Biology subjects

Gimenez, M.

Publications and source records attributed to Gimenez, M..

6 recordsLinked to original sources

bioRxiv2024

KIR3DL1 and Tox identify clonally expanded encephalitogenic neuron-specific CD8+ T cells in autoimmune encephalitis

Autoreactive CD8+ T cells are the principal suspects in autoimmune encephalitis (AIE) with antibodies targeting intracellular neuronal antigens So far, the search for neuron-autoreactive CD8+ T cells has been focused on a few autoantigens and did not yield convincing results. Here, we leveraged natural antigen presentation by hiPSC-derived neurons to look at the global autoreactive CD8+ T cell response, independently of pre-conceived hypothesis of the autoantigens involved in the disease. This unbiased approach allowed for the identification of rare polyclonal neuron-reactive CD8+ T cells in healthy donors, and contrastingly, expanded clonotypes in two patients with anti-Ri AIE. Detailed ex vivo phenotypic characterization of these clonotypes revealed a specific transcriptional program suggestive of a pathogenic potential. In particular, this subset can be identified by the expression of KIR3DL1 and TOX. Strikingly, we could also demonstrate that CD8+ T cells found in the brain of an anti-Ri AIE patient display a similar phenotype associated with cytotoxicity and encephalitogenic features.

immunology↗

bioRxiv2024

Human stem cell derived neurons and astrocytes to detect auto-reactive IgG in neurological diseases

Background and objectivesUp to 46% of patients with presumed autoimmune limbic encephalitis are seronegative for all currently known CNS antigens. We developed a cell-based assay (CBA) to screen for novel neural antibodies in serum and CSF using neurons and astrocytes derived from human induced pluripotent stem cells (hiPSC). MethodsHuman iPSC-derived astrocytes or neurons were incubated with serum/CSF from 99 patients (42 with inflammatory neurological diseases (IND) and 57 with non-IND (NIND)). The IND group included 11 patients with previously established neural antibodies, six with seronegative neuromyelitis optica spectrum disorder (NMOSD), 12 with suspected autoimmune encephalitis/paraneoplastic syndrome (AIE/PNS), and 13 with other IND (OIND). IgG binding to fixed CNS cells was detected using fluorescently-labelled antibodies and analyzed through automated fluorescence measures. IgG neuronal/astrocyte reactivity was further analyzed by flow cytometry. Peripheral blood mononuclear cells (PBMC) were used as CNS-irrelevant control target cells. Reactivity profile was defined as positive using a Robust regression and Outlier removal test with a false discovery rate at 10% following each individual readout. ResultsUsing our CBA, we detected antibodies recognizing hiPSC-derived neural cells in 19/99 subjects. Antibodies bound specifically to astrocytes in nine cases, to neurons in eight cases and to both cell types in two cases, as confirmed by microscopy single-cell analyses. Highlighting the significance of our novel 96-well CBA assay, neural-specific antibody binding was more frequent in IND (15/42) than in NIND patients (4/57) (Fisher test, p=0.0005). Three of three patients with astrocyte- reactive (2 AQP4+ NMO, 1 GFAP astrocytopathy), and 3/4 with intracellular neuron-reactive antibodies (2 Hu+, 1 Ri+ AIE/PNS), as identified in diagnostic laboratories, were also positive with our CBA. Most interestingly, we showed antibody-reactivity in 2/6 seronegative NMOSD, 6/12 probable AIE/PNS, and 1/13 OIND. Flow cytometry using hiPSC-derived CNS cells or PBMC detected antibody binding in 13 versus 0 patients, respectively, establishing the specificity of the detected antibodies for neural tissue. DiscussionOur unique hiPSC-based CBA allows for the screening of novel neuron-/astrocyte-reactive antibodies in patients with suspected immune-mediated neurological syndromes, and negative testing in established routine laboratories, opening new perspectives in establishing early diagnosis of such complex diseases.

neuroscience↗

bioRxiv2023

Unique proteomic signature of JCPyV-infected human astrocytes: from cells to extracellular vesicles

JC polyomavirus (JCPyV) is an opportunistic virus that remains in a latent state in the kidneys of more than half of the human adult population. In rare cases of severe immune suppression, the virus is able to establish a lytic infection of glial cells in the brain, resulting in a debilitating, demyelinating disease known as progressive multifocal leukoencephalopathy (PML). Because of the exceptional species and tissue specificity of the virus, appropriate models of JCPyV infection in the brain are lacking, thus hampering progress towards the development of novel antiviral strategies and biomarkers of disease activity. While PML has traditionally been characterized as a lytic infection of oligodendrocytes, more recent findings suggest an important role for astrocytes during the initial stages of disease. Here, using human induced pluripotent stem cell (hiPSC) derived-astrocytes coupled with a multiparametric approach, we show that 1. JCPyV readily infects and replicates in astrocytes, 2. JCPyV strongly dysregulates the cell biology and 3. these findings adequately reflect ex vivo findings. We perform an in-depth characterization of the effect of JCPyV on the cell proteome over time, demonstrating a strong dysregulation of the cell cycle and activation of the DNA damage response. Furthermore, we show that the proteomic signature observed for infected astrocytes is extended to excreted vesicles, underlining their potential to gain valuable insights into JCPyV propagation in the brain.

neuroscience↗

bioRxiv2022

Improved detection and classification of plasmids from circularized and fragmented assemblies

Plasmids are mobile genetic elements important for bacterial adaptation. The study of plasmids from sequencing data is challenging because short reads produce fragmented assemblies, requiring of subsequent discrimination between chromosome and plasmid sequences. Although circularized assemblies are now possible using long-read data, there is still a need to differentiate plasmids from other circular elements. Here, we present plaSquid, a dockerized tool developed in Nextflow that expands plasmid detection and improves replicon typing and mobility groups classification schemes, outperforming previously available methods in both precision and sensitivity. When applied to [~]10.5 million metagenomic contigs, plaSquid revealed a 2.7-fold increase in plasmid phylogenetic diversity. Also, we used plaSquid to uncover a significant role of plasmids in the widespread distribution of clinically-relevant antimicrobial resistance genes in the built environment, from cities to spacecraft. Together, we present an improved approach to study plasmid biology from fragmented or circularized genomic and metagenomic assemblies.

bioinformatics↗

bioRxiv2021

Rev-erb-α controls skeletal muscle calcium homeostasis through myoregulin repression: implications in Duchenne Muscular Dystrophy

The sarcoplasmic reticulum (SR) plays an important role in calcium homeostasis. SR calcium mishandling is described in pathological conditions such as myopathies. Here, we investigated whether the nuclear receptor Rev-erb- regulates skeletal muscle SR calcium homeostasis. Our data demonstrate that Rev-erb invalidation in mice impairs SERCA-dependent SR calcium uptake. Rev-erb- acts on calcium homeostasis by repressing the SERCA inhibitor Myoregulin, through direct binding to its promoter. Restoration of Myoregulin counteracts the effects of REV-ERB- overexpression on SR calcium content. Interestingly, myoblasts from Duchenne myopathy patients display downregulated REV-ERB expression, whereas pharmacological Rev-erb activation ameliorates SR calcium homeostasis, and improves muscle structure and function in dystrophic mdx/Utr+/- mice. Our findings demonstrate that Rev-erb- regulates muscle SR calcium homeostasis, pointing to its therapeutic interest for mitigating myopathy.

cell biology↗

bioRxiv2021

Disruption of the Pseudomonas aeruginosa Tat system perturbs PQS-dependent quorum sensing and biofilm maturation through loss of the Rieske cytochrome bc1 sub-unit

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via the pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of 2-heptyl-3-hydroxy-4-quinolone (PQS) dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA, rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat secretion substrate responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc1 complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. Thus, lack of the Rieske sub-unit export is clearly responsible for the Tat-mediated perturbation of PQS-dependent QS, the loss of virulence factor production, biofilm eDNA and the tobramycin tolerance of P. aeruginosa biofilms. Author SummaryPseudomonas aeruginosa is a highly adaptable human pathogen responsible for causing chronic biofilm-associated infections. Biofilms are highly refractory to host defences and antibiotics and thus difficult to eradicate. The biofilm extracellular matrix incorporates extracellular DNA (eDNA). This stabilizes biofilm architecture and helps confer tolerance to antibiotics. Since mechanisms that control eDNA release are not well understood, we screened a P. aeruginosa mutant bank for strains with defects in eDNA release and discovered a role for the twin-arginine translocation (Tat) pathway that exports folded proteins across the cytoplasmic membrane. Perturbation of the Tat pathway resulted in defective biofilms susceptible to antibiotic treatment as a consequence of perturbed pseudomonas quinolone (PQS) signalling. This resulted in the failure to produce or release biofilm components including eDNA, phenazines and rhamnolipids as well as microvesicles. Furthermore, we discovered that perturbation of PQS signalling was a consequence of the inability of tat mutants to translocate the Rieske subunit of the cytochrome bc1 complex involved in electron transfer and energy transduction. Given the importance of PQS signalling and the Tat system to virulence and biofilm maturation in P. aeruginosa, our findings underline the potential of the Tat system as a drug target for novel antimicrobial agents.

microbiology↗