20(S)-Protopanaxadiol Exerts Antidepressive Effects in Chronic Corticosterone-Induced Rodent Animal Models as an Activator of Brain-Type Creatine Kinase.

20(S)-Protopanaxadiol (PPD) is one of the bioactive ingredients in ginseng and possesses neuroprotective properties. Brain-type creatine kinase (CK-BB) is an enzyme involved in brain energy homeostasis via the phosphocreatine-creatine kinase system. We previously identified PPD as directly bound to CK-BB and activated its activity in vitro. In this study, we explored the antidepressive effects of PPD that target CK-BB. First, we conducted time course studies on brain CK-BB, behaviors, and hippocampal structural plasticity responses to corticosterone (CORT) administration. Five weeks of CORT injection reduced CK-BB activity and protein levels and induced depression-like behaviors and hippocampal structural plasticity impairment. Next, a CK inhibitor and an adeno-associated virus-targeting CKB were used to diminish CK-BB activity or its expression in the brain. The loss of CK-BB in the brain led to depressive behaviors and morphological damage to spines in the hippocampus. Then, a polyclonal antibody against PPD was used to determine the distribution of PPD in the brain tissues. PPD was detected in the hippocampus and cortex and observed in astrocytes, neurons, and vascular endotheliocytes. Finally, different PPD doses were used in the chronic CORT-induced depression model. Treatment with a high dose of PPD significantly increased the activity and expression of CK-BB after long-term CORT injection. In addition, PPD alleviated the damage to depressive-like behaviors and structural plasticity induced by repeated CORT injection. Overall, our study revealed the critical role of CK-BB in mediating structural plasticity in CORT-induced depression and identified CK-BB as a therapeutic target for PPD, allowing us to treat stress-related mood disorders.

Parallel control of cold-triggered adipocyte thermogenesis by UCP1 and CKB.

That uncoupling protein 1 (UCP1) is the sole mediator of adipocyte thermogenesis is a conventional viewpoint that has primarily been inferred from the attenuation of the thermogenic output of mice genetically lacking Ucp1 from birth (germline Ucp1-/-). However, germline Ucp1-/- mice harbor secondary changes within brown adipose tissue. To mitigate these potentially confounding ancillary changes, we constructed mice with inducible adipocyte-selective Ucp1 disruption. We find that, although germline Ucp1-/- mice succumb to cold-induced hypothermia with complete penetrance, most mice with the inducible deletion of Ucp1 maintain homeothermy in the cold. However, inducible adipocyte-selective co-deletion of Ucp1 and creatine kinase b (Ckb, an effector of UCP1-independent thermogenesis) exacerbates cold intolerance. Following UCP1 deletion or UCP1/CKB co-deletion from mature adipocytes, moderate cold exposure triggers the regeneration of mature brown adipocytes that coordinately restore UCP1 and CKB expression. Our findings suggest that thermogenic adipocytes utilize non-paralogous protein redundancy-through UCP1 and CKB-to promote cold-induced energy dissipation.

Comparative study of aluminum speciation on brain-type creatine kinase: Enzyme kinetic, molecular docking, cellular experiment, and mouse model study.

Brain-type Creatine kinase (CK-BB), which has a high affinity for Aluminum (Al), and its abnormality is closely related to neurodegenerative diseases. In this study, the comparative effect of Al speciation on the bioactivity of CK-BB has been studied by the inhibition kinetics method, molecular docking, cellular experiment, and mouse model study. Results showed that the half-inhibitory concentration of AlCl3 was 0.67 mM, while Al(mal)3 was 3.81 mM. Fluorescence spectra showed that Al(mal)3 had a more substantial effect on the endogenous fluorescence of CK-BB than AlCl3. Molecular docking showed that AlCl3 was closer to the active site of CK-BB. C6 cells were used to explore the enzyme activity and intracellular distribution of CK-BB by AlCl3 or Al(mal)3. AlCl3 treatment may directly affect CK-BB activity and cause insufficient local ATP supply in cells which affected the formation of F-actin and cell morphology. The change in the hydrophobicity of CK-BB induced by Al(mal)3 affected the movement of CK-BB, which subsequently activated thecytochrome C (Cyt C)/Caspase 9/Caspase 3 pathway. Similar results have been found in vivo experiments. This study demonstrated that interaction between Al and CK-BB might be related to the process of Al-induced energy metabolism disorders, in which the Al speciation revealed differentiated toxicity mechanisms.

Creatine kinase B controls futile creatine cycling in thermogenic fat.

Obesity increases the risk of mortality because of metabolic sequelae such as type 2 diabetes and cardiovascular disease1. Thermogenesis by adipocytes can counteract obesity and metabolic diseases2,3. In thermogenic fat, creatine liberates a molar excess of mitochondrial ADP-purportedly via a phosphorylation cycle4-to drive thermogenic respiration. However, the proteins that control this futile creatine cycle are unknown. Here we show that creatine kinase B (CKB) is indispensable for thermogenesis resulting from the futile creatine cycle, during which it traffics to mitochondria using an internal mitochondrial targeting sequence. CKB is powerfully induced by thermogenic stimuli in both mouse and human adipocytes. Adipocyte-selective inactivation of Ckb in mice diminishes thermogenic capacity, increases predisposition to obesity, and disrupts glucose homeostasis. CKB is therefore a key effector of the futile creatine cycle.

Serum and Saliva Concentrations of Biochemical Parameters in Men with Prostate Cancer and Benign Prostate Hyperplasia.

OBJECTIVES: To find suitable biomarkers for diagnosis of prostate cancer (PC) in serum and saliva; also, to evaluate the diagnostic efficacy of saliva in patients with PC. METHODS: This case-control study included 20 patients with PC and 20 patients with benign prostatic hyperplasia (BPH). Blood and saliva were collected from the participants and centrifuged. Serum and supernatant saliva were used for biochemical analysis. We evaluated serum and salivary levels of urea, creatinine, prostate-specific antigen (PSA), creatine kinase BB (CK-BB), zinc, ß-2 microglobulin (B2M), and melatonin. Also, we used Mann-Whitney U testing, Spearman correlation coefficients, and receiver operating characteristic (ROC) analysis to evaluate the data. RESULTS: Serum and salivary concentrations of urea, creatinine, PSA, CK-BB, zinc, and B2M were significantly higher in patients with PC, compared with the BPH group (P <.05). However, serum and salivary concentrations of melatonin were significantly lower in patients with PC, compared with BPH group (P <.05). In both groups, salivary concentrations of all markers were lower (P <.05), compared with those values in serum. We observed positive correlation between serum and salivary concentrations of all markers studied (P <.05). CONCLUSION: From the data, we conclude that investigation using saliva specimens is a noninvasive, simple, and effective tool for screening of biochemical parameters.

GnRH antagonist alters the migration of endometrial epithelial cells by reducing CKB.

Some studies have demonstrated that the implantation rate of fresh transfer cycles is lower in the gonadotropin-releasing hormone antagonist (GnRH-ant) protocol than in the GnRH agonist (GnRH-a) protocol during in vitro fertilization (IVF). This effect may be related to endometrial receptivity. However, the mechanisms are unclear. Here, endometrial tissues obtained from the mid-secretory phase of patients treated with GnRH-a or GnRH-ant protocols and from patients on their natural cycle were assessed. Endometrial expression of B-type creatine kinase (CKB), which plays important roles in the implantation phase, was significantly reduced in the GnRH-ant group. At the same time, expression of the endometrial receptivity marker HOXA10 was considerably reduced in the GnRH-ant group. GnRH-ant exposure in endometrial epithelial cells (EECs) in vitro decreased CKB expression and ATP generation and blocked polymerization of actin. Furthermore, in vitro GnRH-ant-exposed Ishikawa cells showed enhanced F-actin depolymerization, and these effects were rescued by CKB overexpression. Similar effects were observed after CKB knockdown, and these effects were rescued by CKB overexpression. Moreover, cell migration was decreased in CKB-knockdown Ishikawa cells compared with that in control cells, and this effect was also rescued by CKB overexpression. Overall, these findings showed that GnRH-ant affected CKB expression in EECs, resulting in cytoskeletal damage and migration failure. These results provide insight into the roles and molecular mechanisms of GnRH-ant treatment in the endometrium.

PERK-mediated induction of microRNA-483 disrupts cellular ATP homeostasis during the unfolded protein response.

Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), which reduces levels of misfolded proteins. However, if ER homeostasis is not restored and the UPR remains chronically activated, cells undergo apoptosis. The UPR regulator, PKR-like endoplasmic reticulum kinase (PERK), plays an important role in promoting cell death when persistently activated; however, the underlying mechanisms are poorly understood. Here, we profiled the microRNA (miRNA) transcriptome in human cells exposed to ER stress and identified miRNAs that are selectively induced by PERK signaling. We found that expression of a PERK-induced miRNA, miR-483, promotes apoptosis in human cells. miR-483 induction was mediated by a transcription factor downstream of PERK, activating transcription factor 4 (ATF4), but not by the CHOP transcription factor. We identified the creatine kinase brain-type (CKB) gene, encoding an enzyme that maintains cellular ATP reserves through phosphocreatine production, as being repressed during the UPR and targeted by miR-483. We found that ER stress, selective PERK activation, and CKB knockdown all decrease cellular ATP levels, leading to increased vulnerability to ER stress-induced cell death. Our findings identify miR-483 as a downstream target of the PERK branch of the UPR. We propose that disruption of cellular ATP homeostasis through miR-483-mediated CKB silencing promotes ER stress-induced apoptosis.

Differences in structure and function between human and murine tau.

The main difference between the primary structures of human and mouse tau can be found at the N-terminal end of the protein. Residues 17 to 28 in human tau are not present in the mouse form of the molecule. Here we tested the capacity of these human tau residues to bind to specific proteins. Several proteins were observed to bind to these residues. Among those that showed the greatest binding were three related to energetic processes: enolase, glyceraldehyde 3 phosphate dehydrogenase and creatine kinase B. The latter did not bind to tau from brain extracts taken from patients with Alzheimer's disease (AD). This lack of binding could be due to the modification of CKB by oxidation in AD.

Possible ATP trafficking by ATP-shuttles in the olfactory cilia and glucose transfer across the olfactory mucosa.

Odor transduction in the cilia of olfactory sensory neurons involves several ATP-requiring enzymes. ATP is generated by glycolysis in the ciliary lumen, using glucose incorporated from surrounding mucus, and by oxidative phosphorylation in the dendrite. During prolonged stimulation, the cilia maintain ATP levels along their length, by unknown means. We used immunochemistry, RT-PCR, and immunoblotting to explore possible underlying mechanisms. We found the ATP-shuttles, adenylate and creatine kinases, capable of equilibrating ATP. We also investigated how glucose delivered by blood vessels in the olfactory mucosa reaches the mucus. We detected, in sustentacular and Bowman's gland cells, the crucial enzyme in glucose secretion glucose-6-phosphatase, implicating both cell types as putative glucose pathways. We propose a model accounting for both processes.

Creatine kinase in human erythrocytes: A genetic anomaly reveals presence of soluble brain-type isoform.

For maintaining energy homeostasis, creatine kinase (CK) is present at elevated levels in tissues with high and/or fluctuating energy requirements such as muscle, brain, and epithelia, while there is very few CK, if any, in peripheral blood cells. However, an ectopic expression of brain-type creatine kinase (BCK) has been reported for platelets and leukocytes in an autosomal dominant inherited anomaly named CKBE. Here we investigated CK in erythrocytes of CKBE individuals from eight unrelated families. The data revealed a varying but significant increase of CK activity in CKBE individuals as compared to controls, reaching an almost 800-fold increase in two CKBE individuals which also had increased erythrocyte creatine. Immunoblotting with highly specific antibodies confirmed that the expressed CK isoform is BCK. Cell fractionation evidenced soluble BCK, suggesting cytosolic and not membrane localization of erythrocyte CK as reported earlier. These results are discussed in the context of putative CK energy buffering and transfer functions in red blood cells.

Effects of obesity on protein kinase C, brain creatine kinase, transcription, and autophagy in cochlea.

Diet-induced obesity (DIO) has been shown to exacerbate hearing degeneration via increased hypoxia, inflammatory responses, and cell loss via both caspase-dependent and caspase-independent apoptosis signaling pathways. This study aimed to investigate the effects of DIO on the mRNA expressions of protein kinase c-ß (PKC-ß), brain creatine kinase (CKB), transcription modification genes, and autophagy-related genes in the cochlea of CD/1 mice. Sixteen 4-week-old male CD/1 mice were randomly divided into 2 groups. For 16 weeks, the DIO group was fed a high fat diet (60% kcal fat) and the controls were fed a standard diet. Morphometry, biochemistry, auditory brainstem response thresholds, omental fat, and histopathology of the cochlea were compared. Results showed that body weight, body length, body-mass index, omental fat, plasma triglyceride, and auditory brainstem response thresholds were significantly elevated in the DIO group compared with those of the control group. The ratio of vessel wall thickness to radius in the stria vascularis was significantly higher in the DIO group. The cell densities in the spiral ganglion, but not in the spiral prominence, of the cochlea were significantly lower in the DIO group. The expression of histone deacetylation gene 1 (HDAC1) was significantly higher in the DIO group than the control group. However, the expressions of PKC-ß, CKB, HDAC3, histone acetyltransferase gene (P300), lysosome-associated membrane protein 2 (Lamp2), and light chain 3 (Lc3) genes were not significantly different between two groups. These results suggest that DIO might exacerbate hearing degeneration possibly via increased HDAC1 gene expression in the cochlea of CD/1 mice.

[Creatine kinase isoform B distribution in the brain in schizophrenia]. / Raspredelenie izoformy V kreatinkinazy v mozge pri shizofrenii.

AIM: To compare patterns of brain isoform creatine phosphokinase (CPK B) distributions in post-mortem brain from patients with schizophrenia (Sch) and patients with somatic diseases (controls). MATERIAL AND METHODS: Extracts of readily soluble and membrane-associated proteins were prepared from post-mortem samples of prefrontal cortex (Brodmann area 10), anterior (area 24) and posterior (area 23) cingulate cortex, hippocampus and cerebellum cortex from patients with Sch and control group (the samples were matched by age and postmortem interval). CPK enzymatic activity was measured by determination of inorganic phosphate, amounts of immunoreative CPK В were estimated by ECL-Western blotting using monoclonal antibodies. RESULTS: A significant decrease in CPK activity and amounts of immunoreative CPK В was observed in fractions of readily soluble proteins in all studied brain structures of patients with Sch compared to controls (p<0.01). Significant differences in CPK activity were found in membrane-associated protein fractions from the hippocampus (p<0.01), but not from the cingulate cortex (areas 23 and 24), of Sch patients compared with controls, whereas no difference between groups was found in levels of immunoreactive CPK B in membrane-associated protein fractions from the cingulate cortex (areas 23 and 24) and hippocampus. The decrease in the amount of CPK B in the frontal cortex of patients with Sch was confirmed by purification of CPK B active dimer from brain samples of patients with Sch and controls. CONCLUSION: Changes in the levels of CPK brain isoform in the brain of patients with Sch (the decrease in CPK activity and amounts in various brain structures at different extents) lead to the substantial alteration of CPK distribution pattern among the brain areas studied, result in the disturbance of the brain energy metabolism and contribute to Sch pathogenesis.

Cellular compartmentation of energy metabolism: creatine kinase microcompartments and recruitment of B-type creatine kinase to specific subcellular sites.

There is an increasing body of evidence for local circuits of ATP generation and consumption that are largely independent of global cellular ATP levels. These are mostly based on the formation of multiprotein(-lipid) complexes and diffusion limitations existing in cells at different levels of organization, e.g., due to the viscosity of the cytosolic medium, macromolecular crowding, multiple and bulky intracellular structures, or controlled permeability across membranes. Enzymes generating ATP or GTP are found associated with ATPases and GTPases enabling the direct fueling of these energy-dependent processes, and thereby implying that it is the local and not the global concentration of high-energy metabolites that is functionally relevant. A paradigm for such microcompartmentation is creatine kinase (CK). Cytosolic and mitochondrial isoforms of CK constitute a well established energy buffering and shuttling system whose functions are very much based on local association of CK isoforms with ATP-providing and ATP-consuming processes. Here we review current knowledge on the subcellular localization and direct protein and lipid interactions of CK isoforms, in particular about cytosolic brain-type CK (BCK) much less is known compared to muscle-type CK (MCK). We further present novel data on BCK, based on three different experimental approaches: (1) co-purification experiments, suggesting association of BCK with membrane structures such as synaptic vesicles and mitochondria, involving hydrophobic and electrostatic interactions, respectively; (2) yeast-two-hybrid analysis using cytosolic split-protein assays and the identifying membrane proteins VAMP2, VAMP3 and JWA as putative BCK interaction partners; and (3) phosphorylation experiments, showing that the cellular energy sensor AMP-activated protein kinase (AMPK) is able to phosphorylate BCK at serine 6 to trigger BCK localization at the ER, in close vicinity of the highly energy-demanding Ca(2+) ATPase pump. Thus, membrane localization of BCK seems to be an important and regulated feature for the fueling of membrane-located, ATP-dependent processes, stressing again the importance of local rather than global ATP concentrations.

Biomarkers in traumatic brain injury: a review.

Biomarkers allow physiological processes to be monitored, in both health and injury. Multiple attempts have been made to use biomarkers in traumatic brain injury (TBI). Identification of such biomarkers could allow improved understanding of the pathological processes involved in TBI, diagnosis, prognostication and development of novel therapies. This review article aims to cover both established and emerging TBI biomarkers along with their benefits and limitations. It then discusses the potential value of TBI biomarkers to military, civilian and sporting populations and the future hopes for developing a role for biomarkers in head injury management.

Prx II and CKBB proteins interaction under physiologic al and thermal stress conditions in A549 and HeLa cells.

Peroxiredoxins (Prxs) are versatile enzymes that demonstrate various cell functions as peroxidases, protein chaperones, functions of signal modulators and binding partners. It is well established that Prxs can interact with multiple proteins in cells, such as ASK1, Cdk5-p35, JNK, MIF, PDGF, TK R4 and others. In this study, we attempted to evaluate a possible association between ubiquitous Prx II and ATP/ADP buffering enzyme - brain-type creatine kinase (CK BB). Our co-immunoprecipitation (Co-IP) results from the A549 and HeLa cell lysates with overexpressed HA-Prx II and Flag-CK BB have demonstrated strong association between two proteins under non-stressed conditions. This protein interaction was enhanced by the heat treatment with further HA-Prx II precipitation to the immobilized Flag-CK BB depending on the temperature increase. Temperature induced oligomerization of Prx II may contribute to the formation of Prx II conglomerates, which in turn, can associate with CK BB and increase signal intensities on the blotted membranes. Thus, such association and oligomerization of Prx II could take part in recovery and protection of the CK BB enzyme activity from inactivation during heat-induced stress.

Creatine kinase B is necessary to limit myoblast fusion during myogenesis.

Myoblast fusion is critical for proper muscle growth and regeneration. During myoblast fusion, the localization of some molecules is spatially restricted; however, the exact reason for such localization is unknown. Creatine kinase B (CKB), which replenishes local ATP pools, localizes near the ends of cultured primary mouse myotubes. To gain insights into the function of CKB, we performed a yeast two-hybrid screen to identify CKB-interacting proteins. We identified molecules with a broad diversity of roles, including actin polymerization, intracellular protein trafficking, and alternative splicing, as well as sarcomeric components. In-depth studies of α-skeletal actin and α-cardiac actin, two predominant muscle actin isoforms, demonstrated their biochemical interaction and partial colocalization with CKB near the ends of myotubes in vitro. In contrast to other cell types, specific knockdown of CKB did not grossly affect actin polymerization in myotubes, suggesting other muscle-specific roles for CKB. Interestingly, knockdown of CKB resulted in significantly increased myoblast fusion and myotube size in vitro, whereas knockdown of creatine kinase M had no effect on these myogenic parameters. Our results suggest that localized CKB plays a key role in myotube formation by limiting myoblast fusion during myogenesis.

Extracellular metabolic energetics can promote cancer progression.

Colorectal cancer primarily metastasizes to the liver and globally kills over 600,000 people annually. By functionally screening 661 microRNAs (miRNAs) in parallel during liver colonization, we have identified miR-551a and miR-483 as robust endogenous suppressors of liver colonization and metastasis. These miRNAs convergently target creatine kinase, brain-type (CKB), which phosphorylates the metabolite creatine, to generate phosphocreatine. CKB is released into the extracellular space by metastatic cells encountering hepatic hypoxia and catalyzes production of phosphocreatine, which is imported through the SLC6A8 transporter and used to generate ATP­fueling metastatic survival. Combinatorial therapeutic viral delivery of miR-551a and miR-483-5p through single-dose adeno-associated viral (AAV) delivery significantly suppressed colon cancer metastasis, as did CKB inhibition with a small-molecule inhibitor. Importantly, human liver metastases express higher CKB and SLC6A8 levels and reduced miR-551a/miR-483 levels relative to primary tumors. We identify the extracellular space as an important compartment for malignant energetic catalysis and therapeutic targeting.

Accelerated protein damage in brains of PIMT+/- mice; a possible model for the variability of cognitive decline in human aging.

Isoaspartate formation is a common type of protein damage normally kept in check by the repair enzyme protein-L-isoaspartyl methyltransferase (PIMT). Mice with a knockout of the gene (Pcmt1) for this enzyme (KO, -/-) exhibit a pronounced neuropathology with fatal epileptic seizures at 30-60 days. Heterozygous (HZ, +/-) mice have 50% of the PIMT activity found in wild-type (WT, +/+) mice, but appear normal. To see if HZ mice exhibit accelerated aging at the molecular level, we compared brain extracts from HZ and WT mice at 8 months and 2 years with regard to PIMT activity, isoaspartate levels, and activity of an endogenous PIMT substrate, creatine kinase B. PIMT activity declined modestly with age in both genotypes. Isoaspartate was significantly higher in HZ than WT mice at 8 months and more so at 2 years, rising 5× faster in HZ males and 3× faster in females. Creatine kinase activity decreased with age and was always lower in the HZ mice. These findings suggest the individual variation of human PIMT levels may significantly influence the course of age-related central nervous system dysfunction.

Submembranous recruitment of creatine kinase B supports formation of dynamic actin-based protrusions of macrophages and relies on its C-terminal flexible loop.

Subcellular partitioning of creatine kinase contributes to the formation of patterns in intracellular ATP distribution and the fuelling of cellular processes with a high and sudden energy demand. We have previously shown that brain-type creatine kinase (CK-B) accumulates at the phagocytic cup in macrophages where it is involved in the compartmentalized generation of ATP for actin remodeling. Here, we report that CK-B catalytic activity also helps in the formation of protrusive ruffle structures which are actin-dependent and abundant on the surface of both unstimulated and LPS-activated macrophages. Recruitment of CK-B to these structures occurred transiently and inhibition of the enzyme's catalytic activity with cyclocreatine led to a general smoothening of surface morphology as visualized by scanning electron microscopy. Comparison of the dynamics of distribution of YFP-tagged CK-mutants and isoforms by live imaging revealed that amino acid residues in the C-terminal segment (aa positions 323-330) that forms one of the protein's two mobile loops are involved in partitioning over inner regions of the cytosol and nearby sites where membrane protrusions occur during induction of phagocytic cup formation. Although wt CK-B, muscle-type CK (CK-M), and a catalytically dead CK-B-E232Q mutant with intact loop region were normally recruited from the cytosolic pool, no dynamic transition to the phagocytic cup area was seen for the CK-homologue arginine kinase and a CK-B-D326A mutant protein. Bioinformatics analysis helped us to predict that conformational flexibility of the C-terminal loop, independent of conformational changes induced by substrate binding or catalytic activity, is likely involved in exposing the enzyme for binding at or near the sites of membrane protrusion formation.

Proteomics reveals intersexual differences in the rat brain hippocampus.

It is widely accepted that intersexual differences occur in cognitive domains, e.g., in spatial learning and memory. The hippocampus plays important roles in the consolidation of information from short-term memory to long-term memory and spatial navigation. However, it still remains unknown whether the hippocampal proteomic profiling differs between males and females. In this study, we investigated the intersexual differences in protein expression of hippocampi using the two-dimensional electrophoresis analysis. In all, 33 differentially expressed proteins were characterized by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry and validated by Western-blotting analysis. In line with Western-blotting validation, the proteomic identification revealed the overexpression of glial fibrillary acidic protein in female rats' hippocampi, and the overexpression of both creatine kinase B-type and DRP-2 in male rats' hippocampi. The intersexual differences in hippocampal proteomic profiling are probably closely related to those in spatial learning and memory abilities.