Alzheimer's disease (AD) is characterized by amyloid-beta (A␤)-containing plaques, neurofibrillary tangles, and neuron and synapse loss. Tangle formation has been reproduced in P301L tau transgenic pR5 mice, whereas APP sw PS2 N141I double-transgenic APP152 mice develop A␤ plaques. Cross-breeding generates triple transgenic ( triple AD) mice that combine both pathologies in one model. To determine functional consequences of the combined A␤ and tau pathologies, we performed a proteomic analysis followed by functional validation. Specifically, we obtained vesicular preparations from triple AD mice, the parental strains, and nontransgenic mice, followed by the quantitative mass-tag labeling proteomic technique iTRAQ and mass spectrometry. Within 1,275 quantified proteins, we found a massive deregulation of 24 proteins, of which one-third were mitochondrial proteins mainly related to complexes I and IV of the oxidative phosphorylation system (OXPHOS). Notably, deregulation of complex I was tau dependent, whereas deregulation of complex IV was A␤ dependent, both at the protein and activity levels. Synergistic effects of A␤ and tau were evident in 8-month-old triple AD mice as only they showed a reduction of the mitochondrial membrane potential at this early age. At the age of 12 months, the strongest defects on OXPHOS, synthesis of ATP, and reactive oxygen species were exhibited in the triple AD mice, again emphasizing synergistic, age-associated effects of A␤ and tau in perishing mitochondria. Our study establishes a molecular link between A␤ and tau protein in AD pathology in vivo, illustrating the potential of quantitative proteomics.amyloid-beta peptide ͉ electron transport chain ͉ energy metabolism ͉ mitochondrial complexes ͉ tau protein A lzheimer's disease (AD) is a devastating neurodegenerative disorder affecting Ͼ15 million people worldwide (1). The key histopathological features are amyloid-beta (A␤)-containing plaques and microtubule-associated protein tau-containing neurofibrillary tangles (NFTs), along with neuronal and synapse loss in selected brain areas (2, 3). In determining the role of distinct proteins in these processes, traditionally, candidate-driven approaches have been pursued, linking neuronal dysfunction to the distribution of known proteins in healthy compared with degenerating neurons, or in transgenic compared with control brain. In comparison, proteomics offers a powerful nonbiased approach as shown by us previously (4, 5).APP152 (APP/PS2) double-transgenic mice model the A␤ plaque pathology of AD (6); they coexpress the N141I mutant form of PS2 together with the APP sw mutant found in familial cases of AD. The mice display age-related cognitive deficits associated with discrete brain A␤ deposition and inflammation (6). pR5 mice model the tangle pathology of AD (7-9). They express P301L mutant tau found in familial cases of frontotemporal dementia (FTD), a dementia related to AD. The pR5 mice show a hippocampus-and amygdala-dependent behavioral impairment related to AD (10). Crossing of ...

The aim of the present study was to identify the distribution of the second melatonin receptor (MT2) in the human hippocampus of elderly controls and Alzheimer's disease (AD) patients. This is the first report of immunohistochemical MT2 localization in the human hippocampus both in control and AD cases. The specificity of the MT2 antibody was ascertained by fluorescence microscopy using the anti-MT2 antibody in HEK 293 cells expressing recombinant MT2, in immunoblot experiments on membranes from MT2 expressing cells, and, finally, by immunoprecipitation experiments of the native MT2. MT2 immunoreactivity was studied in the hippocampus of 16 elderly control and 16 AD cases. In controls, MT2 was localized in pyramidal neurons of the hippocampal subfields CA1-4 and in some granular neurons of the stratum granulosum. The overall intensity of the MT2 staining was distinctly decreased in AD cases. The results indicate that MT2 may be involved in mediating the effects of melatonin in the human hippocampus, and this mechanism may be heavily impaired in AD.

Evidence suggests that amyloid-beta (Ab) protein is a key factor in the pathogenesis of Alzheimer's disease (AD) and it has been recently proposed that mitochondria are involved in the biochemical pathway by which Ab can lead to neuronal dysfunction. Here we investigated the specific effects of Ab on mitochondrial function under physiological conditions. Mitochondrial respiratory functions and energy metabolism were analyzed in control and in human wild-type amyloid precursor protein (APP) stably transfected human neuroblastoma cells (SH-SY5Y). Mitochondrial respiratory capacity of mitochondrial electron transport chain (ETC) in vital cells was measured with a high-resolution respirometry system (Oxygraph-2k). In addition, we determined the individual activities of mitochondrial complexes I-IV that compose ETC and ATP cellular levels. While the activities of complexes I and II did not change between cell types, complex IV activity was significantly reduced in APP cells. In contrast, activity of complex III was significantly enhanced in APP cells, as compensatory response in order to balance the defect of complex IV. However, this compensatory mechanism could not prevent the strong impairment of total respiration in vital APP cells. As a result, the respiratory control ratio (state3/state4) together with ATP production decreased in the APP cells in comparison with the control cells. Chronic exposure to soluble Ab protein may result in an impairment of energy homeostasis due to a decreased respiratory capacity of mitochondrial electron transport chain which, in turn, may accelerate neurons demise.

The 'in vivo' decoding properties of four tRNAHis isoacceptors, two from Drosophila melanogaster and two from brewer's yeast, were studied after their microinjection, along with turnip yellow mosaic virus (L'YMV) coat protein mRNA, into Xenopus laevis oocytes. The two Drosophila isoacceptors are identical besides containing either a guanosine (G) or the hypermodified nucleoside queuosine (Q) in the wobble position. The brewer's yeast isoacceptors differ by four bases in the anticodon stem, and by one base in the amino acceptor stem. Our results show that, under competing 'in vivo' conditions, the Drosophila tRNAHis with the anticodon GUG clearly prefers the histidine codon CAC to the codon CAU, whereas little preference is observed for the tRNAHis with the anticodon QUG for the codon CAU, and no preference for either codon by the two yeast isoacceptors. Hence, it can be concluded that the presence of the Q-base clearly affects the choice of the codon. This is the first demonstration of an 'in vivo' codon preference by tRNA isoacceptors differing in the modification of the wobble base during the elongation step of protein synthesis. These results imply that one function of the Q-base is at the translational level. Key words: decoding/modification/queuosine/transfer RNA/ wobble base as a sensitive assay system. The two Drosophila tRNAHis differ only by the presence or absence of the Q-base in the wobble position (Altwegg and and unpublished results), whereas the two brewer's yeast tRNAs differ by four bases in the anticodon stem and one base in the amino acid acceptor stem. They do not contain the Q-modification in the wobble position, i.e., they possess identical anticodons (Keith et al., 1983).Turnip yellow mosaic virus (TYMV) coat protein RNA contains three histidine codons (Figure 1): two CAU in the 5' and one CAC in the 3' half of the mRNA (Guilley and Briand, 1978). Cyanogen bromide cleavage of the coat protein at the methionine residues yields three peptides: one large fragment (X) of 135 amino acid residues, containing the two CAU coded histidines, one fragment of 42 amino acids containing no histidine, and a small fragment (Y) of 10 amino acid residues, containing the unique CAC coded histidine. Thus, it is possible to study the incorporation of histidine from each of the two tRNAH1s isoacceptors into the coat protein sites coded by the two histidine codons under competing conditions. TYMV coat protein mRNA, histidinol (inhibits the endogenous synthetase, Hansen et al., 1972), and equal amounts (pmol) of aminoacylated tRNA isoacceptors were injected into Xenopus oocytes and, after 4 h of incubation, the incorporation into the peptides X and Y determined.Since only [3H]histidine labelling provides the necessary specific activity needed for these experiments, only one of the isoacceptors could be labeled. The competing isoacceptor carried an unlabeled histidine. In order to exclude potential artefacts (e.g., heterogeneity of the oocytes) we calculated the percentages and ratios of histidine incorporation f...

The pineal secretory product melatonin has, in addition to regulating retinal, circadian and vascular functions, neuroprotective effects. Blood melatonin levels are often decreased in Alzheimer's disease (AD), a progressively disabling neurodegenerative disorder. In this study we provide the first immunohistochemical evidence for the localization of melatonin 1a-receptor (MT(1)) in aged human hippocampus and a comparison of AD cases. MT(1) was localized to pyramidal neurons in the hippocampal cornu ammonis (CA)1-4 subfields. There was a distinct increase in staining intensity in all AD cases indicating an up-regulation of the receptor, possibly as a compensatory response to impaired melatonin levels in order to augment melatonin's neuroprotective effects.