Biology subjects

Williams, J. T.

Publications and source records attributed to Williams, J. T..

10 recordsLinked to original sources

bioRxiv2025

The synaptic vesicle priming protein Munc13 mediates evoked somatodendritic dopamine release

Midbrain dopamine neurons secrete dopamine from their somata and dendrites in addition to their axonal release. Shared and distinct properties have been proposed for somatodendritic and axonal release, but the mechanisms of somatodendritic release remain unclear. We here used mouse genetics, electrophysiology, and imaging to define roles of the synaptic vesicle priming protein Munc13 in somatodendritic release in comparison to axonal secretion. Munc13 ablation in dopamine neurons decreased evoked but not spontaneous somatodendritic dopamine transmission measured as D2 receptor-mediated currents. Imaging with a fluorescent dopamine sensor confirmed this finding and established comparable importance for Munc13 in somatodendritic and axonal secretion. Pharmacological experiments revealed that release from adrenergic terminals contributes to D2 receptor-mediated currents, and their relative contribution was enhanced after Munc13 knockout. Altogether, these data establish important roles for Munc13 in evoked somatodendritic release. These roles are similar to Munc13 functions in axonal dopamine release and at fast synapses. Spontaneous midbrain dopamine release does not necessitate Munc13 in dopamine neurons and may rely on a release pathway that is independent of the prototypical release machinery employed at synapses.

neuroscience↗

bioRxiv2024

Reductions of Grin2a in adolescent dopamine neurons confers aberrant salience and related psychosis phenotype

Psychosis is a hallmark of schizophrenia. It involves a collection of symptoms that are typically associated with disrupted dopamine signaling and emerges during adolescence or early adulthood. Most schizophrenia-associated genes, however, involve glutamatergic or other ubiquitous targets that do not explain the latent expression of psychosis or dopaminergic abnormalities. Here, we describe an etiologically relevant model for the adolescent onset of dopamine-related dysfunction in schizophrenia. We focused on GRIN2A, the gene encoding the GluN2A subunit of the NMDA receptor, as both the common loss-of-function variants and the rare missense variants in this gene are risk factors for schizophrenia. We find that GluN2A levels distinctly decline in dopamine neuron-containing regions throughout adolescence while remaining stable in other regions. This suggested that adolescent dopamine neurons may be particularly vulnerable to further reductions in GluN2A caused by a damaging variant of GRIN2A. Consistent with this idea, we find that selective knockout of Grin2a in adolescent rat dopamine neurons results in a psychosis-relevant behavioral phenotype. This manipulation also reduced dopamine release in response to unexpected outcomes in young adults, reflective of prediction error signaling abnormalities observed in the clinical population. These data provide mechanistic insight into how GRIN2A mutations may contribute to the delayed onset of dopamine-related symptoms and provide a model for identifying course altering treatments for schizophrenia.

neuroscience↗

bioRxiv2023

Improved dual-color GRAB sensors for monitoring dopaminergic activity in vivo

Dopamine (DA) plays multiple roles in a wide range of physiological and pathological processes via a vast network of dopaminergic projections. To fully dissect the spatiotemporal dynamics of DA release in both dense and sparsely innervated brain regions, we developed a series of green and red fluorescent GPCR activation-based DA (GRABDA) sensors using a variety of DA receptor subtypes. These sensors have high sensitivity, selectivity, and signal-to-noise properties with subsecond response kinetics and the ability to detect a wide range of DA concentrations. We then used these sensors in freely moving mice to measure both optogenetically evoked and behaviorally relevant DA release while measuring neurochemical signaling in the nucleus accumbens, amygdala, and cortex. Using these sensors, we also detected spatially resolved heterogeneous cortical DA release in mice performing various behaviors. These next-generation GRABDA sensors provide a robust set of tools for imaging dopaminergic activity under a variety of physiological and pathological conditions.

neuroscience↗

bioRxiv2023

A Novel, Uniquely Efficacious Type of CFTR Corrector with Complementary Mode of Action

Three distinct pharmacological corrector types (I, II, III) with different binding sites and additive behaviour only partially rescue the F508del-CFTR folding and trafficking defect observed in cystic fibrosis. Here, we describe novel, uniquely effective, macrocyclic CFTR correctors that were additive to the known corrector types, thus exerting a new, complementary "type-IV" corrector mechanism. Macrocycles achieved wildtype-like folding efficiency of F508del-CFTR at the endoplasmic reticulum and normalized CFTR currents in reconstituted patient-derived bronchial epithelium. Using photo-activatable macrocycles, docking studies and site-directed mutagenesis a highly probable binding site and pose for type-IV correctors was identified in a cavity between lasso helix-1 (Lh1) and transmembrane helix-1 of membrane spanning domain-1 (MSD1), distinct from the known corrector binding sites. Since only F508del-CFTR fragments spanning from Lh1 until MSD2 responded to type-IV correctors, these likely promote co-translational assembly of Lh1, MSD1, and MSD2. Remarkably, previously corrector-resistant CFTR folding mutations were also robustly rescued, suggesting substantial therapeutic potential for this novel type-IV corrector mechanism. TeaserA novel type of macrocyclic CFTR corrector with new binding site, complementary mode of action and unique folding / trafficking efficacy is described.

pharmacology and toxicology↗

bioRxiv2022

Gait Abnormalities and Aberrant D2 Receptor Expression and Signaling in a Mouse Model of the Human Pathogenic Mutation DRD2I212F

A dopamine D2 receptor mutation was recently identified in a family with a novel hyperkinetic movement disorder (Mov Disord 36: 729-739, 2021). That allelic variant D2-I212F is a constitutively active and G protein-biased receptor. We now describe mice engineered to carry the D2-I212F variant, Drd2I212F. The mice exhibited gait abnormalities resembling those in other mouse models of chorea and/or dystonia, and had decreased striatal D2 receptor expression. Electrically evoked IPSCs in midbrain dopamine neurons and striatum from Drd2I212F mice exhibited slow onset and decay compared to wild type mice. In the presence of dopamine, current decay initiated by photolytic release of sulpiride from CyHQ-sulpiride was slower in midbrain slices from Drd2I212F mice than Drd2+/+ mice. Furthermore, in contrast to wild type mice in which dopamine is more potent at neurons in the nucleus accumbens than in the dorsal striatum, reflecting activation of Go vs. Gi1, dopamine had similar potencies in those two brain regions of Drd2I212F mice. Repeated cocaine treatment, which decreases dopamine potency in the nucleus accumbens of wild type mice, had no effect on dopamine potency in Drd2I212F mice. The results demonstrate the utility of this mouse model for investigating the role of pathogenic DRD2 variants in early-onset hyperkinetic movement disorders.

neuroscience↗

bioRxiv2021

Synaptotagmin-7 enhances phasic dopamine release

Dopamine released from substantia nigra pars compacta (SNc) neurons modulates movement, motivation, and reward. In addition to their tonic firing pattern, dopamine neurons also fire high-frequency bursts that cause superlinear increases in dopamine release. To study this poorly understood form of short-term plasticity, we used the fluorescent dopamine sensor dLight1.3b to examine the role of the calcium-binding protein synaptotagmin-7 (SYT7). We report that SYT7 mediates a hidden component of facilitation, which was unmasked by lowering initial release probability, or by low-frequency stimulation of nerve terminals. In Syt7 KO neurons, there was profound synaptic depression that significantly reduced release during stimulations that mimic in vivo firing patterns of SNc neurons. D2-mediated inhibitory postsynaptic currents in the SNc revealed a similar role for SYT7 in somatodendritic release. Our results indicate that SYT7 drives short-term facilitation of release from dopamine neurons, which likely underlies frequency-dependence of dopamine signaling in vivo.

neuroscience↗

bioRxiv2020

Artemisia annua and Artemisia afra extracts exhibit strong bactericidal activity against Mycobacterium tuberculosis

Ethnopharmacological relevanceEmergence of drug-resistant and multidrug-resistant Mycobacterium tuberculosis (Mtb) strains is a major barrier to tuberculosis (TB) eradication, as it leads to longer treatment regimens and in many cases treatment failure. Thus, there is an urgent need to explore new TB drugs and combinations, in order to shorten TB treatment and improve outcomes. Here, we evaluate the potential of two medicinal plants, Artemisia annua, a natural source of artemisinin (AN), and Artemisia afra, as sources of novel antitubercular agents. Aim of the studyOur goal was to measure the activity of A. annua and A. afra extracts against Mtb as potential natural and inexpensive therapies for TB treatment, or as sources of compounds that could be further developed into effective treatments. Materials and MethodsThe minimum inhibitory concentrations (MICs) of A. annua and A. afra dichloromethane extracts were determined, and concentrations above the MICs were used to evaluate their ability to kill Mtb and Mycobacterium abscessus in vitro. ResultsPrevious studies showed that A. annua and A. afra inhibit Mtb growth. Here, we show for the first time that Artemisia extracts have a strong bactericidal activity against Mtb. The killing effect of A. annua was much stronger than equivalent concentrations of pure AN, suggesting that A. annua extracts kill Mtb through a combination of AN and additional compounds. A. afra, which produces very little AN, displayed bactericidal activity against Mtb that was substantial but weaker than that of A. annua. In addition, we measured the activity of Artemisia extracts against Mycobacterium abscessus. Interestingly, we observed that while A. annua is not bactericidal, it inhibits growth of M. abscessus, highlighting the potential of this plant in combinatory therapies to treat M. abscessus infections. ConclusionOur results indicate that Artemisia extracts have an enormous potential for treatment of TB and M. abscessus infections, and that these plants contain bactericidal compounds in addition to AN. Combination of extracts with existing antibiotics may not only improve treatment outcomes but also reduce the emergence of resistance to other drugs.

microbiology↗

bioRxiv2019

A DNA barcode reference library of the French Polynesian shore fishes

The emergence of DNA barcoding and metabarcoding opened new ways to study biological diversity, however, the completion of DNA barcode libraries is fundamental for such approaches to succeed. This dataset is a DNA barcode reference library (fragment of Cytochrome Oxydase I gene) for 2,190 specimens representing at least 540 species of shore fishes collected over 10 years at 154 sites across the four volcanic archipelagos of French Polynesia; the Austral, Gambier, Marquesas and Society Islands, a 5,000,000 km2 area. At present, 65% of the known shore fish species of these archipelagoes possess a DNA barcode associated with preserved, photographed, tissue sampled and cataloged specimens, and extensive collection locality data. This dataset represents one of the most comprehensive DNA barcoding efforts for a vertebrate fauna to date. Considering the challenges associated with the conservation of coral reef fishes and the difficulties of accurately identifying species using morphological characters, this publicly available library is expected to be helpful for both authorities and academics in various fields.

zoology↗

bioRxiv2019

Identification of new MmpL3 inhibitors by untargeted and targeted mutant screens defines MmpL3 domains with differential resistance

The Mycobacterium tuberculosis (Mtb) mycolic acid flippase MmpL3 has been the proposed target for multiple inhibitors with diverse chemical scaffolds. This diversity in chemical scaffolds has made it difficult to predict compounds that inhibit MmpL3 without whole genome sequencing of isolated resistant mutants. Here we describe the identification of four new inhibitors that select for resistance mutations in mmpL3. Using these resistant mutants, we conducted a targeted whole-cell phenotypic screen of 163 novel Mtb growth inhibitors for differential growth inhibition of wild type Mtb as compared to a pool of twenty-four unique mmpL3 mutants. The screen successfully identified six additional putative MmpL3 inhibitors. The compounds were bactericidal both in vitro and against intracellular Mtb. Mtb cells treated with these compounds were shown to accumulate trehalose monomycolate and have reduced levels of trehalose dimycolate, supporting MmpL3 as the target. The inhibitors were mycobacteria specific with several also showing activity against the non-tuberculosis mycobacterial species M. abscessus. Cluster analysis of cross resistance profiles generated by dose response experiments for each combination of 13 MmpL3 inhibitors against each of the 24 mmpL3 mutants defined two clades of inhibitors and two clades of mmpL3 mutants. Pairwise combination studies of the inhibitors revealed interactions that were specific to the clades identified in the cross-resistance profiling. Additionally, modeling of resistance substitutions to the MmpL3 crystal structure revealed clade specific localization of the residues to specific domains of MmpL3, with the clades showing differential resistance. Several compounds exhibited high solubility and stability in microsomes and low cytotoxicity in macrophages, supporting their further development. The combined study of multiple mutants and novel compounds provides new insights into structure-function interactions of MmpL3 and small molecule inhibitors.

microbiology↗

bioRxiv2018

Cellular tolerance at the μ-opioid receptor is phosphorylation dependent

The role of phosphorylation of the -opioid receptor (MOR) in desensitization, internalization and long-term tolerance was examined in locus coeruleus (LC) neurons. Viral expression of wild type (exWT) and mutant MORs, where all phosphorylation sites on the C-terminus (Total Phosphorylation Deficient (TPD)) were mutated to alanine, were examined in a MOR knockout rat. Both expressed receptors acutely activate potassium conductance similar to endogenous receptors in wild type animals. The exWT receptors, like endogenous receptors, displayed signs of tolerance after chronic morphine treatment. There was however a loss of agonist-induced desensitization and internalization in experiments with the TPD receptors. In addition the development of tolerance was not observed in the TPD receptors following chronic morphine treatment. The results indicate a key role of C-terminal phosphorylation in the expression of acute desensitization, trafficking and long-term tolerance to morphine.

neuroscience↗