Human brain nucleoside diphosphate kinase activity is decreased in Alzheimer's disease and Down syndrome

In brain, nucleoside diphosphate kinase (NDPK) and its coding gene, nm23, have been implicated to modulate neuronal cell proliferation, differentiation, and neurite outgrowth. However, a role of NDPK in neurodegenerative diseases has not been reported yet. Using proteomics techniques, we evaluated the protein levels of NDPK-A in seven brain regions from patients with Alzheimer's disease (AD) and Down syndrome (DS) showing AD-like neuropathology. NDPK-A was significantly decreased in brain regions (frontal, occipital, and parietal cortices) of both disorders. Due to the limitation of brain samples, the activity of NDPK was measured in three brain regions (frontal cortex, temporal cortex, and cerebellum). The specific activity of NDPK was significantly decreased in AD (frontal cortex) and DS (frontal and temporal cortices). Since NDPK-B could also drive the activity of NDPK, protein expression levels of both NDPK-A and NDPK-B were studied in frontal cortex by Western blot analysis. NDPK-A was significantly decreased in AD, which was consistent with the results of proteomics. However, NDPK-A was slightly decreased in DS and protein expression levels of NDPK-B in both DS and AD were moderately decreased, without reaching statistical significance. We propose that oxidative modification of NDPK could lead to the decreased activity of NDPK and, subsequently, influence several neuronal functions in neurodegenerative diseases as multifunctional enzyme through several mechanisms.     

The reduction of NADH ubiquinone oxidoreductase 24- and 75-kDa subunits in brains of patients with Down syndrome and Alzheimer's disease

NADH: ubiquinone oxidoreductase (complex I), one of the most complicated multi-protein enzyme complexes, is important for energy metabolism because it is the initial enzyme of the mitochondrial respiratory chain. Deficiency of complex I is frequently found in various tissues of patients with neurodegenerative disease. Here we studied the protein levels of complex I 24- and 75-kDa subunits in several brain regions from patients with Down syndrome (DS) and Alzheimer's disease (AD). We determined protein levels of complex I 24-, 75-kDa subunits and mitochondrial marker proteins mitochondrial matrix protein P1 (hsp60) and aconitate hydratase from seven brain regions of patients with DS, AD and controls. Proteins were separated by two-dimensional (2-D) gel electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Complex I 24-kDa subunit was significantly reduced in occipital cortex and thalamus in patients with DS and temporal and occipital cortices in patients with AD. Complex I 75-kDa subunit was significantly reduced in brain regions from patients with DS (temporal, occipital and caudate nucleus) and AD (parietal cortex). Reductions of two subunits of complex I may lead to the impairment of energy metabolism and result in neuronal cell death (apoptosis), a hallmark of both neurodegenerative disorders.     

Cortical Cytochrome Oxidase Activity Is Reduced in Alzheimer's Disease

Abstract: A defect in energy metabolism may play a role in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease. In the present study, we examined the activities of the enzymes that catalyze oxidative phosphorylation in frontal, temporal, parietal, and occipital cortex from Alzheimer's disease patients and age-matched controls. Complex I and complex II–III activities showed a small decrease in occipital cortex, but were unaffected in the other cortical areas. The most consistent change was a significant decrease of cytochrome oxidase (complex IV) activity of 25–30% in the four cortical regions examined. These results provide further evidence of a cytochrome oxidase defect in Alzheimer's disease postmortem brain tissue. A deficiency in this key energy-metabolizing enzyme could lead to a reduction in energy stores and thereby contribute to the neurodegenerative process.     

Neuroendocrine-specific protein C, a marker of neuronal differentiation, is reduced in brain of patients with Down syndrome and Alzheimer's disease

Neuroendocrine-specific protein C (NSP-C) is found in neural and neuroendocrine cells and associated with the endoplasmic reticulum. Its expression was found to correlate with the degree of neuronal differentiation. As the neuropathological findings in Down syndrome (DS) includes deficits of differentiation, and we detected a downregulated sequence with 100% homology with NSP-C homolog mRNA in temporal cortex of patients with DS as well as Alzheimer's disease (AD) using differential display-polymerase chain reaction (DD-PCR), we decided to examine the protein levels of NSP-C in temporal, frontal cortex and cerebellum of DS and AD. To normalize NSP-C versus neuronal density, we also determined neuron-specific enolase (NSE) levels and calculated the ratios. NSP-C was significantly reduced in DS (temporal and frontal cortex) and AD (frontal cortex) compared to controls. The significant decrease of NSP-C in DS was even more pronounced when related to NSE levels. Impaired differentiation in DS brain may well be due to absolutely and relatively decreased NSP-C levels in temporal and frontal cortex. As NSP-C was also reduced in AD frontal cortex, NSP-C deficits in these disorders may be reflecting neurodegenerative changes rather than a primary and specific finding of DS or AD pathogenesis.     

Chronic overexpression of the calcineurin inhibitory gene DSCR1 (Adapt78) is associated with Alzheimer's disease

The DSCR1 (Adapt78) gene was independently discovered as a resident of the "Down syndrome candidate region"and as an "adaptive response"shock or stress gene that is transiently induced during oxidative stress. Recently the DSCR1 (Adapt78) gene product was discovered to be an inhibitor of the serine/threonine phosphatase, calcineurin, and its signaling pathways. We hypothesized that DSCR1 (Adapt78) might also be involved in the development of Alzheimer's disease. To address this question we first studied DSCR1 (Adapt78) in multiple human tissues and found significant expression in brain, spinal cord, kidney, liver, mammary gland, skeletal muscle, and heart. Within the brain DSCR1 (Adapt78) is predominantly expressed in neurons within the cerebral cortex, hippocampus, substantia nigra, thalamus, and medulla oblongata. When we compared DSCR1 (Adapt78) mRNA expression in post-mortem brain samples from Alzheimer's disease patients and individuals who had died with no Alzheimer's diagnosis, we found that DSCR1 (Adapt78) mRNA levels were about twice as high in age-matched Alzheimer's patients as in controls. DSCR1 (Adapt78) mRNA levels were actually three times higher in patients with extensive neurofibrillary tangles (a hallmark of Alzheimer's disease) than in controls. In comparison, post-mortem brain samples from Down syndrome patients (who suffer Alzheimer's symptoms) also exhibited DSCR1 (Adapt78) mRNA levels two to three times higher than controls. Using a cell culture model we discovered that the amyloid beta(1-42) peptide, which is a major component of senile plaques in Alzheimer's, can directly induce increased expression of DSCR1 (Adapt78). Our findings associate DSCR1 (Adapt78) with such major hallmarks of Alzheimer's disease as amyloid protein, senile plaques, and neurofibrillary tangles.     

Neuronal localization of amyloid beta protein precursor mRNA in normal human brain and in Alzheimer''s disease.

Clones for the amyloid beta protein precursor gene were isolated from a cDNA library prepared from the frontal cortex of a patient who had died with a histologically confirmed diagnosis of Alzheimer's disease; they were used to investigate the tissue and cellular distribution of amyloid beta protein precursor mRNA in brain tissues from control patients and from Alzheimer's disease patients. Amyloid beta protein precursor mRNA was expressed in similar amounts in all control human brain regions examined, but a reduction of the mRNA level was observed in the frontal cortex from patients with Alzheimer's disease. By in situ hybridization amyloid beta protein precursor mRNA was present in granule and pyramidal cell bodies in the hippocampal formation and in pyramidal cell bodies in the cerebral cortex. No specific labelling of glial cells or endothelial cells was found. The same qualitative distribution was observed in tissues from control patients and from patients with Alzheimer's disease. Senile plaque amyloid thus probably derives from neurones. The tissue distribution of amyloid beta protein precursor mRNA and its cellular localization demonstrate that its expression is not confined to the brain regions and cells that exhibit the selective neuronal death characteristic of Alzheimer's disease.     

Influence of Region-Specific Alterations of Neuropeptidase Content on the Catabolic Fates of Neuropeptides in Alzheimer's Disease

Abstract: We established the cartography of 11 exo- and endopeptidases in the frontal and parietal cortices and in the cerebellum of brains of patients diagnosed with a senile dementia of the Alzheimer's type (SDAT). Comparison with those of four subjects who had died without known neurologic or psychiatric illness indicated that there existed a region-specific alteration of the peptidase contents in the disease. In the frontal area of SDAT brains, postproline dipeptidyl aminopeptidase and aminopeptidase M activities were significantly reduced. In the parietal cortex of SDAT brain, activities of three additional endopeptidases—angiotensin-converting enzyme, proline endopeptidase, and endopeptidase 24.15—were also drastically reduced. In contrast, the cerebellum displayed a set of proteolytic activities that remained unaffected in SDAT brain. The putative influence of the disease on the catabolic fates of neurotensin, neuropeptide Y, and somatostatin(1–14) was investigated. Neurotensin was catabolized at identical rates in the frontal and parietal cortices in nondemented and SDAT brains. In contrast, neuropeptide Y metabolism was slowed down in SDAT brains in the frontal but not in the parietal cortex. Finally, the degradation velocities of somatostatin(1–14) were lowered in both cortical areas of SDAT brains. It is interesting that, by means of specific peptidase inhibitors, we demonstrated that endopeptidase 24.15 participated in somatostatin(1–14) inactivation in the parietal but not in the frontal cortex. It is suggested that the lowering of the rate of somatostatin(1–14) inactivation in the parietal cortex of SDAT brains likely results from the depletion of endopeptidase 24.15 in this brain region.     

Phosphatidylinositol Kinase Is Reduced in Alzheimer''s Disease

Phosphatidylinositol (PI) kinase and PI phosphate (PIP) kinase activities were measured in postmortem samples of brain tissue from patients with Alzheimer's disease and nondemented control subjects. A membrane-free cytosolic fraction from four neocortical locations, with exogenous inositol lipids as the substrate, was used. Tissue from patients with Alzheimer's disease was characterized by reduced PIP formation; the reduction was 50% in prefrontal cortex, temporal cortex, and parietal cortex and 40% in precentral gyrus. In contrast, no alterations were found in PI bisphosphate formation in these four neocortical locations. The specific changes in PI kinase but not PIP kinase activity suggest that the findings may have functional relevance to the involvement of brain membrane processes in Alzheimer's disease.     

Ceruloplasmin immunoreactivity in neurodegenerative disorders

Ceruloplasmin (CP) is a 132kd cuproprotein which, together with transferrin, provides the majority of anti-oxidant capacity in serum. Increased iron deposition and lipid peroxidation in the basal ganglia of subjects with hereditary CP deficiency suggest that CP may serve as an anti-oxidant in the brain as well. The present study compared CP immunoreactivity in brain specimens from normal controls and subjects with neurodegenerative disorders (Alzheimer's disease [AD], Parkinson's disease [PD], progressive supranuclear palsy [PSP], and Huntington's disease [HD]) (n = 5 per group). The relative intensity of neuronal CP staining and the numbers of CP-stained neurons per 25x microscope field were determined in hippocampus (CA1, subiculum, and parahippocampal gyrus), parietal cortex, frontal cortex, substantia nigra, and caudate. CP was detected in both neurons and astrocytes in all specimens, and in senile plaques and occasional neurofibrillary tangles in AD brain. Neuronal CP staining intensity tended to increase in most AD brain regions, but was statistically significant vs controls only in the CA1 region of hippocampus (p = .016). Neuronal CP staining in brain specimens from other neurodegenerative disorders showed a slight but nonsignificant increase vs controls. The numbers of CP-stained neurons per field did not differ between the various neurodegenerative disorders and controls. These results suggest that a modest increase in neuronal CP content is present in the AD brain, and lesser elevations in neuronal CP occur in the other neurodegenerative disorders in this study. Though CP functions as both an acute phase protein and an anti-oxidant in peripheral tissues, whether it does so in the brain remains to be determined.     

Cellular ageing of Alzheimer's disease and Down syndrome cells in culture.

In Alzheimer's disease, the typical clinical symptoms and the pathological findings are restricted to the nervous system. Nevertheless, like in some other neurologic-metabolic disorders, several alterations are found in peripheral tissues. The aim of this study was to examine whether cellular properties which can be studied in vitro on skin fibroblast cultures obtained from Alzheimer's disease patients differ from those of age-matched controls. Down syndrome patients were also included, since the same neuropathological findings are present in nearly 100% of Down syndrome patients. Since Alzheimer's disease is an age-related disorder, we examined the growth characteristics of skin fibroblast cultures. The in vitro senescence of cultured fibroblasts is widely accepted as a model for in vivo ageing. Normal growth properties were found. We can conclude that there is no premature ageing in Alzheimer's disease nor in Down syndrome and that the abnormalities found in peripheral tissues are related to the disease itself. The beta amyloid precursor protein (beta APP) has been shown to have adhesive interactions. We therefore investigated several parameters of adhesion in the skin fibroblast cultures: adhesion to a fibronectin coat, adhesion to extracellular matrix of Alzheimer's disease cultures and semi-quantification of adhesion-related molecules (beta 1-integrin, cell surface proteoglycans, extracellular matrix proteoglycans, extracellular matrix fibronectin). No significant difference was found in the parameters examined.     

Activity of phosphatidylethanolamine-N-methyltransferase in brain affected by Alzheimer's disease

To determine whether phospholipid abnormality in Alzheimer's disease is associated with modification of phosphatidylethanolamine-N-methyltransferase, the activity of the enzyme was analysed in the frontal and occipital cortex of the brain from patients with Alzheimer's disease and from aged-matched control. The optimum pH for phosphatidylethanolamine-N-methyltransferase in human brain was 9.0. The enzyme activity was stimulated by detergent TWEEN 20 but inhibited by Triton X-100. Neither magnesium dependence nor chemical methylation was found. A decrease in activity of phosphatidylethanolamine-N-methyltransferase was observed in the frontal cortex of brain affected with Alzheimer's disease. The addition of exogenous phosphatidylethanolamine resulted in no modification in the methylation rate as compared with that of endogenous PE. The addition of phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine resulted in significantly increased rates of methylation in brain tissues. However, the increased rate of phosphatidylethanolamine-N-methyltransferase activity stimulated by exogenous phospholipids was lower in the frontal cortex of brains with Alzheimer's disease when compared to the normals and there was no difference in the occipital cortex between Alzheimer's disease and the control. It is plausible that the decreased activity of phosphatidylethanolamine-N-methyltransferase and its low compensating ability could relate to the modification of phosphatidylcholine in brain tissues from Alzheimer's disease patients.     

High levels of synaptosomal Na(+)-Ca(2+) exchangers (NCX1, NCX2, NCX3) co-localized with amyloid-beta in human cerebral cortex affected by Alzheimer's disease

Synaptosomal expression of NCX1, NCX2, and NCX3, the three variants of the Na(+)-Ca(2+) exchanger (NCX), was investigated in Alzheimer's disease parietal cortex. Flow cytometry and immunoblotting techniques were used to analyze synaptosomes prepared from cryopreserved brain of cognitively normal aged controls and late stage Alzheimer's disease patients. Major findings that emerged from this study are: (1) NCX1 was the most abundant NCX isoform in nerve terminals of cognitively normal patients; (2) NCX2 and NCX3 protein levels were modulated in parietal cortex of late stage Alzheimer's disease: NCX2 positive terminals were increased in the Alzheimer's disease cohort while counts of NCX3 positive terminals were reduced; (3) NCX1, NCX2 and NCX3 isoforms co-localized with amyloid-beta in synaptic terminals and all three variants are up-regulated in nerve terminals containing amyloid-beta. Taken together, these data indicate that NCX isoforms are selectively regulated in pathological terminals, suggesting different roles of each NCX isoform in Alzheimer's disease terminals.     

Cortical distribution of gangliosides in Alzheimer's disease.

In Alzheimer's disease, all ganglio-series gangliosides (GM1, GD1a, GD1b and GT1b) were found to be decreased in temporal and frontal cortex, and nucleus basalis of Meynert. In addition, in Alzheimer's disease simple gangliosides (GM2, GM3) were elevated in frontal and parietal cortex, possibly correlating to accelerated lysosomal degradation of gangliosides and/or astrogliosis occurring during neuronal death.     

Endocytic adaptor protein intersectin 1 forms a complex with microtubule stabilizer STOP in neurons

Intersectin 1 (ITSN1) is a multidomain adaptor protein that functions in clathrin-mediated endocytosis and signal transduction. This protein is highly abundant in neurons and is implicated in Down syndrome, Alzheimer's disease and, possibly, other neurodegenerative disorders. Here we used an in vitro binding assay combined with MALDI-TOF mass spectrometry to identify novel binding partners of ITSN1. We found that the neuron-specific isoform of the stable tubule-only polypeptide (STOP) interacts with SH3A domain of ITSN1. STOP and ITSN1 were shown to form a complex in vivo and to partially co-localize in rat primary hippocampal neurons. As STOP is a microtubule-stabilizing protein that is required for several forms of synaptic plasticity in the hippocampus, identification of this interaction raises the possibility of ITSN1 participation in this process.     

Intact cannabinoid CB1 receptors in the Alzheimer's disease cortex

The cannabinoid CB1 receptor has gained much attention as a potential pharmacotherapeutic target in various neurodegenerative diseases including Alzheimer's disease (AD). However, the relation of CB1 receptors to cognitive function in AD is at present unclear. In this study, postmortem brain tissues from a cohort of prospectively assessed, neuropathologically confirmed AD patients and aged controls were used to measure CB1 receptors by immunoblotting, and a subset of subjects also had [(3)H]SR141716A binding. Correlational analyses were then performed for the neurochemical and cognitive data. We found that CB1 receptor levels in were unchanged AD in the brain regions assessed (frontal cortex, anterior cingulate gyrus, hippocampus, caudate nucleus). Within the AD group, frontal cortical CB1 immunoreactivity correlated with cognitive scores assessed within a year of death. Our study suggests that CB1 receptors are intact in AD and may play a role in preserving cognitive function. Therefore, CB1 receptors should be further assessed as a potential therapeutic target in AD.     

A gel-based proteomic method reveals several protein pathway abnormalities in fetal Down syndrome brain

A large series of protein pathway components have been shown to be dysregulated in Down syndrome (DS) brain. No information about pathomechanisms linked to the trisomic state can be obtained from adult DS brain, however, as neurodegeneration occurs from the fourth decade. The aim of the study was to search for protein dysregulation in fetal DS brain before neurodegenerative changes are observed. Proteins were extracted from fetal DS and control frontal cortex, run on 2-DE, followed by quantification of protein spots with subsequent nano-ESI-LC-MS/MS analysis using an ion trap. Aberrant expression of proteins tropomodulin-2, tubulin alpha 1A chain, and alpha-internexin may indicate disturbed synaptic plasticity; fatty acid binding protein 7 suggests impaired maintenance of neuroepithelial cells; and creatine kinase B may reflect defective energy metabolism. RNA binding protein 4B derangement may represent impaired splicing, altered retrotransposon gag domain-containing protein 1 levels may be pointing to altered retrotransposition, and level changes of the potassium-chloride transporter solute carrier family 12 member 7 may lead to impaired ion fluxes with electrophysiological consequences. Taken together, aberrant protein levels from several pathways in fetal DS are challenging as well as fertilizing the area of research and providing the basis for additional neurochemical and functional studies.     

3H]-ketanserin binding sites in different psychiatric disorders

The results of the present study showed the presence of a high-affinity and saturable binding of [3H]-ketanserin to frontal and parietal brain membranes obtained postmortem from bipolar, depressed, schizophrenic patients and normal controls. The human brain samples (60 frontal cortex and 51 parietal cortex), were donated by the Stanley Foundation Brain Collection. The overall data showed that normal controls, depressed and schizophrenic patients had a higher density in the frontal than in the parietal cortex, while bipolar patients did not show any difference. When the data were analysed according to the two hemispheres, some additional, intriguing observations were made: it emerged that [3H]-ketanserin binding sites did not show any difference in the two frontal cortices, while they were less represented in the right parietal cortex of normal and bipolar patients and more dense in schizophrenic patients. In conclusion, our study has demonstrated the presence of heterogenous alterations of [3H]-ketanserin binding sites in healthy controls and different psychiatric disorders that may be of help in a further elucidation of the specific role that 5-HT(2A) receptors may play in these disorders.     

Human brain gangliosides in development, aging and disease.

In this study, brain gangliosides in prenatal and postnatal human life and Alzheimer's disease were analyzed. Immunohistochemically, the presence of the "c"-series of gangliosides (GQ1c) was only registered in the embryonic brain at 5 weeks of gestation. Biochemical results indicated a two-fold increase in ganglioside concentration in the human cortex between 16 and 22 weeks of gestation. The increasing ganglioside concentration was based on an increasing GD1a ganglioside fraction in all regions analyzed except in the cerebellar cortex, which was characterized by increasing GT1b. During prenatal human development, regional differences in ganglioside composition could only be detected between the cerebrum ("a"-pathway) and the cerebellum ("b"-pathway). Between birth and 20-30 years of age, a cerebral neocortical difference of ganglioside composition occurred, characterized by the lowest GD1a in visual cortex. Analyzing the composition of gangliosides in cortical regions during aging, they were observed to follow region-specific alterations. In the frontal cortex, there was a greater decrease in GD1a and GM1 than in GT1b and GD1b, but in the occipital (visual) cortex there was no change in individual gangliosides. In hippocampus, GD1a moderately decreased, whereas other fractions were stable. In the cerebellar cortex, GD1b and GT1b fractions decreased with aging. In Alzheimer's disease, we found all ganglio-series gangliosides (GM1, GD1a, GD1b, GT1b) to be decreased in regions (temporal and frontal cortex and nucleus basalis of Meynert) involved in pathogenesis of disease. In addition, in Alzheimer's disease we found simple gangliosides (GN2, GM3) to be elevated in the frontal and parietal cortex, which might correlate accelerated lysosomal degradation of gangliosides and/or astrogliosis occurring during neuronal death.     

Heterologous expression and folding analysis of a beta-tubulin isotype from the Antarctic ciliate Euplotes focardii.

Mammalian tubulins and actins attain their native conformation following interactions with CCT (the cytosolic chaperonin containing t-complex polypeptide 1). To study the beta-tubulin folding in lower eukaryotes, an isotype of beta-tubulin (beta-T1) from the Antarctic ciliate Euplotes focardii, was expressed in Escherichia coli. Folding analysis was performed by incubation of the 35S-labeled, denatured beta-T1 in the presence, or absence, of purified rabbit CCT and cofactor A, a polypeptide that stabilizes folded monomeric beta-tubulin. We show for the first time in protozoa that beta-tubulin folding is assisted by CCT and requires cofactor A. In addition, we observed that E. focardiibeta-T1 competes with human beta5 tubulin isotype for binding to CCT. The affinity of CCT to E. focardiibeta-T1 and beta5 tubulin are compared. Finally, the mitochondrial chaperonin mt-cpn60 binds to beta-T1 but is unable to release it in a native or quasi-native state.     

iTRAQ analysis of complex proteome alterations in 3xTgAD Alzheimer's mice: understanding the interface between physiology and disease

Alzheimer's disease (AD) is characterized by progressive cognitive impairment associated with accumulation of amyloid beta-peptide, synaptic degeneration and the death of neurons in the hippocampus, and temporal, parietal and frontal lobes of the cerebral cortex. Analysis of postmortem brain tissue from AD patients can provide information on molecular alterations present at the end of the disease process, but cannot discriminate between changes that are specifically involved in AD versus those that are simply a consequence of neuronal degeneration. Animal models of AD provide the opportunity to elucidate the molecular changes that occur in brain cells as the disease process is initiated and progresses. To this end, we used the 3xTgAD mouse model of AD to gain insight into the complex alterations in proteins that occur in the hippocampus and cortex in AD. The 3xTgAD mice express mutant presenilin-1, amyloid precursor protein and tau, and exhibit AD-like amyloid and tau pathology in the hippocampus and cortex, and associated cognitive impairment. Using the iTRAQ stable-isotope-based quantitative proteomic technique, we performed an in-depth proteomic analysis of hippocampal and cortical tissue from 16 month old 3xTgAD and non-transgenic control mice. We found that the most important groups of significantly altered proteins included those involved in synaptic plasticity, neurite outgrowth and microtubule dynamics. Our findings have elucidated some of the complex proteome changes that occur in a mouse model of AD, which could potentially illuminate novel therapeutic avenues for the treatment of AD and other neurodegenerative disorders.