Biology subjects

Chaplot, K.

Publications and source records attributed to Chaplot, K..

5 recordsLinked to original sources

bioRxiv2026

Neuro-glial lipid imbalance in a Drosophila model of Amyotrophic Lateral Sclerosis 8

Membrane Contact sites (MCS) have emerged as physiologically relevant zones that coordinate inter-organelle communication and cellular function. VAPB, an ER-resident MCS tethering protein, plays a central role in regulating MCSs through its numerous protein interactors, thereby influencing cellular homeostasis. A pathogenic missense VAPBP56S mutation causes familial Amyotrophic Lateral Sclerosis 8 (ALS8) in humans, with progressive degeneration of motor neurons. The precise mechanisms underlying the motor neurodegeneration remain poorly understood. In this study, we examine lipid imbalance in the brain of a Drosophila model of ALS8 (VAPBP58S). Specifically, we find that lipid homeostasis is disrupted in an age-dependent manner. Strikingly, cholesterol esters and sphingolipids show an age-dependent increase, while cholesterol shows a decrease. Intriguingly, from a cellular perspective, despite the accumulation of triacylglycerols (TAGs) in the brains of VAPBP58S animals, the increased neutral lipid species do not correlate with lipid droplets (LDs), which are fewer in density and smaller in size. Lipid imbalance and progressive motor dysfunction in VAPBP58S animals can be reversed by expressing VAPBWT, suggesting a relationship between VAPB activity and lipid flux. To uncover VAPBs role in lipid homeostasis, we modulate VAPB activity in neurons and glia to dissect out tissue-specific roles. We find that both cell types contribute to lipid homeostasis in differential ways. In glia, LD flux is strongly dependent on VAPB activity, a dependence further recapitulated in cultured human cell lines, suggesting evolutionary conservation of the regulatory mechanism. Thus, we hypothesise that lipid dysregulation constitutes a critical pathogenic feature of ALS8, with the VAPBP56S allele disrupting lipid homeostasis in the neuro-glial axis. Summary StatementThe ER-membrane tethering protein VAPB regulates lipid homeostasis

neuroscience↗

bioRxiv2025

Rapid generation of ventral A9-like dopaminergic neurons from patterned iPSCs

In vitro modelling of highly vulnerable nigral dopaminergic (DA) neuronal subtypes in Parkinsons disease (PD), is necessary for studying disease mechanisms. Here, we optimized a new approach by expressing the pioneer neurogenic transcription factor, Achaete-scute-like 1 (Ascl1), implicated in determining dopaminergic fate. Sequential small-molecule patterning of iPSCs into early floor plate mesencephalic progenitors, followed by inducible Ascl1 expression, rapidly differentiates midbrain DA neurons. Immunocytochemistry and transcriptomic analysis of these patterned Ascl1-driven DA neurons (PA-DANs) confirmed midbrain-lineage specificity. Importantly, we found an enrichment of DA subpopulations that corresponded to the adult human ventral SOX6-positive A9 DA subtypes vulnerable in PD. Furthermore, we combined these ventral A9-like PA-DANs with human iPSC-derived midbrain astrocytes and microglia in defined ratios to generate mature 3D A9-like assembled organoids that display characteristic spontaneous neuronal activity and electrical propagation along the axon. Our method efficiently generates a mature and functional A9-like DA neuronal platform to study PD. HighlightsO_LISequential midbrain patterning and Ascl1 expression accelerates DA differentiation C_LIO_LIPA-DANs resemble human adult ventral A9-like DA subtypes vulnerable in PD C_LIO_LI3D assembled organoids show mature identity of PA-DANs, iAstrocytes and iMicroglia C_LIO_LIPA-DANs matured in 3D organoids show neuronal network activity within weeks C_LI eTOC blurbIn this study, Ullian and colleagues have developed a rapid method to differentiate dopaminergic neurons, using small molecules to generate early floor plate mesencephalic progenitors from human iPSCs and sequentially expressing a pioneer transcription factor, Ascl1, that accelerates uniform dopaminergic neurogenesis. Patterned Ascl1-driven dopaminergic neurons (PA-DANs) in 2D and 3D assembled organoids serve as a platform to study Parkinsons disease O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/685897v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1d83eb3org.highwire.dtl.DTLVardef@1fc7fa5org.highwire.dtl.DTLVardef@2033e3org.highwire.dtl.DTLVardef@2e8fa3_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2020

A protease protection assay for the detection of internalized alpha-synuclein pre-formed fibrils

Alpha-synuclein pre-formed fibrils (PFFs) represent a promising model system for the study of cellular processes underlying cell-to-cell transmission of alpha-synuclein proteopathic aggregates. However, the ability to differentiate the fate of internalized PFFs from those which remain in the extracellular environment remains limited due to the propensity for PFFs to adhere to the cell surface. Removal of PFFs requires repeated washing and/or specific quenching of extracellular fluorescent PFF signals. In this paper we present a new method for analyzing the fate of internalized alpha-synuclein. We inserted a tobacco etch virus (TEV) protease cleavage site between alpha-synuclein and green fluorescent protein and subjected cells to brief treatment with TEV protease after incubation with tagged PFFs. As the TEV protease is highly specific, non-toxic, and active under physiological conditions, protection from TEV cleavage can be used to distinguish internalized PFFs from those which remain attached to the cell surface. Using this experimental paradigm, downstream intracellular events can be analyzed via live or fixed cell microscopy as well as by Western blotting. We suggest that this method will be useful for understanding the fate of PFFs after endocytosis under various experimental manipulations.

neuroscience↗

bioRxiv2020

Phase separation of proSAAS into spheres results in core sequestration of TDP-43216-414 aggregates

As neurons age, protein homeostasis becomes less efficient, resulting in misfolding and aggregation. Chaperone proteins perform vital functions in the maintenance of cellular proteostasis, and chaperone-based therapies that promote sequestration of toxic aggregates may prove useful in blocking the development of neurodegenerative disease. We previously demonstrated that proSAAS, a small secreted neuronal protein, exhibits potent chaperone activity against protein aggregation in vitro, and blocks the cytotoxic effects of amyloid and alpha synuclein oligomers in cell culture models. We now examine whether cytoplasmic expression of proSAAS results in interaction with protein aggregates in this cellular compartment. We used site-directed mutagenesis, confocal microscopy, in vitro aggregation assays, and functional assays to investigate the interaction of proSAAS with TDP-43 and other known aggregating proteins. We report that expression of proSAAS within the cytoplasm generates dense, membrane-less 2 m proSAAS spheres which progressively fuse to form larger spheres, suggesting liquid droplet-like properties. ProSAAS spheres selectively accumulate a C-terminally truncated fluorescently-tagged form of TDP-43216-414, initiating its cellular redistribution by sequestration within the sphere core; these TDP-43216-414 -containing spheres also exhibit dynamic fusion. Removal of either the predicted -helix (37-70) composed of hydrophobic and charged amino acids or the stretch of amino acids encompassing the conserved hydrophobic region and the positively charged furin site (163-189) inhibits the ability of proSAAS both to form spheres and to encapsulate TDP-43 aggregates. As a functional output, we demonstrate that proSAAS expression results in cytoprotection against full-length TDP-43 toxicity in yeast. In summary, the normally secreted neuronal chaperone proSAAS, when expressed in the cytoplasm unexpectedly phase-separates to form spherical liquid-like condensates that undergo dynamic fusion. We conclude that cyto-proSAAS acts as a functional holdase for cytoplasmic TDP-43216-414 molecules via this phase-separation property, representing a cytoprotectant whose unusual biochemical properties can potentially be exploited in the design of therapeutic molecules.

neuroscience↗

bioRxiv2018

SOD1 activity thresholds and TOR signalling modulate VAP(P58S) aggregation via ROS-induced proteasomal degradation in a Drosophila model of Amyotrophic Lateral Sclerosis

Familial Amyotrophic Lateral Sclerosis (F-ALS) is an incurable, late onset motor neuron disease, linked strongly to various causative genetic loci. ALS8 codes for a missense mutation, P56S, in VAMP-associated Protein B (VAPB) that causes the protein to misfold and form cellular aggregates. Uncovering genes and mechanisms that affect aggregation dynamics would greatly help increase our understanding of the disease and lead to potential therapeutics.\n\nHere, we develop a quantitative high-throughput, Drosophila S2R+ cell-based kinetic assay coupled with fluorescent microscopy to score for genes involved in the modulation of aggregates of fly ortholog, VAP(P58S), tagged with GFP. As proof of principle, we conducted a targeted RNAi screen against 900 genes, consisting of VAP genetic interactors, other ALS loci, as also genes involved in proteostasis. The screen identified 150 hits that modify aggregation, including the ALS loci SOD1, TDP43 and also genes belonging to the TOR pathway.\n\nTo validate these modifiers, we developed a system to measure the extent of VAP(P58S) aggregation in the Drosophila third instar larval brain using the UAS-GAL4 system, followed by quantitative imaging of cellular inclusions. We find that reduction of SOD1 activity or decreased TOR signalling reduces aggregation. Interestingly, we find that increase in cellular reactive oxygen species (ROS) levels, assessed by measuring oxidation of cellular lipids and proteins, in response to SOD1 knockdown or by inhibition of TOR signalling appears to be the trigger for clearing of aggregates. The mechanism of aggregate clearance is, primarily, the proteasomal machinery, and not autophagy. Increase in VAP, but not VAP(P58S) levels, appears to elevate ROS, which may in turn regulate VAP transcription in a feedback loop.\n\nWe have thus uncovered an interesting interplay between SOD1, ROS and TOR signalling that regulates the dynamics of VAP aggregation. Mechanistic processes underlying such cellular regulatory networks will lead us to a better understanding of initiation and progression of ALS.

neuroscience↗