Disturbed energy metabolism and muscular dystrophy caused by pure creatine deficiency are reversible by creatine intake.

Creatine (Cr) plays an important role in muscle energy homeostasis by its participation in the ATP-phosphocreatine phosphoryl exchange reaction mediated by creatine kinase. Given that the consequences of Cr depletion are incompletely understood, we assessed the morphological, metabolic and functional consequences of systemic depletion on skeletal muscle in a mouse model with deficiency of l-arginine:glycine amidinotransferase (AGAT(-/-)), which catalyses the first step of Cr biosynthesis. In vivo magnetic resonance spectroscopy showed a near-complete absence of Cr and phosphocreatine in resting hindlimb muscle of AGAT(-/-) mice. Compared with wild-type, the inorganic phosphate/ß-ATP ratio was increased fourfold, while ATP levels were reduced by nearly half. Activities of proton-pumping respiratory chain enzymes were reduced, whereas F(1)F(0)-ATPase activity and overall mitochondrial content were increased. The Cr-deficient AGAT(-/-) mice had a reduced grip strength and suffered from severe muscle atrophy. Electron microscopy revealed increased amounts of intramyocellular lipid droplets and crystal formation within mitochondria of AGAT(-/-) muscle fibres. Ischaemia resulted in exacerbation of the decrease of pH and increased glycolytic ATP synthesis. Oral Cr administration led to rapid accumulation in skeletal muscle (faster than in brain) and reversed all the muscle abnormalities, revealing that the condition of the AGAT(-/-) mice can be switched between Cr deficient and normal simply by dietary manipulation. Systemic creatine depletion results in mitochondrial dysfunction and intracellular energy deficiency, as well as structural and physiological abnormalities. The consequences of AGAT deficiency are more pronounced than those of muscle-specific creatine kinase deficiency, which suggests a multifaceted involvement of creatine in muscle energy homeostasis in addition to its role in the phosphocreatine-creatine kinase system.

Advantages of indium-tin oxide-coated glass slides in correlative scanning electron microscopy applications of uncoated cultured cells.

A method of direct visualization by correlative scanning electron microscopy (SEM) and fluorescence light microscopy of cell structures of tissue cultured cells grown on conductive glass slides is described. We show that by growing cells on indium-tin oxide (ITO)-coated glass slides, secondary electron (SE) and backscatter electron (BSE) images of uncoated cells can be obtained in high-vacuum SEM without charging artefacts. Interestingly, we observed that BSE imaging is influenced by both accelerating voltage and ITO coating thickness. By combining SE and BSE imaging with fluorescence light microscopy imaging, we were able to reveal detailed features of actin cytoskeletal and mitochondrial structures in mouse embryonic fibroblasts. We propose that the application of ITO glass as a substrate for cell culture can easily be extended and offers new opportunities for correlative light and electron microscopy studies of adherently growing cells.

Activation of glucose transport and AMP-activated protein kinase during muscle contraction in adenylate kinase-1 knockout mice.

AIM: Recently it was reported that adenylate kinase-1 knockout mice (AK(-/-)) exhibit elevated rates of glucose uptake following repeated contractions and hypoxia, but the mechanism was not investigated. The purpose of the present study was to measure the changes in glucose transport and AMP-activated protein kinase (AMPK) phosphorylation/activity following repeated contractions in isolated muscles from AK(-/-) mice. METHODS: Extensor digitorum longus muscles underwent an intense stimulation protocol that decreased force to less than 10% of initial by the end of 10 min. Glucose uptake was measured with 2-deoxy-D-[1,2-(3)H]glucose. RESULTS: Muscle glucose uptake in the basal state was identical between control and AK(-/-) mice and increased twofold in both groups during contraction. The general antioxidant: N-acetylcysteine, decreased contraction-mediated glucose uptake by 30% in both groups. AMPK activity and phosphorylation were similar in the two groups in the basal state and, surprisingly, after contraction as well (approximately threefold increase). Both groups exhibited marked decreases in adenosine triphosphate following contraction (60-70% depletion), which coincided with stoichiometric increases in the content of inosine monophosphate, an indirect marker of AMP production. Adenylate kinase activity averaged 2081 +/- 106 micromol min(-1) (g dry wt)(-1) for control and 37 +/- 10 for AK(-/-) muscles; the activity in the AK(-/-) muscle is likely accounted for by isoforms other than AK1. CONCLUSION: In conclusion, AK(-/-) mice have a normal capacity for contraction-mediated glucose uptake. This appears to occur via increases in AMP and reactive oxygen species that result in the activation of AMPK.

An adenylate kinase is involved in KATP channel regulation of mouse pancreatic beta cells.

AIMS/HYPOTHESIS: In a previous study, we demonstrated that a creatine kinase (CK) modulates K(ATP) channel activity in pancreatic beta cells. To explore phosphotransfer signalling pathways in more detail, we examined whether K(ATP) channel regulation in beta cells is determined by a metabolic interaction between adenylate kinase (AK) and CK. METHODS: Single channel activity was measured with the patch-clamp technique in the inside-out (i/o) and open-cell attached (oca) configuration. RESULTS: The ATP sensitivity of K(ATP) channels was higher in i/o patches than in permeabilised beta cells (oca). One reason for this observation could be that the local ATP:ADP ratio in the proximity of the channels is determined by factors not active in i/o patches. AMP (0.1 mmol/l) clearly increased open channel probability in the presence of ATP (0.125 mmol/l) in permeabilised cells but not in excised patches. This suggests that AK-catalysed ADP production in the vicinity of the channels is involved in K(ATP) channel regulation. The observation that the stimulatory effect of AMP on K(ATP) channels was prevented by the AK inhibitor P (1),P (5)-di(adenosine-5')pentaphosphate (Ap(5)A; 20 micromol/l) and abolished in the presence of the non-metabolisable ATP analogue adenosine 5'-(beta,gamma-imido)triphosphate tetralithium salt (AMP-PNP; 0.12 mmol/l) strengthens this idea. In beta cells from AK1 knockout mice, the effect of AMP was less pronounced, though not completely suppressed. The increase in K(ATP) channel activity induced by AMP in the presence of ATP was outweighed by phosphocreatine (1 mmol/l). We suggest that this is due to an elevation of the ATP concentration by CK. CONCLUSIONS/INTERPRETATION: We propose that phosphotransfer events mediated by AK and CK play an important role in determining the effective concentrations of ATP and ADP in the microenvironment of pancreatic beta cell K(ATP) channels. Thus, these enzymes determine the open probability of K(ATP) channels and eventually the actual rate of insulin secretion.

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.

Transgenic mouse models for myotonic dystrophy type 1 (DM1).

The study of animal models for myotonic dystrophy type 1 (DM1) has helped us to 'de- and reconstruct' our ideas on how the highly variable multisystemic constellation of disease features can be caused by only one type of event, i.e., the expansion of a perfect (CTG)(n) repeat in the DM1 locus on 19q. Evidence is now accumulating that cell type, cell state and species dependent activities of the DNA replication/repair/recombination machinery contribute to the intergenerational and somatic behavior of the (CTG)(n) repeat at the DNA level. At the RNA level, a gain-of-function mechanism, with dominant toxic effects of (CUG)(n) repeat containing transcripts, probably has a central role in DM1 pathology. Parallel study of DM2, a closely related form of myotonic dystrophy, has revealed a similar mechanism, but also made clear that part of the attention should remain focused on a possible role for candidate loss-of-function genes from the DM1 locus itself (like DMWD, DMPK and SIX5) or elsewhere in the genome, to find explanations for clinical aspects that are unique to DM1. This review will focus on new insight regarding structure-function features of candidate genes involved in DM1 pathobiology, and on the mechanisms of expansion and disease pathology that have now partly been disclosed with the help of transgenic animal models.

Presence of (phospho)creatine in developing and adult skeletal muscle of mice without mitochondrial and cytosolic muscle creatine kinase isoforms.

We assessed the relationship between phosphocreatine (PCr) and creatine (Cr) content and creatine kinase (CK) activity in skeletal muscle of mice. The PCr and total Cr (tCr) concentrations, as well as CK activity, in hindlimb muscles of mice, with or without the cytosolic and mitochondrial isoforms of muscle creatine kinase (wild-type or CK--/-- mice), were determined by in vivo magnetic resonance (MR) spectroscopy and by biochemical means during postnatal growth and adulthood. In wild-type muscle the [tCr], PCr/ATP ratio and CK activity increased rapidly in the first 4-7 weeks. Remarkably, CK--/-- mice showed a similar increase in the PCr/ATP ratio during the first month in the presence of only minor brain-type BB-CK activity. Uptake of Cr in muscle was seemingly unrelated to CK activity as tCr increased in the same way in the muscles of both mouse types. At older ages the PCr/ATP ratio decreased in CK--/-- muscles, in contrast to wild-type where it still slowly increased, whereas [tCr] was similar for muscle of both mouse types. Using a new in vivo MR approach with application of [4-13C]Cr, a lower PCr/tCr ratio was also observed in CK--/-- muscle. From these data it follows that in vivo global ATP levels at rest are similar in the presence or absence of CK. Although Cr could still be converted to PCr in mature CK--/-- muscle, the immediate availability of PCr decreased, and PCr became partly inconvertible at older ages. Apparently, catalysis of the CK reaction by BB-CK, although significant in muscles of newborn mice, gradually declines to very low levels in adulthood. Part or all of this BB-CK may arise from satellite cells fusing with myotubes, a process that is most active during the first months of life. Finally, our observation that the MR and chemical assessment of muscle [tCr] and PCr/tCr ratio were similar for all mice does not support the existence of a significant MR-invisible or immobile pool of Cr, with a role for CK in this phenomenon.

Developmental dyslexics show altered allocation of attention in visual classification tasks.

OBJECTIVES: Event-related brain potentials (ERPs) were recorded to investigate allocation of attention in adult developmental dyslexics. SUBJECTS AND METHODS: Twelve adult developmental dyslexics and 12 matched normal controls performed three visual choice reaction tasks. In the passive oddball condition, subjects watched two different simple visual stimuli presented with 87.5 and 12.5% probability. In the active oddball condition, participants responded to the low-probability target stimulus. In the active 50/50-condition, both stimuli were presented with 50% probability and a response was required to the target stimulus only. RESULTS: No group differences emerged for performance, P300 latency or laterality and for N200 amplitude, latency or laterality. An enhancement of P300 amplitude with a frontal distribution was found for NoGo (standard)-stimuli in both active conditions for the dyslexic sample. CONCLUSION: Results are discussed in the context of deviances in allocation of attentional resources in dyslexic readers.

Altered brain phosphocreatine and ATP regulation when mitochondrial creatine kinase is absent.

In cerebral gray matter, ATP concentration is closely maintained despite rapid, large increases in turnover and low substrate reserves. As seen in vivo by (31)P nuclear magnetic resonance (NMR) spectroscopy, brain ATP is stable early in seizures, a state of high energy demand, and in mild hypoxia, a state of substrate deficiency. Like other tissues with high and variable ATP turnover, cerebral gray matter has high phosphocreatine (PCr) concentration and both cytosolic and mitochondrial creatine kinase (UbMi-CK) isoenzymes. To understand the physiology of brain creatine kinases, we used (31)P NMR to study PCr and ATP regulation during seizures and hypoxia in mice with targeted deletion of the UbMi-CK gene. The baseline CK reaction rate constant (k) was higher in mutants than wild-types. During seizures, PCr and ATP decreased in mutants but not in wild-types. The k-value for the CK catalyzed reaction rate increased in wild-types but not in the mutants. Hypoxic mutants and wild-types showed similar PCr losses and stable ATP. During recovery from hypoxia, brain PCr and ATP concentrations returned to baseline in wild-types but were 20% higher than baseline in the mutants. We propose that UbMi-CK couples ATP turnover to the CK catalyzed reaction rate and regulates ATP concentration when synthesis is increased.

Discrepant target detection and action monitoring in obsessive-compulsive disorder.

Obsessive-compulsive disorder (OCD) has been related to altered mechanisms of action monitoring and target detection, and it has been hypothesized that hyperactive striatal-cortical circuits constitute the underlying pathophysiology. This study used event-related brain potentials (ERPs) to explore this hypothesis. A choice reaction time experiment was carried out in a group of OCD patients and a normal comparison group. The P3b component of the ERP to targets was taken as an indicator of the target-evaluation process and the response-locked error-related negativity (ERN) served as an indicator of action monitoring. We hypothesized that the OCD group would show a shortened P3b latency and an amplitude-enhanced ERN. Consistent with our expectations, the P3b latency was shorter and the ERN amplitude was higher in the OCD group. Unexpectedly, we also observed a prolonged ERN latency in the OCD group and a more posterior topography of this component. The data provide partial support for the hypothesis of a hyperactive neural network in OCD. In addition the disorder must involve pathophysiological processes that are presumably related to other aspects of its complex and heterogeneous clinical hallmarks.

Changes in mRNA expression profile underlie phenotypic adaptations in creatine kinase-deficient muscles.

We have studied the mechanisms that regulate the remodeling of the glycolytic, mitochondrial and structural network of muscles of creatine kinase M (M-CK)/sarcomeric mitochondrial creatine kinase (ScCKmit) knockout mice by comparison of wild-type and mutant mRNA profiles on cDNA arrays. The magnitudes of changes in mRNA levels were most prominent in M-CK/ScCKmit (CK(-/-)) double mutants but did never exceed those of previously observed changes in protein level for any protein examined. In gastrocnemius of CK(-/-) mice we measured a 2.5-fold increase in mRNA level for mitochondrial encoded cytochrome c oxidase (COX)-III which corresponds to the increase in protein content. The level of the nuclear encoded mRNAs for COX-IV, H(+)-ATP synthase-C, adenine nucleotide translocator-1 and insulin-regulatable glucose transporter-4 showed a 1.5-fold increase, also in agreement with protein data. In contrast, no concomitant up-regulation in mRNA and protein content was detected for the mitochondrial inorganic phosphate-carrier, voltage-dependent anion channel and certain glycolytic enzymes. Our results reveal that regulation of transcript level plays an important role, but it is not the only principle involved in the remodeling of mitochondrial and cytosolic design of CK(-/-) muscles.

Changes in glycolytic network and mitochondrial design in creatine kinase-deficient muscles.

Skeletal muscles respond with high plasticity to pathobiological conditions or changes in physiological demand by remodeling cytoarchitectural and metabolic characteristics of individual myocytes. We have previously shown that muscles of mice without mitochondrial and/or cytosolic creatine kinases (ScCKmit(-/-) and/or M-CK(-/-)) partly compensate for the defect(s) by redirecting metabolic pathways and ultrastructural characteristics. Here, we show by semiquantitative Western blot analysis that the compensatory changes involve mutation- and fiber-type-specific coordinated regulation of divergent but functionally coupled groups of proteins. Fast-twitch gastrocnemius muscle of CK(--/--) mice display a two- to fourfold upregulation of mitochondrial cytochrome c oxidase, inorganic phosphate carrier, adenine nucleotide translocator, and voltage-dependent anion channel proteins. In parallel, cytosolic myoglobin is upregulated. Slow-twitch soleus muscle responds with changes in the glycolytic enzyme pattern, including a shift in lactate dehydrogenase isoenzyme composition. Adaptations in the network for oxidative adenosine triphosphate (ATP) production are already apparent at 17 days of age.

Deficit in automatic sound-change detection may underlie some music perception deficits after acute hemispheric stroke.

Music perception deficits following acute neurological damage are thought to be rare. By a newly devised test battery of music-perception skills, however, we were able to identify among a group of 12 patients with acute hemispheric stroke six patients with music perception deficits (amusia) while six others had no such deficits. In addition we recorded event-related brain potentials (ERPs) in a passive listening task with frequent standard and infrequent pitch deviants designed to elicit the mismatch negativity (MMN). The MMN in the patients with amusia was grossly reduced, while the non-amusic patients and control subjects had MMNs of equal size. These data show that amusia is quite common in unselected stroke patients. The MMN reduction suggests that amusia is related to unspecific automatic stimulus classification deficits in these patients.

Altered inhibition of motor responses in Tourette Syndrome and Obsessive-Compulsive Disorder.

OBJECTIVES: The Gilles de la Tourette Syndrome (TS) and Obsessive Compulsive Disorder (OCD) have been shown to display impaired cognitive and motor inhibition. This study investigated inhibitory mechanisms of motor responses in order to expand the understanding of sensorimotor integration processes in both disorders. We hypothesized that both patient groups would display altered frontal inhibitory activity. MATERIAL AND METHODS: To this end event-related brain potentials (ERPs) were recorded in a STOP-paradigm in groups of TS and OCD patients and in a control group. The paradigm required the execution of a motor response after a "go" signal was given and the occasional suppression of this response after a second "stop" signal occurred. RESULTS: Behavioral parameters and Lateralized Readiness Potential (LRP) confirmed that both patient groups were well able to initiate motor responses. "Go" and "stop" stimuli elicited an enhanced frontal negative activity in both patient groups. In addition, "stop" stimuli were associated with a frontal shift of the NoGo-Anteriorization (NGA) in the TS group but not in the OCD group. CONCLUSIONS: The data are interpreted to indicate altered frontal inhibitory functions. Similarities and dissimilarities between the findings for TS and OCD are discussed with respect to other pathophysiologic aspects of the disorders.

Adenylate kinase phosphotransfer communicates cellular energetic signals to ATP-sensitive potassium channels.

Transduction of energetic signals into membrane electrical events governs vital cellular functions, ranging from hormone secretion and cytoprotection to appetite control and hair growth. Central to the regulation of such diverse cellular processes are the metabolism sensing ATP-sensitive K+ (K(ATP)) channels. However, the mechanism that communicates metabolic signals and integrates cellular energetics with K(ATP) channel-dependent membrane excitability remains elusive. Here, we identify that the response of K(ATP) channels to metabolic challenge is regulated by adenylate kinase phosphotransfer. Adenylate kinase associates with the K(ATP) channel complex, anchoring cellular phosphotransfer networks and facilitating delivery of mitochondrial signals to the membrane environment. Deletion of the adenylate kinase gene compromised nucleotide exchange at the channel site and impeded communication between mitochondria and K(ATP) channels, rendering cellular metabolic sensing defective. Assigning a signal processing role to adenylate kinase identifies a phosphorelay mechanism essential for efficient coupling of cellular energetics with K(ATP) channels and associated functions.

Oxazepam alters action monitoring.

RATIONALE: Oxazepam has been demonstrated to slow reaction times and increase the rate of omission errors in attentional experiments. This suggests that action monitoring might also be impaired. OBJECTIVES: The present study used the event-related brain potential (ERP) technique to investigate this hypothesis. The P3b component to targets was taken as an indicator of the target evaluation process, and the response-locked error-related negativity (ERN) served as an indicator of action monitoring. We hypothesized that the amplitudes of ERN and P3b would be reduced as an effect of oxazepam. METHODS: A simple "oddball" reaction time experiment was conducted in a double-blind crossover study of 30 mg oxazepam versus placebo. In order to investigate variations in attentional allocation, separate experimental runs were undertaken with target frequencies of 50% and 80%. RESULTS: ERN and P3b amplitudes were lower in the 80% target condition than in the 50% condition. Oxazepam did not affect behavioral parameters but was associated with an ERN of lower amplitude than the placebo condition. ERN amplitude variations between target conditions remained unchanged. CONCLUSIONS: Although the intake of 30 mg oxazepam did not impair behavioral performance, measures of the electrophysiological recordings show that action monitoring processes were altered. We argue that this may be related to the anxiolytic properties of the drug and may constitute an important causal factor for behavioral impairments after the intake of oxazepam.

Electrophysiological measures and dual-task performance in Tourette syndrome indicate deficient divided attention mechanisms.

Tourette syndrome has been associated with impairments of attentional functions such as distractability, even in subjects without co-morbid attention deficit hyperactivity disorder. Based on the results of earlier research we hypothesized that Tourette syndrome patients might employ altered control mechanisms of attentional processes and have concurrent difficulties in allocating their attentional resources among competing tasks. Event-related brain potentials (ERPs) were recorded in a group of Tourette syndrome patients and in a matched control group during a dual task experiment. This experiment required the simultaneous detection of visual and auditory target stimuli which were manipulated to yield two different difficulty levels each of which were varied orthogonally. The behavioural parameters confirmed the intended performance differences between difficult-to-detect targets and easy-to-detect targets. This was paralleled by lower amplitudes and longer latencies of the corresponding P3b-ERP subcomponents. Although Tourette syndrome patients were unimpaired in overall performance they showed an increased interference of visual task demands with auditory target perception. In parallel they also exhibited a reduced amplitude of the P3b component to auditory targets. The findings show that Tourette syndrome patients are not generally impaired in their dual task performance. The allocation of attentional resources to competing tasks however, is altered. We speculate that this may be related to deficient inhibitory functions.

Differential effects of emotional content on event-related potentials in word recognition memory.

The aim of this study was to delineate the influence of the emotional content of stimuli to be remembered on the recognition performance of normal subjects by means of the event-related potential (ERP) technique. When words are presented repeatedly, brain responses to repeated and recognized items are characterized by a more positive waveform, referred to as "old/new effect". Words judged for their emotional connotation ("negative", "positive" and "neutral") were presented successively on a video monitor to subjects, who had the task to indicate whether a given word occurred for the first ("new") or second ("old") time within the list by pressing one of two buttons. For each word category, the ERPs of the old words were more positive compared to those of the new items from about 250 ms after stimulus. The old/new effect was significantly enhanced for the negative and positive items compared to the neutral stimuli between 450 and 650 ms after stimulus pointing to a significant influence of the emotional content of words on verbal memory processes. This paradigm appears to be feasible to investigate interactions of emotion and cognition in psychiatric patients.

Differences in brain potentials to open and closed class words: class and frequency effects.

Closed class (determiners, pronouns, conjunctions, prepositions etc. ) and open class (nouns, verbs, adjectives, adverbs) words have different linguistic functions and have been proposed to be processed by different neural systems. Here, event-related potentials (ERPs) were recorded in young German-speaking subjects while they read closed class and open class words flashed upon a video-screen. In the first experiment closed class words were sorted into four different frequency categories and open class words into three categories. The words were presented in a list with the subjects' task to detect occasional non-words. A centroparietal negativity (N400) with a peak latency of about 400 ms varied in amplitude as a function of frequency in both classes. The N400 in closed class items, however, was considerably smaller than that in open class words of similar frequency. A left anterior negativity (N280/LPN) showed some degree of frequency-sensitivity regardless of word class. Only for the very high frequency closed class words a frontal negativity with an onset of about 400 ms was obtained (N400-700). This N400-700 effect was replicated in the second study, in which medium frequency closed and open class words and very high frequency closed class words were presented at the fifth position of simple German sentences. It is suggested that neither N400 nor the left anterior negativity (N280/LPN) distinguish qualitatively between the two word classes and thus claims about different brain systems involved in the processing of open and closed class words are not substantiated electrophysiologically. The N400-700 effect is possibly related to specific grammatical functions of some closed class items, such as determiners.