Zinc metallothionein imported into liver mitochondria modulates respiration

Metallothionein (MT) localizes in the intermembrane space of liver mitochondria as well as in the cytosol and nucleus. Incubation of intact liver mitochondria with physiological, micromolar concentrations of MT leads to the import of MT into the mitochondria where it inhibits respiration. This activity is caused by the N-terminal β-domain of MT; in this system, the isolated C-terminal α-domain is inactive. Free zinc inhibits respiration at concentrations commensurate with the zinc content of either MT or the isolated β-domain, indicating that MT inhibition involves zinc delivery to mitochondria. Respiratory inhibition of uncoupled mitochondria identifies the electron transfer chain as the primary site of inhibition. The apoform of MT, thionein, is an endogenous chelating agent and activates zinc-inhibited respiration with a 1:1 stoichiometry ([zinc binding sites]/[zinc]). Carbamoylation of the lysines of MT significantly attenuates the inhibitory effect, suggesting that these residues are critical for the passage of MT through the outer mitochondrial membrane. Such an import pathway has been proposed for other proteins that also lack a mitochondrial targeting sequence, e.g., apocytochrome c, and possibly Cox17, a mitochondrial copper chaperone that is the only protein known so far to exhibit significant primary sequence homology to MT. The presence and respiratory inhibition of MT in liver, but not heart, mitochondria suggest a hitherto unknown biological modulating activity of MT in cellular respiration and energy metabolism in a tissue-specific manner.

Changes in global gene expression patterns during development and maturation of the rat kidney

We set out to define patterns of gene expression during kidney organogenesis by using high-density DNA array technology. Expression analysis of 8,740 rat genes revealed five discrete patterns or groups of gene expression during nephrogenesis. Group 1 consisted of genes with very high expression in the early embryonic kidney, many with roles in protein translation and DNA replication. Group 2 consisted of genes that peaked in midembryogenesis and contained many transcripts specifying proteins of the extracellular matrix. Many additional transcripts allied with groups 1 and 2 had known or proposed roles in kidney development and included LIM1, POD1, GFRA1, WT1, BCL2, Homeobox protein A11, timeless, pleiotrophin, HGF, HNF3, BMP4, TGF-α, TGF-β2, IGF-II, met, FGF7, BMP4, and ganglioside-GD3. Group 3 consisted of transcripts that peaked in the neonatal period and contained a number of retrotransposon RNAs. Group 4 contained genes that steadily increased in relative expression levels throughout development, including many genes involved in energy metabolism and transport. Group 5 consisted of genes with relatively low levels of expression throughout embryogenesis but with markedly higher levels in the adult kidney; this group included a heterogeneous mix of transporters, detoxification enzymes, and oxidative stress genes. The data suggest that the embryonic kidney is committed to cellular proliferation and morphogenesis early on, followed sequentially by extracellular matrix deposition and acquisition of markers of terminal differentiation. The neonatal burst of retrotransposon mRNA was unexpected and may play a role in a stress response associated with birth. Custom analytical tools were developed including “The Equalizer” and “eBlot,” which contain improved methods for data normalization, significance testing, and data mining.

A cardiac myocyte vascular endothelial growth factor paracrine pathway is required to maintain cardiac function

The role of the cardiac myocyte as a mediator of paracrine signaling in the heart has remained unclear. To address this issue, we generated mice with cardiac myocyte-specific deletion of the vascular endothelial growth factor gene, thereby producing a cardiomyocyte-specific knockout of a secreted factor. The hearts of these mice had fewer coronary microvessels, thinned ventricular walls, depressed basal contractile function, induction of hypoxia-responsive genes involved in energy metabolism, and an abnormal response to β-adrenergic stimulation. These findings establish the critical importance of cardiac myocyte-derived vascular endothelial growth factor in cardiac morphogenesis and determination of heart function. Further, they establish an adult murine model of hypovascular nonnecrotic cardiac contractile dysfunction.

Calorie restriction lowers body temperature in rhesus monkeys, consistent with a postulated anti-aging mechanism in rodents.

Many studies of caloric restriction (CR) in rodents and lower animals indicate that this nutritional manipulation retards aging processes, as evidenced by increased longevity, reduced pathology, and maintenance of physiological function in a more youthful state. The anti-aging effects of CR are believed to relate, at least in part, to changes in energy metabolism. We are attempting to determine whether similar effects occur in response to CR in nonhuman primates. Core (rectal) body temperature decreased progressively with age from 2 to 30 years in rhesus monkeys fed ad lib (controls) and is reduced by approximately 0.5 degrees C in age-matched monkeys subjected to 6 years of a 30% reduction in caloric intake. A short-term (1 month) 30% restriction of 2.5-year-old monkeys lowered subcutaneous body temperature by 1.0 degrees C. Indirect calorimetry showed that 24-hr energy expenditure was reduced by approximately 24% during short-term CR. The temporal association between reduced body temperature and energy expenditure suggests that reductions in body temperature relate to the induction of an energy conservation mechanism during CR. These reductions in body temperature and energy expenditure are consistent with findings in rodent studies in which aging rate was retarded by CR, now strengthening the possibility that CR may exert beneficial effects in primates analogous to those observed in rodents.

Increased tricarboxylic acid cycle flux in rat brain during forepaw stimulation detected with 1H[13C]NMR.

NMR spectroscopy was used to test recent proposals that the additional energy required for brain activation is provided through nonoxidative glycolysis. Using localized NMR spectroscopic methods, the rate of C4-glutamate isotopic turnover from infused [1-(13)C]glucose was measured in the somatosensory cortex of rat brain both at rest and during forepaw stimulation. Analysis of the glutamate turnover data using a mathematical model of cerebral glucose metabolism showed that the tricarboxylic acid cycle flux [(V(TCA)] increased from 0.49 +/- 0.03 at rest to 1.48 +/- 0.82 micromol/g/min during stimulation (P < 0.01). The minimum fraction of C4-glutamate derived from C1-glucose was approximately 75%, and this fraction was found in both the resting and stimulated rats. Hence, the percentage increase in oxidative cerebral metabolic rate of glucose use (CMRglc) equals the percentage increases in V(TCA) and cerebral metabolic rate of oxygen consumption (CMRO2). Comparison with previous work for the same rat model, which measured total CMRglc [Ueki, M., Linn, F. & Hossman, K. A. (1988) J. Cereb. Blood Flow Metab. 8, 486-4941, indicates that oxidative CMRglc supplies the majority of energy during sustained brain activation.

Imaging of H217O distribution in the brain of a live rat by using proton-detected 17O MRI

Imaging of H217O has a number of important applications. Mapping the distribution of H217O produced by oxidative metabolism of 17O-enriched oxygen gas may lead to a new method of metabolic functional imaging; regional cerebral blood flow also can be measured by measuring the H217O distribution after the injection of 17O-enriched physiological saline solution. Previous studies have proposed a method for indirect detection of 17O. The method is based on the shortening of the proton T2 in H217O solutions, caused by the residual 17O-1H scalar coupling and transferred to the bulk water via fast chemical exchange. It has been shown that the proton T2 of H217O solutions can be restored to that of H216O by irradiating the resonance frequency of the 17O nucleus. The indirect 17O image thus is obtained by taking the difference between two T2-weighted spin-echo images: one acquired after irradiation of the 17O resonance and one acquired without irradiation. It also has been established that, at relatively low concentrations of H217O, the indirect method yields an image that quantitatively reflects the H217O distribution in the sample. The method is referred to as PRIMO (proton imaging of oxygen). In this work, we show in vivo proton images of the H217O distribution in a rat brain after an i.v. injection of H217O-enriched physiological saline solution. Implementing the indirect detection method in an echo-planar imaging sequence enabled obtaining H217O images with good spatial and temporal resolution of few seconds.

Role of brain allopregnanolone in the plasticity of γ-aminobutyric acid type A receptor in rat brain during pregnancy and after delivery

The relation between changes in brain and plasma concentrations of neurosteroids and the function and structure of γ-aminobutyric acid type A (GABAA) receptors in the brain during pregnancy and after delivery was investigated in rats. In contrast with plasma, where all steroids increased in parallel, the kinetics of changes in the cerebrocortical concentrations of progesterone, allopregnanolone (AP), and allotetrahydrodeoxycorticosterone (THDOC) diverged during pregnancy. Progesterone was already maximally increased between days 10 and 15, whereas AP and allotetrahydrodeoxycorticosterone peaked around day 19. The stimulatory effect of muscimol on 36Cl− uptake by cerebrocortical membrane vesicles was decreased on days 15 and 19 of pregnancy and increased 2 days after delivery. Moreover, the expression in cerebral cortex and hippocampus of the mRNA encoding for γ2L GABAA receptor subunit decreased during pregnancy and had returned to control values 2 days after delivery. Also α1,α2, α3, α4, β1, β2, β3, and γ2S mRNAs were measured and failed to change during pregnancy. Subchronic administration of finasteride, a 5α-reductase inhibitor, to pregnant rats reduced the concentrations of AP more in brain than in plasma as well as prevented the decreases in both the stimulatory effect of muscimol on 36Cl− uptake and the decrease of γ2L mRNA observed during pregnancy. These results indicate that the plasticity of GABAA receptors during pregnancy and after delivery is functionally related to fluctuations in endogenous brain concentrations of AP whose rate of synthesis/metabolism appears to differ in the brain, compared with plasma, in pregnant rats.

Cyp7b, a novel brain cytochrome P450, catalyzes the synthesis of neurosteroids 7α-hydroxy dehydroepiandrosterone and 7α-hydroxy pregnenolone

Steroids produced locally in brain (neurosteroids), including dehydroepiandrosterone (DHEA), influence cognition and behavior. We previously described a novel cytochrome P450, Cyp7b, strongly expressed in rat and mouse brain, particularly in hippocampus. Cyp7b is most similar to steroidogenic P450s and potentially could play a role in neurosteroid metabolism. To examine the catalytic activity of the enzyme mouse Cyp7b cDNA was introduced into a vaccinia virus vector. Extracts from cells infected with the recombinant showed NADPH-dependent conversion of DHEA (Km, 13.6 μM) and pregnenolone (Km, 4.0 μM) to slower migrating forms on thin layer chromatography. The expressed enzyme was less active against 25-hydroxycholesterol, 17β-estradiol and 5α-androstane-3β,17β-diol, with low to undetectable activity against progesterone, corticosterone, and testosterone. On gas chromatography and mass spectrometry of the Cyp7b metabolite of DHEA the retention time and fragmentation patterns were identical to those obtained with authentic 7α-hydroxy DHEA. The reaction product also comigrated on thin layer chromatography with 7α-hydroxy DHEA but not with 7β-hydroxy DHEA; when [7α-3H]pregnenolone was incubated with Cyp7b extracts the extent of release of radioactivity into the medium suggested that hydroxylation was preferentially at the 7α position. Brain extracts also efficiently liberated tritium from [7α-3H]pregnenolone and converted DHEA to a product with a chromatographic mobility indistinguishable from 7α-hydroxy DHEA. We conclude that Cyp7b is a 7α-hydroxylase participating in the synthesis, in brain, of neurosteroids 7α-hydroxy DHEA, and 7α-hydroxy pregnenolone.

Brain-wave representation of words by superposition of a few sine waves

Data from three previous experiments were analyzed to test the hypothesis that brain waves of spoken or written words can be represented by the superposition of a few sine waves. First, we averaged the data over trials and a set of subjects, and, in one case, over experimental conditions as well. Next we applied a Fourier transform to the averaged data and selected those frequencies with high energy, in no case more than nine in number. The superpositions of these selected sine waves were taken as prototypes. The averaged unfiltered data were the test samples. The prototypes were used to classify the test samples according to a least-squares criterion of fit. The results were seven of seven correct classifications for the first experiment using only three frequencies, six of eight for the second experiment using nine frequencies, and eight of eight for the third experiment using five frequencies.

Effect of rabies virus infection on gene expression in mouse brain

A variety of molecular genetic approaches were used to study the effect of rabies virus (RV) infection on host gene expression in mouse brain. The down-regulation of gene expression was found to be a major effect of RV infection by using subtraction hybridization. However, a combination of techniques identified approximately 39 genes activated by infection. These included genes involved in regulation of cell metabolism, protein synthesis, synaptic activity, and cell growth and differentiation. Northern blot analysis to monitor temporal activation of several of these genes following infection revealed essentially two patterns of activation: (i) an early response with up-regulation beginning within 3 days after infection and correlating with transcription of RV nuclear protein; and (ii) a late response with enhanced expression occurring at days 6–7 after infection and associated with peak RV replication. The gene activation patterns and the known functions of their products suggest that a number of host genes may be involved in the replication and spread of RV in the brain.

Cerebral energetics and the glycogen shunt: Neurochemical basis of functional imaging

Positron-emission tomography and functional MRS imaging signals can be analyzed to derive neurophysiological values of cerebral blood flow or volume and cerebral metabolic consumption rates of glucose (CMRGlc) or oxygen (CMRO2). Under basal physiological conditions in the adult mammalian brain, glucose oxidation is nearly complete so that the oxygen-to-glucose index (OGI), given by the ratio of CMRO2/CMRGlc, is close to the stoichiometric value of 6. However, a survey of functional imaging data suggests that the OGI is activity dependent, moving further below the oxidative value of 6 as activity is increased. Brain lactate concentrations also increase with stimulation. These results had led to the concept that brain activation is supported by anaerobic glucose metabolism, which was inconsistent with basal glucose oxidation. These differences are resolved here by a proposed model of glucose energetics, in which a fraction of glucose is cycled through the cerebral glycogen pool, a fraction that increases with degree of brain activation. The “glycogen shunt,” although energetically less efficient than glycolysis, is followed because of its ability to supply glial energy in milliseconds for rapid neurotransmitter clearance, as a consequence of which OGI is lowered and lactate is increased. The value of OGI observed is consistent with passive lactate efflux, driven by the observed lactate concentration, for the few experiments with complete data. Although the OGI changes during activation, the energies required per neurotransmitter release (neuronal) and clearance (glial) are constant over a wide range of brain activity.

Involvement of the amygdala in memory storage: Interaction with other brain systems

There is extensive evidence that the amygdala is involved in affectively influenced memory. The central hypothesis guiding the research reviewed in this paper is that emotional arousal activates the amygdala and that such activation results in the modulation of memory storage occurring in other brain regions. Several lines of evidence support this view. First, the effects of stress-related hormones (epinephrine and glucocorticoids) are mediated by influences involving the amygdala. In rats, lesions of the amygdala and the stria terminalis block the effects of posttraining administration of epinephrine and glucocorticoids on memory. Furthermore, memory is enhanced by posttraining intra-amygdala infusions of drugs that activate β-adrenergic and glucocorticoid receptors. Additionally, infusion of β-adrenergic blockers into the amygdala blocks the memory-modulating effects of epinephrine and glucocorticoids, as well as those of drugs affecting opiate and GABAergic systems. Second, an intact amygdala is not required for expression of retention. Inactivation of the amygdala prior to retention testing (by posttraining lesions or drug infusions) does not block retention performance. Third, findings of studies using human subjects are consistent with those of animal experiments. β-Blockers and amygdala lesions attenuate the effects of emotional arousal on memory. Additionally, 3-week recall of emotional material is highly correlated with positron-emission tomography activation (cerebral glucose metabolism) of the right amygdala during encoding. These findings provide strong evidence supporting the hypothesis that the amygdala is involved in modulating long-term memory storage.

Blood flow and oxygen delivery to human brain during functional activity: Theoretical modeling and experimental data

Coupling of cerebral blood flow (CBF) and cerebral metabolic rate for oxygen (CMRO2) in physiologically activated brain states remains the subject of debates. Recently it was suggested that CBF is tightly coupled to oxidative metabolism in a nonlinear fashion. As part of this hypothesis, mathematical models of oxygen delivery to the brain have been described in which disproportionately large increases in CBF are necessary to sustain even small increases in CMRO2 during activation. We have explored the coupling of CBF and oxygen delivery by using two complementary methods. First, a more complex mathematical model was tested that differs from those recently described in that no assumptions were made regarding tissue oxygen level. Second, [15O] water CBF positron emission tomography (PET) studies in nine healthy subjects were conducted during states of visual activation and hypoxia to examine the relationship of CBF and oxygen delivery. In contrast to previous reports, our model showed adequate tissue levels of oxygen could be maintained without the need for increased CBF or oxygen delivery. Similarly, the PET studies demonstrated that the regional increase in CBF during visual activation was not affected by hypoxia. These findings strongly indicate that the increase in CBF associated with physiological activation is regulated by factors other than local requirements in oxygen.

Effects of anesthesia on functional activation of cerebral blood flow and metabolism

Functional brain mapping based on changes in local cerebral blood flow (lCBF) or glucose utilization (lCMRglc) induced by functional activation is generally carried out in animals under anesthesia, usually α-chloralose because of its lesser effects on cardiovascular, respiratory, and reflex functions. Results of studies on the role of nitric oxide (NO) in the mechanism of functional activation of lCBF have differed in unanesthetized and anesthetized animals. NO synthase inhibition markedly attenuates or eliminates the lCBF responses in anesthetized animals but not in unanesthetized animals. The present study examines in conscious rats and rats anesthetized with α-chloralose the effects of vibrissal stimulation on lCMRglc and lCBF in the whisker-to-barrel cortex pathway and on the effects of NO synthase inhibition with NG-nitro-l-arginine methyl ester (l-NAME) on the magnitude of the responses. Anesthesia markedly reduced the lCBF and lCMRglc responses in the ventral posteromedial thalamic nucleus and barrel cortex but not in the spinal and principal trigeminal nuclei. l-NAME did not alter the lCBF responses in any of the structures of the pathway in the unanesthetized rats and also not in the trigeminal nuclei of the anesthetized rats. In the thalamus and sensory cortex of the anesthetized rats, where the lCBF responses to stimulation had already been drastically diminished by the anesthesia, l-NAME treatment resulted in loss of statistically significant activation of lCBF by vibrissal stimulation. These results indicate that NO does not mediate functional activation of lCBF under physiological conditions.

Peripheral anti-Aβ antibody alters CNS and plasma Aβ clearance and decreases brain Aβ burden in a mouse model of Alzheimer's disease

Active immunization with the amyloid β (Aβ) peptide has been shown to decrease brain Aβ deposition in transgenic mouse models of Alzheimer's disease and certain peripherally administered anti-Aβ antibodies were shown to mimic this effect. In exploring factors that alter Aβ metabolism and clearance, we found that a monoclonal antibody (m266) directed against the central domain of Aβ was able to bind and completely sequester plasma Aβ. Peripheral administration of m266 to PDAPP transgenic mice, in which Aβ is generated specifically within the central nervous system (CNS), results in a rapid 1,000-fold increase in plasma Aβ, due, in part, to a change in Aβ equilibrium between the CNS and plasma. Although peripheral administration of m266 to PDAPP mice markedly reduces Aβ deposition, m266 did not bind to Aβ deposits in the brain. Thus, m266 appears to reduce brain Aβ burden by altering CNS and plasma Aβ clearance.