New insights into the early mechanisms of epileptogenesis in a zebrafish model of Dravet syndrome.

OBJECTIVE: To pinpoint the earliest cellular defects underlying seizure onset (epileptogenic period) during perinatal brain development in a new zebrafish model of Dravet syndrome (DS) and to investigate potential disease-modifying activity of the 5HT2 receptor agonist fenfluramine. METHODS: We used CRISPR/Cas9 mutagenesis to introduce a missense mutation, designed to perturb ion transport function in all channel isoforms, into scn1lab, the zebrafish orthologue of SCN1A (encoding voltage-gated sodium channel alpha subunit 1). We performed behavioral analysis and electroencephalographic recordings to measure convulsions and epileptiform discharges, followed by single-cell RNA-Seq, morphometric analysis of transgenic reporter-labeled γ-aminobutyric acidergic (GABAergic) neurons, and pharmacological profiling of mutant larvae. RESULTS: Homozygous mutant (scn1labmut/mut ) larvae displayed spontaneous seizures with interictal, preictal, and ictal discharges (mean = 7.5 per 20-minute recording; P < .0001; one-way analysis of variance). Drop-Seq analysis revealed a 2:1 shift in the ratio of glutamatergic to GABAergic neurons in scn1labmut/mut larval brains versus wild type (WT), with dynamic changes in neuronal, glial, and progenitor cell populations. To explore disease pathophysiology further, we quantified dendritic arborization in GABAergic neurons and observed a 40% reduction in arbor number compared to WT (P < .001; n = 15 mutant, n = 16 WT). We postulate that the significant reduction in inhibitory arbors causes an inhibitory to excitatory neurotransmitter imbalance that contributes to seizures and enhanced electrical brain activity in scn1labmut/mut larvae (high-frequency range), with subsequent GABAergic neuronal loss and astrogliosis. Chronic fenfluramine administration completely restored dendritic arbor numbers to normal in scn1labmut/mut larvae, whereas similar treatment with the benzodiazepine diazepam attenuated seizures, but was ineffective in restoring neuronal cytoarchitecture. BrdU labeling revealed cell overproliferation in scn1labmut/mut larval brains that were rescued by fenfluramine but not diazepam. SIGNIFICANCE: Our findings provide novel insights into early mechanisms of DS pathogenesis, describe dynamic cell population changes in the scn1labmut/mut brain, and present first-time evidence for potential disease modification by fenfluramine.

Single-cell transcriptomics reveals distinct inflammation-induced microglia signatures.

Microglia are specialized parenchymal-resident phagocytes of the central nervous system (CNS) that actively support, defend and modulate the neural environment. Dysfunctional microglial responses are thought to worsen CNS diseases; nevertheless, their impact during neuroinflammatory processes remains largely obscure. Here, using a combination of single-cell RNA sequencing and multicolour flow cytometry, we comprehensively profile microglia in the brain of lipopolysaccharide (LPS)-injected mice. By excluding the contribution of other immune CNS-resident and peripheral cells, we show that microglia isolated from LPS-injected mice display a global downregulation of their homeostatic signature together with an upregulation of inflammatory genes. Notably, we identify distinct microglial activated profiles under inflammatory conditions, which greatly differ from neurodegenerative disease-associated profiles. These results provide insights into microglial heterogeneity and establish a resource for the identification of specific phenotypes in CNS disorders, such as neuroinflammatory and neurodegenerative diseases.

Application of an Enhanced Version of Recursive Operability Analysis for Combustible Dusts Risk Assessment.

Organic dust explosions were and are still today a critical issue in the food, pharmaceutical, and fine chemical industry. Materials such as flour, corn starch, sugar and APIs represent a cause of severe accidents. In this framework, we investigated a modified version of Recursive Operability Analysis-Incidental Sequence Diagrams (ROA-ISD), called ROA Plus-ISD, specifically tailored to describe industrial processes involving organic combustible dusts. Compared to more classical techniques such as Hazard and Operability (HazOp), ROA-ISD allows for a direct generation of fault trees, providing a useful tool to connect Qualitative with Quantitative Risk Analysis (QRA). ROA Plus-ISD is very similar to ROA-Cause Consequence Diagrams (CCD), which has already proven to be an effective tool to perform both risk assessment on existing plants and reconstructing already occurred accidents, given its logical structure and width of the application fields. In this work, we modified specific parts of the standard ROA-CCD method: (1) the Failure Mode and Operability Analysis (FMEA) database has been structured in order to retrieve the well-known explosion pentagon (for dusts) and all the instruments, devices, apparatuses and controllers typical of industries which process organic dusts; (2) a new comprehensive list of process variables has been compiled. In this way, it is possible to tailor the information required for the generation of the fault trees concerning top events involving mainly dust explosions and fires. This method has been implemented in order to reconstruct the dynamics of the February 2008 Imperial Sugar refinery plant accident (Port Wentworth, GA, USA). Results demonstrated the applicability of the enhanced method by highlighting the criticalities of the process already showed by a previously detailed reconstruction performed by the Chemical Safety Board.