Expression of brain-type creatine kinase and ubiquitous mitochondrial creatine kinase in the fetal rat brain: Evidence for a nuclear energy shuttle

To test the hypothesis that embryonic brain cells utilize a creatine phosphate energy shuttle, we examined the pattern of creatine kinase (CK) isoform expression and localization in the fetal rat brain. Moderate levels of CK activity are present at embryonic day 14 (7 U/mg protein) and decrease slightly until 3 days postpartum followed by a rapid, fourfold up- regulation to adult levels by 1 month (18 U/mg protein). In parallel with changes in enzyme activity, there is a biphasic and coordinate pattern of expression of brain-type CK (BCK) and ubiquitous mitochondrial CK (uMtCK) determined by nondenaturing electrophoresis and immunoblot analysis. The localization of CK isoforms was examined by immunocytochemistry, and, during the fetal period, BCK and uMtCK immunoreactivity was detected throughout the central and peripheral nervous system, especially in neuroepithelial regions of the cerebral vesicles and spinal cord. In large cells within the olfactory neuroepithelium and ventral spinal cord, differential compartmentation of CK isoforms was evident, with BCK localized primarily in cell nuclei, whereas uMtCK immunoreactivity was present in the cell body (but not within nuclei). In olfactory bulb neuroepithelium, both isoforms were expressed in the middle zone of the germinal layer associated with DNA synthesis. In embryonic skeletal and cardiac muscle, which also express BCK, the same compartmentation of BCK was seen, with BCK localized primarily in the cell nucleus of cardiac and skeletal myoblasts. These results demonstrate a coordinate pattern of expression and compartmentation of BCK and uMtCK isoforms in the fetal brain that, in some cells, provides the anatomic basis for a nuclear energy shuttle. © 1995 Wiley-Liss, Inc.     

Compartment of brain-type cretine kinase and ubiquitous mitochondrial cretine kinase in neurons: Evidence for a cretine phosphate energy shuttle in adult rat brain

Multiple isoforms of cretine kinase (CK) are expressed in specific cell types as part of an energy delivery or shuttle system. To test hypothesis that neurons utilize a cretine phosphate energy shuttle, we examined the pattern of CK isoform expression and localization in adult rat brain. Two isoforms of CK are present in brain extracts, “brain-type,” or BCK, and the ubiquitin form of the mitochondrial CK (uMtCK), as detected by enzyme activity following nondenaturing electrophoresis and by Western blotting following denaturing electrophorsis. In formalin-fixed and parafinn-embedded sections of rat brain, uMtCK immunostaining is detected in the somata of all Golgi type I neurons in the cerebellum, pontine reticular formation, red nucleus, hippocampus, and cerebral cortex. Immunostainig for uMtCK appears throughout the cell body but not in nuclei. BCK immunostaining is also present in somata of Golgi type I neurons in the cerebellum, red nucleus, and pons and is distributed throughout the cell body and within nuclei. BCK immunostaining also appeared in neuronal processes and is concentrated in the molecular layers of the cerebellum and the hippocampus and in cortical pyramidal cell dendrites. These results demonstrate a coordinate pattern of expression and compartmentation of BCK and MtCK isoform in neurons, which provides an anatomic basis for the transfer of metabolic energy via a cretine phosphate energy shuttle. © 1994 Wiley-Liss, Inc.     

Brain creatine phosphate and creatine kinase in mice fed an analogue of creatine

Brain phosphocreatine (PCr) concentration and creatine kinase (CK) activity have been studied by 31P nuclear magnetic resonance (NMR) spectroscopy in mice fed an analogue of creatine, beta-guanidinopropionic acid (GPA). The phosphorylated analogue (GPAP), which almost completely replaces PCr in skeletal muscle, is a poor substrate for CK. Mice, which received GPA in food (2%) and water (0.5%) for up to 9 months beginning at 35 days of age, were normal in appearance and activity. Maximal brain GPAP concentration, reached after two weeks of feedings, was approximately equal to the concentration of PCr. The concentration of PCr decreased at least 20% relative to that of the nucleoside triphosphates. When GPA feedings were stopped, GPAP disappeared in about 20 days from skeletal muscle, but only after 40-50 days from brain. Steady-state NMR saturation transfer studies showed a markedly reduced chemical exchange rate from PCr to ATP in brains of GPA-fed mice. These results suggest a compartmentation of brain PCr. The GPA-accessible PCr compartment has a slow rate of PCr turnover compared to skeletal muscle. The slow reaction rate of the GPA-inaccessible PCr as a CK substrate is consistent with the hypothesis that this residual PCr is the same compartment which is stable in hypoxic or seizing animals.     

Opposite transitions of chick brain catalytically active cytosolic creatine kinase isoenzymes during development

Postnatally the rat brain synthesizes catalytic forms of muscle type (MM) and heart type (MB) creatine kinase (CK), besides the supposedly sole type vertebrate brain-specific (BB) CK. We intended to demonstrate that in Rhode Island chicken brain, cytosolic (c) CK isoenzymatic transitions, (for example BB–CK is followed by the appearance of MB–CK and MM–CK during muscle differentiation), can also occur during development and aging. Cytosolic post 125 000×g, mitochondrial CK-free, brain samples were obtained for zone electrophoresis separation and identification of catalytically active cCK isoforms. BB–CK was never found during chicken brain ontogeny. Against the accepted view, an opposite isoenzyme transition pattern from MM through BB–CK was found in the chicken embryonic brain from the very early stages of development up to day 2 post-hatching. At very early stages of chicken brain ontogeny constitutive MM– and MB–CK isoenzymes were present before the advent of creatine. It seems to be that typical and atypical brain MM– and MB–CK could be working as ATPases in the absence of creatine before embryonic stage 28 (day 5.5) and/or such CK isoforms may begin to form part of the slow component b in developing early neurons and later in the nuclei of glial cells to be used by the CK/phosphocreatine (PC) system as the neural tissues mature. The post-hatching transition pattern showed simultaneous expression of more than one CK isoenzyme within the same neural sample as in post-natal rat brain, presumably due to regional differential transphosphorylation requirements. Strain-dependent enzymatic specific activities have been reported in several species. Since equivalent values of brain CK specific activity were obtained previously from the embryonic plateau phase of CK activity during White Leghorn development, and those from Rhode Island brain neurons cultured 11 days, we compared if, in vivo, a similar brain CK specific activity pattern was physiologically equivalent during Rhode Island and White Leghorn chicken ontogeny. We found quantitatively different strain-specific CK specific activity patterns during this period. Rhode Island brain CK activity values were approximately 4.5-fold those of White Leghorn ones. This indicates that production of energy from anaerobic metabolism and transphosphorylation by the CK/PC system to synthesize ATP more efficiently is strain-specific. In Rhode Islands, there was an age-dependent increase of CK specific activity, mostly in older animals (440% above the value found during the embryonic plateau), when the Krebs cycle and glycolysis lose capacity. During adult life and aging, under physiological conditions, the three CK isoenzymes may participate in diverse functions of the different cell compartments of brain glia and neurons with regard to their high and fluctuating energy demands that are not completely covered by anaerobic and aerobic glycolisis.     

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.     

Proton magnetic resonance spectroscopic creatine correlates with creatine transporter protein density in rat brain

Creatine (Cr) is an amino acid, which upon phosphorylation is utilized as an energy reservoir in cells with high-energy demand. The ongoing catabolism of creatine to creatinine requires a permanent creatine replenishment into the cells. Because neurons themselves cannot synthesize creatine, they have to take it up via the creatine transporter (CrT). Thus, the concentration of intracellular Cr available for the Cr/PCr shuttle system depends on the expression level of CrT protein. The proton magnetic resonance spectroscopy (MRS) creatine peak (total creatine=tCr) constitutes of two metabolites, namely Cr and phosphocreatine (PCr). We have quantified the level of CrT protein expression with western blotting and compared it to tCr content as estimated by in vitro MRS in Sprague-Dawley rats. Under the assumption of hemispheric symmetry, we took identical samples from left and right hemisphere, which were used for in vitro MRS (tCr) and for western blotting (CrT), respectively. Altogether, it was possible to take 90 corresponding brain samples from 31 animals. A Pearson linear regression analysis for CrT and tCr revealed p<0.0001, explaining 14% of the variance. Since MR-detectable alterations of tCr in the human brain are widespread (e.g. in most major psychiatric disorders proton MRS detectable tCr alterations have been described as regionally and usually state dependent) it is stringent to elucidate their meaning. An influence of tCr on the brain's energy regulating system seems plausible.     

Brain creatine kinase and creatine transporter proteins in normal and creatine-treated rabbit pups

Systemic creatine (Cr) supplementation increases brain phosphocreatine (PCr) and prevents hypoxic seizures in 15-day-old rabbits. Between 5 and 30 days of age during normal development, rabbit gray matter mitochondrial creatine kinase (Mi-CK) increases 400% while cytosolic CK (BB-CK) increases 60%. In white matter, both isoenzymes show smaller, similar increases (40%) during this period. The Cr transporter protein decreases 60% between 5 and 15 days in both regions. In vivo CK rate constants measured by (31)P nuclear magnetic resonance increase 30% between 10 and 20 days, and then fall 50% between 20 and 30 days in predominantly gray matter slices. Similar maturational changes are seen in predominantly white matter slices. Injecting Cr at 15 days does not significantly change BB-CK or Mi-CK isoenzymes or the in vivo CK reaction rate constants. Thus, the largest change in the CK system associated with suppression of hypoxic seizures in Cr-treated rabbits is increased PCr in gray and white matter.     

Stabilization of ubiquitous mitochondrial creatine kinase preprotein by APP family proteins

Amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease (AD). However, the physiological role of APP and its family members is still unclear. To gain insights into APP function, we used a proteomic approach to identify APP interacting proteins. We report here for the first time a direct interaction between the C-terminal region of APP family proteins and ubiquitous mitochondrial creatine kinase (uMtCK). This interaction was confirmed in vitro as well as in cultured cells and in brain. Interestingly, expression of full-length and C-terminal domain of APP family proteins stabilized uMtCK preprotein in cultured cells. Our data suggest that APP may regulate cellular energy levels and mitochondrial function via a direct interaction and stabilization of uMtCK.     

Creatine promotes the GABAergic phenotype in human fetal spinal cord cultures

In the present study, we investigated the expression pattern of cytosolic brain specific-BB-CK and ubiquitous mitochondrial-creatine kinases (uMt-CK) in developing human spinal cord. Consequently, we studied the effects of creatine treatment on cultured fetal human spinal cord tissue. We found that both CK isoforms were expressed in fetal spinal cord at all time points investigated (5 to 11.5 weeks post conception) and correspondingly specific CK activity was detected. Chronic creatine exposure resulted in significantly higher densities of GABA-immunoreactive neurons in the cultures, while total neuronal cell density was not altered, suggesting a differentiation inducing mechanism of creatine supplementation. Taken together, our observations favour the view that the creatine phosphocreatine system plays an important role in the developing CNS.     

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 left arrow over right arrow 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.     

A position-dependent transgene reveals patterns of gene expression in the developing brain

We have made a detailed analysis of the developing mouse brain using a transgene (HSVtk-lacZ), whose expression is dependent on its unique site of integration. The position-dependent transgene expression defines a novel pattern of gene expression in the developing mouse brain. The transgene is first transcribed on day 10 of gestation in a small cluster of cells in the developing hind brain. Extensive expression is subsequently seen in a number of developing brain structures (cortex, cerebellum, hippocampus), but postnatally this becomes restricted to cell types within distinct anatomical foci principally associated with the olfactory system. Expression in the olfactory system is maintained even into adulthood. Hence, the expression of this transgene is confined to cell types which are known from other studies to sustain developmental plasticity in embryos and in adults. It will be of interest to analyse the transgene pre-integration site and determine if endogenous genes at this locus are also expressed in these cells and if they have a role in maintaining their developmental potential.     

Developmental expression of histone deacetylase 11 in the murine brain

Recent studies indicate that neural cell development in the central nervous system (CNS) correlates with a reduction in acetylation of histone core proteins. Moreover, histone hypoacetylation is thought to be important to oligodendrocyte lineage development. The mechanisms mediating the reduction in acetylation during postnatal neural development remain to be defined. To begin to understand these mechanisms, we investigated the expression of histone deacetylase 11 (HDAC11), a newly identified HDAC, in mouse brain during postnatal development. We show that HDAC11 was widely expressed in the brain and that this expression gradually increased in a region-specific pattern between birth and 4 weeks of age. At the cellular level HDAC11 protein was predominately localized in the nuclei of mature oligodendrocytes but only minimally in astrocytes. Although dentate gyrus granule neurons abundantly expressed HDAC11, granule neuron precursors in the subgranule layer exhibited little HDAC11 immunoreactivity. Double-immunostaining of the corpus callosum and dentate gyrus demonstrated that HDAC11 and Ki67, a cell-proliferating marker, are rarely colocalized in same cells. Our data show that HDAC11 was expressed in the developing brain in a temporal and spatial pattern that correlates with the maturation of neural cells, including cells of the oligodendrocyte lineage. These findings support a role for HDAC11 in CNS histone deacetylation and the development of oligodendrocytes and neurons during postnatal development.     

Expression of the long form of leptin receptor (Ob-Rb) mRNA in the brain of mouse embryos and newborn mice

The long form of the leptin receptor (Ob-Rb) has a cytoplasmic domain which activates the JAK-STAT signal transduction pathway. It is related to appetite and energy expenditure and is expressed in various parts of the brain in adults. In embryos, however, the detailed distribution of Ob-Rb expression sites and the function of the leptin-Ob-Rb system remain unclear, although leptin is detected in human cord plasma and leptin mRNA is detected in mouse embryos. In this study, we investigated the Ob-Rb mRNA expression pattern in the brains of mouse embryos and newborn mice by RT-PCR and in situ hybridization. At embryonic day 10.5 (E10.5), Ob-Rb mRNA was already detected in the brain by RT-PCR. By in situ hybridization, Ob-Rb mRNA was observed in the ventricular zone of the rhombencephalon at E11.5. At E12.5, it was also expressed in the ventricular zone of the telencephalon, mesencephalon and cerebellar primordium. From E14. 5 it was expressed in the cortical plate of the telencephalon and the ventricular zone of the thalamus. At E16.5, it was expressed in the premamillary hypothalamic nucleus, superficial gray matter of the superior colliculus, external germinal and Purkinje cell layers of the cerebellum, and facial nucleus. At E18.5, it was expressed in the arcuate nucleus and ventromedial hypothalamic nucleus. These results suggest that the leptin-Ob-Rb system is related to brain development.     

Expression of prostaglandin H synthase-2 in human brain tumors

Numerous studies have demonstrated that prostaglandin H synthase-2 (PHS-2) is involved in gastrointestinal carcinogenesis, and that nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit PHS, can reduce the risk of colon cancer. In brain tumors, elevated prostaglandin production and its correlation to anaplastic grade of gliomas have been demonstrated. To determine whether the increased prostaglandin production is due to enhanced expression of PHS-2 and whether the up-regulation of PHS-2 has any correlation to histopathological findings in brain tumors, we evaluated the profile of PHS expression in several human glioma cell lines and surgical specimens from patients with various types of brain tumors. In glioma cell lines, five out of six cell lines showed constitutive expression of PHS-2, whereas PHS-1 was weakly expressed in all of them. All surgical specimens, except an ependymoma, which expressed both isozymes equally, expressed PHS-2 mRNA predominantly. Immunohistochemistry of various types of brain tumors, including six glioblastomas, nine astrocytomas, six meningiomas, five medulloblastomas, four craniopharyngiomas, three ependymomas, three neurinomas, two oligodendrogliomas, two malignant lymphomas, two dysembryoplastic neuroepitherial tumors and one metastatic brain tumor showed PHS-2 staining in most cases. In gliomas, astrocytomas (grade 2 and 3) were strongly stained, but the staining intensity of glioblastomas was relatively weak. Meningiomas and a metastatic brain tumor were also strongly stained. Our data thus suggest that most brain tumors express PHS-2, which may also play a role in tumorigenesis in the brain.     

In vivo phosphocreatine and ATP in piglet cerebral gray and white matter during seizures

The creatine kinase (CK) reaction is thought to be important in coupling ATP metabolism and regulating ADP concentration in tissues with high and variable ATP turnover, including cerebral gray matter (GM). There is low phosphocreatine (PCr), low CK reaction rates, and high mitochondrial CK (MiCK) isoenzyme activity in GM compared to white matter (WM). To compare the CK reaction in GM and WM when ATP metabolism is high, CK reactants and reaction rates were measured in predominantly GM and WM slices in vivo in 2 and 14-day old piglets during pentylenetetrazole (PTZ) seizures using ³¹P nuclear magnetic resonance (NMR) 1-dimensional chemical shift imaging (CSI). Arterial pressure, temperature, and blood gasses were stable at both ages. Before seizures, the PCr/nucleoside triphosphate (NTP) ratio was higher in WM than GM at both ages with a developmental increase seen in WM. The CK reaction rate constant increased in both regions between 2 and 14 days. During seizures, PCr/NTP increased in GM at 14 days due to increased PCr while the ratio and PCr decreased in WM. The NTP was more stable in WM and GM at both ages. The CK reaction rate decreased in both regions more at 2 than at 14 days. Thus, brain ATP, deduced from NTP, is stable during seizures in the piglet. In GM stable ATP is associated with a unique increase in PCr concentration.     

Quantitation of ATP-binding cassette subfamily-A transporter gene expression in primary human brain cells

Five ATP-binding cassette (ABC) subfamily-A transporters (ABCA1, ABCA2, ABCA3, ABCA7 and ABCA8) are expressed in the brain. These transporters may regulate brain lipid transport; however, their relative expression level in isolated human brain cells is unknown. We developed real-time polymerase chain reaction assays to quantify the expression of these genes in human neurons, astrocytes, oligodendrocytes, microglia and cell lines. Neurons expressed predominantly ABCA1 and ABCA3; astrocytes ABCA1, ABCA2 and ABCA3; microglia ABCA1 and oligodendrocytes ABCA2 and ABCA3. Although ABCA7 and ABCA8 expression was relatively low in all cells, the highest expression occurred in microglia and neurons, respectively. ABCA gene expression in the NTERA-2 and MO3.13 cell lines closely resembled the ABCA expression pattern of primary neurons and oligodendrocytes, respectively.     

Regional expression of 5-HT1B receptor mRNA in the human brain

The regional mRNA expression pattern of 5-HT(1B) receptors has been extensively characterized in the rodent and guinea pig brain, but a detailed mapping of the 5-HT(1B) receptor mRNA expression in the human brain has not previously been performed. In the present study, the mRNA expression of 5-HT(1B) receptors was analyzed using in situ hybridization histochemistry and whole hemisphere sections of the human postmortem brain. The mRNA expression was compared with the autoradiographic distribution of 5-HT(1B) receptors. High levels of mRNA expression were found in the striatum, cortex, lateral geniculate nucleus, and raphe nuclei. The expression was higher in ventral than in dorsal striatal regions and was absent from the substantia nigra and pallidum, where high levels of 5-HT(1B) receptors were found. A layer-specific expression pattern was observed in cortical regions. The results extend previous knowledge about the localization of the 5-HT(1B) receptor in the human brain. This study provides evidence of a mismatch of the regional expression of 5-HT(1B) receptor mRNA and the 5-HT(1B) receptor distribution in human brain, similar to what has been demonstrated in other species. This is in line with the localization of this receptor subtype in nerve terminals. The results give support to species differences in the cortical mRNA expression pattern of this receptor subtype.     

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.