Biology subjects

O'Reilly, M.

Publications and source records attributed to O'Reilly, M..

10 recordsLinked to original sources

bioRxiv2025

Redefining catecholaminergic polymorphic ventricular tachycardia (CPVT) as a neurocardiac condition

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome characterised by adrenergic activity-induced sudden cardiac death. It is most often caused by mutations in the RYR2 gene encoding ryanodine receptor 2 (RyR2), which is essential for intracellular calcium handling. Research has traditionally focused on the consequences of mutations at the cardiomyocyte level. However, RyR2 is also expressed in neuronal tissue, and patients often present with clinical signs of autonomic dysfunction. Here, we assessed if there is a neuronal phenotype in this classically cardiac condition. Using the established CPVT mouse model Ryr2-R2474S, we found that RyR2 is abundantly expressed in stellate ganglia neurons (SGNs) - the adrenergic neurons that project directly to the myocardium and modulate heart function. We revealed that in isolated Ryr2-R2474S SGNs there is altered calcium homeostasis suggestive of intracellular calcium leakage, and increased neurite projections when maintained in culture. We furthermore showed that hearts of Ryr2-R2474S mice are sympathetically hyper-innervated, with an increased heterogeneity in innervation within the myocardium. This led to changes in the quantities of neurotransmitters and their metabolites within the heart, indicating increased norepinephrine turnover. CPVT may therefore be redefined as a neurocardiac disorder, with neuronal dysregulation being a prominent component of the disease. In addition to cardiomyocytes, RYR2 mutations also affect stellate ganglia neuron function and cardiac innervation, with potential implications for arrhythmogenesis, risk stratification and therapy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/635037v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1494446org.highwire.dtl.DTLVardef@1218190org.highwire.dtl.DTLVardef@c11db9org.highwire.dtl.DTLVardef@8674aa_HPS_FORMAT_FIGEXP M_FIG C_FIG In wildtype hearts, activation of sympathetic neurons leads to their release of norepinephrine (NE), which binds to beta adrenergic receptors ({beta}-ARs) on the cardiomyocyte membrane. The resultant initiation of intracellular signalling cascades involving cyclic AMP (cAMP) leads to reduced inhibition of the sarcoendoplasmic reticulum (SR) calcium ATPase (SERCA) pump and consequent calcium overload in the SR. This triggers the opening of ryanodine receptor 2 (RyR2), causing SR calcium to be released into the cytoplasmic space resulting in a calcium wave which triggers contraction. In CPVT hearts, there is increased sympathetic neuron innervation of the ventricular myocardium. When cardiac sympathetic nerves are activated, there is an enhanced release of NE. This excess NE causes increased activation of {beta}-ARs and an augmented intracellular cAMP response, which in turn leads to a greater reduction of the inhibition of SERCA, augmented SR calcium overload, and a heightened propensity for spontaneous calcium leakage to trigger a calcium wave, delayed afterdepolarisations, and arrhythmias. Figure created with BioRender.com.

physiology↗

bioRxiv2024

Electrophysiological comparison of left versus right stellate ganglia neurons

BackgroundThe stellate ganglia of the peripheral autonomic nervous system innervate the heart and continuously fine-tune cardiac function to meet physiological demands. The right stellate ganglion (RSG) predominantly innervates the sinoatrial node and has functional effects on chronotropy/heart rate, whereas the left stellate ganglion (LSG) has predominance in the ventricular myocardium and impacts inotropy/contractility. Whilst the innervation patterns and functional consequences of block and stimulation are well-documented, basic electrophysiological characterisation and single-cell comparison of RSG and LSG neurons has not been performed. In addition, sex differences in stellate ganglion action potential (AP) parameters may exist, but remain as yet unknown. Methods/ResultsHere we characterise the electrical properties of enzymatically isolated mouse stellate ganglia neurons using the patch clamp technique. Using 500 ms depolarising pulses of varying amplitude, we provide detailed characterisation of basic AP properties and their correlations. We reveal that there are two populations of neurons in terms of their AP firing properties (phasic or tonic firing), with the majority (67%) firing with a phasic pattern. When all recordings were pooled, tonic neurons had a significantly larger AP amplitude (85 {+/-} 3.0 vs 76 {+/-} 2.4 mV) and overshoot (28 {+/-} 1.8 vs 19 {+/-} 1.8 mV) compared to phasic neurons (P<0.05). Moreover, phasic neurons did not fire spontaneously, whereas 50% of tonic neurons did, and more often presented with anodal break excitation (P<0.05). When male vs female neurons were compared, males had a more negative minimum diastolic potential (MDP; -55 {+/-} 1.7 vs -47 {+/-} 3.0 mV, P<0.05). When LSG vs RSG neurons were compared, the RSG had a more negative resting membrane potential (Vrest; -60 {+/-} 1.5 vs -54 {+/-} 1.3 mV, P<0.05). All other AP parameters did not differ significantly between these groups. ConclusionsThe RSG and LSG contain a similar proportion of phasic and tonic firing neurons. A significant difference was observed in the Vrest of RSG vs LSG neurons, and in the MDP of male vs female neurons. However, all other AP parameters were similar. This suggests that the LSG and RSG can be combined irrespective of sex when investigating the electrophysiological properties of these distinct anatomical structures in healthy and disease conditions.

physiology↗

bioRxiv2023

LRRK2 kinase inhibition reverses G2019S mutation-dependent effects on tau pathology spread

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinsons disease (PD). These mutations elevate LRRK2 kinase activity, making LRRK2 kinase inhibitors an attractive therapeutic target. LRRK2 kinase activity has been consistently linked to specific cell signaling pathways, mostly related to organelle trafficking and homeostasis, but its relationship to PD pathogenesis has been more difficult to define. LRRK2-PD patients consistently present with loss of dopaminergic neurons in the substantia nigra but show variable development of Lewy body or tau tangle pathology. Animal models carrying LRRK2 mutations do not develop robust PD-related phenotypes spontaneously, hampering the assessment of LRRK2 inhibitors efficacy against disease processes. We hypothesized that mutations in LRRK2 may not be directly related to a single disease pathway, but instead may elevate susceptibility to multiple disease processes, depending on the disease trigger. To test this hypothesis, we have previously evaluated progression of -synuclein and tau pathologies following injection of proteopathic seeds. We demonstrated that transgenic mice overexpressing mutant LRRK2 show alterations in the brain-wide progression of pathology, especially at older ages. Here, we assess tau pathology progression in relation to long-term LRRK2 kinase inhibition. Wildtype or LRRK2G2019S knock-in mice were injected with tau fibrils and treated with control diet or diet containing LRRK2 kinase inhibitor MLi-2 targeting the IC50 or IC90 of LRRK2 for 3 to 6 months. Mice were evaluated for tau pathology by brain-wide quantitative pathology in 665 brain regions and subsequent linear diffusion modeling of progression. Consistent with our previous work, we found systemic alterations in the progression of tau pathology in LRRK2G2019S mice that were most pronounced at 6 months. Importantly, LRRK2 kinase inhibition reversed these effects in LRRK2G2019S mice, but had minimal effect in wildtype mice, suggesting that LRRK2 kinase inhibition is likely to reverse specific disease processes in G2019S mutation carriers, but additional work may be necessary to determine the potential effect in non-carriers. This work supports a protective role of LRRK2 kinase inhibition in G2019S carriers and provides a rational workflow for systematic evaluation of brain-wide phenotypes in therapeutic development.

neuroscience↗

bioRxiv2023

Modelling drug responses and evolutionary dynamics using triple negative breast cancer patient-derived xenografts

Triple negative breast cancers (TNBC) exhibit inter- and intra-tumour heterogeneity, which is reflected in diverse drug responses and interplays with tumour evolution. Here, we use TNBC patient-derived tumour xenografts (PDTX) as a platform for co-clinical trials to test their predictive value and explore the molecular features of drug response and resistance. Patients and their matched PDTX exhibited mirrored drug responses to neoadjuvant therapy in a clinical trial. In parallel, additional clinically-relevant treatments were tested in PDTXs in vivo to identify alternative effective therapies for each PDTX model. This framework establishes the foundation for anticipatory personalised therapies for those patients with resistant or relapsed tumours. The PDTXs were further explored to model PDTX- and treatment-specific behaviours. The dynamics of drug response were characterised at single-cell resolution revealing a novel mechanism of response to olaparib. Upon olaparib treatment PDTXs showed phenotypic plasticity, including transient activation of the immediate-early response and irreversible sequential phenotypic switches: from epithelial to epithelial-mesenchymal-hybrid states, and then to mesenchymal states. This molecular mechanism was exploited ex vivo by combining olaparib and salinomycin (an inhibitor of mesenchymal-transduced cells) to reveal synergistic effects. In summary, TNBC PDTXs have the potential to help design individualised treatment strategies derived from model-specific evolutionary insights.

cancer biology↗

bioRxiv2022

Reference compounds for characterizing cellular injury in high-content cellular morphology assays

Robust, generalizable approaches to identify compounds efficiently with undesirable mechanisms of action in complex cellular assays remain elusive. Such a process would be useful for hit triage during high-throughput screening and, ultimately, predictive toxicology during drug development. We generated cell painting and cellular health profiles for 218 prototypical cytotoxic and nuisance compounds in U-2 OS cells in a concentration-response format. A diversity of compounds causing cellular damage produced bioactive cell painting morphologies, including cytoskeletal poisons, genotoxins, nonspecific electrophiles, and redox-active compounds. Further, we show that lower quality lysine acetyltransferase inhibitors and nonspecific electrophiles can be distinguished from more selective counterparts. We propose that the purposeful inclusion of cytotoxic and nuisance reference compounds such as those profiled in this Resource will help with assay optimization and compound prioritization in complex cellular assays like cell painting.

pharmacology and toxicology↗

bioRxiv2022

Male sex hormone and reduced plakoglobin jointly impair atrial conduction and cardiac sodium currents

Androgenic anabolic steroids (AAS) are commonly abused by young men. Male sex associates with earlier manifestation of common and rare cardiac conditions including atrial fibrillation and arrhythmogenic right ventricular cardiomyopathy (ARVC). Clinical data suggest an atrial involvement in ARVC. The disease is caused by desmosomal gene defects such as reduced plakoglobin expression. Analysis of clinical records from 146 ARVC patients identified male preponderance and increased prevalence of atrial arrhythmias in patients with definite ARVC. Definite patients displayed ECG changes suggesting atrial remodelling. To study mechanisms of atrial remodelling due to desmosomal vulnerability and AAS, young adult male mice, heterozygously deficient for plakoglobin (Plako+/-) and wildtype (WT) littermates, were chronically exposed to 5-dihydrotestosterone (DHT) or placebo. DHT increased atrial expression of pro-hypertrophic, fibrotic and inflammatory transcripts. DHT caused atrial conduction slowing, decreased peak sodium current density, reduced action potential amplitude and lowered the peak depolarisation rate in Plako+/- but not WT atria. Super-resolution microscopy revealed a reduction in Nav1.5 clustering in Plako+/- atrial cardiomyocytes following DHT exposure. These data reveal that AAS combined with plakoglobin deficiency cause pathological atrial electrical remodelling in young male hearts. AAS abuse may increase the risk of atrial myopathy in males with desmosomal gene variants.

molecular biology↗

bioRxiv2022

Increased early sodium current provokes familial atrial fibrillation and reduces effectiveness of sodium channel block

(1) AimsAtrial fibrillation (AF) is the most common cardiac arrhythmia. Pathogenic variants in genes encoding ion channels are associated with familial AF. The point mutation M1875T in the SCN5A gene, which encodes the -subunit of the cardiac sodium channel Nav1.5, has been associated with increased atrial excitability and familial AF. (2) MethodsWe designed a new murine model carrying the Scn5a-M1875T mutation enabling us to study the effects of the Nav1.5 mutation in detail in vivo and in vitro using patch clamp and microelectrode recording of atrial cardiomyocytes, optical mapping, ECG, echocardiography, gravimetry, histology and biochemistry. (3) ResultsAtrial cardiomyocytes from newly generated adult Scn5a-M1875T+/- mice showed a selective increase in the early (peak) cardiac sodium current, larger action potential amplitude and a faster peak upstroke velocity. Conduction slowing caused by the sodium channel blocker flecainide was less pronounced in Scn5a-M1875T+/- compared to wildtype atria. Overt hypertrophy or heart failure in Scn5a-M1875T+/- mice could be excluded. (4) ConclusionThe Scn5a-M1875T point mutation causes gain-of-function of the cardiac sodium channel. Our results suggest increased atrial peak sodium current as a potential trigger for increased atrial excitability and thus AF. Whats newO_LIThe point mutation M1875T in the C-terminal domain of the cardiac sodium channel Nav1.5 causes an increase in early peak sodium current in left atria. C_LIO_LIThe observed changes induced by this point mutation suggest an increase in peak sodium current as a cause of familial atrial fibrillation (AF). C_LIO_LIOur findings provide a possible explanation for the variable effectiveness of sodium channel blockers in patients with AF. Carriers of such sodium channel gain-of-function mutations may benefit more from tailored treatments. C_LI Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

molecular biology↗

bioRxiv2020

Effects of sustained inflation pressure during neonatal cardiopulmonary resuscitation of asphyxiated piglets

ObjectiveSustained inflation (SI) during chest compression (CC = CC+SI) significantly reduces time to return of spontaneous circulation (ROSC) compared to 3:1 compression-to-ventilation ratio during neonatal resuscitation. However, the optimal peak inflation pressure (PIP) of SI during CC+SI to improve ROSC and hemodynamic recovery in severely asphyxiated piglets is unknown. AimTo examine if different PIPs of SI during CC+SI will improve ROSC and hemodynamic recovery in severely asphyxiated piglets. Intervention and measurementsTwenty-nine newborn piglets (1-3 days old) were anesthetized, intubated, instrumented and exposed to 30-min normocapnic hypoxia followed by asphyxia. Piglets were randomized into four groups: CC+SI with a PIP of 10 cmH2O (CC+SI_PIP_10, n=8), a PIP of 20 cmH2O (CC+SI_PIP_20, n=8), a PIP of 30 cmH2O (CC+SI_PIP_30, n=8), and a sham-operated control group (n=5). Heart rate, arterial blood pressure, carotid blood flow, cerebral oxygenation, and respiratory parameters were continuously recorded throughout the experiment. Main resultsBaseline parameters were similar between all groups. There was no difference in asphyxiation (duration and degree) between intervention groups. PIP correlated positively with tidal volume and inversely with exhaled CO2 during cardiopulmonary resuscitation. Time to ROSC and rate of ROSC were similar between piglets resuscitated with CC+SI_PIP_10, CC+SI_PIP_20, and CC+SI_PIP_30 cmH2O: median (IQR) 75 (63-193) sec, 94 (78-210) sec, and 85 (70-90) sec; 5/8 (63%), 7/8 (88%), and 3/8 (38%) (p=0.56 and p=0.12, respectively). All piglets that achieved ROSC survived to four hours post-resuscitation. Piglets resuscitated with CC+SI_PIP_30 cmH2O exhibited increased concentrations of pro-inflammatory cytokines interleukin-1{beta} and tumour necrosis factor- in the frontoparietal cerebral cortex (both p<0.05 vs. sham-operated controls). ConclusionsIn asphyxiated term newborn piglets resuscitated by CC+SI, the use of different PIPs resulted in similar time to ROSC, but PIP at 30 cmH2O showed a larger VT delivery, lower exhaled CO2 and increased tissue inflammatory markers in the brain.

scientific communication and education↗

bioRxiv2019

Longitudinal single cell transcriptomics reveals Krt8+ alveolar epithelial progenitors in lung regeneration

Lung injury activates quiescent stem and progenitor cells to regenerate alveolar structures. The sequence and coordination of transcriptional programs during this process has largely remained elusive. Using single cell RNA-seq, we first generated a whole-organ birds-eye view on cellular dynamics and cell-cell communication networks during mouse lung regeneration from [~]30,000 cells at six timepoints. We discovered an injury-specific progenitor cell state characterized by Krt8 in flat epithelial cells covering alveolar surfaces. The number of these cells peaked during fibrogenesis in independent mouse models, as well as in human acute lung injury and fibrosis. Krt8+ progenitors featured a highly distinct connectome of receptor-ligand pairs with endothelial cells, fibroblasts, and macrophages. To sky dive into epithelial differentiation dynamics, we sequenced >30,000 sorted epithelial cells at 18 timepoints and computationally derived cell state trajectories that were validated by lineage tracing genetic reporter mice. Airway stem cells within the club cell lineage and alveolar type-2 cells underwent transcriptional convergence onto the same Krt8+ progenitor cell state, which later resolved by terminal differentiation into alveolar type-1 cells. We derived distinct transcriptional regulators as key switch points in this process and show that induction of TNF-alpha/NFkappaB, p53, and hypoxia driven gene expression programs precede a Sox4, Ctnnb1, and Wwtr1 driven switch towards alveolar type-1 cell fate. We show that epithelial cell plasticity can induce non-gradual transdifferentiation, involving intermediate progenitor cell states that may persist and promote disease if checkpoint signals for terminal differentiation are perturbed.

systems biology↗

bioRxiv2016

BaalChIP: Bayesian analysis of allele-specific transcription factor binding in cancer genomes

Allele-specific measurements of transcription factor binding from ChIP-seq data are key to dissecting the allelic effects of non-coding variants and their contribution to phenotypic diversity. However, most methods to detect allelic imbalance assume diploid genomes. This assumption severely limits their applicability to cancer samples with frequent DNA copy number changes. Here we present a Bayesian statistical approach called BaalChIP to correct for the effect of background allele frequency on the observed ChIP-seq read counts. BaalChIP allows the joint analysis of multiple ChIP-seq samples across a single variant and outperforms competing approaches in simulations. Using 548 ENCODE ChIP-seq and 6 targeted FAIRE-seq samples we show that BaalChIP effectively corrects allele-specific analysis for copy number variation and increases the power to detect putative cis-acting regulatory variants in cancer genomes.

cancer biology↗