Biology subjects

Merle, L.

Publications and source records attributed to Merle, L..

4 recordsLinked to original sources

bioRxiv2026

LUCID-EV: a robust and quantitative bioluminescent assay for the detection of EV cytosolic delivery in the absence of VSV-G expression

Extracellular vesicles are key intercellular messengers that modulate the function of target cells by carrying effectors, either at their surface or in their lumen. In the latter case, their action depends on the ability to deliver their content into the cytosol of target cells. How efficiently EVs deliver their content upon interaction with their target cell is thus a central question for understanding the functional impact of this mode of action. To address this question, signal-driven bimolecular interactions between two partners located respectively in the EV lumen and the target cell cytosol have become a widely used strategy to detect the cytosolic delivery EV content. However, the detection of cytosolic delivery with these assays was often tributary to the artificial enhancement of the fusion between EV and cell membranes, through for instance VSV-G fusogenic protein expression. Here we provide a robust and quantitative LUCiferase-based complementation assay (HiBiT/LgBiT), to quantify the Internalization and cytosolic Delivery of EV content: LUCID-EV. By optimizing the signal-to-noise ratio of the assay, the method for loading HiBiT fragment into EVs (fusion to a lipid-binding domain rather than to tetraspanins), and the intracellular position of LgBiT (associated to membranes), we could quantify cytosolic delivery from various non-VSV-G-expressing EVs into target immune dendritic cells. Importantly, this delivery did not involve the acidic late endosomes environment required for VSV-G-dependent EV cytosolic delivery. The limited efficacy of the process highlights the need for highly sensitive assays like the one described here. Further development of the LUCID-EV assay could help identifying EV/target cells pairs with enhanced cytosolic delivery properties and characterize the cellular route for delivery.

cell biology↗

bioRxiv2024

Olfactory and Trigeminal Routes of HSV-1 CNS Infection with Regional Microglial Heterogeneity

Herpes simplex virus type 1 (HSV-1) primarily targets the oral and nasal epithelia before establishing latency in the trigeminal and other peripheral ganglia (TG). HSV-1 can also infect and go latent in the central nervous system (CNS) independent of latency in the TGs. Recent studies suggest entry to the CNS via two distinct routes: the TG-brainstem connection and olfactory nerve; however, to date, there is no characterization of brain regions targeted during HSV-1 primary infection. Furthermore, the immune response by microglia may also contribute to the heterogeneity between different brain regions. However, the response to HSV-1 by microglia has not been characterized in a region-specific manner. This study investigated the time course of HSV-1 spread within the olfactory epithelium (OE) and CNS following intranasal inoculation and the corresponding macrophage/microglial response in a C57BL/6 mouse model. We found an apical to basal spread of HSV-1 within the OE and underlying tissue accompanied by an inflammatory response of macrophages. OE Infection was followed by infection of a small subset of brain regions targeted by the TG in the brainstem, as well as other cranial nerve nuclei, including the vagus and hypoglossal nerve. Furthermore, other brain regions were positive for HSV-1 antigens, such as the locus coeruleus (LC), raphe nucleus (RaN), and hypothalamus, while sparing the hippocampus and cortex. Within each brain region, microglia activation also varied widely. These findings provide critical insights into the region-specific dissemination of HSV-1 within the CNS, elucidating potential mechanisms linking viral infection to neurological and neurodegenerative diseases. ImportanceThis study sheds light on how herpes simplex virus type 1 (HSV-1) spreads within the brain after infecting the nasal passages. Our data reveals the distinct pattern of HSV-1 through the brain during a non-encephalitic infection. Furthermore, microglial activation was also temporally and spatially specific, with some regions of the brain having sustained microglial activation even in the absence of viral antigen. Previous reports have identified specific regions of the brain found to be positive for HSV-1 infection; however, to date, there has not been a concise investigation of the anatomical spread of HSV-1 and the regions of the brain consistently vulnerable to viral entry and spread. Understanding these region-specific differences in infection and immune response is crucial because it links HSV-1 infection to potential triggers for neurological and neurodegenerative diseases.

neuroscience↗

bioRxiv2022

Signatures for Viral Infection and Inflammation in the Proximal Olfactory System in Familial Alzheimer's Disease

ObjectiveAlzheimers disease (AD) is characterized by loss of smell and olfactory system pathology that precedes the diagnosis of dementia. Understanding these early processes can potentially identify diagnostic and therapeutic targets to slow AD progression. Here we analyzed differential gene and protein expression in the olfactory bulb (OB) and olfactory tract (OT) of familial AD (FAD) individuals carrying the autosomal dominant presenilin 1 E280A paisa mutation and age-matched controls. MethodsFormalin-fixed, paraffin-embedded sections containing both the OB and OT from 6 FAD individuals and 6 age-matched controls were obtained. Tissue morphology and composition were characterized by immunohistochemistry using antibodies against the myelin marker proteolipid protein (PLP), amyloid-beta (A{beta}), and microglia/macrophage markers Iba1 and CD68, respectively. OB and OT were analyzed separately by targeted RNA sequencing of the whole human transcriptome (BioSpyder TempO-Seq); ingenuity pathway analysis and R-computational program were used to identify differentially expressed genes and pathways between groups. The nanoString spatial proteomics assay for 88 proteins, including markers for AD and immune responses, was used to complement gene expression findings. ResultsCompared to control OT, FAD OT had significantly increased immunostaining for A{beta} and CD68 in the high and low myelinated regions, as well as increased immunostaining for Iba1 in the high myelinated region only; both control and FAD OT samples had similar total area of high and low myelinated regions. In FAD samples, RNA sequencing showed a transcription profile consistent with: (1) viral infection in the OB; (2) inflammation in the OT that carries information via entorhinal cortex from the OB to hippocampus, a brain region essential for learning and memory; and (3) decreased oligodendrocyte deconvolved transcripts, indicating dysregulation of myelination. Interestingly, spatial proteomic analysis confirmed altered myelination in the OT of FAD individuals, implying dysfunction of communication between the OB and hippocampus. ConclusionsThese findings raise the possibility that viral infection and associated inflammation and dysregulation of myelination of the olfactory system may disrupt downstream hippocampal function, contributing to acceleration of FAD progression.

neuroscience↗

bioRxiv2020

Transcriptional profiling reveals TRPM5-expressing cells involved in viral infection in the olfactory epithelium

BackgroundUnderstanding viral infection of the olfactory epithelium is essential because the olfactory nerve is an important route of entry for viruses to the central nervous system. Specialized chemosensory epithelial cells that express the transient receptor potential cation channel subfamily M member 5 (TRPM5) are found throughout the airways and intestinal epithelium and are involved in responses to viral infection. ResultsHerein we performed deep transcriptional profiling of olfactory epithelial cells sorted by flow cytometry based on the expression of mCherry as a marker for olfactory sensory neurons and for eGFP in OMP-H2B::mCherry/TRPM5-eGFP transgenic mice (Mus musculus). We find profuse expression of transcripts involved in inflammation, immunity and viral infection in TRPM5-expressing microvillous cells. ConclusionOur study provides new insights into a potential role for TRPM5-expressing microvillous cells in viral infection of the olfactory epithelium. We find that, as found for solitary chemosensory cells (SCCs) and brush cells in the airway epithelium, and for tuft cells in the intestine, the transcriptome of TRPM5-expressing microvillous cells indicates that they are likely involved in the inflammatory response elicited by viral infection of the olfactory epithelium.

neuroscience↗