Biology subjects

Ashcroft, F. M.

Publications and source records attributed to Ashcroft, F. M..

7 recordsLinked to original sources

bioRxiv2026

Single-Cell Multimodal Profiling Reveals Maternal Diabetes-Nkx2-5 Interaction in Congenital Heart Disease in a Genetic Model Recapitulating Human Embryopathies

The etiological complexity of congenital heart disease (CHD) arises from the interaction between genetic and environmental factors. We hypothesized that environmental exposure could potentiate a genetic susceptibility, leading to an increased incidence of specific cardiac defects. To test this hypothesis, we used betaV59M+ female mice exhibiting severe hyperglycemia (>28 mmol/L) without dyslipidemia, a model we characterized that faithfully recapitulates human diabetic embryopathy. These females were crossed with males haploinsufficient for Nkx2-5, a transcription factor essential for cardiac development and implicated in human CHD. Our results show that the combination of genetic susceptibility and a hyperglycemic maternal environment (GxE) leads to a higher frequency and severity of cardiac malformations, including ventricular septal defects specifically involving the muscular septum, univentricular morphologies, and dilation with thinning of the myocardium. These structural abnormalities were associated with transcriptional changes affecting the contractile identity of the chamber myocardium. Single-nucleus multiomic profiling (snRNA-seq and snATAC-seq) of the E9.5 cardiac region revealed that the interaction between maternal diabetes and Nkx2-5 haploinsufficiency profoundly reshapes transcriptional and chromatin accessibility landscapes. These changes affect not only metabolic pathways, but also cell cycle progression in second heart field (SHF) progenitors, likely contributing to the observed structural cardiac defects. Multiomic integration further identifies a disrupted indirect Nkx2-5-Aldh1a2 regulatory axis in posterior SHF progenitors, while demonstrating that maternal diabetes selectively affects a restricted subset of Nkx2-5-dependent regulatory elements associated with cardiomyocyte maturation. Our findings establish a comprehensive single-cell multiomic resource that will facilitate the integration of human GWAS and other genomic datasets, enabling the identification and functional interpretation of disease-associated loci.

developmental biology↗

bioRxiv2026

Hyperglycaemia reversibly disrupts circadian behaviour in mice

Disrupted sleep and circadian rhythms are among the most common features of diabetes, and are known to raise the risk of developing the disease. Whether the relationship also runs the other way, with diabetes itself disrupting the clock, has remained unresolved. Using an inducible mouse model of diabetes, in which hyperglycaemia can be induced and then reversed within the same animal, we show that elevated blood glucose is a reversible cause of circadian disruption. Raising blood glucose reduced the amplitude of circadian rest-activity rhythms by 50%, in proportion to the degree of hyperglycaemia, and shortened the free-running period of the clock. Restoring normoglycaemia reversed both effects. Diabetic animals had a delayed phase of activity onset, i.e. later chronotype, and re-entrained more slowly after a simulated jet lag. However, phase shifting to nocturnal light pulses was intact, suggesting normal photic responses. Brain glucose and O-GlcNAcylation, a nutrient-sensitive protein modification, were both elevated in diabetic animals, showing that peripheral hyperglycaemia reaches the brain and alters its metabolic state, and providing a molecular readout of that exposure. Together, these findings establish that a peripheral metabolic state is a reversible determinant of circadian rest-activity behaviour, completing a bidirectional link between blood glucose and the clock and arguing that the two must be treated together rather than in isolation.

neuroscience↗

bioRxiv2023

KATP channel mutation disrupts hippocampal network activity and nocturnal γ shifts

ATP-sensitive potassium (KATP) channels enable ATP to control the membrane potential and insulin secretion. Humans affected by severe activating mutations in KATP channels suffer from developmental delay, epilepsy and neonatal diabetes (DEND syndrome). While the diabetes in DEND syndrome is well understood, the pathophysiology of the neurological symptoms remains unclear. We hypothesized that parvalbumin-positive interneurons (PV-INs) are key for the pathophysiology and found, by using electrophysiology, that expressing the DEND mutation Kir6.2-V59M selectively in PV-INs reduced intrinsic gamma frequency preference and short-term depression as well as disturbed cognition-associated gamma oscillations and hippocampal sharp waves. Furthermore, risk of seizures is increased and day-night shift in gamma activity disrupted. Thus, PV-INs play a key role in DEND syndrome and this provides a framework for establishing treatment options. One Sentence SummaryOveractive KATP channels in PV-interneurons disturb cellular behaviour and cognition-associated network oscillations.

neuroscience↗

bioRxiv2021

The dynamic interplay of PIP2 and ATP in the regulation of the KATP channel

ATP-sensitive potassium (KATP) channels couple the intracellular ATP concentration to insulin secretion. KATP channel activity is inhibited by ATP binding to the Kir6.2 tetramer and activated by phosphatidylinositol-4,5-bisphosphate (PIP2). Here, we use molecular dynamics (MD) simulation, electrophysiology and fluorescence spectroscopy to show that ATP and PIP2 occupy different binding pockets that share a single amino acid residue, K39. When both ligands are present, K39 shows a greater preference to co-ordinate with PIP2 than ATP. A neonatal diabetes mutation at K39 (K39R) increases the number of hydrogen bonds formed between K39 and PIP2, reducing ATP inhibition. We also find direct effects on nucleotide binding from mutating E179, a residue proposed to interact with PIP2. Our work suggests PIP2 and ATP interact allosterically to regulate KATP channel activity.

biochemistry↗

bioRxiv2020

Evaluating Inositol phospholipid interactions with Inward Rectifier Potassium Channels and characterising their role in Disease

Membrane proteins are frequently modulated by specific protein-lipid interactions. The activation of human inward rectifying potassium (hKir) channels by phosphoinositides (PI) has been well characterised. Here, we apply a coarse-grained molecular dynamics free-energy perturbation (CG-FEP) protocol to capture the energetics of binding of PI lipids to hKir channels. By using either a single- or multi-step approach, we establish a consistent value for the binding of PIP2 to hKir channels, relative to the binding of the bulk phosphatidylcholine phospholipid. Furthermore, by perturbing amino acid side chains on hKir6.2, we show that the neonatal diabetes mutation E179K increases PIP2 affinity, while the congenital hyperinsulinism mutation K67N results in a reduced affinity. We show good agreement with electrophysiological data where E179K exhibits a reduction in neomycin sensitivity, implying that PIP2 binds more tightly E179K channels. This illustrates the application of CG-FEP to compare affinities between lipid species, and for annotating amino acid residues.

biochemistry↗

bioRxiv2019

Nucleotide inhibition of the pancreatic ATP-sensitive K+ channel explored with patch-clamp fluorometry

Pancreatic ATP-sensitive K+ channels (KATP) comprise four inward rectifier subunits (Kir6.2), each associated with a sulphonylurea receptor (SUR1). ATP/ADP binding to Kir6.2 shuts KATP. Mg-nucleotide binding to SUR1 stimulates KATP. In the absence of Mg2+, SUR1 increases the apparent affinity for nucleotide inhibition at Kir6.2 by an unknown mechanism. We simultaneously measured channel currents and nucleotide binding to Kir6.2. Fits to combined data sets suggest that KATP closes with only one nucleotide molecule bound. A Kir6.2 mutation (C166S) that increases channel activity did not affect nucleotide binding, but greatly perturbed the ability of bound nucleotide to inhibit KATP. Mutations at position K205 in SUR1 affected both nucleotide affinity and the ability of bound nucleotide to inhibit KATP. This suggests a dual role for SUR1 in KATP inhibition, both in directly contributing to nucleotide binding and in stabilising the nucleotide-bound closed state.

biophysics↗

bioRxiv2018

Activation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide binding

The response of ATP-sensitive K+ channels (KATP) to cellular metabolism is coordinated by three classes of nucleotide binding site (NBS). We used a novel approach involving labeling of intact channels in a native, membrane environment with a non-canonical fluorescent amino acid and measurement (using FRET with fluorescent nucleotides) of steady-state and time-resolved nucleotide binding to dissect the role of NBS2 of the accessory SUR1 subunit of KATP in channel gating. Binding to NBS2 was Mg2+-independent, but Mg was required to trigger a conformational change in SUR1. Mutation of a lysine (K1384A) in NBS2 that coordinates bound nucleotides increased the EC50 for trinitrophenyl-ADP binding to NBS2, but only in the presence of Mg2+, indicating that this mutation disrupts the ligand-induced conformational change. Comparison of nucleotide-binding with ionic currents suggests a model in which each nucleotide binding event to NBS2 of SUR1 is independent and promotes KATP activation by the same amount.

biophysics↗