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Transcriptome-To-Metabolome™ Biosimulation Reveals Human Hippocampal Hypometabolism with Age and Alzheimer’s Disease

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Abstract

The authors had validated a proprietary method, Transcriptome-To-Metabolome™ (TTM™) Biosimulation, for using the transcriptome to determine parameters for kinetic biosimulation of 16 core metabolic pathways. In vivo and in silico evidence confirmed that hippocampal cholesterol metabolism decreases with aging and increases with Alzheimer’s disease (AD). The molecular studies on aging primate and human hippocampus, including AD samples, provided internal validations on the biosimulations, while evidence from the literature, bibliome, provided external validations. This study extends the investigations with the TTM™ Biosimulations into the changes in these 16 metabolic pathways in aging male human hippocampus and for stages of AD. The authors report robust hippocampal hypometabolism in the fifth to tenth decade of life involving glucose and lipid metabolism in male humans. These findings are validated externally from the bibliome. Several changes in AD are demonstrated to be exaggerations or deviations of very late stage changes of normal aging among these pathways.

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... The TTM TM method is patent pending (Phelix 2011;Phelix et al. 2011;Valdez et al. 2011), wherein the gene expression level (see above) values are used to derive a set of k-values via globalisation (Fundel et al. 2008) to be used as a tool. Each (GSM) sample generated its own unique set of k-values that were input into separate COPASI â TTM TM model files. ...
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Declining cognitive performance is associated with increasing age, even in the absence of overt pathological processes. We and others have reported that declining cognitive performance is associated with age-related changes in brain glucose utilization, long-term potentiation and paired-pulse facilitation, protein expression, neurotransmitter levels, and trophic factors. However, it is unclear whether these changes are causes or symptoms of the underlying alterations in dendritic and synaptic morphology that occur with age. In this study, we examined the hippocampal proteome for age- and cognition-associated changes in behaviorally stratified young and old rats, using two-dimensional in-gel electrophoresis and MS/MS. Comparison of old cognitively intact with old cognitively impaired animals revealed additional changes that would not have been detected otherwise. Interestingly, not all age-related changes in protein expression were associated with cognitive decline, and distinct differences in protein expression were found when comparing old cognitively intact with old cognitively impaired rats. A large number of protein changes with age were related to the glycolysis/gluconeogenesis pathway. In total, the proteomic changes suggest that age-related alterations act synergistically with other perturbations to result in cognitive decline. This study also demonstrates the importance of examining behaviorally-defined animals in proteomic studies, as comparison of young to old animals regardless of behavioral performance would have failed to detect many cognitive impairment-specific protein expression changes evident when behavioral stratification data were used.
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Brain ischemia was produced by bilateral ligation of the common carotid arteries of spontaneously hypertensive rats. The concentrations of fructose 2,6-bisphosphate and other glycolytic intermediates as well as of pyridine and adenine nucleotides were measured in frozen brain samples. In contrast to the decrease reported in hepatocytes under anoxic conditions, the fructose 2,6-bisphosphate content was increased by 20-30% during the early stages of ischemia. Elevation in fructose 1,6-bisphosphate level and lactate formation followed the rise in fructose 2,6-bisphosphate content, a finding suggesting that this compound plays a key role in the compensatory acceleration of glycolysis under ischemic conditions in vivo.
Article
We describe light- and electron-microscopically a new type of intracytoplasmatic inclusions within cell processes of the cerebral cortex and the underlying white matter. These structures measure 5-50 micron in diameter and consist almost exclusively of densely packed alpha- or beta-glycogen granules, which never occur together in any single structure. Within their periphery, electron-dense amorphous spots and cell organelles, especially mitochondria, were seen. No membrane-bound glycogen was observed. We propose to call them granular glycogen bodies. They occur in 4 of 7 examined postmortem specimens of the cerebral cortex of people older than 60 years of age. They were not found in 4 younger controls aged 26-48. Their appearance may reflect a distinct turnover disorder of carbohydrate metabolism, which becomes manifest under diverse pathologic conditions and in the normal aging process.
Article
Brain energy state and glycolytic metabolites were measured in young (6 month) and aged (28 month) male rats under normoxic (70% nitrous oxide, 30% oxygen) or hypoxic (PaO2 = 25 mm Hg) test conditions. Hypoxic ischemia was induced in one cerebral hemisphere by ligation of one carotid artery. Under normoxic test conditions brain energy metabolite concentrations were similar between young and aged rats. Brain tissue glucose, glycogen, glucose-6-phosphate and critic acid cycle intermediate concentrations were decreased in aged rats during normoxia while fructose-6-phosphate and pyruvate were increased. Decreases in brain energy state and increases in lactate/pyruvate ratios were significant in both young and aged rats during hypoxia and were greater in aged animals in hypoxic-ischemic tissues. These results indicate that brain energy state is normal in aged rats under normoxic conditions but that hypoxic-ischemia produces a greater degree of brain energy failure compared to younger animals.
Article
Histochemical investigations were made upon activities of succinic dehydro-genase, lactic dehydrogenase, nicotinamide adenine dinucleotide diaphorase, cytochrome oxidase and glucose-6-phosphate dehydrogenase, and contents of copper and of nucleic acids in the diencephalon of rats in reference to aging. 1) In the diencephalon of senescent rats, a slight decrease of activity of glucose-6-phosphate dehydrogenase and a slight increase in the activities of both succinic dehydrogenase and cytochrome oxidase in the supraoptic and paraventricular nuclei were found. 2) A deposition of copper was increased with aging in the stratum periventriculare hypothalami, fimbria hippocampi and habenular nucleus. 3) A decrease of cytoplasmic RNA content of the diencephalon and an increase of RNA in the supraoptic nucleus as well as paraventricular nucleus were found with advancing age.
Article
The greatest advantage of histochemistry as applied to the study of ageing, is that focal alterations and changes in heterogenity at the tissue and cellular levels can be analysed by light and electron microscopy. The major disadvantage of this technique, however, has been that in the past results have not been easy to quantify except by insensitive and rather subjective means. Sensitivity can be increased by applying histochemistry at the electron microscope level but amounts of reaction product are difficult to quantify accurately by eye using either a light microscope or electron microscope. This contrasts, of course with biochemical determinations carried out on homogenates where highly accurate measurements of enzyme activity can be made to within picogram amounts. However, when as in ageing, a number of crucial alterations are focal or heterogeneous in nature, it is not helpful to be limited to whole-organ homogenates, or even cell fractions, since the value obtained will be an average for the wide range of cell types and sub-types present. The ideal situation would be to develop techniques to allow such accurate and highly sensitive determination of reaction but within single cells. Some considerable progress has already been made towards this aim and such quantitative histochemical techniques are beginning to be applied to the study of enzyme and nucleic acid alterations in ageing (Campbell & Stoward, 1981; Middleton & Gahan, 1982). Although it is still in its developmental stages, this type of quantitative measurement at the cellular level will provide the means of analysing heterogeneity at the light microscope level in the future. Similar analysis of heterogeneity at the subcellular level awaits the development of techniques for the morphometric analysis of reaction product at the electron microscope level. With respect to the results already obtained by applying histochemistry to the study of ageing in mammals, the same conclusions can be drawn as after the analysis of biochemical results; there is no simple, general pattern of alteration with age. On the contrary, changes tend to be organ, tissue or cell-type specific. In some cases, clear pathological changes are seen associated with senescence and these are predictably accompanied by histochemical alterations at these foci, for instance in the senile plaques of the brain, intimal cushions of the arteries and cataracts of the lens. In other organs characteristic focal changes are seen which correlate with a loss of function, such as in the periportal regions of liver lobules, in type II skeletal muscle fibres and in parietal cells of gastric glands. In other cases an increase in the heterogeneity of expression of enzyme activity is seen within a single cell type within an organ, such as in hepatocytes and suggests a possible increase in phenotypic instability with age. To date, the range of cellular constituents that has been studied histochemically during ageing has been relatively narrow and although some organs have been studied extensively, others have been neglected. It is to be hoped that, with the number of reliable histochemical techniques now available, in the future whole enzymes chains or at least the important rate-limiting steps within these chains, may be studied with respect to ageing, which may provide more insight into alteration in any particular cellular function. Indeed, several lines of approach are waiting to be fully exploited. One is to concentrate, on a particular cellular organelle and study it in detail to determine whether subtle changes in substrate utilization are crucial, for instance with respect to phosphatase actions at the plasma membrane or oxidative enzymes in the mitochondria. Another approach, of particular current interest, would be to expand on techniques for the study of enzymes linked with the production and removal of free radicals, such as cytochrome P450 and superoxide dismutase. An imbalance in this system could exert detrimental effects on key molecular structures, cause lipid peroxidation, cross-link proteins, split polypeptides and cause changes in DNA. Finally, in the light of biochemical evidence of decline and delay in the responsiveness of some enzymes to hormones, histochemical techniques could be applied effectively to determine whether there is a cellular component to this type of alteration. If so, histochemistry is the technique of choice to determine whether this is a homogeneous effect or whether alterations in the responsiveness of certain key cell populations are responsible for the age-associated decline in the ability of an organism to adapt to stress and changes in the environment.
Article
The activities of glycolytic enzymes were determined in human autoptic temporal lobes from patients with different forms of dementia. For some enzymes (hexokinase, phosphofructokinase and phosphoglycerate mutase) the effect seen in dementia can be regarded as an intensification of the normal ageing affect. For other enzymes (aldolase, phosphoglucose isomerase, triosephosphate isomerase and lactate dehydrogenase) no changes in enzyme activities corresponding to those found in dementia are observed in the normal ageing process. These effects are most pronounced in the non-vascular Alzheimer cases. With the exception of triosephosphate isomerase and lactate dehydrogenase, enzyme activity is also reduced in bronchopneumonia. The effects of dementia and bronchopneumonia on the activities of glycolytic enzymes in human autoptic brain tissue are often difficult to distinguish.
Article
The incorporation of cytidine-containing precursors (CDP-Cho and CDP-Etn) into the main phospholipid classes of cellular fractions enriched in neurons and glial cells from whole rat brains of different ages was examined. The rate of synthesis of choline phosphoglycerides in neuronal homogenates significantly decreased with age up to 18 months; after this time no additional decrease was found. The decrease of CDP-Etn incorporation in neurons was found to be less significantly affected by age up to 18 months, but the enzymic activity decreased after 18 months of age. No changes were found in the corresponding glial activity at any age. Biochemical phenomena that occur in 18-month-old rat brain (aged animals) were compared with phenomena occurring in 2-month-old rat brain (adult animals). No significant variations of lipid composition were found in neurons from either 18-month-old or 2-month-old rat brain. These results, together with some kinetic parameters, suggest that ethanolamine and choline phosphotransferases are affected differently by aging.
Article
Measurements have been made of the activity of the enzymes of the glycolytic, pentose phosphate and lipogenic pathways and of some marker enzymes of the tricarboxylic acid cycle in brains of rats aged between 20 days and 24 months. In general, the activity of the most enzymes measured was unchanged by aging but exceptions to this were increases of hexokinase, glucose-6-phosphate dehydrogenase and 'malic enzyme' and decreases of ATP-citrate lyase, acetyl-CoA carboxylase and fatty acid synthetase. An exceptionally large (2-fold) increase in the activity of cytosolic glycerol 3-phosphate dehydrogenase was noted. These changes are considered in relation to the overall metabolic activity of the brain.
Article
Localization of mRNA encoding for the enzyme hexokinase and its regulation in aged animals was carried out in rat brain using the in situ hybridization technique. The highest levels of the hybridization signal were observed in the olfactory bulb, piriform cortex, tenia tecta, hippocampus and granular cells of the cerebellum. Other brain areas and nuclei including cerebral cortex, thalamus, hypothalamus, substantia nigra, subiculum, choroid plexus and superior colliculus displayed moderate to low density of transcripts. Correlation between relative hexokinase content and levels of its mRNA was found only for some brain regions such as caudate-putamen, geniculate nucleus, ventral and lateral thalamic nuclei, superior colliculus and granular cells of the cerebellum. In the cerebral cortex and hippocampus of old animals the expression of hexokinase was significantly increased at 18 and 24 months of age. From the present data we conclude that although hexokinase is an ubiquitous enzyme, sites of synthesis display a discrete and uneven localization in rat CNS and expression, in the aging brain, might be regulated to compensate for reduced oxidative phosphorylation in the brain tissue.
Article
Although the abnormal gene products responsible for several hereditary neurodegenerative disorders caused by repeat CAG trinucleotides have been identified, the mechanism by which the proteins containing the expanded polyglutamine domains cause cell death is unknown. The observation that several of the mutant proteins interact in vitro with the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) suggests that interaction between the different gene products and GAPDH might damage brain neurons. To measure the activity of GAPDH in postmortem brain of patients with CAG repeat disorders. Activity of GAPDH was measured in morphologically affected and unaffected brain areas of patients with 4 different CAG repeat disorders (Huntington disease, spinocerebellar ataxia 1 [SCA1], SCA2, and SCA3-Machado-Joseph disease), in brains of patients with Friedreich ataxia (a GAA repeat disorder) and Alzheimer disease, and in brains of matched control subjects. Brain GAPDH activity was normal in all groups with the exception of a slight but statistically significant region-specific reduction in the patients with Huntington disease (caudate nucleus, -12%) and Alzheimer disease (temporal cortex, -19%). The presence of the polyglutamine-containing proteins in CAG repeat disorders does not result in substantial irreversible inactivation or in increased activity of GAPDH in human brain.
Article
Advanced glycation end products (AGEs) are known to accumulate in long-lived tissue proteins during normal ageing. In this study, we examined the expression of AGEs in human hippocampus using immunohistochemistry and determined its utility for estimating the age of cadavers of unknown age. Hippocampus tissues were obtained at autopsy from 31 individuals, including 10 fire victims, aged 0--96 years within 3 days postmortem. Immunostaining using anti-AGE antibody demonstrated that the perikarya of pyramidal neurones in the hippocampus was immunoreactive for the anti-AGE antibody, and the immunoreactivity was increased with age. Quantitative analysis of the AGE-immunoreactivity in the pyramidal neurones of the CA4 region revealed a significant correlation between the AGE-immunoreactivity and the age in nonfire death cases with a correlation coefficient of 0.91 (P < 0.01). The significant correlation could be obtained even in fire death cases affected by the unusual environmental condition. These results suggest that the immunohistochemical analysis of AGEs in human hippocampus may be useful for the age estimation of cadavers with unknown age.
Article
Alzheimer's disease is associated with markedly impaired cerebral glucose metabolism as detected by reduced cortical desoxyglucose utilization, by altered activities of key glycolytic enzymes or by reduced densities of cortical glucose transporter subtypes. To determine whether formation and/or deposition of beta-amyloid plays a role in the pathology of glucose metabolism, transgenic Tg2576 mice that overexpress the Swedish mutation of the human amyloid precursor protein and demonstrate a progressive, age-related cortical and hippocampal deposition of beta-amyloid plaques, were used to study expression and activity of key enzymes of brain glycolysis (phosphofructokinase, PFK) and glyconeogenesis (fructose1,6-bisphosphatase; FbPase). Quantitative RT-PCR revealed high expression levels of both C- and M-type PFK mRNA in non-transgenic mouse cerebral cortex, whilst there was little expression of the L-type. In 24-month-old transgenic Tg2576 mouse cortex, but not in 7-, 13-, and 17-month-old mice, the copy number of PFK-C mRNA was significantly reduced in comparison to non-transgenic littermates, while the mRNA level of the other PFK isoforms and FbPase did not differ between transgenic and non-transgenic tissue samples. In situ hybridization in brain sections from aged Tg2576 mice revealed reduced PFK-C mRNA expression in beta-amyloid plaque-associated neurons and upregulation in reactive astrocytes surrounding beta-amyloid deposits. The decreased PFK-C protein level detected by Western analysis in cerebral cortical tissue from 24-month-old transgenic Tg2576 mice was accompanied by reduced enzyme activity of PFK in comparison to non-transgenic littermates. Our data demonstrate that impairment of cerebral cortical glucose metabolism occurs only due to the long-lasting high beta-amyloid burden. This results from a reduction in glycolytic activity in beta-amyloid plaque-associated neurons and a concomitant upregulation in reactive, plaque-surrounding astrocytes.
Article
The concentration and metabolism of the primary carbohydrate store in the brain, glycogen, is unknown in the conscious human brain. This study reports the first direct detection and measurement of glycogen metabolism in the human brain, which was achieved using localized 13C NMR spectroscopy. To enhance the NMR signal, the isotopic enrichment of the glucosyl moieties was increased by administration of 80 g of 99% enriched [1-13C]glucose in four subjects. 3 h after the start of the label administration, the 13C NMR signal of brain glycogen C1 was detected (0.36+/-0.07 micromol/g, mean+/-S.D., n=4). Based on the rate of 13C label incorporation into glycogen and the isotopic enrichment of plasma glucose, the flux through glycogen synthase was estimated at 0.17+/-0.05 micromol/(gh). This study establishes that brain glycogen can be measured in humans and indicates that its metabolism is very slow in the conscious human. The noninvasive detection of human brain glycogen opens the prospect of understanding the role and function of this important energy reserve under various physiological and pathophysiological conditions.
A review of available information on over-all cerebral blood flow and oxygen consumption in man obtained by means of the nitrous oxide technique reveals a distinct correlation of these functions with age.There is a rapid fall in the circulation and oxygen utilization of the brain from childhood through adolescence followed by a more gradual but progressive reduction throughout the remaining age span.The factors responsible for these changes and whether one or the other is primary appear to be suitable subjects for continued investigation.
Article
Methylglyoxal (MG) is one of the most powerful glycating agents of proteins and other important cellular components and has been shown to be toxic to cultured cells. Under hyperglycaemic conditions, an increase in the concentration of MG has been observed in human body fluids and tissues that seems to be responsible for diabetic complications. Recent data suggest that diabetes may cause impairment of cognitive processes, according to a mechanism involving both oxidative stress and advanced glycation end product (AGE) formation. In this work, we explored the molecular mechanism underlying MG toxicity in neural cells, by investigating the effect of MG on both the interleukin-1beta (IL-1beta), as the major inducer of the acute phase response, and the nervous growth factor (NGF) expression. Experiments were performed on cultured neural cells from rat hippocampus, being this brain region mostly involved in cognitive processes and, therefore, possible target of diabetes-mediated impairment of cognitive abilities. Results show that MG treatment causes in hippocampal neural cells extensive, oxidative stress-mediated cell death, in consequence of a strong catalase enzymatic activity and protein inhibition. MG also causes a very significant increase in both transcript and protein expression of the NGF as well as of the pro-inflammatory cytokine IL-1beta. MG co-treatment with the antioxidant N-acetylcysteine (NAC) completely abrogates the observed effects. Taken together, these data demonstrate that hippocampal neurons are strongly susceptible to MG-mediated oxidative stress.
Article
Plasmalogens (Pls) are phospholipids containing a vinyl-ether bond at the sn-1 position of the glycerol backbone. They represent between 1/2 and 2/3 of the ethanolamine phospholipids in the brain. During aging, the Pls content in human brain falls down. However, the role of Pls metabolism-related enzymes in the regulation of Pls levels remains to be determined. Dihydroxyacetone phosphate acyltransferase (DHAP-AT) is the enzyme involved in the first step of Pls biosynthesis. In the brain, a phospholipase A2, which selectively acts on Pls, has been isolated (Pls-PLA2s). In this work, we aimed to evaluate the impact of DHAP-AT (a key enzyme of Pls biosynthesis) and Pls-PLA2 (a specific Pls degradation enzyme) on the evolution of Pls content in the rat brain during aging. The influence of n-3 fatty acid intake was also evaluated. Littermates from two generations of n-3 deficient rats were fed an equilibrated diet containing either alpha-LNA alone or with two doses of DHA. After weaning, 3, 9 or 21 months of diet, rats were sacrificed. Enzymatic assays were performed, Pls levels were assessed and the sn-2 position of ethanolamine Pls was analyzed. DHAP-AT activity significantly increased between weaning and 3 months with a concomitant increase of brain Pls, which reached maximal levels after 9 months. Then, Pls levels and DHAP-AT activity significantly decreased while Pls-PLA2s activity significantly increased. Dietary n-3 fatty acids had no effect on DHAP-AT activity and on Pls levels. In conclusion, the increase of brain Pls content in the first part of the life may be related to the high increase of DHAP-AT activity, probably stimulated by DHA. In aged animals, the decrease of Pls levels may mainly be caused to an increase of their degradation by Pls-PLA2. Dietary DHA may not oppose the physiologic aging.
Article
This longitudinal study used FDG-PET imaging to predict and monitor cognitive decline from normal aging. Seventy-seven 50-80-year-old normal (NL) elderly received longitudinal clinical examinations over 6-14 years (561 person-years, mean per person 7.2 years). All subjects had a baseline FDG-PET scan and 55 subjects received follow-up PET exams. Glucose metabolic rates (MRglc) in the hippocampus and cortical regions were examined as predictors and correlates of clinical decline. Eleven NL subjects developed dementia, including six with Alzheimer's disease (AD), and 19 declined to mild cognitive impairment (MCI), on average 8 years after the baseline exam. The baseline hippocampal MRglc predicted decline from NL to AD (81% accuracy), including two post-mortem confirmed cases, from NL to other dementias (77% accuracy), and from NL to MCI (71% accuracy). Greater rates of hippocampal and cortical MRglc reductions were found in the declining as compared to the non-declining NL. Hippocampal MRglc reductions using FDG-PET during normal aging predict cognitive decline years in advance of the clinical diagnosis. Future studies are needed to increase preclinical specificity in differentiating dementing disorders.
Article
The vesicular monoamine transporter 2 (VMAT2) sequesters monoamines into synaptic vesicles in preparation for neurotransmission. Samples of cerebellum, cortex, hippocampus, substantia nigra and striatum from VMAT2-deficient mice were compared to age-matched control mice. Multivariate statistical analyses of (1)H NMR spectral profiles separated VMAT2-deficient mice from controls for all five brain regions. Although the data show that metabolic alterations are region- and age-specific, in general, analyses indicated decreases in the concentrations of taurine and creatine/phosphocreatine and increases in glutamate and N-acetyl aspartate in VMAT2-deficient mouse brain tissues. This study demonstrates the efficacy of metabolomics as a functional genomics phenotyping tool for mouse models of neurological disorders, and indicates that mild reductions in the expression of VMAT2 affect normal brain metabolism.
Article
We used comparative proteomic techniques to identify aging-related brain proteins in normal mice from neonate to old age. By 2-dimensional electrophoresis (2-DE), matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and peptide mass fingerprint (PMF) analysis, 39 proteins were identified, among which 6 stayed unchanged since 3 months, 6 increased and 27 decreased in various manners during aging. They are mainly involved in processes usually with destructive changes during aging, such as metabolism, transport, signaling, stress response and apoptosis. The 27 proteins' decrease may be responsible for brain aging. In particular, decrease of proteasome alpha subunits 3/6, ubiquitin carboxyl-terminal esterase L3, valosin-containing protein and calreticulin may be responsible for the declination of protein quality control; glutamate dehydrogenase 1, isocitrate dehydrogenase 1 and ubiquinol cytochrome c reductase core protein 2 for the shortage of energy and reducing agent; ubiquitin-conjugating enzyme E2N and heterogeneous nuclear ribonucleoprotein A2/B1 for the increase of DNA damage and transcription detuning; calbindin 1 and amphiphysin for the disturbance of synaptic transport and ion signals. The six proteins' increase may be involved in anti-aging processes. In particular, transketolase, mitochondrial creatine kinase 1 and ribosomal protein L37 may help to enhance energy metabolism; triosephosphate isomerase 1 may help to resist oxidative stress. Moreover, most of these proteins were found for the first time to be involved in the natural senescence of brain, which would provide new clues about the mechanism of brain aging.