Frank Oerlemans

Fmr1 knockout mice: A model to study fragile X mental retardation

Male patients with fragile X syndrome lack FMR1 protein due to silencing of the FMR1 gene by amplification of a CGG repeat and subsequent methylation of the promoter region. The absence of FMR1 protein leads to mental retardation, aberrant behavior, and macroorchidism. Hardly anything is known about the physiological function of FMR1 and the pathological mechanisms leading to these symptoms. Therefore, we designed a knockout model for the fragile X syndrome in mice. The knockout mice lack normal Fmr1 protein and show macroorchidism, learning deficits, and hyperactivity. Consequently, this knockout mouse may serve as a valuable tool in the elucidation of the physiological role of FMR1 and the mechanisms involved in macroorchidism, abnormal behavior, and mental retardation.

Skeletal muscles of mice deficient in muscle creatine kinase lack burst activity

To understand the physiological role of the creatine kinase-phosphocreatine (CK-PCr) system in muscle bioenergetics, a null mutation of the muscle CK (M-CK) gene was introduced into the germline of mice. Mutant mice show no alterations in absolute muscle force, but lack the ability to perform burst activity. Their fast-twitch fibers have an increased intermyofibrillar mitochondrial volume and an increased glycogenolytic/glycolytic potential. PCr and ATP levels are normal in resting M-CK-deficient muscles, but rates of high energy phosphate exchange between PCr and ATP are at least 20-fold reduced. Strikingly, PCr levels decline normally during muscle exercise, suggesting that M-CK-mediated conversion is not the only route for PCr utilization in active muscle.

Altered Ca2+ responses in muscles with combined mitochondrial and cytosolic creatine kinase deficiencies

We have blocked creatine kinase (CK)-mediated phosphocreatine (PCr) -->/<-- ATP transphosphorylation in skeletal muscle by combining targeted mutations in the genes encoding mitochondrial and cytosolic CK in mice. Contrary to expectation, the PCr level was only marginally affected, but the compound was rendered metabolically inert. Mutant muscles in vivo showed significantly impaired tetanic force output, increased relaxation times, altered mitochondrial volume and location, and conspicuous tubular aggregates of sarcoplasmic reticulum membranes, as seen in myopathies with electrolyte disturbances. In depolarized myotubes cultured in vitro, CK absence influenced both the release and sequestration of Ca2+. Our data point to a direct link between the CK-PCr system and Ca2+-flux regulation during the excitation and relaxation phases of muscle contraction.

Creatine kinase B-driven energy transfer in the brain is important for habituation and spatial learning behaviour, mossy fibre field size and determination of seizure susceptibility

Creatine kinases are important in maintaining cellular‐energy homeostasis, and neuroprotective effects have been attributed to the administration of creatine and creatine‐like compounds. Herein we examine whether ablation of the cytosolic brain‐type creatine kinase (B‐CK) in mice has detrimental effects on brain development, physiological integrity or task performance. Mice deficient in B‐CK (B‐CK–/–) showed no gross abnormalities in brain anatomy or mitochondrial ultrastructure, but had a larger intra‐ and infrapyramidal mossy fibre area. Nuclear magnetic resonance spectroscopy revealed that adenosine triphosphate (ATP) and phosphocreatine (PCr) levels were unaffected, but demonstrated an apparent reduction of the PCr ⇆ ATP phosphorus exchange capacity in these mice. When assessing behavioural characteristics B‐CK–/– animals showed diminished open‐field habituation. In the water maze, adult B‐CK–/– mice were slower to learn, but acquired the spatial task. This task performance deficit persisted in 24‐month‐old, aged B‐CK–/– mice, on top of the age‐related memory decline normally seen in old animals. Finally, a delayed development of pentylenetetrazole‐induced seizures (creating a high‐energy demand) was observed in B‐CK–/– mice. It is suggested that the persistent expression of the mitochondrial isoform ubiquitous mitochondrial CK (UbCKmit) in the creatine/phospho‐creatine shuttle provides compensation for the loss of B‐CK in the brain. Our studies indicate a role for the creatine–phosphocreatine/CK circuit in the formation or maintenance of hippocampal mossy fibre connections, and processes that involve habituation, spatial learning and seizure susceptibility. However, for fuelling of basic physiological activities the role of B‐CK can be compensated for by other systems in the versatile and robust metabolic‐energy network of the brain.

Adenylate kinase 1 gene deletion disrupts muscle energetic economy despite metabolic rearrangement

Efficient cellular energy homeostasis is a critical determinant of muscle performance, providing evolutionary advantages responsible for species survival. Phosphotransfer reactions, which couple ATP production and utilization, are thought to play a central role in this process. Here, we provide evidence that genetic disruption of AK1‐catalyzed β‐phosphoryl transfer in mice decreases the potential of myofibers to sustain nucleotide ratios despite up‐regulation of high‐energy phosphoryl flux through glycolytic, guanylate and creatine kinase phosphotransfer pathways. A maintained contractile performance of AK1‐deficient muscles was associated with higher ATP turnover rate and larger amounts of ATP consumed per contraction. Metabolic stress further aggravated the energetic cost in AK1−/− muscles. Thus, AK1‐catalyzed phosphotransfer is essential in the maintenance of cellular energetic economy, enabling skeletal muscle to perform at the lowest metabolic cost.

Brain-type creatine kinase has a crucial role in osteoclast-mediated bone resorption

Osteoclasts differentiate from precursor cells of the monocyte-macrophage lineage and subsequently become activated to be competent for bone resorption through programs primarily governed by receptor activator of nuclear factor-κB ligand in cooperation with macrophage colony–stimulating factor. Proteins prominently expressed at late phases of osteoclastogenesis and with a supportive role in osteoclast function are potential therapeutic targets for bone-remodeling disorders. In this study, we used a proteomics approach to show that abundance of the brain-type cytoplasmic creatine kinase (Ckb) is greatly increased during osteoclastogenesis. Decreasing Ckb abundance by RNA interference or blocking its enzymatic activity with a pharmacological inhibitor, cyclocreatine, suppressed the bone-resorbing activity of osteoclasts grown in vitro via combined effects on actin ring formation, RhoA GTPase activity and vacuolar ATPase function. Activities of osteoclasts derived from Ckb−/− mice were similarly affected. In vivo studies showed that Ckb−/− mice were better protected against bone loss induced by ovariectomy, lipopolysaccharide challenge or interleukin-1 treatment than wild-type controls. Furthermore, administration of cyclocreatine or adenoviruses harboring Ckb small hairpin RNA attenuated bone loss in rat and mouse models. Our findings establish an important role for Ckb in the bone-resorbing function of osteoclasts and underscore its potential as a new molecular target for antiresorptive drug development.

Structural and behavioural consequences of double deficiency for creatine kinases BCK and UbCKmit

The cytosolic brain-type creatine kinase (BCK) isoform and the mitochondrial ubiquitous creatine kinase (UbCKmit) isoform are both important for the maintenance and distribution of cellular energy in neurons and astrocytes. Previously, we reported that mice deficient for BCK or UbCKmit each showed a surprisingly mild phenotype, probably due to reciprocal functional compensation by the remaining creatine kinase. This study shows that adult male mice lacking both creatine kinase isoforms (CK--/-- double knockout mice) have a reduced body weight, and demonstrate a severely impaired spatial learning in both a dry and a wet maze, lower nestbuilding activity and diminished acoustic startle reflex responses when compared to age-matched male wildtype mice with the same genetic background. In contrast, their visual and motor functions, exploration behaviour, prepulse inhibition and anxiety-related responses were not changed, suggesting no global deficit in sensorimotor function, hearing or motivation. Morphological analysis of CK--/-- double knockout brains revealed a reduction of approximately 7% in wet brain weight and hippocampal size, a approximately 15% smaller regio-inferior and relatively larger supra-pyramidal, and intra-infra-pyramidal mossy fiber areas. These results suggest that lack of both brain specific creatine kinase isoforms renders the synaptic circuitry in adult brain less efficient in coping with sensory or cognitive activity related challenges.

CYTOARCHITECTURAL AND METABOLIC ADAPTATIONS IN MUSCLES WITH MITOCHONDRIAL AND CYTOSOLIC CREATINE KINASE DEFICIENCIES

We have blocked creatine kinase (CK) mediated phosphocreatine (PCr) ⇄ ATP transphosphorylation in mitochondria and cytosol of skeletal muscle by knocking out the genes for the mitochondrial (ScCKmit) and the cytosolic (M-CK) CK isoforms in mice. Animals which carry single or double mutations, if kept and tested under standard laboratory conditions, have surprisingly mild changes in muscle physiology. Strenuous ex vivo conditions were necessary to reveal that MM-CK absence in single and double mutants leads to a partial loss of tetanic force output. Single ScCKmit deficiency has no noticeable effects but in combination the mutations cause slowing of the relaxation rate. Importantly, our studies revealed that there is metabolic and cytoarchitectural adaptation to CK defects in energy metabolism. The effects involve mutation type-dependent alterations in the levels of AMP, IMP, glycogen and phosphomonoesters, changes in activity of metabolic enzymes like AMP-deaminase, alterations in mitochondrial volume and contractile protein (MHC isoform) profiles, and a hyperproliferation of the terminal cysternae of the SR (in tubular aggregates). This suggests that there is a compensatory resiliency of loss-of-function and redirection of flux distributions in the metabolic network for cellular energy in our mutants.

Cerebral creatine kinase deficiency influences metabolite levels and morphology in the mouse brain: a quantitative in vivo 1H and 31P magnetic resonance study.

Creatine kinase (CK)‐catalysed ATP–phosphocreatine (PCr) exchange is considered to play a key role in energy homeostasis of the brain. This study assessed the metabolic and anatomical consequences of partial or complete depletion of this system in transgenic mice without cytosolic B‐CK (B‐CK–/–), mitochondrial ubiquitous CK (UbCKmit–/–), or both isoenzymes (CK –/–), using non‐invasive quantitative magnetic resonance (MR) imaging and spectroscopy. MR imaging revealed an increase in ventricle size in a subset of B‐CK–/– mice, but not in animals with UbCKmit or compound CK mutations. Mice lacking single CK isoenzymes had normal levels of high‐energy metabolites and tissue pH. In the brains of CK double knockouts pH and ATP and Pi levels were also normal, even though PCr had become completely undetectable. Moreover, a 20–30% decrease was observed in the level of total creatine and a similar increase in the level of neuronal N‐acetyl‐aspartate compounds. Although CKs themselves are not evenly distributed throughout the CNS, these alterations were uniform and concordant across different brain regions. Changes in myo‐inositol and glutamate peaks did appear to be mutation type and brain area specific. Our results challenge current models for the biological significance of the PCr–CK energy system and suggest a multifaceted role for creatine in the brain.

Increased OXPHOS activity precedes rise in glycolytic rate in H-RasV12/E1A transformed fibroblasts that develop a Warburg phenotype

BackgroundThe Warburg phenotype in cancer cells has been long recognized, but there is still limited insight in the consecutive metabolic alterations that characterize its establishment. We obtained better understanding of the coupling between metabolism and malignant transformation by studying mouse embryonic fibroblast-derived cells with loss-of-senescence or H-RasV12/E1A-transformed phenotypes at different stages of oncogenic progression.ResultsSpontaneous immortalization or induction of senescence-bypass had only marginal effects on metabolic profiles and viability. In contrast, H-RasV12/E1A transformation initially caused a steep increase in oxygen consumption and superoxide production, accompanied by massive cell death. During prolonged culture in vitro, cell growth rate increased gradually, along with tumor forming potential in in vitro anchorage-independent growth assays and in vivo tumor formation assays in immuno-deficient mice. Notably, glucose-to-lactic acid flux increased with passage number, while cellular oxygen consumption decreased. This conversion in metabolic properties was associated with a change in mitochondrial NAD+/NADH redox, indicative of decreased mitochondrial tricarboxic acid cycle and OXPHOS activity.ConclusionThe high rate of oxidative metabolism in newly transformed cells is in marked contrast with the high glycolytic rate in cells in the later tumor stage. In our experimental system, with cells growing under ambient oxygen conditions in nutrient-rich media, the shift towards this Warburg phenotype occurred as a step-wise adaptation process associated with augmented tumorigenic capacity and improved survival characteristics of the transformed cells. We hypothesize that early-transformed cells, which potentially serve as founders for new tumor masses may escape therapies aimed at metabolic inhibition of tumors with a fully developed Warburg phenotype.