Biology subjects

Woods, G.

Publications and source records attributed to Woods, G..

6 recordsLinked to original sources

bioRxiv2025

A pain-reducing Kv6.4 variant spares other Kv6 channels, offering a target for uterine pain

Uterine pain conditions such as dysmenorrhea and endometriosis are highly prevalent, poorly managed, and associated with long-term impacts on womens health. The identification of a rare KCNG4 variant (rs140124801; p.Val419Met) previously linked to reduced labor pain suggests Kv6.4 (encoded by KCNG4) may play a role in visceral nociception and offer a new target for non-opioid uterine pain relief. We analyzed UK Biobank data to evaluate clinical phenotypes associated with rare single nucleotide polymorphisms (SNPs) in the conserved TVGYG selectivity filter motif of the Kv6 family wherein p.Val419Met is located. Functional consequences of these variants were assessed using immunofluorescence in SHSY5Y cells to examine membrane trafficking, co-immunoprecipitation to investigate interactions between Kv6 subunits and Kv2.1, and single-cell RNA sequencing to determine expression patterns in mouse sensory neurons. Our genetic analysis identified 5,816 individuals heterozygous and 28 homozygous for the p.Val419Met variant, as well as 292 heterozygous carriers of the p.Thr418Met variant, all located in the Kv6.4 subunit. Neither variant was associated with increased risk for general, neurological, or pain-related disorders, even in homozygous p.Val419Met carriers, supporting a favorable safety profile. Kv6.4Val419Met has a dominant negative effect on wild type Kv6.4. However, this effect is specific to Kv6.4 as, in SHSY5Y cells, co-expression of Kv6.4Val419Met along with Kv6.1, Kv6.2 or Kv6.3, showed no effects on the efficient membrane localization of Kv6.1-3. In contrast, Kv6.4Thr418Met does not interfere with Kv6.4 trafficking or its heteromerization with Kv2.1. Additionally, In SHSY5Y cells, the equivalent p.Val419Met substitution, when introduced into Kv6.1, Kv6.2 and Kv6.3, disrupts their membrane localization, noting that these variants have never been reported. Co-immunoprecipitation shows that Kv6.4 does not interact with other Kv6 subunits and transcriptomic analysis shows that Kv6.4 is expressed in a distinct subset of mouse lumbar dorsal root ganglion neurons innervating pelvic organs. Our findings show that the Kv6.4Val419Met variant selectively disrupts Kv6.4 function without affecting other family members and is not linked to adverse phenotypes. This finding supports Kv6.4 as a highly selective and functionally distinct Kv6 subunit with no widespread deleterious effects on other Kv6 subunits, making it an attractive candidate for therapeutic targeting for uterine pain.

genetics↗

bioRxiv2025

An Autophagy-Inducing Compound Reduces Tau Pathology, Microglia Reactivity, and Rescues Working Memory in 3xTg AD Mice

BackgroundRecent advancements in Alzheimers disease (AD) therapeutics have validated the use of amyloid beta (A{beta})-clearing antibodies, which reduce A{beta} pathology but leave other disease hallmarks largely unaddressed. Since AD involves multiple pathological processes, additional strategies are needed to target complementary mechanisms. One such target is autophagy, a lysosomal mediated degradation pathway essential for cellular homeostasis that removes toxic protein aggregates and damaged organelles. This process is implicated in AD, as impaired lysosomal function promotes A{beta} and tau accumulation. Our laboratory recently identified a novel natural mitophagy-inducing compound (MIC) that may serve as a therapeutic intervention for AD. MethodsWe evaluated the effects of MIC in aged 3xTgAD mice, a transgenic model displaying both A{beta} and tau pathology. Mice received either standard diet or diet containing MIC beginning at age 4 months until 20 months on alternating weeks. Age-matched non-transgenic (NonTg) controls were included under standard and MIC-supplemented diets to assess compound effects during normal aging. Neuropathological changes were assessed using immunohistochemistry (IHC) for A{beta}, phosphorylated tau (pTau), and microglial reactivity. Cognitive performance was evaluated using the Morris Water Maze (MWM) to assess spatial learning and memory and the Y-maze to measure working memory. ResultsAt 20 months of age, our neuropathological assessment showed that 3xTgAD mice fed an MIC-supplemented diet had a significant reduction in pTau accumulation and microglial reactivity, although A{beta} burden remained unchanged. At 15 months, MIC diet also improved spatial learning and memory in aged NonTg controls but not in 3xTgAD mice. However, in younger 8-month-old 3xTgAD mice, MIC restored working memory performance to NonTg levels, indicating an age-dependent therapeutic response. ConclusionMIC emerges as a potential modulator of tau pathology and neuroinflammation. As a naturally derived compound, MIC offers potential for combination therapy with FDA-approved A{beta}-clearing antibodies, enabling a multimodal approach to AD treatment that addresses amyloid, tau, and microglia-related pathology.

neuroscience↗

bioRxiv2025

Painless Nav1.7 mutations reveal function-critical residuesin the outer vestibule and N-terminus

Chronic pain is a common condition, placing a large cost on society and a heavy burden on the individual. Nav1.7 has emerged as a non-redundant and axiomatically important part of human pain pathways. Biallelic loss-of-function (LOF) mutations in SCN9A, encoding voltage-gated sodium channel Nav1.7, cause the Mendelian disorder Congenital Insensitivity to Pain (CIP). Studying novel missense mutations in this channel has the potential to uncover unrealised functions of critical residues that could enable new strategies for analgesia. Here we describe detailed functional studies of six SCN9A missense variants found in individuals with typical SCN9A-CIP: c.224T>C/p.Leu75Pro, c.1025C>T/p.Thr342Met, c.2686C>T/p.Arg896Trp, c.2732G>A/p.Arg911His, c.5059G>C/p.Ala1687Pro, and c.5173G>C/p.Gly1725Arg. While all variants were extremely rare and predicted to alter function, current tools cannot specify the extent or direction of change in function, nor provide information about the mechanism(s) of pathogenicity. Stable cell lines were generated for each variant, using the isogenic Jump-In T-REx HEK293 system. We investigated how each variant caused molecular dysfunction using multiple approaches: sequence conservation analysis and 3D modelling to examine the structural importance of the affected residues; automated whole-cell patch clamp electrophysiology to assess channel biophysics; and finally, immunocytochemistry along with cell-surface biotinylation to detect any alterations in subcellular localisation. Our studies revealed that all mutations reached the cell membrane but caused a complete LOF through small localised structural changes in the extracellular vestibule, ion selectivity filter and N-terminus that abolished sodium conductance. Functional analysis remains the most reliable method of classifying SCN9A missense variants and can help to pinpoint important subdomains. Online SummaryVoltage-gated sodium channel Nav1.7 is a key modulator of nociceptor excitability and associated with congenital painlessness. Functional analysis and in silico modelling reveal critical roles for residues in the extracellular vestibule, ion selectivity filter and unexpectedly, the N-terminus.

molecular biology↗

bioRxiv2024

Two distinct mechanisms for Nav1.7 null analgesia

Genetic deletion and pharmacological inhibition are distinct approaches to unravelling pain mechanisms, identifying targets and developing new analgesics. Both approaches have been applied to the voltage-gated sodium channels Nav1.7 and Nav1.8. Genetic deletion of Nav1.8 in mice leads to a loss of pain, and antagonists are effective analgesics. Complete embryonic loss of Nav1.7 in humans or in mouse sensory neurons leads to profound analgesia substantially mediated by endogenous opioid signaling, and anosmia that is opioid independent. Autonomic function appears to be normal. Adult deletion of Nav1.7 in sensory neurons also leads to analgesia with diminished sensory neuron excitability but there is no opioid component of analgesia. Pharmacological inhibition of Nav1.7 leads to dramatic side-effects on the autonomic nervous system. Here we compare and contrast the distinct embryonic and adult null mechanisms of Nav1.7 loss-of-function analgesia. We describe an endogenous opioid mechanism of analgesia that provides new opportunities for therapeutic intervention and pain relief. SummaryIn contrast to Nav1.8, Nav1.7, a genetically validated human pain target is unsuitable for small molecule drug development because of its wide spread expression both centrally and peripherally.

neuroscience↗

bioRxiv2021

EROS-mediated control of NOX2 and P2X7 biosynthesis

EROS (Essential for Reactive Oxygen Species) protein is indispensable for expression of gp91phox, the catalytic core of the phagocyte NADPH oxidase. EROS deficiency in humans is a novel cause of the severe immunodeficiency, chronic granulomatous disease (CGD), but its mechanism of action was unknown until now. We elucidate the role of EROS, showing it acts at the earliest stages of gp91phox maturation. It binds the immature 58kDa gp91phox directly, preventing gp91phox degradation and allowing glycosylation via the oligosaccharyltransferase (OST) machinery and the incorporation of the heme prosthetic groups essential for catalysis. EROS also regulates the purine receptors P2X7 and P2X1 through direct interactions and P2X7 is almost absent in EROS deficient mouse and human primary cells. Accordingly, lack of EROS results in markedly abnormal P2X7 signalling, inflammasome activation and T cell responses. The loss of both ROS and P2X7 signalling leads to resistance to influenza infection. Our work identifies EROS as a highly selective chaperone for key proteins in innate and adaptive immunity and a rheostat for immunity to infection. It has profound implications for our understanding of immune physiology, ROS dysregulation and possibly gene therapy.

immunology↗

bioRxiv2020

Post release immune responses of Tasmanian devils vaccinated with an experimental devil facial tumour disease vaccine

Disease is increasingly becoming a driver of wildlife population declines and extinction risk. Vaccines have been one of the most successful health interventions in human history, but few have been tested for mitigating wildlife disease. The transmissible cancer, devil facial tumour disease (DFTD), triggered the Tasmanian devils (Sarcophilus harrisii) inclusion on the international endangered species list. Development of a protective DFTD vaccine would provide a valuable management approach for conservation of the species. In 2016, 33 devils from a DFTD-free insurance population were given an experimental DFTD vaccination prior to their release on the north coast of Tasmania. The release site was already home to an incumbent population of devils, including some individuals with DFTD. To determine the efficacy of the vaccination protocol and the longevity of the response it induced, six trapping trips took place at the site over the 2.5 years following release. Eight of the 33 vaccinated devils were re-trapped, and six of those developed DFTD within the monitoring period. Despite the apparent lack of protection provided by the vaccine for the re-trapped devils, we observed several signs of immune activation not usually found in unvaccinated devils. Firstly, sera collected from the eight devils showed that anti-DFTD antibodies persisted for up to two years post vaccination. Secondly, tumour infiltrating lymphocytes were found in three out of four biopsies collected from vaccinated devils which contrasts with the "immune deserts" typical of DFTs; only one out of twenty incumbent devils with DFTD trapped during the same period had a tumour biopsy exhibiting immune cell infiltrate. Thirdly, immunohistochemical analysis of tumour biopsies from the vaccinated devils identified the functional immune molecules associated with antigen presenting cells (MHC-II) and T cells (CD3), and the immune checkpoint molecule PD-1, all associated with anti-tumour immunity in other species. These results correlate with our previous study on captive devils in which a prophylactic vaccine primed the devil immune system and, following DFTD challenge and tumour growth, immunotherapy induced complete tumour regressions. The field trial results presented here provide further evidence that the devil immune system can be primed to recognise DFTD cells, but additional immune manipulation could be needed for complete protection or induction of tumour regressions.

immunology↗