubule scaffold

Centrioles and basal bodies fascinate by their spectacular architecture, featuring an arrangement of nine microtubule triplets into an axial symmetry, whose biogenesis relies on yet elusive mechanisms. However, the recent discovery of new tubulins, such as δ-, ɛ-, or η-tubulin, could constitute a breakthrough for deciphering the assembly steps of this unconventional microtubule scaffold. Here, we report the functional analysis in vivo of ɛ-tubulin, based on gene silencing in Paramecium, which demonstrates that this protein, which localizes at the basal bodies, is essential for the assembly and anchorage of the centriolar microtubules

Identification and Characterization of Known Biallelic Mutations in the IFT27 (BBS19) Gene in a Novel Family With Bardet-Biedl Syndrome

Bardet-Biedl syndrome (BBS; MIM 209900) is a rare ciliopathy characterized by retinitis pigmentosa, postaxial polydactyly, obesity, hypogonadism, cognitive impairment and kidney dysfunction. Mutations in 22 BBS genes have been identified to cause the disease. We report a family with typical BBS features (retinitis pigmentosa, postaxial polydactyly, obesity, cognitive impairment, and atrioventricular septal defect) mutated in IFT27/BBS19. IFT27 is part of the Intraflagellar transport (IFT), a bidirectional mechanism allowing the protein motility within the cilia. Using whole exome sequencing, two compound heterozygous mutations were found in the proband (NM_006860.4:c.[104A > G];[349+1G > T], p.[Tyr35Cys];[?]) consistent with the expected autosomal recessive inheritance mode. These two mutations have already been reported but independently in other families and lacking either familial segregation or functional validation. This is the third report of IFT27 mutations in BBS patients confirming IFT27 as a BBS gene (BBS19). Mutations in IFT genes (IFT27, IFT172 and IFT74) confirm the IFT-pathway as a pathomechanism for BBS

A New SLC10A7 Homozygous Missense Mutation Responsible for a Milder Phenotype of Skeletal Dysplasia With Amelogenesis Imperfecta

International audienceAmelogenesis imperfecta (AI) is a heterogeneous group of rare inherited diseases presenting with enamel defects. More than 30 genes have been reported to be involved in syndromic or non-syndromic AI and new genes are continuously discovered (Smith et al., 2017). Whole-exome sequencing was performed in a consanguineous family. The affected daughter presented with intra-uterine and postnatal growth retardation, skeletal dysplasia, macrocephaly, blue sclerae, and hypoplastic AI. We identified a homozygous missense mutation in exon 11 of SLC10A7 (NM_001300842.2: c.908C>T; p.Pro303Leu) segregating with the disease phenotype. We found that Slc10a7 transcripts were expressed in the epithelium of the developing mouse tooth, bones undergoing ossification, and in vertebrae. Our results revealed that SLC10A7 is overexpressed in patient fibroblasts. Patient cells display altered intracellular calcium localization suggesting that SLC10A7 regulates calcium trafficking. Mutations in this gene were previously reported to cause a similar syndromic phenotype, but with more severe skeletal defects (Ashikov et al., 2018;Dubail et al., 2018). Therefore, phenotypes resulting from a mutation in SLC10A7 can vary in severity. However, AI is the key feature indicative of SLC10A7 mutations in patients with skeletal dysplasia. Identifying this important phenotype will improve clinical diagnosis and patient management

A New SLC10A7 Homozygous Missense Mutation Responsible for a Milder Phenotype of Skeletal Dysplasia With Amelogenesis Imperfecta

Amelogenesis imperfecta (AI) is a heterogeneous group of rare inherited diseases presenting with enamel defects. More than 30 genes have been reported to be involved in syndromic or non-syndromic AI and new genes are continuously discovered (Smith et al., 2017). Whole-exome sequencing was performed in a consanguineous family. The affected daughter presented with intra-uterine and postnatal growth retardation, skeletal dysplasia, macrocephaly, blue sclerae, and hypoplastic AI. We identified a homozygous missense mutation in exon 11 of SLC10A7 (NM_001300842.2: c.908C>T; p.Pro303Leu) segregating with the disease phenotype. We found that Slc10a7 transcripts were expressed in the epithelium of the developing mouse tooth, bones undergoing ossification, and in vertebrae. Our results revealed that SLC10A7 is overexpressed in patient fibroblasts. Patient cells display altered intracellular calcium localization suggesting that SLC10A7 regulates calcium trafficking. Mutations in this gene were previously reported to cause a similar syndromic phenotype, but with more severe skeletal defects (Ashikov et al., 2018;Dubail et al., 2018). Therefore, phenotypes resulting from a mutation in SLC10A7 can vary in severity. However, AI is the key feature indicative of SLC10A7 mutations in patients with skeletal dysplasia. Identifying this important phenotype will improve clinical diagnosis and patient management

Proteasome subunit variants cause neurosensory syndrome combining deafness and cataract due to proteotoxic stress

The ubiquitin–proteasome system degrades ubiquitin‐modified proteins to maintain protein homeostasis and to control signalling. Whole‐genome sequencing of patients with severe deafness and early‐onset cataracts as part of a neurological, sensorial and cutaneous novel syndrome identified a unique deep intronic homozygous variant in the PSMC3 gene, encoding the proteasome ATPase subunit Rpt5, which lead to the transcription of a cryptic exon. The proteasome content and activity in patient\u27s fibroblasts was however unaffected. Nevertheless, patient\u27s cells exhibited impaired protein homeostasis characterized by accumulation of ubiquitinated proteins suggesting severe proteotoxic stress. Indeed, the TCF11/Nrf1 transcriptional pathway allowing proteasome recovery after proteasome inhibition is permanently activated in the patient\u27s fibroblasts. Upon chemical proteasome inhibition, this pathway was however impaired in patient\u27s cells, which were unable to compensate for proteotoxic stress although a higher proteasome content and activity. Zebrafish modelling for knockout in PSMC3 remarkably reproduced the human phenotype with inner ear development anomalies as well as cataracts, suggesting that Rpt5 plays a major role in inner ear, lens and central nervous system development

Serotonin 3A Receptor Subtype as an Early and Protracted Marker of Cortical Interneuron Subpopulations

To identify neocortical neurons expressing the type 3 serotonergic receptor, here we used transgenic mice expressing the enhanced green fluorescent protein (GFP) under the control of the 5-HT3A promoter (5-HT3A:GFP mice). By means of whole-cell patch-clamp recordings, biocytin labeling, and single-cell reversed-transcriptase polymerase chain reaction on acute brain slices of 5-HT3A:GFP mice, we identified 2 populations of 5-HT3A-expressing interneurons within the somatosensory cortex. The first population was characterized by the frequent expression of the vasoactive intestinal peptide and a typical bipolar/bitufted morphology, whereas the second population expressed predominantly the neuropeptide Y and exhibited more complex dendritic arborizations. Most interneurons of this second group appeared very similar to neurogliaform cells according to their electrophysiological, molecular, and morphological properties. The combination of 5-bromo-2-deoxyuridine injections with 5-HT3A mRNA detection showed that cortical 5-HT3A interneurons are generated around embryonic day 14.5. Although at this stage the 5-HT3A receptor subunit is expressed in both the caudal ganglionic eminence and the entopeduncular area, homochronic in utero grafts experiments revealed that cortical 5-HT3A interneurons are mainly generated in the caudal ganglionic eminence. This protracted expression of the 5-HT3A subunit allowed us to study specific cortical interneuron populations from their birth to their final functional phenotype

Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Distress Syndrome associated with COVID-19: An Emulated Target Trial Analysis.

RATIONALE: Whether COVID patients may benefit from extracorporeal membrane oxygenation (ECMO) compared with conventional invasive mechanical ventilation (IMV) remains unknown. OBJECTIVES: To estimate the effect of ECMO on 90-Day mortality vs IMV only Methods: Among 4,244 critically ill adult patients with COVID-19 included in a multicenter cohort study, we emulated a target trial comparing the treatment strategies of initiating ECMO vs. no ECMO within 7 days of IMV in patients with severe acute respiratory distress syndrome (PaO2/FiO2 <80 or PaCO2 ≥60 mmHg). We controlled for confounding using a multivariable Cox model based on predefined variables. MAIN RESULTS: 1,235 patients met the full eligibility criteria for the emulated trial, among whom 164 patients initiated ECMO. The ECMO strategy had a higher survival probability at Day-7 from the onset of eligibility criteria (87% vs 83%, risk difference: 4%, 95% CI 0;9%) which decreased during follow-up (survival at Day-90: 63% vs 65%, risk difference: -2%, 95% CI -10;5%). However, ECMO was associated with higher survival when performed in high-volume ECMO centers or in regions where a specific ECMO network organization was set up to handle high demand, and when initiated within the first 4 days of MV and in profoundly hypoxemic patients. CONCLUSIONS: In an emulated trial based on a nationwide COVID-19 cohort, we found differential survival over time of an ECMO compared with a no-ECMO strategy. However, ECMO was consistently associated with better outcomes when performed in high-volume centers and in regions with ECMO capacities specifically organized to handle high demand. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)