Biology subjects

Keijer, J.

Publications and source records attributed to Keijer, J..

6 recordsLinked to original sources

bioRxiv2025

Increased ambient temperature mitigates pathology in the Ndufs4-/- mouse model of Leigh Syndrome

Leigh syndrome (LS) is a devastating mitochondrial disease (MD) for which there is no treatment. Together with Leigh-like syndrome (LLS), LS constitutes part of the Leigh syndrome spectrum (LSS) disorders, which are the most frequent manifestation of a primary mitochondrial disease (MD) in children. Ndufs4-/- mice are a widely used animal model to study LS pathophysiology and interventions. These mice display an isolated mitochondrial complex I deficiency and a brain-specific pathomechanism. Similar to other mouse models of human disease, Ndufs4-/- mice are routinely housed and studied at a sub-thermoneutral ambient temperature. This means that these mice experience chronic cold-stress, which potentially aggravates disease symptoms and reduces their translational value. Here, we provide evidence that housing Ndufs4-/- mice at 26 {degrees}C instead of 20 {degrees}C increases their skin, core and brain temperature. At this higher temperature, Ndufs4-/- mice displayed lower energy expenditure and, importantly, a longer lifespan, pathology reversal in specific brain regions, as well as increased voluntary locomotor activity. We conclude that ambient temperature is a previously overlooked but highly relevant disease modifier in Ndufs4-/- mice. Given the reduced mitochondrial energy production and aberrant thermoregulation in LSS and other MD patients, our findings suggest that reducing energy requirements might be of therapeutic value and/or contribute to an improved quality of life in these patients. In a broader sense, our results advocate the use of (more) thermoneutral housing to evaluate pathomechanisms and intervention strategies in murine models of human disease. SignificanceWe conclude that ambient temperature is a previously overlooked but highly relevant disease modifier in Ndufs4-/- mice. Given the reduced mitochondrial energy production and aberrant thermoregulation in LS and other MD patients, our findings suggest that reducing energy requirements might be of therapeutic value and/or contribute to an improved quality of life in these patients. In a broader sense, our results clearly demonstrate why it is essential to use a (more) thermoneutral housing to evaluate pathomechanisms and intervention strategies in translational research with murine models of human disease.

neuroscience↗

bioRxiv2025

Dual effects of nicotinamide on aging-related arrhythmia: protective at low dose, proarrhythmic at high doses

BackgroundCardiac arrhythmia and dysfunction progressively increase with age, both of which are closely associated with a decline in NAD+. Supplementation with nicotinamide (NAM), a critical NAD+ precursor, has shown protection in experimental models of aging-related cardiac diseases, including atrial fibrillation (AF) and heart failure, especially heart failure with preserved ejection fraction. With the potential rise of NAM in treatment of various cardiometabolic diseases, its dose-dependent cardiac-specific adverse effects and underlying mechanisms warrant investigation. MethodsThe effects of NAM on aging-related arrhythmia and cardiac dysfunction were assessed using ex vivo Langendorff mouse hearts and adult Drosophila heart preparations. Different doses of NAM (10-100 mM) were tested for their impact on the contractility of HL-1 cardiomyocytes, lifespan and cardiac function of Drosophila, as well as arrhythmia susceptibility of ex vivo mouse hearts. Acetylation of sarcoplasmic/endoplasmic reticulum Ca{superscript 2} ATPase 2a (SERCA2a) was measured by immunoprecipitation followed by Western blotting. ResultsAcute perfusion with 10 mM NAM had limited influence on aging-related AF susceptibility in ex vivo mouse hearts. Short-term dietary intervention with 10 mM NAM in late-life protected against aging-induced cardiac arrhythmia and contractile dysfunction exclusively in male Drosophila. In contrast, life-long exposure or NAM concentrations above 20 mM led to dose-dependent adverse cardiac effects, including impaired contractility in HL-1 cardiomyocyte and shortened lifespan in Drosophila, with increased arrhythmia observed in both models. In ex vivo mouse hearts, 100 mM NAM increased SERCA2a acetylation, suggesting inhibition of sirtuin1 and impaired calcium handling, which likely underlies the observed effects of high-dose NAM on arrhythmia and cardiac dysfunction. ConclusionsNAM exhibits a narrow therapeutic window in aging-related cardiac dysfunction and arrhythmia, with its efficacy highly dependent on dose, duration, and biological context. While a moderate dose in late-life may be protective, chronic or excessive intake of NAM can induce arrythmia and impair cardiac function, likely through disruption of the SERCA2a activity. These findings underscore the importance of cautious and context-specific application of NAM in clinical settings.

physiology↗

bioRxiv2024

A novel and robust method for assessing mitochondrial (dys)function in healthy and diseased frozen cardiac tissue

Cardiovascular diseases are often associated with impairment in mitochondrial function detected by reduced mitochondrial oxygen consumption using high-resolution respirometry. However, existing respirometry protocols are limited by the necessity for fresh tissue samples. This study developed a method with tailored substrate-inhibitor titration (TSIT) of mitochondrial electron transport complexes (ETC) to measure mitochondrial function in frozen cardiac samples using high-resolution respirometry. Briefly, acetyl-CoA was added to fuel the tricarboxylic acid (TCA) cycle for NADH production, enabling complex I (CI)-linked respiratory assessment. NADH was then added to measure maximum CI-linked respiratory capacity, followed by rotenone and succinate to assess complex II (CII)-linked respiratory capacity. TSIT detected mitochondrial functional differences between frozen atrial and ventricular tissue, with comparable results as measured in fresh samples. It also detected cardiac mitochondrial dysfunction across various (patho)physiological mouse models (including aging, ischemia reperfusion, obesity, and CI deficiency) as well as in frozen human donor samples, highlighting its clinical potential. Furthermore, we showed the first evidence for supercomplexes (SCs) formation between ETC-SCs and the TCA cycle metabolon, underpinning TSIT feasibility. In conclusion, we established a novel, robust, sensitive and translational method (TSIT) for assessing mitochondrial (dys)function in frozen cardiac samples from various species, enabling flexible analysis of mitochondrial function in both laboratory and clinical settings.

physiology↗

bioRxiv2023

SIRT5 variants from patients with mitochondrial disease are associated with reduced SIRT5 stability and activity, but not with neuropathology

SIRT5 is a sirtuin deacylase that represents the major activity responsible for removal of negatively-charged lysine modifications, in the mitochondrial matrix and elsewhere in the cell. In benign cells and mouse models, under basal non-stressed conditions, the phenotypes of SIRT5 deficiency are generally quite subtle. Here, we identify two homozygous SIRT5 variants in human patients suffering from severe mitochondrial disease. Both variants, P114T and L128V, are associated with reduced SIRT5 protein stability and impaired biochemical activity, with no evidence of neomorphic or dominant negative properties. The crystal structure of the P114T enzyme was solved and shows only subtle deviations from wild-type. Via CRISPR-Cas9, we generate a mouse model that recapitulates the human P114T mutation; homozygotes show reduced SIRT5 levels and activity, but no obvious metabolic abnormalities, neuropathology or other gross evidence of severe disease. We conclude that these human SIRT5 variants most likely represent severe hypomorphs, and are likely not the primary pathogenic cause of the neuropathology observed in the patients.

genetics↗

bioRxiv2022

The genome-wide mutational consequences of DNA hypomethylation

DNA methylation is important for establishing and maintaining cell identity and for genomic stability. This is achieved by regulating the accessibility of regulatory and transcriptional elements and the compaction of subtelomeric, centromeric, and other inactive genomic regions. Carcinogenesis is accompanied by a global loss in DNA methylation, which facilitates the transformation of cells. Cancer hypomethylation may also cause genomic instability, for example through interference with the protective function of telomeres and centromeres. However, understanding the role(s) of hypomethylation in tumor evolution is incomplete because the precise mutational consequences of global hypomethylation have thus far not been systematically assessed. Here we made genome-wide inventories of all possible genetic variation that accumulates in single cells upon the long-term global hypomethylation by CRISPR/CAS9-mediated conditional knockdown of DNMT1. Depletion of DNMT1 results in a genomewide reduction in DNA methylation levels. Hypomethylated cells show reduced proliferation rates, reactivation of the inactive X-chromosome and abnormal nuclear morphologies. Prolonged hypomethylation is accompanied by increased chromosomal instability. However, there is no increase in mutational burden, enrichment for certain mutational signatures or structural changes to the genome. We conclude that the primary consequence of global hypomethylation is chromosomal instability and does not necessarily lead to other small-scale or structural mutational effects.

genomics↗

bioRxiv2021

OCRbayes: A Bayesian hierarchical modeling framework for Seahorse extracellular flux oxygen consumption rate data analysis

BackgroundMitochondrial dysfunction is involved in many complex diseases. Efficient and accurate evaluation of mitochondrial functionality is crucial for understanding pathology as well as facilitating novel therapeutic developments. As a popular platform, Seahorse extracellular flux (XF) analyzer is widely used for measuring mitochondrial oxygen consumption rate (OCR) in living cells. A hidden feature of Seahorse XF OCR data is that it has a complex data structure, caused by nesting and crossing between measurement cycles, wells and plates. Surprisingly, statistical analysis of Seahorse XF data has not received sufficient attention, and current methods completely ignore the complex data structure, impairing the robustness of statistical inference. ResultsTo rigorously incorporate the complex structure into data analysis, here we developed a Bayesian hierarchical modeling framework, OCRbayes, and demonstrated its applicability based on analysis of published data sets. ConclusionsWe showed that OCRbayes can analyze Seahorse XF OCR experimental data derived from either single or multiple plates. Moreover, OCRbayes has potential to be used for diagnosing patients with mitochondrial diseases.

systems biology↗