Gerda Huber

Apoptotic Cell Death Induced by β-Amyloid1–42 Peptide Is Cell Type Dependent

Abstract: β‐Amyloid peptide (Aβ), a proteolytic fragment of the β‐amyloid precursor protein, is a major component of senile plaques in the brain of Alzheimers disease patients. This neuropathological feature is accompanied by increased neuronal cell loss in the brain and there is evidence that Aβ is directly neurotoxic. In the present study reduced cell viability in four different neuroblastoma cell types was observed after treatment with human Aβ1–42 for 1 day. Of the cell types tested rat PC12 and human IMR32 cells were most susceptible to Aβ toxicity. Chromosomal condensation and fragmentation of nuclei were seen in PC12, NB2a, and B104 cells but not in IMR32 cells irrespective of their high sensitivity to Aβ. Electrophoretic analysis of cellular DNA confirmed internucleosomal DNA fragmentation typical for apoptosis in all cell types except IMR32. These findings suggest that the form of Aβ‐induced cell death (necrosis or apoptosis) may depend on the cell type.

Central adenosine A2A receptors: an overview

Abstract Recent advances in molecular biology, biochemistry, cell biology and behavioral pharmacology together with the development of more selective ligands to the various adenosine receptors have increased our understanding of the functioning of central adenosine A 2A receptors. The A 2A receptor is one of four adenosine receptors found in the brain. Its expression is highest in striatum, nucleus accumbens and olfactory tubercles, although it also occurs in neurons and microglia in most other brain regions. The receptor has seven transmembrane domains and couples via Gs to adenyl cyclase stimulation. Antagonistic interactions between A 2A receptors and dopamine D 2 receptors have been described, as stimulation of the A 2A receptor leads to a reduction in the affinity of D 2 receptors for D 2 receptor agonists. The A 2A receptor is thought to play a role in a number of physiological responses and pathological conditions. Indeed, A 2A receptor antagonists may be useful for the treatment of acute and chronic neurodegenerative disorders such as cerebral ischemia or Parkinsons disease. A 2A receptor agonists may treat certain types of seizures or sleep disorders. This review discusses the characteristics, distribution, pharmacochemical properties and regulation of central A 2A receptors, as well as A 2A receptor-mediated behavioural responses and their potential role in various neuropsychiatric disorders.

β‐Amyloid Precursor Protein Isoforms in Various Rat Brain Regions and During Brain Development

Abstract: To address the question of the possible functions of different Alzheimers disease β‐amyloid precursor protein (β‐APP) isoforms in the brain, we studied their expression at different times during postnatal rat brain development and in various regions of the adult rat brain. Polyclonal antibodies directed to two peptide antigens were used. The majority of all β‐APP forms was found to be soluble as revealed by western blot analysis. The highest level of most β‐APP forms was reached in the second postnatal week, which is the time of brain maturation and completion of synaptic connections. Strikingly high concentrations of the Kunitz protease inhibitor‐containing β‐APP were present in the adult olfactory bulb, where continuous synaptogenesis occurs in the adult animal. These findings support the idea of an involvement of β‐APPs in the processes of cell differentiation and, probably, in the establishment of synaptic contacts.

cDNA cloning of a novel secreted isoform of the human receptor for advanced glycation end products and characterization of cells co-expressing cell-surface scavenger receptors and Swedish mutant amyloid precursor protein.

The receptor for advanced glycation end products (RAGE) has been proposed as a cell surface receptor that binds amyloid-beta protein (Abeta), thereby triggering its cytotoxic effects [S.D. Yan, X. Chen, J. Fu, M. Chen, H. Zhu, A. Roher, T. Slattery, L. Zhao, M. Nagashima, J. Morser, A. Migheli, P. Nawroth, D. Stern, A.M. Schmidt, RAGE and amyloid-beta peptide neurotoxicity in Alzheimers disease, Nature 382 (1996) 685-691.]. A cDNA library of human lung was screened for RAGE with an appropriate hybridization probe. In addition to cell surface RAGE, one clone was found which encodes a new version of RAGE, termed hRAGEsec, which lacks the 19 amino acids of the membrane-spanning region and is therefore secreted. Comparison with the genomic sequence revealed that the synthesis of the secreted isoform requires alternative splicing. The deduced protein sequence of the mature hRAGEsec consists of 321 amino acids with a predicted molecular mass of 35.66 kDa. The pattern of expression of hRAGEsec in human brain was analyzed by in situ hybridization histochemistry. The most intense expression of the gene in contrast to cell surface RAGE was detected in hippocampal CA3 pyramidal cells, dentate gyrus granule cells, cortical neurons as well as glial cells in white matter. To investigate the interaction between Abeta and RAGE and another scavenger receptor, SRA, under physiological conditions, they were co-expressed with human betaAPP(695)-SFAD in a human cell and the level of Abeta in the condition medium was assessed by immunoprecipitation and enzyme-linked immunosorbent assay (ELISA) analysis. A nearly 100% reduction of Abeta from the conditioned medium of hRAGE cells and approximately 40% reduction from the SRA-cells implied that hRAGE could be a prominent cell surface receptor interacting with Abeta.

Molecular Cloning of Microtubule-Associated Protein 1 (MAP1A) and Microtubule-Associated Protein 5 (MAP1B): Identification of Distinct Genes and Their Differential Expression in Developing Brain

Abstract: cDNA clones encoding microtubule‐associated proteins 1 (MAP1/MAP1A) and 5 (MAP5/MAP1B) were isolated and have been used to study their structural relationship as well as their regulated expression in developing rat brain. cDNA clones specific for MAP1 hybridized to a single 10‐kb rat brain mRNA, and analysis of genomic DNA by Southern blotting indicated the existence of a single MAP1 gene. A second set of cDNAs specific for MAP5 hybridized to a single 11‐kb mRNA in rat brain and also detected a single gene. By analysis of hybrid mouse‐hamster cell lines, the MAP1 gene was located to mouse chromosome 2, designated Mtap‐1, and the MAP5 gene to chromosome 13, designated Mtap‐5 MAP1 and MAP5 mRNAs were expressed with different temporal patterns during rat brain development that mirrored the appearance of their protein products, suggesting that expression of these proteins is under transcriptional control. These results taken together demonstrate that although MAP1 and MAP5 have some properties that are similar, they are structurally distinct proteins whose transcription is differently regulated from separate genes.

Rat Brain Glyceraldehyde‐3‐Phosphate Dehydrogenase Interacts with the Recombinant Cytoplasmic Domain of Alzheimer's β‐Amyloid Precursor Protein

Abstract: Abundant senile plaques are a histological hallmark in the brain of Alzheimers disease patients. Such plaques consist of, among many other constituents, aggregated βA4 amyloid peptide. This peptide is derived from an amyloid precursor protein (APP) by irregular proteolytic processing and is considered to be involved in the development of Alzheimers disease. To study possible interactions of brain proteins with 0A4 amyloid or other fragments of APP, βA4 amyloid and βA4 amyloid extended to the C‐terminus of APP were recombinantly produced as fusion proteins termed “Amy” and “AmyC,” respectively. Using Amy and AmyC affinity chromatography, a 35‐kDa protein from rat brain was isolated that bound tightly to AmyC but not to Amy, thus indicating an interaction of the protein with the C‐terminus of APP. This 35‐kDa protein was identified as the glycolytic enzyme gIyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). Binding of GAPDH to AmyC but not to Amy was confirmed by gel filtration. Although AmyC slightly reduced the Vmax of GAPDH, the same reduction was observed in the presence of Amy. These findings suggest that the interaction of the cytoplasmic domain of APP with GAPDH is unlikely to influence directly the rate of glycolysis but may serve another function.

Involvement of amyloid precursor protein in memory formation in the rat: an indirect antibody approach

The potential role of different isoforms of amyloid precursor proteins (APPs) in memory and learning processes was investigated in rats using antibodies differentiating between APP isoforms containing or lacking the Kunitz protease inhibitor (KPI) domain. Rats received intracerebroventricular injection of control immunoglobulins (IgGs), anti-KPI-APP (= anti-P3) or anti-N-terminus-APP (= anti-P4). No immediate effects on learning of a passive avoidance task were observed; however, performance evaluated 1 day later was somewhat impaired by the anti-P3 antibody (reacting with APPKPI) and even more markedly impaired by the anti-P4 antibody (reacting with both APP695 and APPKPI) relative to control antibody. The antibodies did not affect performance of an active avoidance task acquired prior to the experimental treatment or active avoidance learning 8 days post-treatment. These results suggest that blockade of APP by an antibody injected intracerebroventricularly impairs the consolidation and/or retrieval of memory in rats.

Controlling Polymerization of β-Amyloid and Prion-derived Peptides with Synthetic Small Molecule Ligands

The Alzheimer β-amyloid peptide (Aβ) and a fragment of the prion protein have the capacity of forming amyloid-like fibrils when incubated under physiological conditions in vitro. Here we show that a small amyloid ligand, RO-47-1816/001, enhances this process severalfold by binding to amyloid molecules and apparently promote formation of the peptide-to-peptide bonds that join the monomers of the amyloid fibrils. This effect could be antagonized by other ligands, including analogues of RO-47-1816/001, as well as the structurally unrelated ligand Congo red. Analogues of RO-47-1816/001 with low affinity for amyloid did not display any antagonistic effect. In conclusion, these data suggest that synthetic molecules, and possibly also small natural substances present in the brain, may act in a chaperone-like fashion, promoting Aβ polymerization and growth of amyloid fibrils in vitro and possibly also in vivo. Furthermore, we demonstrate that small organic molecules can be used to inhibit the action of amyloid-enhancing compounds.

Synaptic β-amyloid precursor proteins increase with learning capacity in rats

Abstract The precursor proteins of Alzheimers disease β -amyloid peptide, the β -amyloid precursor protein isoforms, comprise a family of neuronal proteins with synaptic localization whose physiological roles in brain are poorly understood. One possible role for synaptic proteins is involvement in neuronal plasticity. After exposure to an enriched environment compared to impoverished conditions, rats exhibited superior cognitive capacity. Up to ∼four-fold increased overall levels of β -amyloid precursor proteins were found in cortical/subcortical tissue of the enriched animals displaying significantly more synapses immunoreactive for the different β -amyloid precursor protein isoforms ( β -amyloid precursor protein 695 - and β -amyloid precursor protein 751/770 ) in hippocampus and adjacent occipital cortex. This correlation thus provides in vivo evidence for an association of β -amyloid precursor proteins with plastic changes induced by complex environment with consequences for cognitive functions and suggests that impaired β -amyloid precursor protein metabolism at synapses might contribute to brain dysfunction in Alzheimers disease.

Lack of β-Amyloidosis in transgenic mice expressing low levels of familial Alzheimer's disease missense mutations

Point mutations within the beta-amyloid precusor protein (beta-APP) gene known to segregate with Alzheimers disease in certain families were introduced into human beta-APP cDNAs and expressed under the control of a neuron-specific enolase (NSE) promoter in mice. The transgenic animals exhibited transgene expression predominantly in neocortex and hippocampus where the levels were maximally 1.3-fold of those of wild-type mouse beta-APP. Quantitative immunoblot analysis in homozygous mice carrying different missense mutations showed slightly increased alpha-secretory processing. In V7171 mice compared to nontransgenic mice there was more alpha-secretory beta-APP (beta-APPsec) in cortex/hippocampus, less in cerebellum, and no difference in midbrain/brain stem. In none of the transgenic animals tested was a 4 kDa amyloid fragment detected by Western blotting of brain extracts, immunohistochemistry, or by 125I-A beta-binding onto brain sections. No glial reaction was observed. Behavioral analysis of mice carrying the V7171 mutation showed no appreciable deficit in comparison to wild-type mice. Together, these data suggest that low levels of expression of mutated beta-APP in 10-12-month-old transgenic mouse brains result in slightly more beta-APPsec, and are insufficient to induce amyloidogenic processing and AD-like pathology.