Biology subjects

Sharrock, A. V.

Publications and source records attributed to Sharrock, A. V..

4 recordsLinked to original sources

bioRxiv2025

Targeted ablation and regeneration of enteric nervous system neurons in zebrafish

The enteric nervous system (ENS) is the intrinsic nervous system of the gut and regulates essential gut functions, including motility, digestion, and immune response, ensuring gut homeostasis. ENS dysfunction or loss is associated with gastrointestinal disorders such as Hirschsprung disease (HSCR). Currently, surgery is the only treatment for HSCR, but it often has lifelong, severe complications. Restoring missing ENS neurons by stimulating endogenous neuronal regeneration presents a promising therapeutic approach for ENS disease. To reveal the cellular-molecular mechanisms regulating neuronal regeneration we study a species capable of robust ENS restoration, the zebrafish. For this, we developed a chemogenetic ablation model in zebrafish using the Gal4/UAS NTR 2.0 system for targeted ENS neuron ablation. Spatially and temporally controlled neuronal death was confirmed by morphological changes, quantification of neuronal loss, and TUNEL assays. We observed an acute immune response that normalizes at 1 day of treatment. Quantification of regenerated neurons demonstrated complete restoration of ENS neuron numbers to control levels by 9 days post treatment, with recovery of gut motility. Among the regenerated neurons, nitrergic, cholinergic and VIPergic subtypes showed full recovery, whereas serotonergic neurons only displayed partial recovery, indicating subtype-specific differences in regenerative capacity and/or timing of cell replacement. Our study establishes a robust platform for dissecting the cellular-molecular mechanisms of ENS regeneration to develop potential treatment approaches for ENS-related diseases.

neuroscience↗

bioRxiv2023

Molecular regulation of retinal regeneration is context specific

Many genes are known to regulate retinal regeneration following widespread tissue damage. Conversely, genes controlling regeneration following limited retinal cell loss, akin to disease conditions, are undefined. Combining a novel retinal ganglion cell (RGC) ablation-based glaucoma model, single cell omics, and rapid CRISPR/Cas9-based knockout methods to screen 100 genes, we identified 18 effectors of RGC regeneration kinetics. Surprisingly, 32 of 33 previously known/implicated regulators of retinal tissue regeneration were not required for RGC replacement; 7 knockouts accelerated regeneration, including sox2, olig2, and ascl1a. Mechanistic analyses revealed loss of ascl1a increased "fate bias", the propensity of progenitors to produce RGCs. These data demonstrate plasticity and context-specificity in how genes function to control regeneration, insights that could help to advance disease-tailored therapeutics for replacing lost retinal cells. One sentence summaryWe discovered eighteen genes that regulate the regeneration of retinal ganglion cells in zebrafish.

neuroscience↗

bioRxiv2023

A metagenomic library cloning strategy that promotes high-level expression of captured genes to enable efficient functional screening

Functional screening of environmental DNA (eDNA) libraries is a potentially powerful approach to discover enzymatic "unknown unknowns", but is usually heavily biased toward the tiny subset of genes preferentially transcribed and translated by the screening strain. We have overcome this by preparing an eDNA library via partial digest with restriction enzyme Fatl (cuts CATG), causing a substantial proportion of ATG start codons to be precisely aligned with strong plasmid-encoded promoter and ribosome-binding sequences. Whereas we were unable to select nitroreductases from standard metagenome libraries, our Fatl strategy yielded 21 nitroreductases spanning eight different enzyme families, each conferring resistance to the nitro-antibiotic niclosamide and sensitivity to the nitro-prodrug metronidazole. We showed expression could be improved by co-expressing rare tRNAs and encoded proteins purified directly using an embedded Hisg-tag. In a transgenic zebrafish model of metronidazole-mediated targeted cell ablation, our lead MhqN-family nitroreductase proved [~]5- fold more effective than the canonical nitroreductase NfsB.

biochemistry↗

bioRxiv2020

Large-scale Phenotypic Drug Screen Identifies Neuroprotectants in Zebrafish and Mouse Models of Retinitis Pigmentosa

Retinitis pigmentosa (RP) and associated inherited retinal diseases (IRDs) are caused by rod photoreceptor degeneration, necessitating therapeutics promoting rod photoreceptor survival. To address this, we tested compounds for neuroprotective effects in zebrafish and mouse RP models, reasoning drugs effective across species may translate better clinically. We first performed a large-scale phenotypic drug screen using a larval zebrafish model of inducible RP. 2,934 compounds, mostly human-approved drugs, were tested across six concentrations. Statistically, 113 compounds achieved "hit" status. Secondary tests of 42 high-priority hits confirmed eleven lead compounds. Nine leads were then evaluated in mouse RP models, with six exhibiting neuroprotective effects. An analysis of potential mechanisms of action suggested complementary activities. Paired lead compound assays in zebrafish showed additive neuroprotective effects for the majority. These results highlight the value of cross-species phenotypic drug discovery and suggest combinatorial drug therapies may provide enhanced therapeutic benefits for patients with RP and IRDs.

neuroscience↗