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Kir6.2 channel activity is regulated by interaction of transmembrane domains 1 and 2 through I167 in the bundle-crossing gate.
ATP-sensitive potassium (KATP) channel in pancreatic β-cells is composed of four pore-forming inward rectifier potassium (Kir) 6.2 subunits and four regulatory sulfonylurea receptor (SUR) 1 subunits and regulate insulin secretion. Kir6.2 consists of a N-terminal region, an outer transmembrane helix (TM1), an intramembrane region that functions as a potassium selectivity filter, an inner transmembrane helix (TM2) that forms a bundle-crossing gate, and a C-terminal cytoplasmic domain. Mutations in the Kir6.2 subunit can cause neonatal diabetes with severe neurological features (DEND syndrome). The DEND syndrome-inducing I167L mutation of Kir6.2 increases the open probability (Po) of the KATP channel. To investigate the gating mechanism impacted by this mutation in Kir6.2 alone, we used C-terminus-truncated Kir6.2 channels to ascertain the impact of I167 mutations on Po in Kir6.2 channels in the absence of SUR1. We found that I167L and I167F mutations showed an increased Po while the Po of other mutations (I167A, I167V) were unchanged when compared to wild-type channels. By mutating residues in TM1 (W68, L72, F75) that may interact with I167, we found that a double mutation of I167L and F75A normalized the Po. These results would suggest that I167 may play an important role in stabilizing the open state of Kir6.2 channels.
Mitochondrial origins of the pressure to sleep.
To gain a comprehensive, unbiased perspective on molecular changes in the brain that may underlie the need for sleep, we have characterized the transcriptomes of single cells isolated from rested and sleep-deprived flies. Here we report that transcripts upregulated after sleep deprivation, in sleep-control neurons projecting to the dorsal fan-shaped body1,2 (dFBNs) but not ubiquitously in the brain, encode almost exclusively proteins with roles in mitochondrial respiration and ATP synthesis. These gene expression changes are accompanied by mitochondrial fragmentation, enhanced mitophagy and an increase in the number of contacts between mitochondria and the endoplasmic reticulum, creating conduits3,4 for the replenishment of peroxidized lipids5. The morphological changes are reversible after recovery sleep and blunted by the installation of an electron overflow6,7 in the respiratory chain. Inducing or preventing mitochondrial fission or fusion8-13 in dFBNs alters sleep and the electrical properties of sleep-control cells in opposite directions: hyperfused mitochondria increase, whereas fragmented mitochondria decrease, neuronal excitability and sleep. ATP concentrations in dFBNs rise after enforced waking because of diminished ATP consumption during the arousal-mediated inhibition of these neurons14, which augments their mitochondrial electron leak7. Consistent with this view, uncoupling electron flux from ATP synthesis15 relieves the pressure to sleep, while exacerbating mismatches between electron supply and ATP demand (by powering ATP synthesis with a light-driven proton pump16) precipitates sleep. Sleep, like ageing17,18, may be an inescapable consequence of aerobic metabolism.
Accurate Paediatric Brain Tumour Classification Through Improved Quantitative Analysis of 1H MR Imaging and Spectroscopy.
Multimodality imaging is an emerging research topic in neuro-oncology for its potential of being able to demonstrate tumours in a more comprehensive manner. Diffusion-weighted magnetic resonance imaging (dMRI) and proton magnetic resonance spectroscopy (1H-MRS) allow inferring tissue cellularity and biochemical properties, respectively. Combining dMRI and 1H-MRS may provide more accurate diagnosis for paediatric brain tumours than only one modality. This retrospective study collected 1.5-T clinical 1H-MRS and dMRI from 32 patients to assess paediatric brain tumour classification with combined dMRI and 1H-MRS. Specifically, spectral noise of 1H-MRS was suppressed before calculating metabolite concentrations. Extracted radiomic features were apparent diffusion coefficient (ADC) histogram features through dMRI and metabolite concentrations through 1H-MRS. These features were put together and then ranked according to the multiclass area under the curve (mAUC) and selected for tumour classification through machine learning. Tumours were precisely typed by combining noise-suppressed 1H-MRS and dMRI, and the cross-validated accuracy was improved to be 100% according to naïve Bayes. The finally selected radiomic biomarkers, which showed the highest diagnostic ability, were ADC fifth percentile (mAUC = 0.970), myo-inositol (mAUC = 0.952), combined glutamate and glutamine (mAUC = 0.853), total creatine (mAUC = 0.837) and glycine (mAUC = 0.815). The study indicates combining MR imaging and spectroscopy can provide better diagnostic performance than single-modal imaging.
Immunoassay for pyruvate kinase M1/2 as an Alzheimer's biomarker in CSF.
Alzheimer's disease (AD) is characterized by amyloid-beta plaques and tau tangles in the brain, but these markers alone do not predict disease progression. The intersection of these pathologies with other processes including metabolic changes may contribute to disease progression. Brain glucose metabolism changes are among the earliest detectable events in AD. Pyruvate kinase (PKM) has been implicated as a potential biomarker to track these metabolic changes. We have developed an enzyme-linked immunosorbent assay (ELISA) to assess PKM levels in cerebrospinal fluid (CSF). First, we verified the relationship of CSF PKM levels with cognitive decline, revealing a correlation between elevated CSF PKM levels and accelerated cognitive decline in preclinical AD patients in a tau-dependent manner. We developed the ELISA using two PKM-specific antibodies and validated it through quality control steps, indicating robust quantification of PKM. We showed that ELISA measurements of PKM correlate with mass spectrometry values in matching samples. When tested on an independent cohort, the assay confirmed elevation of PKM in AD. These findings support the use of PKM as a potential biomarker for tracking early metabolic changes in AD, offering a novel tool for investigating metabolic alterations and their intersection with other underlying pathologies in AD progression.
Efficient in vivo targeting of the myocardial scar using Moloney murine leukaemia virus complexed with nanoparticles
AbstractDuring myocardial infarction, native myocardium is replaced by a fibrous scar, impairing cardiac pump function and leading to potentially life‐threatening ventricular tachycardias. Gene therapy‐based targeting of the cardiac scar is essential for short‐ and long‐term treatment of post‐infarct sequelae. However, there are currently no effective methods to target and transduce cardiac (myo)fibroblasts (FB) in vivo. Therefore, Moloney murine leukaemia virus (MMLV) encoding for the fluorescent reporter mCherry complexed with magnetic nanoparticles (MNP) in combination with magnetic steering was tested. This approach strongly increased the transduction rate of FB by four‐fold in vitro. Additionally, injection of MMLV/MNP complexes into the forming scar during exposure of the heart to a magnetic field to increase virus dwell‐time 3 days after left ventricular cryo‐injury, a time when FB proliferation in the infarct peaks, yielded efficient transduction of resident FB. We further assessed the functional impact of overexpressing the gap junction protein connexin 43 (Cx43). MMLV‐mediated Cx43 overexpression (MMLV‐Cx43) in FB increased the formation of functional gap junctions in vitro and substantially lowered post‐cryo‐injury ventricular tachycardia incidence (by 50%), as demonstrated by in vivo electrophysiological testing 2 and 8 weeks after MMLV/MNP injection into the lesion. This anti‐arrhythmic effect was probably the result of a decrease in the heterogeneity of conduction around and across the scar, as observed by optical mapping in isolated hearts overexpressing Cx43. Thus, MMLV/MNP complexes combined with magnetic steering offer an efficient strategy for targeting and transducing FB in vitro and in vivo, allowing modulation of the functional properties of cardiac scars. imageKey points Genetic targeting and efficient transduction of (myo)fibroblasts (FB) in vivo are necessary to modify the properties of cardiac scars for therapeutic benefit. However, this has proven highly inadequate because of a lack of suitable viral vectors. We complexed Moloney murine leukaemia virus (MMLV) with magnetic nanoparticles (MNP) and applied a magnetic field to achieve prominent in vitro transduction of FB. Magnet‐assisted in vivo injections of MMLV/MNP complexes into the developing scar enabled efficient transduction of cardiac tissue‐resident FB. MMLV‐Cx43‐based overexpression of connexin 43 increased the density of functional gap junctions in FB in vitro. Following direct injection of the virus into the developing cardiac scar in a murine cryo‐lesion model, electrophysiological in vivo testing showed that the incidence of ventricular tachycardia was reduced by 50% at 2 and 8 weeks post‐treatment with MMLV. Our approach enables efficient targeting and transduction of FB in the cardiac scar, providing a blueprint for translation.
A missense mutation in zinc finger homeobox‐3 (ZFHX3) impedes growth and alters metabolism and hypothalamic gene expression in mice
AbstractA protein altering variant in the gene encoding zinc finger homeobox‐3 (ZFHX3) has recently been associated with lower BMI in a human genome‐wide association study. We investigated metabolic parameters in mice harboring a missense mutation in Zfhx3 (Zfhx3Sci/+) and looked for altered in situ expression of transcripts that are associated with energy balance in the hypothalamus to understand how ZFHX3 may influence growth and metabolic effects. One‐year‐old male and female Zfhx3Sci/+ mice weighed less, had shorter body length, lower fat mass, smaller mesenteric fat depots, and lower circulating insulin, leptin, and insulin‐like growth factor‐1 (IGF1) concentrations than Zfhx3+/+ littermates. In a second cohort of 9–20‐week‐old males and females, Zfhx3Sci/+ mice ate less than wildtype controls, in proportion to body weight. In a third cohort of female‐only Zfhx3Sci/+ and Zfhx3+/+ mice that underwent metabolic phenotyping from 6 to 14 weeks old, Zfhx3Sci/+ mice weighed less and had lower lean mass and energy expenditure, but fat mass did not differ. We detected increased expression of somatostatin and decreased expression of growth hormone‐releasing hormone and growth hormone‐receptor mRNAs in the arcuate nucleus (ARC). Similarly, ARC expression of orexigenic neuropeptide Y was decreased and ventricular ependymal expression of orphan G protein‐coupled receptor Gpr50 was decreased. We demonstrate for the first time an energy balance effect of the Zfhx3Sci mutation, likely by altering expression of key ARC neuropeptides to alter growth, food intake, and energy expenditure.
Decreasing HepG2 Cytotoxicity by Lowering the Lipophilicity of Benzo[d]oxazolephosphinate Ester Utrophin Modulators.
Utrophin modulation is a disease-modifying therapeutic strategy for Duchenne muscular dystrophy that would be applicable to all patient populations. To improve the suboptimal profile of ezutromid, the first-in-class clinical candidate, a second generation of utrophin modulators bearing a phosphinate ester moiety was developed. This modification significantly improved the physicochemical and ADME properties, but one of the main lead molecules was found to have dose-limiting hepatotoxicity. In this work we describe how less lipophilic analogues retained utrophin modulatory activity in a reporter gene assay, upregulated utrophin protein in dystrophic mouse muscle cells, but also had improved physicochemical and ADME properties. Notably, ClogP was found to directly correlate with pIC50 in HepG2 cells, hence leading to a potentially safer toxicological profiles in this series. Compound 21 showed a balanced profile (H2K EC50: 4.17 μM, solubility: 477 μM, mouse hepatocyte T 1/2 > 240 min) and increased utrophin protein 1.6-fold in a Western blot assay.
2-Arylbenzo[d]oxazole Phosphinate Esters as Second-Generation Modulators of Utrophin for the Treatment of Duchenne Muscular Dystrophy.
Utrophin modulation is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD), which should be applicable to all patient populations. Following on from ezutromid, the first-generation utrophin modulator, we describe the development of a second generation of utrophin modulators, based on the bioisosteric replacement of the sulfone group with a phosphinate ester and substitution of the metabolically labile naphthalene with a haloaryl substituent. The improved physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties, further reflected in the enhanced pharmacokinetic profile of the most advanced compounds, 30 and 27, led to significantly better in vivo exposure compared to ezutromid and alleviation of the dystrophic phenotype in mdx mice. While 30 was found to have dose-limiting hepatotoxicity, 27 and its enantiomers exhibited limited off-target effects, resulting in a safe profile and highlighting their potential utility as next-generation utrophin modulators suitable for progression toward a future DMD therapy.
A genetic modifier suggests that endurance exercise exacerbates Huntington's disease
Polyglutamine expansions in the huntingtin gene cause Huntington's disease (HD). Huntingtin is ubiquitously expressed, leading to pathological alterations also in peripheral organs. Variations in the length of the polyglutamine tract explain up to 70% of the age-at-onset variance, with the rest of the variance attributed to genetic and environmental modifiers. To identify novel disease modifiers, we performed an unbiased mutagenesis screen on an HD mouse model, identifying a mutation in the skeletal muscle voltage-gated sodium channel (Scn4a, termed 'draggen' mutation) as a novel disease enhancer. Double mutant mice (HD; Scn4aDgn/+) had decreased survival, weight loss and muscle atrophy. Expression patterns show that the main tissue affected is skeletal muscle. Intriguingly, muscles from HD; Scn4aDgn/+ mice showed adaptive changes similar to those found in endurance exercise, including AMPK activation, fibre type switching and upregulation of mitochondrial biogenesis. Therefore, we evaluated the effects of endurance training on HD mice. Crucially, this training regime also led to detrimental effects on HD mice. Overall, these results reveal a novel role for skeletal muscle in modulating systemic HD pathogenesis, suggesting that some forms of physical exercise could be deleterious in neurodegeneration.
Disruption of the homeodomain transcription factor orthopedia homeobox (Otp) is associated with obesity and anxiety
Objective Genetic studies in obese rodents and humans can provide novel insights into the mechanisms involved in energy homeostasis. Methods In this study, we genetically mapped the chromosomal region underlying the development of severe obesity in a mouse line identified as part of a dominant N-ethyl-N-nitrosourea (ENU) mutagenesis screen. We characterized the metabolic and behavioral phenotype of obese mutant mice and examined changes in hypothalamic gene expression. In humans, we examined genetic data from people with severe early onset obesity. Results We identified an obese mouse heterozygous for a missense mutation (pR108W) in orthopedia homeobox (Otp), a homeodomain containing transcription factor required for the development of neuroendocrine cell lineages in the hypothalamus, a region of the brain important in the regulation of energy homeostasis. OtpR108W/+ mice exhibit increased food intake, weight gain, and anxiety when in novel environments or singly housed, phenotypes that may be partially explained by reduced hypothalamic expression of oxytocin and arginine vasopressin. R108W affects the highly conserved homeodomain, impairs DNA binding, and alters transcriptional activity in cells. We sequenced OTP in 2548 people with severe early-onset obesity and found a rare heterozygous loss of function variant in the homeodomain (Q153R) in a patient who also had features of attention deficit disorder. Conclusions OTP is involved in mammalian energy homeostasis and behavior and appears to be necessary for the development of hypothalamic neural circuits. Further studies will be needed to investigate the contribution of rare variants in OTP to human energy homeostasis.
Comprehensive Energy Balance Measurements in Mice.
In mice with altered body composition, establishing whether it is food intake or energy expenditure, or both, that is the major determinant resulting in changed energy balance is important. In order to ascertain where the imbalance is, the acquisition of reproducible data is critical. Therefore, here we provide detailed descriptions of how to determine energy balance in mice. This encompasses protocols for establishing energy intake from home cage measurement of food intake, determining energy lost in feces using bomb calorimetry, and using equations to calculate parameters such as energy intake (EI), digested energy intake (DEI), and metabolisable energy intake (MEI) to determine overall energy balance. We also discuss considerations that should be taken into account when planning these experiments, including diet and sample sizes. © 2016 by John Wiley & Sons, Inc.