Biology subjects

Shields, R.

Publications and source records attributed to Shields, R..

4 recordsLinked to original sources

bioRxiv2025

Inhibitory GABAergic neuron loss due to oxidative damage during ex vivo acute brain slice preparation influences genesis and dynamics of epileptiform activity

Ex vivo acute brain slice is a popular technique in neuroscience research. Since its inception five decades ago, many variations of the brain slice preparation method have emerged. While all variations are currently used by many labs throughout the world, no study has comprehensively examined the impact of these variation on the quality of the acute brain slice preparation. In this study, we comprehensively examined the effect of animal sacrifice methods (decapitation or transcardial perfusion) and cutting solution (normal or sucrose artificial cerebrospinal fluid) on brain slice preparation. Neuronal population was quantified by immunohistochemistry against various neuronal markers. Neuronal dynamics was evaluated by in vitro electrophysiology using two acute epilepsy models - zero-magnesium and 4-aminopyridine. To modulate the stress incurred in acute brain slice preparation, we administered antioxidants or HIF-1 inhibitor, chrysin, to the cutting solution. The method of brain slice preparation significantly affected the quality of the brain slice preparation. In general, the combination of transcardial perfusion and sucrose artificial cerebrospinal fluid produces the optimal brain slice preparation. This is evidenced by a preservation of inhibitory GABAergic neurons in slices prepared with this combination. We subsequently found that this loss of inhibitory GABAergic neurons significantly influenced the genesis and dynamics of induced acute epileptiform activity. The slices with preserved inhibition had less successful induction of acute epileptiform activity and a seizure-like event that is typical of those induced in brain with preserved inhibition. Finally, we found that loss of inhibitory GABAergic neurons during brain slice preparation is primarily due to oxidative damage. Limiting oxidative stress is an effective neuroprotection strategy to prevent loss of inhibition in brain slice preparation. In conclusion, consideration of brain slice preparation method is crucial in preservinginhibitory GABAergic neurons and the degree of inhibition in ex vivo acute brain slice preparation.

neuroscience↗

bioRxiv2023

Intranasal CRMP2-Ubc9 Inhibitor Regulates NaV1.7 to Alleviate Trigeminal Neuropathic Pain

Dysregulation of voltage-gated sodium NaV1.7 channels in sensory neurons contributes to chronic pain conditions, including trigeminal neuropathic pain. We previously reported that chronic pain results in part from increased SUMOylation of collapsin response mediator protein 2 (CRMP2), leading to an increased CRMP2/NaV1.7 interaction and increased functional activity of NaV1.7. Targeting this feed-forward regulation, we developed compound 194, which inhibits CRMP2 SUMOylation mediated by the SUMO-conjugating enzyme Ubc9. We further demonstrated that 194 effectively reduces the functional activity of NaV1.7 channels in dorsal root ganglia neurons and alleviated inflammatory and neuropathic pain. Here, we employed a comprehensive array of investigative approaches, encompassing biochemical, pharmacological, genetic, electrophysiological, and behavioral analyses, to assess the functional implications of NaV1.7 regulation by CRMP2 in trigeminal ganglia (TG) neurons. We confirmed the expression of Scn9a, Dpysl2, and UBE2I within TG neurons. Furthermore, we found an interaction between CRMP2 and NaV1.7, with CRMP2 being SUMOylated in these sensory ganglia. Disrupting CRMP2 SUMOylation with compound 194 uncoupled the CRMP2/NaV1.7 interaction, impeded NaV1.7 diffusion on the plasma membrane, and subsequently diminished NaV1.7 activity. Compound 194 also led to a reduction in TG neuron excitability. Finally, when intranasally administered to rats with chronic constriction injury of the infraorbital nerve (CCI-ION), 194 significantly decreased nociceptive behaviors. Collectively, our findings underscore the critical role of CRMP2 in regulating NaV1.7 within TG neurons, emphasizing the importance of this indirect modulation in trigeminal neuropathic pain.

neuroscience↗

bioRxiv2022

Activation of TnSmu1, an integrative and conjugative element, is lethal to Streptococcus mutans

Integrative and conjugative elements (ICEs) are chromosomally encoded mobile genetic elements that can transfer DNA between bacterial strains. Recently, as part of efforts to determine hypothetical gene functions, we have discovered an important regulatory module encoded on an ICE known as TnSmu1 on the Streptococcus mutans chromosome. The regulatory module consists of a cI-like repressor with a helix-turn-helix DNA binding domain immRSmu (immunity repressor) and a metalloprotease immASmu (anti-repressor). It is not possible to create an in-frame deletion mutant of immRSmu and repression of immRSmu with CRISPRi causes substantial cell defects. We used a bypass of essentiality (BoE) screen to discover genes that allow deletion of the regulatory module. This revealed that conjugation genes, located within TnSmu1, can restore the viability of an immRSmu mutant. Deletion of immRSmu also leads to production of a circular intermediate form of TnSmu1, that is also inducible by the genotoxic agent mitomycin C. To gain further insights into potential regulation of TnSmu1 by ImmRSmu and broader effects on S. mutans UA159 physiology we used CRISPRi and RNA-seq. Strongly induced genes included all the TnSmu1 mobile element, genes involved in amino acid metabolism, transport systems, and a Type I-C CRISPR-Cas system. Lastly, bioinformatic analysis shows that the TnSmu1 mobile element and its associated genes are well distributed across S. mutans isolates. Taken together, our results show that activation of TnSmu1 is controlled by the immRASmu module, and that activation is deleterious to S. mutans, highlighting the complex interplay between mobile elements and their host.

microbiology↗

bioRxiv2018

Competence Inhibition by the XrpA Peptide Encoded Within the comX Gene of Streptococcus mutans

Streptococcus mutans displays complex regulation of natural genetic competence. Competence development in S. mutans is controlled by a peptide derived from ComS (XIP); which along with the cytosolic regulator ComR controls the expression of the alternative sigma factor comX, the master regulator of competence development. Recently, a gene embedded within the coding region of comX was discovered and designated xrpA (comX regulatory peptide A). XrpA was found to be an antagonist of ComX, but the mechanism was not established. In this study, we reveal through both genomic and proteomic techniques that XrpA is the first describe negative regulator of ComRS systems in streptococci. Transcriptomic and promoter activity assays in the {Delta}xrpA strain revealed an up-regulation of genes controlled by both the ComR- and ComX-regulons. An in vivo protein crosslinking and in vitro fluorescent polarization assays confirmed that the N-terminal region of XrpA were found to be sufficient in inhibiting ComR-XIP complex binding to ECom-box located within the comX promoter. This inhibitory activity was sufficient for decreases in PcomX activity, transformability and ComX accumulation. XrpA serving as a modulator of ComRS activity ultimately results in changes to subpopulation behaviors and cell fate during competence activation.\n\nABBREVIATED SUMMARYStreptococcus mutans displays complex regulation of natural genetic competence, highlighted by a novel gene, xrpA, embedded within the coding region for the master regulator ComX. We show that XrpA modulates ComRS-dependent activation of comX expression, resulting in changes to sub-population behaviors, including cell lysis. XrpA is the first described inhibitor of a ComRS system and, because it is unique to S. mutans it may be targetable to prevent diseases caused by this pathogen.

microbiology↗