Neuron–Astrocyte Interactions,Pyruvate Carboxylation and the Pentose Phosphate Pathway in the Neonatal Rat Brain

Glucose and acetate metabolism and the synthesis of amino acid neurotransmitters, anaplerosis, glutamate-glutamine cycling and the pentose phosphate pathway (PPP) have been extensively investigated in the adult, but not the neonatal rat brain. To do this, 7 day postnatal (P7) rats were injected with [1-¹³C]glucose and [1,2-¹³C]acetate and sacrificed 5, 10, 15, 30 and 45 min later. Adult rats were injected and sacrificed after 15 min. To analyse pyruvate carboxylation and PPP activity during development, P7 rats received [1,2-¹³C]glucose and were sacrificed 30 min later. Brain extracts were analysed using ¹H- and ¹³C-NMR spectroscopy. Numerous differences in metabolism were found between the neonatal and adult brain. The neonatal brain contained lower levels of glutamate, aspartate and N-acetylaspartate but similar levels of GABA and glutamine per mg tissue. Metabolism of [1-¹³C]glucose at the acetyl CoA stage was reduced much more than that of [1,2-¹³C]acetate. The transfer of glutamate from neurons to astrocytes was much lower while transfer of glutamine from astrocytes to glutamatergic neurons was relatively higher. However, transport of glutamine from astrocytes to GABAergic neurons was lower. Using [1,2-¹³C]glucose it could be shown that despite much lower pyruvate carboxylation, relatively more pyruvate from glycolysis was directed towards anaplerosis than pyruvate dehydrogenation in astrocytes. Moreover, the ratio of PPP/glucose-metabolism was higher. These findings indicate that only the part of the glutamate-glutamine cycle that transfers glutamine from astrocytes to neurons is operating in the neonatal brain and that compared to adults, relatively more glucose is prioritised to PPP and pyruvate carboxylation. Our results may have implications for the capacity to protect the neonatal brain against excitotoxicity and oxidative stress.     

Glucose and Intermediary Metabolism and Astrocyte–Neuron Interactions Following Neonatal Hypoxia–Ischemia in Rat

Neonatal hypoxia–ischemia (HI) and the delayed injury cascade that follows involve excitotoxicity, oxidative stress and mitochondrial failure. The susceptibility to excitotoxicity of the neonatal brain may be related to the capacity of astrocytes for glutamate uptake. Furthermore, the neonatal brain is vulnerable to oxidative stress, and the pentose phosphate pathway (PPP) may be of particular importance for limiting this kind of injury. Also, in the neonatal brain, neurons depend upon de novo synthesis of neurotransmitters via pyruvate carboxylase in astrocytes to increase neurotransmitter pools during normal brain development. Several recent publications describing intermediary brain metabolism following neonatal HI have yielded interesting results: (1) Following HI there is a prolonged depression of mitochondrial metabolism in agreement with emerging evidence of mitochondria as vulnerable targets in the delayed injury cascade. (2) Astrocytes, like neurons, are metabolically impaired following HI, and the degree of astrocytic malfunction may be an indicator of the outcome following hypoxic and hypoxic-ischemic brain injury. (3) Glutamate transfer from neurons to astrocytes is not increased following neonatal HI, which may imply that astrocytes fail to upregulate glutamate uptake in response to the massive glutamate release during HI, thus contributing to excitotoxicity. (4) In the neonatal brain, the activity of the PPP is reduced following HI, which may add to the susceptibility of the neonatal brain to oxidative stress. The present review aims to discuss the metabolic temporal alterations observed in the neonatal brain following HI.     

Copper Modulation of Ion Channels of PrP[106–126] Mutant Prion Peptide Fragments

We have shown previously that the protease-resistant and neurotoxic prion peptide fragment PrP[106-126] of human PrP incorporates into lipid bilayer membranes to form heterogeneous ion channels, one of which is a Cu(2+)-sensitive fast cation channel. To investigate the role of PrP[106-126]'s hydrophobic core, AGAAAAGA, on its ability to form ion channels and their regulation with Cu(2+), we used the lipid-bilayer technique to examine membrane currents induced as a result of PrP[106-126] (AA/SS) and PrP[106-126] (VVAA/SSSS) interaction with lipid membranes and channel formation. Channel analysis of the mutant (VVAAA/SSS), which has a reduced hydrophobicity due to substitution of hydrophobic residues with the hydrophilic serine residue, showed a significant change in channel activity, which reflects a decrease in the beta-sheet structure, as shown by CD spectroscopy. One of the channels formed by the PrP[106-126] mutant has fast kinetics with three modes: burst, open and spike. The biophysical properties of this channel are similar to those of channels formed with other aggregation-prone amyloids, indicating their ability to form the common beta sheet-based channel structure. The current-voltage (I-V) relationship of the fast cation channel, which had a reversal potential, E(rev), between -40 and -10 mV, close to the equilibrium potential for K(+) ( E(K) = -35 mV), exhibited a sigmoidal shape. The value of the maximal slope conductance (g(max)) was 58 pS at positive potentials between 0 and 140 mV. Cu(2+) shifted the kinetics of the channel from being in the open and "burst" states to the spike mode. Cu(2+) reduced the probability of the channel being open (P(o)) and the mean open time (T(o)) and increased the channel's opening frequency (F(o)) and the mean closed time (T(c)) at a membrane potential ( V(m)) between +20 and + 140 mV. The fact that Cu(2+) induced changes in the kinetics of this channel with no changes in its conductance, indicates that Cu(2+) binds at the mouth of the channel via a fast channel block mechanism. The Cu(2+)-induced changes in the kinetic parameters of this channel suggest that the hydrophobic core is not a ligand Cu(2+) site, and they are in agreement with the suggestion that the Cu(2+)-binding site is located at M(109) and H(111) of this prion fragment. Although the data indicate that the hydrophobic core sequence plays a role in PrP[106-126] channel formation, it is not a binding site for Cu(2+). We suggest that the role of the hydrophobic region in modulating PrP toxicity is to influence PrP assembly into neurotoxic channel conformations. Such conformations may underlie toxicity observed in prion diseases. We further suggest that the conversions of the normal cellular isoform of prion protein (PrP(c)) to abnormal scrapie isoform (PrP(Sc)) and intermediates represent conversions to protease-resistant neurotoxic channel conformations.     

Host–Parasite Interactions and the Evolution of Gene Expression

Interactions between hosts and parasites provide an ongoing source of selection that promotes the evolution of a variety of features in the interacting species. Here, we use a genetically explicit mathematical model to explore how patterns of gene expression evolve at genetic loci responsible for host resistance and parasite infection. Our results reveal the striking yet intuitive conclusion that gene expression should evolve along very different trajectories in the two interacting species. Specifically, host resistance loci should frequently evolve to co-express alleles, whereas parasite infection loci should evolve to express only a single allele. This result arises because hosts that co-express resistance alleles are able to recognize and clear a greater diversity of parasite genotypes. By the same token, parasites that co-express antigen or elicitor alleles are more likely to be recognized and cleared by the host, and this favours the expression of only a single allele. Our model provides testable predictions that can help interpret accumulating data on expression levels for genes relevant to host−parasite interactions.     

Expression of GDNF and GDNFR-α mRNAs in Muscles of Patients with Motor Neuron Diseases

The mRNA expression levels of GDNF, GDNFR- and RET were examined in the muscles of amyotrophic lateral screlosis (ALS) and X-linked spinal and bulbar muscular atrophy (SBMA). GDNF mRNA levels were significantly elevated to variable extent in the diseased muscles compared to control muscles, although they were not specific to the type of the diseases. The diseased muscles also have a different expression pattern of GDNF mRNA isoforms from controls. GDNF mRNA expression, however, tended to reduce in advanced muscle pathology. On the other hand, GDNFR- mRNA levels were not changed significantly on expression levels in the diseased muscles. In situ hybridization study revealed that GDNF and GDNFR- mRNAs were localized in subsarcolemmal space of muscle cells. RET mRNA was not detected in control nor diseased muscles. These results suggest that the elevated muscle GDNF acts as a trophic signal for motor neurons of motor neuron diseases, implying a possible therapeutic implication of GDNF to this type of diseases.(Drs. Yamamoto, N. Mitsuma, Inukai, Ito, Li, Sobue)     

Transformation and Functional Expression of the rFCA-RRM2 Gene in Rice

The primary aim of the present study was to investigate the overexpression of the rice (Oryza sativa L. subsp, japonica var. Zhonghua 11) flowering control gene (rFCA-RRM2) in monocotyledonous model rice. Constitutive expression of rFCA-RRM2 from the Actl-5 rice promoter caused late flowering in transgenic rice and increased grain weight that was more than 50% higher than that of control plants, which is the first demonstration of rFCA-RRM2 being able to increase rice production. Late flowering was accompanied by strong phenotype and some morphological modifications. These observations suggest that rFCA-RRM2 is a useful tool for phenotype improvement and yield enhancement in cereal crops.     

Chronomics of Pressure Overload–Induced Cardiac Hypertrophy in Mice Reveals Altered Day/Night Gene Expression and Biomarkers of Heart Disease

There is critical demand in contemporary medicine for gene expression markers in all areas of human disease, for early detection of disease, classification, prognosis, and response to therapy. The integrity of circadian gene expression underlies cardiovascular health and disease; however time-of-day profiling in heart disease has never been examined. We hypothesized that a time-of-day chronomic approach using samples collected across 24-h cycles and analyzed by microarrays and bioinformatics advances contemporary approaches, because it includes sleep-time and/or wake-time molecular responses. As proof of concept, we demonstrate the value of this approach in cardiovascular disease using a murine Transverse Aortic Constriction (TAC) model of pressure overload–induced cardiac hypertrophy in mice. First, microarrays and a novel algorithm termed DeltaGene were used to identify time-of-day differences in gene expression in cardiac hypertrophy 8 wks post-TAC. The top 300 candidates were further analyzed using knowledge-based platforms, paring the list to 20 candidates, which were then validated by real-time polymerase chain reaction (RTPCR). Next, we tested whether the time-of-day gene expression profiles could be indicative of disease progression by comparing the 1- vs. 8-wk TAC. Lastly, since protein expression is functionally relevant, we monitored time-of-day cycling for the analogous cardiac proteins. This approach is generally applicable and can lead to new understanding of disease. (Author correspondence: tmartino@uoguelph.ca)     

Cloning of Rat Vitamin K-Dependent γ-Glutamyl Carboxylase and Developmentally Regulated Gene Expression in Postimplantation Embryos

Vitamin K-dependent carboxylase catalyzes the posttranslational modification of glutamate to γ-carboxyglutamate (Gla) in its substrates, the vitamin K-dependent proteins (VKDPs). This modification is required for the activities of the VKDPs. Recent evidence demonstrates previously unrecognized roles for VKDPs as signaling molecules important in the regulation of cell growth, adhesion, and apoptosis, suggesting developmental functions for VKDPs and hence the carboxylase. The tissue distribution and functions of carboxylase in development are unknown. In this study, we isolated and characterized the full-length cDNA encoding the rat carboxylase and analyzed, at the cellular level, the expression of this gene in rat embryos byin situhybridization. We demonstrate that the expression of this gene is highly regulated in a developmental and tissue-specific manner. Hepatocytes, the major site of synthesis of VKDPs of blood coagulation, express carboxylase mRNA late in gestation, in contrast to the central nervous system, mesenchymal, and skeletal tissues which express carboxylase mRNA early during rat embryogenesis. The tissue-specific temporal expression of the carboxylase gene during embryogenesis indicates that vitamin K-dependent carboxylation and the formation of Gla is developmentally regulated. These studies suggest that vitamin K-dependent carboxylation is an important modulator of embryonic VKDP function.     

Altered Protease–Activated Receptor-1 Expression and Signaling in a Malignant Pleural Mesothelioma Cell Line,NCI-H28, with Homozygous Deletion of the β-Catenin Gene

Protease activated receptors (PARs) are G-protein coupled receptors that are activated by an unique proteolytic mechanism. These receptors play crucial roles in hemostasis and thrombosis but also in inflammation and vascular development. PARs have also been implicated in tumor progression, invasion and metastasis. In this study, we investigated expression and signaling of PAR₁ in nonmalignant pleural mesothelial (Met-5A) and malignant pleural mesothelioma (NCI-H28) cells. We found that the expression level of PAR₁ was markedly higher in NCI-H28 cells compared to Met-5A and human primary mesothelial cells. Other three malignant pleural mesothelioma cell lines, i.e. REN, Ist-Mes2, and Mero-14, did not show any significant PAR₁ over-expression compared to Met-5A cell line. Thrombin and PAR₁ activating peptides enhanced Met-5A and NCI-H28 cell proliferation but in NCI-H28 cells higher thrombin concentrations were required to obtain the same proliferation increase. Similarly, thrombin caused extracellular signal-regulated kinase 1/2 activation in both cell lines but NCI-H28 cells responded at higher agonist concentrations. We also determined that PAR₁ signaling through G_q and G_12/13 proteins is severely altered in NCI-H28 cells compared to Met-5A cells. On the contrary, PAR₁ signaling through G_i proteins was persistently maintained in NCI-H28 cells. Furthermore, we demonstrated a reduction of cell surface PAR₁ expression in NCI-H28 and malignant pleural mesothelioma REN cells. Thus, our results provide evidences for dysfunctional PAR₁ signaling in NCI-H28 cells together with reduced plasma membrane localization. The role of PAR₁ in mesothelioma progression is just emerging and our observations can promote further investigations focused on this G-protein coupled receptor.     

Altered Expression of 14-3-3ζ Protein in Spinal Cords of Rat Fetuses with Spina Bifida Aperta

Background

A large number of studies have confirmed that excessive apoptosis is one of the reasons for deficient neuronal function in neural tube defects (NTDs). A previous study from our laboratory used 2-D gel electrophoresis to demonstrate that 14-3-3ζ expression was low in the spinal cords of rat fetuses with spina bifida aperta at embryonic day (E) 17. As a member of the 14-3-3 protein family, 14-3-3ζ plays a crucial role in the determination of cell fate and anti-apoptotic activity. However, neither the expression of 14-3-3ζ in defective spinal cords, nor the correlation between 14-3-3ζ and excessive apoptosis in NTDs has been fully confirmed.

Methodology/Principal Findings

We used immunoblotting and quantitative real-time PCR (qRT-PCR) to quantify the expression of 14-3-3ζ and double immunofluorescence to visualize 14-3-3ζ and apoptosis. We found that, compared with controls, 14-3-3ζ was down-regulated in spina bifida between E12 and E15. Excessive apoptotic cells and low expression of 14-3-3ζ were observed in the dorsal region of spinal cords with spina bifida during the same time period. To initially explore the molecular mechanisms of apoptosis in NTDs, we investigated the expression of microRNA-7 (miR-7), microRNA-375 (miR-375) and microRNA-451 (miR-451), which are known to down-regulate 14-3-3ζ in several different cell types. We also investigated the expression of p53, a molecule that is downstream of 14-3-3ζ and can be down-regulated by it. We discovered that, in contrast to the reduction of 14-3-3ζ expression, the expression of miR-451, miR-375 and p53 increased in spina bifida rat fetuses.

Conclusions/Significance

These data suggest that the reduced expression of 14-3-3ζ plays a role in the excessive apoptosis that occurs in spina bifida and may be partly regulated by the over-expression of miR-451 and miR-375, and the consequent up-regulation of p53 might further promote apoptosis in spina bifida.     

Supplementation of Substrate Uptake Gene Enhances the Expression of rhIFN-β in High Cell Density Fed-Batch Cultures of Escherichia coli

Over-expression of recombinant proteins in Escherichia coli triggers a metabolic stress response which causes a sharp decline in both growth and product formation rates post induction. We identified a key down-regulated substrate utilization gene, glycerol kinase (glpK), whose up-regulation could help alleviate this stress response. In a proof of principal study conducted in shake flask cultures, the glpK gene under the “ara” promoter in a pPROLar.A122 vector was co-transformed along with the recombinant interferon-β (rhIFN-β) gene in a pET22b vector into E. coli BL-21(DE3) cells. Co-expression of glpK improved the expression levels of rhIFN-β in glycerol containing medium, while no such gain was observed in medium without glycerol. This study was extended to high cell density fed-batch cultures where exponential feeding of complex substrates was done to increase biomass and hence product titers. For this we first constructed a modified E. coli strain BL-21(glpK ⁺) where the glpK gene was inserted downstream of the ibpA promoter in the host chromosome. There was a significant improvement in growth as well as expression levels of rhIFN-β in this modified strain when the feed medium contained high glycerol. A final product concentration of 4.8 g/l of rhIFN-β was obtained with the modified strain which was 35 % higher than the control.     

δ-Opioid Receptor Activation and MicroRNA Expression of the Rat Cortex in Hypoxia

Prolonged hypoxic/ischemic stress may cause cortical injury and clinically manifest as a neurological disability. Activation of the δ-opioid receptor (DOR) may induce cortical protection against hypoxic/ischemic insults. However, the mechanisms underlying DOR protection are not clearly understood. We have recently found that DOR activation modulates the expression of microRNAs (miRNAs) in the kidney exposed to hypoxia, suggesting that DOR protection may involve a miRNA mechanism. To determine if the miRNAs expressed in the cortex mediated DOR neuroprotection, we examined 19 miRNAs that were previously identified as hypoxia- and DOR-regulated miRNAs in the kidney, in the rat cortex treated with UFP-512, a potent and specific DOR agonist under hypoxic condition. Of the 19 miRNAs tested, 17 were significantly altered by hypoxia and/or DOR activation with the direction and amplitude varying depending on hypoxic duration and times of DOR treatment. Expression of several miRNAs such as miR-29b, -101b, -298, 324-3p, -347 and 466b was significantly depressed after 24 hours of hypoxia. Similar changes were seen in normoxic condition 24 hours after DOR activation with one-time treatment of UFP-512. In contrast, some miRNAs were more tolerant to hypoxic stress and showed significant reduction only with 5-day (e.g., miR-31 and -186) or 10-day (e.g., miR-29a, let-7f and -511) exposures. In addition, these miRNAs had differential responses to DOR activation. Other miRNAs like miRs-363* and -370 responded only to the combined exposure to hypoxia and DOR treatment, with a notable reduction of >70% in the 5-day group. These data suggest that cortical miRNAs are highly yet differentially sensitive to hypoxia. DOR activation can modify, enhance or resolve the changes in miRNAs that target HIF, ion transport, axonal guidance, free radical signaling, apoptosis and many other functions.     

Astrocyte–Endotheliocyte Axis in the Regulation of the Blood–Brain Barrier

Neurochemical Research - The evolution of blood–brain barrier paralleled centralisation of the nervous system: emergence of neuronal masses required control over composition of the...     

Disialogangliosides and TNFα Alter Gene Expression for Cytokines and Chemokines in Primary Brain Cell Cultures

Gangliosides have long been implicated in multiple pathologies affecting the central nervous system. Empirical studies have suggested the possibility that gangliosides, particularly GD3, work in tandem with pro-inflammatory cytokines, especially tumor necrosis factor alpha (TNFα), to initiate or facilitate cell death in the CNS. As a step toward unraveling the metabolic pathways activated in the pathogenesis of brain cell death, we have surveyed gene expression for a host of cytokines and chemokines in primary brain cell cultures exposed to GD3, GD1b, and TNFα for 24 h. An initial screen of 98 genes on a focused mini-array revealed the expression of at least 28 genes related to cell growth, death, or inflammation in our system of mixed cells cultured from neonatal rat brains. Clear evidence of a differential response to the gangliosides or TNFα was seen in 12 genes. Quantitative PCR was used to validate the response of six of these genes. We found that both GD3 and GD1b, but not TNFα, up-regulated expression of macrophage inflammatory protein 3 (MIP3A) and interleukin-1 receptor 1 (IL1R1), but down-regulated fibroblast growth factor 13 (FGF13). The expression of FGF receptor activating protein 1 (FRAG1) and interleukin-3 receptor alpha (IL3RA) was down-regulated by GD3. Exposure to TNFα resulted in a dramatic up-regulation of IL3RA and chemokine ligand 2 (CCL2), both of which have been implicated in multiple sclerosis. Our results provide strong evidence that the expression of these genes might be critical links in the metabolic cascades leading to cell degeneration and death in the brain.     

Melatonin and Environmental Lighting Regulate ALA‐S Gene Expression and So Porphyrin Biosynthesis in the Rat Harderian Gland

The main porphyrin in rodent Harderian glands (HGs) is the heme precursor protoporphyrin IX (PPIX). Rhythmic variations in PPIX levels have yet to be studied in rodent HGs. Moreover, the mode of regulation of heme biosynthesis in this organ is poorly documented in the rat. The aim of this study was to determine day‐night PPIX levels as well as day‐night activity and mode of expression of the porphyrinogenic enzymes δ‐aminolevulinate synthase (ALA‐S) and ferrochelatase (Fech) in the rat HG. The mRNA expression of ABCG2/Bcrp1 was also investigated. Male Wistar rats acclimatized to 12 h light (L): 12 h dark (D) cycles were sacrificed in the middle of both the L and D spans, and HG and liver tissues were collected. We report here that the HG contains an extremely high level of PPIX, 630‐ to 670‐fold higher than in the liver, without a day‐night difference, which is the consequence of both low Fech gene expression (5‐ to 7‐fold lower than in the liver) and ALA‐S over‐expression (4‐ to 7‐fold higher in the HG than liver). Fech and PPIX transporter ABCG2/Bcrp1 do not exhibit day‐night variation, whereas HG ALA‐S levels are significantly higher during the scotophase. Interestingly, when melatonin (10 mg/kg) is administered in the middle of the light phase, it increases ALA‐S mRNA levels in the HG to the ones observed during the middle of the D span. Continuous light exposure abolishes the day‐night ALA‐S variation in the HG that is observed under standard 12 L∶12 D conditions. Our results suggest that melatonin and environmental lighting regulate ALA‐S gene expression in the rat HG.     

Expression of the Cloned Hemagglutinin–Neuraminidase Gene of the Newcastle Disease Virus in Mouse Myeloma Cells

Vaccination with autologous cancer cells expressing a potent foreign antigen is promising for immunotherapy of tumors. A construct was obtained to transfect cancer cells with the hemagglutinin–neuraminidase (HN) gene of the Newcastle disease virus (NDV). Specific primers were designed, and the HN cDNA was amplified from RNA isolated from the allantoic fluid of NDV-infected embryonated chicken eggs. The amplified fragment was cloned in pCR2.1, sequenced, and recloned in expression vector pCDNA3.1/Zeo(+). The resulting construct was used to transfect mouse myeloma cells SP2/0. Production of HN was checked by ELISA and by a neuraminidase activity assay. Cell agglutination on ice was proposed as a test for surface HN.