Biology subjects

Brocard, J.

Publications and source records attributed to Brocard, J..

11 recordsLinked to original sources

bioRxiv2026

How flat is your sample? An opportunistic survey of 3D tilt in public fluorescence microscopy data

Sample planarity is rarely monitored in fluorescence microscopy quality control, yet focal plane deviations across the field of view are a potential source of measurement error. Here I describe FlatStat, a tool that estimates sample tilt automatically from any 3D fluorescence stack, without prior knowledge of sample content, by fitting a plane to the Z-map of maximum intensity. Applied to an Argolight calibration slide and biological samples on a laser-scanning confocal system, FlatStat yielded reproducible slope and direction measurements attributable to the instrument rather than the sample. To establish community reference values, FlatStat was extended to Python and applied opportunistically to 1204 image stacks from 22 projects in the Image Data Resource, yielding 4670 tilt measurements. Slopes spanned several orders of magnitude across projects; inter-channel coherence confirmed that measured tilt reflects physical stage and mounting geometry rather than channel-specific biological topography. Unfortunately, instrument and sample preparation metadata were largely absent from the corpus, limiting causal inference. Finally, controlled tilt experiments on fluorescent beads showed that chromatic shift increased modestly with tilt ([~]57 nm over the full range tested), while lateral and axial resolutions were essentially unaffected.

bioinformatics↗

bioRxiv2026

CACNA2D4 variants are associated with exertional heat stroke susceptibility

Exertional Heat Stroke (EHS) is a life-threatening disease defined by severe hyperthermia and sudden neurological dysfunction in healthy individuals exposed to intense physical exercise. As is the case with some forms of malignant hyperthermia, mutations in genes involved in skeletal muscle contraction could predispose some patients to EHS. However, the genetic basis of EHS remains poorly documented. Here we identify EHS-associated variants in the CACNA2D4 gene encoding the Cav Ca2+ channel subunit, 2{delta}4, and describe their impact in heterologous expression systems and a knock-in mutant mouse strain heterozygous for the S299R variant (S299R+/-). In transfected cells, each CACNA2D4 variant, including S299R, affected the trafficking and function of 2{delta}4 and its regulation of the Cav1.1 channel. S299R+/-mice showed EHS-like crisis with signs of rhabdomyolysis and an elevation in core body temperature when submitted to an intense exercise protocol. We show that a CACNA2D4 transcript is expressed in mouse skeletal muscle and that the presence of the S299R variant in 2{delta}4 induces a modification in the calcium flux triggered by muscle cell depolarization. Altogether, our data reveal an unexpected connection between CACNA2D4 gene variants and pathophysiological alterations in skeletal muscle Ca2+ signaling that may increase EHS susceptibility.

genetics↗

bioRxiv2024

Thyroid hormones act as a timer for the postnatal maturation of parvalbumin neurons in mouse neocortex

Thyroid hormones (THs) are required for proper maturation of parvalbumin-expressing GABAergic interneurons in the mouse neocortex, which function is essential to maintain a proper balance between neuronal excitation and inhibition. However, the timeline of this TH action has yet to be elucidated. Here, we used several mouse models expressing dominant negative mutations of the TH nuclear receptor 1 (TR1), to better define the time window during which THs promote the maturation of parvalbumin neurons. The action of THs during the first three postnatal weeks appears to be necessary to initiate the elaboration of a specialized extracellular matrix called the perineuronal net. Transcriptome analysis identified a small set of putative target genes for TR1, which are at the origin of the postnatal remodeling of the repertoire of ions channels and the elaboration of perineuronal nets. These data suggest that THs act as a timer to define the temporal boundaries of the critical period of heightened cortical plasticity, which plays a fundamental role in the development of neuronal circuits. Significance statementThyroid hormones exert a major influence on late neurodevelopment, acting directly on gene expression by binding to nuclear receptors present in all cell types. This study reports the discovery of a direct link between the transcriptional regulation exerted by the thyroid hormones in GABAergic neurons and the timing of the critical period of heightened plasticity. This provides a general framework to understand the pathology associated with altered thyroid hormone signaling.

developmental biology↗

bioRxiv2024

Long-term live imaging, cell identification and cell tracking in regenerating crustacean legs

High resolution live imaging of regeneration presents unique challenges, due to the nature of the specimens (large mobile animals), the duration of the process (spanning days or weeks), and the fact that cellular resolution must be achieved without damage caused by lengthy exposures to light. Building on previous work that allowed us to image different parts of the process of leg regeneration in the crustacean Parhyale hawaiensis, we present here a method for live imaging that captures the entire process of leg regeneration, spanning up to 10 days, at cellular resolution. Our method includes (1) mounting and long-term live imaging of regenerating legs under conditions that yield high spatial and temporal resolution but minimise photodamage, (2) fixing and in situ staining of the regenerated legs that were imaged, to identify cell fates, and (3) computer-assisted cell tracking to determine the cell lineages and progenitors of identified cells. The method is optimised to limit light exposure while maximising tracking efficiency. Combined with appropriate cell-type-specific markers, this method may allow the description of cell lineages for every regenerated cell type in the limb.

developmental biology↗

bioRxiv2024

Hepatitis E Virus-induced antiviral response by plasmacytoid dendritic cells is modulated by the ORF2 protein

Type I and III interferons (IFN-I/III) are critical to protect the host during viral infection. Previous studies have shown that IFN-mediated antiviral responses against hepatitis E virus (HEV) are suppressed and defeated by viral escape mechanisms at play in infected hepatocytes. Here, we studied the anti-HEV function of IFN secreted by plasmacytoid dendritic cells (pDCs), which are specialized producers of IFNs. We showed that pDCs co-cultured with HEV-replicating cells secreted IFN in a cell-to-cell contact-dependent manner. Pharmacological inhibitor and antibodies targeting contact proteins revealed that pDC response against HEV required the endosomal nucleic-acid sensor TLR7 and adhesion molecules, such as ICAM-I and L{beta}2-integrin. IFNs secreted by pDCs reduced viral spread. Intriguingly, ORF2, the capsid protein of HEV, can be produced in various forms by the infected cells. During infection, a fraction of the intracellular ORF2 protein localizes into the nucleus while another ORF2 fraction packages viral genomes to produce infectious virions. In parallel, glycosylated forms of ORF2 are also massively secreted by infected cells. Using viral genome expressing ORF2 mutants, we showed that glycosylated ORF2 forms contribute to better recognition of infected cells by pDCs via regulation of contacts between infected cells and pDCs. ORF2 forms may thus modulate pDC-mediated anti-HEV response. Together, our results suggest that liver-resident pDCs, which exhibit comparable IFN-producing ability as blood-derived pDCs, may be essential to control HEV replication.

immunology↗

bioRxiv2024

Functional benefit of CRISPR/Cas9-induced allele deletion for RYR1 dominant mutation

More than 700 pathogenic or probably pathogenic variations have been identified in the RYR1 gene causing various myopathies collectively known as "RYR1-related myopathies". Currently, there is no treatment for these myopathies, and gene therapy stands out as one of the most promising approaches. In the context of a dominant form of Central Core Disease due to a RYR1 mutation, we aimed at showing the functional benefit of inactivating specifically the mutated RYR1 allele by guiding CRISPR/Cas9 cleavages onto frequent single nucleotide polymorphisms (SNPs) segregating on the same chromosome. Whole-genome sequencing was used to pinpoint SNPs localized on the mutant RYR1 allele and identified specific CRISPR/Cas9 guide-RNAs. Lentiviruses encoding these guide-RNAs and the SpCas9 nuclease were used to transduce immortalized patient muscle cells, inducing the specific deletion of the mutant RYR1 allele. The efficiency of the deletion was assessed at both DNA and RNA levels and at the functional level after monitoring calcium release induced by the stimulation of the RyR1-channel. This study provides in-cellulo proof of concept regarding the benefits of mutant RYR1 allele deletion, in the case of a dominant RYR1 mutation, from both a molecular and functional perspective. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/576997v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@484a3borg.highwire.dtl.DTLVardef@1a842org.highwire.dtl.DTLVardef@cda3d5org.highwire.dtl.DTLVardef@befd91_HPS_FORMAT_FIGEXP M_FIG C_FIG eTOC synopsisMutations in the RYR1 gene, encoding a calcium channel required for muscle contraction, cause severe myopathies. In this study, Marty and colleagues demonstrate the functional benefit of suppression of a mutant RYR1 allele using CRISPR/Cas9, in the case of a dominant mutation, leaving the wild type allele alone.

neuroscience↗

bioRxiv2023

Sperm motility restoration in mice suffering from oligo-astheno-teratozoospermia by in vivo injection and electroporation of naked mRNA

Oligo-astheno-teratozoospermia (OAT), a recurent cause of male infertility, is the most frequent disorder of spermatogenesis with a probable genetic cause. Patients and mice bearing mutations in the ARMC2 gene have a decreased sperm concentration, and individual sperm show multiple morphological defects and a lack of motility - a canonical OAT phenotype. Intracytoplasmic sperm injection (ICSI) is required to treat such a condition but it is associated with a small increase in birth defects in comparison to pregnancies not involving assisted conception. Consequently, new targeted treatments are needed to restore fertility. Here, a combination of in vivo injection and electroporation of capped and poly-A-tailed naked mRNA is tested as a strategy to treat ARMC2-related infertility in mouse. mRNAs coding for several reporter genes are tested and the efficiency and the kinetic of expression are assessed using in vivo and in vitro 2D and 3D imaging experiments. We show that mRNA-coded reporter proteins are detected for up to 3 weeks in germ cells, making the use of mRNA possible to treat infertility. We compare these results with those obtained with a non-integrative plasmid Enhanced Episomal Vector (EEV), which induces low and transient expression in spermatogenic cells. Consequently, injection and electroporation of naked mRNA-Armc2 into the testes of Armc2-deficient males were performed and we show the presence of normal and motile sperm in the epididymis. These motile sperm were able to produce embryos by IVF and ICSI. This study demonstrates, for the first time, that mRNA electroporation can restore sperm motility and partially fertilizing ability, providing a proof-of-concept for mRNA-based strategies to correct monogenic causes of male infertility and opening new avenues for male infertility treatment.

molecular biology↗

bioRxiv2022

Huntingtin-KIF1A-mediated axonal transport of synaptic vesicle precursors influences synaptic transmission and motor skill learning in mice

Neurotransmitters are released at synapses by synaptic vesicles (SVs), which originate from SV precursors (SVPs) that have traveled along the axon. Because each synapse maintains a pool of SVs, only a small fraction of which are released, it is unclear whether axonal transport of SVPs modifies synaptic function. Here, studying the corticostriatal network both in microfluidic devices and in mice, we find that phosphorylation of the Huntingtin protein (HTT) causes it to recruit the kinesin motor KIF1A, which in turn increases axonal transport of SVPs and synaptic glutamate release. In mice, constitutive HTT phosphorylation leads to SV over-accumulation at synapses, increases the probability of SV release, and impairs motor skill learning on the rotating rod. Silencing KIF1A in these mice restored SV transport and motor skill learning to wild-type levels. Axonal SVP transport within the corticostriatal network thus influences synaptic plasticity and motor skill learning.

neuroscience↗

bioRxiv2021

Impaired α-tubulin re-tyrosination leads to synaptic dysfunction and is a feature of Alzheimer's disease

In neurons, dynamic microtubules play regulatory roles in neurotransmission and synaptic plasticity. While stable microtubules contain detyrosinated tubulin, dynamic microtubules are composed of tyrosinated tubulin, suggesting that the tubulin tyrosination/detyrosination (Tyr/deTyr) cycle modulates microtubule dynamics and synaptic function. In the Tyr/deTyr cycle, the C-terminal tyrosine of -tubulin is re-added by tubulin-tyrosine-ligase (TTL). Here we show that TTL+/- mice exhibit decreased tyrosinated microtubules, synaptic plasticity and memory deficits, and that reduced TTL expression is a feature of sporadic and familial Alzheimers disease (AD), with human APPV717I neurons having less dynamic microtubules. We find that spines visited by dynamic microtubules are more resistant to Amyloid{beta}1-42 and that TTL, by promoting microtubule entry into spines, prevents A{beta}1-42-induced spine pruning. Our results demonstrate that the Tyr/deTyr cycle regulates synaptic plasticity, is protective against spine injury, and that tubulin re-tyrosination is lost in AD, providing evidence that a defective Tyr/deTyr cycle may contribute to neurodegeneration.

neuroscience↗

bioRxiv2020

In vivo RyR1 reduction in muscle triggers a core-like myopathy

Some mutations in the RYR1 gene lead to congenital myopathies, through reduction in this calcium channel expression level, but the functional whole organism consequences of reduction in RyR1 amount have never been studied. We have developed and characterized a mouse model with inducible muscle specific RYR1 deletion. Recombination in the RYR1 gene resulted in a progressive reduction in the protein amount and was associated with a progressive muscle weakness and atrophy. Calcium fluxes in isolated muscle fibers were accordingly reduced. Alterations in the muscle structure were observed, with fibers atrophy, abnormal mitochondria distribution, membrane remodeling, associated with increase in the expression level of many proteins and inhibition of the autophagy process. This model demonstrates that RyR1 reduction is sufficient to recapitulate most features of Central Core Disease, and accordingly similar alterations were observed in muscle biopsies from Central Core Disease patients, pointing to common pathophysiological mechanisms related to RyR1 reduction.

neuroscience↗

bioRxiv2020

AutoNeuriteJ: An ImageJ plugin for measurement and classification of neuritic extensions

Morphometry characterization is an important procedure in describing neuronal cultures and identifying phenotypic differences. This task usually requires labor-intensive measurements and the classification of numerous neurites from large numbers of neurons in culture. To automate these measurements, we wrote AutoNeuriteJ, an imageJ/Fiji plugin that measures and classifies neurites from a very large number of neurons. We showed that AutoNeuriteJ is able to detect variations of neuritic growth induced by several compounds known to affect the neuronal growth. In these experiments measurement of more than 5000 mouse neurons per conditions was obtained within a few hours. Moreover, by analyzing mouse neurons deficient for the microtubule associated protein 6 (MAP6) and wild type neurons we illustrate that AutoNeuriteJ is capable to detect subtle phenotypic difference in axonal length. Overall the use of AutoNeuriteJ will provide rapid, unbiased and accurate measurement of neuron morphologies.

bioinformatics↗