Experimental traumatic brain injury in rats stimulates the expression, production and activity of Alzheimer's disease beta-secretase (BACE-1).

Traumatic brain injury (TBI) is a risk factor for the development of Alzheimer's disease (AD). After a traumatic brain injury depositions of amyloid beta (Abeta) in the brain parenchyma were found. In this study we investigated the expression pattern of beta-secretase (BACE-1) in ipsi- or contralateral hippocampus and cortex following controlled cortical TBI in rats. BACE-1 mRNA levels, estimated by real time RT-PCR, were elevated 24 h post injury, and persisting up to 72 h, in the ipsi- and contralateral hippocampus and cerebral cortex as compared to the sham-treated animals (p<0.01). The TBI-induced changes in BACE-1 mRNA are due to enhanced hippocampal and cortical expression of BACE-1 mRNA in neurons and reactive astrocytes as revealed by in situ hybridization. The alterations in hippocampal BACE-1 mRNA levels are accompanied by corresponding increases in BACE-1 protein levels in ipsi- and contralateral hippocampus and ipsilateral cortex as demonstrated by Western blot analysis. In contrast, in the contralateral cortex only a weak increase of traumatically induced BACE-1 protein production was found. The activity of BACE-1 as measured by the formation of the cleavage product of amyloid beta precursor protein, transiently increased up to 48 h after injury, but returned to basal level 7 days post injury. This study demonstrates that the beta-secretase is stimulated following TBI and may suggest a mechanism for the temporal increase of Abeta levels observed in patients with brain trauma.

Cortical glucose metabolism is altered in aged transgenic Tg2576 mice that demonstrate Alzheimer plaque pathology.

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.

Expression of beta-Secretase mRNA in the brain of rats with immunohistochemical destruction of basal forebrain cholinergic system.

We studied properties of cloned BACE mRNA (beta-site of the enzyme cleaving amyloid precursor protein) and evaluated the possibility of using this clone for identification and/or prediction of neurodegenerative disorders associated with cholinergic deficiency. Wistar rats subjected to immunohistochemical destruction of the basal forebrain cholinergic system were used as the experimental model. Nonradioactive in situ hybridization and immunohistochemical visualization of the astroglia revealed strong hybridization signal of BACE mRNA in neurons of the cortex, hippocampus, and thalamus. Astrocytes remained unstained. Immunohistochemical destruction of the basal forebrain produced no significant changes in the distribution of BACE mRNA.

Deafferentation of the septo-hippocampal pathway in rats as a model of the metabolic events in Alzheimer's disease.

Changes in the metabolic activity within the brain of patients suffering from Alzheimer's disease (AD) were investigated and compared with biochemical alterations in the hippocampus induced by fimbria/fornix transection in the rat. The deafferentation of the hippocampus results in a degeneration of cholinergic septo-hippocampal terminals accompanied by a persistent decrease of choline acetyltransferase (ChAT) and acetylcholine esterase (AChE) activities similar to the cholinergic malfunction in AD. In the animal model the [3H]-cytochalasin B binding to the glucose transporters was elevated up to the day 7 after surgery as was the activity of the phosphofructokinase (PFK) on day 3. A reactive astrogliosis could be evidenced by the upregulation of glial fibrillary acidic protein (GFAP). An increase of the PFK activity was also found in AD being accompanied by enhanced level of GFAP as well. A higher concentration of mRNA for all three isoenzymes of PFK was shown by reverse transcription (RT)-real time polymerase chain reaction (PCR) amplification. However, the pattern of PFK isoenzyme proteins and mRNAs did neither change in diseased human nor in the lesioned rat brain. The activities of the mitochondrial enzymes pyruvate dehydrogenase complex (PDHC) and cytochrome c oxidase (CO) were diminished in the lesioned rat hippocampus on day 7 as well as in AD brain. Subcellular fractionation showed that the activity of these enzymes was affected in the synaptosomal as well as in the extrasynaptosomal mitochondria indicating a loss of neuronal input and also a vulnerability of intrinsic hippocampal neurons and/or non-neuronal cells. The recovery of the mitochondrial enzyme activity in the animal model at later post lesion intervals may be the result of compensatory responses of surviving cells or of sprouting of other non-affected inputs. It is concluded that common metabolic mechanisms may underlie the concurrent degenerative and repair processes in the denervated hippocampus and the diseased Alzheimer brain.

Expression of fructose-1,6-bisphosphatase mRNA isoforms in normal and basal forebrain cholinergic lesioned rat brain.

Fructose-1,6-bisphosphatase is one of the key enzymes in the gluconeogenic pathway predominantly occurring in liver, kidney and muscle. In the brain, fructose-1,6-bisphosphatase has been suggested to be an astrocyte-specific enzyme but the functional importance of glyconeogenesis in the brain is still unclear. To further elucidate the cellular source of fructose-1,6-bisphosphatase in the brain, non-radioactive in situ hybridizations were performed using digoxigenin-labeled RNA probes based on the sequence of recently cloned rat liver and muscle fructose-1,6-bisphosphatase cDNAs. In situ hybridization using a riboprobe for the liver isoform revealed a location of the hybridization signal mainly in neurons, while rat muscle fructose-1,6-bisphosphatase mRNA was detected in both neurons and astrocytes in the hippocampal formation and in layer I of the cerebral cortex.RT-PCR using RNA preparations of rat astrocytes, neurons, and adult whole brain demonstrated a localization of liver fructose-1,6-bisphosphatase mRNA isoform in neurons but not in astrocytes. The muscle fructose-1,6-bisphosphatase mRNA isoform could be detected by RT-PCR in total rat brain, astrocytic, and neuronal mRNA preparations. The isoforms of fructose-1,6-bisphosphatase mRNA seemingly demonstrate a distinct cellular expression pattern in rat brain suggesting a role of glyconeogenesis in both neurons and glial cells.

Expression of beta-secretase mRNA in transgenic Tg2576 mouse brain with Alzheimer plaque pathology.

On the basis of the recent cloning of the beta-secretase, the beta-site amyloid precursor protein (APP)-cleaving enzyme (BACE), (Science, 286 (1999) 735), digoxigenin-labelled riboprobes were generated to localize the cellular expression pattern of BACE mRNA in brain sections of transgenic Tg2576 mice, overexpressing the Swedish mutation of the APP695 isoform. Non-radioactive in situ hybridization in combination with immunohistochemistry to identify the cell types and beta-amyloid deposits revealed strong BACE mRNA hybridization signals in neurons of the cerebral cortex, hippocampal formation, thalamus and cholinergic basal forebrain nuclei, while astrocytes did not display any labeling. Neurons surrounding beta-amyloid deposits did not demonstrate altered expression level of BACE mRNA as compared to neurons in cortical areas that are free of beta-amyloid deposits, and the regional expression pattern of BACE mRNA did not correlate with the distribution of beta-amyloid deposits. These data suggest that high level of expression of BACE mRNA is not necessarily related to enhanced deposition of beta-amyloid plaques. To elucidate those factors that contribute to beta-amyloid plaque deposition in a particular region, the transgenic Tg2576 mouse may represent an appropriate tool.

Altered phosphofructokinase mRNA levels but unchanged isoenzyme pattern in brains from patients with Alzheimer's disease.

In order to find out whether the increased phosphofructokinase (PFK) activities observed in brains from Alzheimer's disease (AD) patients are associated with alterations in PFK mRNA levels, we determined total PFK mRNA and the three different PFK isoenzyme mRNAs in AD and control patients by ribonuclease protection assay (RPA) and quantitative RT-PCR. PFK mRNA levels were found increased in some brain areas in AD patients. While all three PFK isoenzyme mRNAs were detectable in every studied brain sample, no changes of the PFK isoenzyme pattern were observed in patients with AD.

Activities of key glycolytic enzymes in the brains of patients with Alzheimer's disease.

The activities of hexokinase, aldolase, pyruvate kinase, lactate dehydrogenase and glucose 6-phosphate dehydrogenase were determined in brains of patients with Alzheimer's disease (AD) and in age matched controls. For pyruvate kinase and lactate dehydrogenase a significant increase in specific activity was found in frontal and temporal cortex of AD brains, while the activities of aldolase and hexokinase are not changed. Glucose 6-phosphate dehydrogenase activity was significantly reduced in hippocampus. The increase of some glycolytic enzyme activities is correlated with increased contents of lactate dehydrogenase and glial fibrillary acidic protein (GFAP) in homogenates of frontal and temporal cortex and elevated phosphofructokinase (PFK) and GFAP in astrocytes from the same brain areas. The data extend previous findings on an increase in brain PFK specific activity in AD and suggest that the increased activity of some glycolytic enzymes may be, at least in part, the result of the reactive astrocytosis developing in the course of AD.

Cellular distribution of 6-phosphofructo-1-kinase isoenzymes in rat brain.

In the brain, all three isoenzyme types [muscle (M), liver (L), and brain (C)] of 6-phosphofructo-1-kinase (PFK; EC 2.7.1.11) occur, forming a complex mixture of homo- and heterotetramers. The PFK isoenzyme pattern of the different brain cell types is yet unknown. In the present study, we investigated the distribution of the PFK isoenzyme subunits in primary and secondary cell cultures and in bulk-isolated cells of rat brain by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. All three PFK isoenzymes are present in all cell types but in different proportions. The cellular distribution of the PFK isoenzymes in situ was studied immunohistochemically with different polyclonal antisera against purified rat PFKs. The monospecific antibody against M-type PFK stained preferentially the perinuclear areas of neurons and glial cells. The antibodies that in immunoblots detected mainly the L-type PFK showed a characteristic staining in only the cytoplasma and the processes of cells, whereas the C-type antibodies almost homogeneously stained whole cell bodies as well as large dendrites. Because the PFK isoenzymes differ with respect to their allosteric properties, their differential distribution in different brain cells might be of importance for the regulation of brain glycolysis in the different cellular compartments of the brain.

Changes of activity and isozyme pattern of phosphofructokinase in the brains of patients with Alzheimer's disease.

A severe reduction of the in vivo cerebral glucose consumption rate is generally found in patients with Alzheimer's disease. In postmortem studies changes in the activities of key regulatory glycolytic enzymes, including 6-phosphofructokinase (PFK), have been reported in Alzheimer's disease brains, but the results obtained so far are inconsistent and controversial. We reevaluated the activity of PFK in brain tissue from clinically and neuropathologically confirmed cases of Alzheimer's disease using optimized tissue disintegration and assay methods and determined the PFK isozyme pattern. PFK activity in brains from patients with Alzheimer's disease was significantly increased in frontal and temporal cortex and unchanged in the other brain areas studied when compared with control brains. All three PFK isozymes were detected in each of the brain areas studied. In brains of Alzheimer's disease patients the level of the C-type PFK was slightly reduced at the expense of the M- and L-type subunits. The data presented do not support the results of other groups, which reported up to a 90% reduction of PFK activity in Alzheimer's disease. In contrast, the data presented clearly rule out the suggestion that changes of PFK activity might be one of the causes for the reduced glucose consumption in Alzheimer's disease brains.

Interaction of rat brain phosphofructokinase with Alzheimer's beta A4-amyloid.

One of the key functional disturbances in incipient dementia of Alzheimer type is the reduction of cerebral glucose utilization. Morphologically the brains of Alzheimer patients are characterized by multiple depositions of beta A4-amyloid mainly within extracellular senile plaques and in the walls of cerebral blood vessels, but also attached to intracellular neurofibrillary tangles. Intracellular beta A4-amyloid may bind to other cellular components. The interaction of beta A4-amyloid with phosphofructokinase, one of the key enzymes in glycolysis, was studied in vitro under various conditions. beta A4-amyloid was found to bind in nanomolar concentrations to phosphofructokinase and decrease its activity. Binding was demonstrated by enzyme linked immunoassays and by gel filtration studies. Inactivation of phosphofructokinase by beta A4-amyloid could only partially be prevented by fructose 6-phosphate. In control experiments no interaction was detectable between lactate dehydrogenase and beta A4-amyloid.

Overexpression of catalytically active yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase in Escherichia coli.

E. coli expression plasmids for yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase (EC 3.1.3.11) as wild-type enzyme and as lacZ fusion protein have been constructed from a pUC vector and a fragment of genomic yeast DNA. Both proteins were overexpressed in E. coli strain TG2 as enzymatically active soluble forms and purified to homogeneity. While the wild-type enzyme is indistinguishable from the authentic yeast enzyme with respect to molecular size, specific activity and kinetic properties, the lacZ fusion protein behaves differently. Being a tetramer like the wild-type enzyme, the specific activity of the purified fusion protein is lower than that of the native enzyme. In contrast to the wild-type enzyme the fusion fructose-1,6-bisphosphatase is not inhibited by excess substrate. Inhibition of the fusion protein by the most potent allosteric effectors of fructose-1,6-bisphosphatase, AMP and fructose 2,6-bisphosphate, is weaker than observed with the wild-type enzyme. The fusion protein but not the wild-type enzyme was found to bind to immobilized Procion Navy H-ER. This was employed to purify the fusion fructose-1,6-bisphosphatase by affinity chromatography. Polyclonal antibodies raised in rabbits against the fusion enzyme were found to cross-react with the wild-type enzyme, but not with E. coli proteins. Both fructose-1,6-bisphosphatases complement the fructose-1,6-bisphosphatase mutant DF656 of E. coli.

Expression of cGMP-dependent protein kinase in Escherichia coli.

Cyclic GMP-dependent protein kinase (cGMP kinase) is involved in the relaxation of smooth muscle. The enzyme has been cloned and expressed in eukaryotic cell lines but so far not in prokaryotic cells. Three vectors were constructed for the expression of I alpha cGMP kinase in Escherichia coli. Transformation with the pET3a/cgk vector which uses the T7 RNA polymerase/promotor system resulted in efficient accumulation of cGMP kinase. Most of the protein was in an insoluble and catalytic inactive form. Various solubilization and refolding conditions did not yield an active enzyme. A small fraction of the cGMP kinase was present in the soluble cell extract. This fraction bound cGMP with high affinity but had no cGMP stimulated kinase activity. To prevent aggregation two additional vectors were constructed. (I) A bacterial leader sequence, which directs the export of proteins into the periplasmic space, was fused to the amino-terminus of the cGMP kinase. (II) A gram/gram+ shuttle vector for expression under the control of the tac promotor was used. Both constructs directed the synthesis of an insoluble and inactive cGMP kinase. These results suggest that large amounts of cGMP kinase can be expressed in E. coli, but mainly in an insoluble and inactive form. In contrast to eukaryotic cells, bacteria may lack systems for correct protein folding and/or posttranslational modification that are crucial for the productive folding and/or activation of cGMP kinase.

Kinetics of 6-phosphofructo-1-kinase from a yeast mutant.

The steady state kinetics of 6-phosphofructo-1-kinase was determined in a cell-free extract obtained from a yeast mutant (DFY 250) and compared with the kinetic properties of the enzyme of a wild-type strain (DFY 1). 6-Phosphofructo-1-kinase from the DFY 250 strain shows a complex kinetic behaviour, which is qualitatively similar to, but quantitatively different from, that of normal yeast 6-phosphofructo-1-kinase. The mutant enzyme has a lower affinity to its activators fructose 6-phosphate, fructose 2,6-bisphosphate and AMP. The inhibiting effect of ATP on the mutant 6-phosphofructo-1-kinase is substantially weaker than on the wild-type enzyme. A complex interaction between fructose 6-phosphate and fructose 2,6-bisphosphate at the 6-phosphofructo-1-kinase from strain DFY 250 is reflected by a remarkable substrate inhibition by fructose 6-phosphate even at saturating fructose 2,6-bisphosphate. The kinetic data were fitted to different variants of the Monod-Wyman-Changeux model by nonlinear regression analysis. It turned out that the influence of fructose 6-phosphate, ATP, AMP and fructose 2,6-bisphosphate on the activity of 6-phosphofructo-1-kinase from wild-type and DFY 250 strain could be described by rate equations of essentially the same structure.