Proteolytical processing of mutated human amyloid precursor protein in transgenic mice.

The evidence that betaA4 is central to the pathology of Alzheimer's disease (AD) came from the identification of several missense mutations in the amyloid precursor protein (APP) gene co-segregating with familial AD (FAD). In an attempt to study the proteolytical processing of mutated human APP in vivo, we have created transgenic mice expressing the human APP695 isoform with four FAD-linked mutations. Expression of the transgene was controlled by the promoter of the HMG-CR gene. Human APP is expressed in the brain of transgenic mice as shown by Western blot and immunohistology. The proteolytic processing of human APP in the transgenic mice leads to the generation of C-terminal APP fragments as well as to the release of betaA4. Despite substantial amounts of betaA4 detected in the brain of the transgenic mice, neither signs of Alzheimer's disease-related pathology nor related behavioural deficits could be demonstrated.

Identification of different ? amyloid cDNAs cloned from the brain of a patient with sporadic alzheimer's disease.

Neurofibrillary tangles and senile plaques, two neuropathological markers of Alzheimer's disease, may both contain peptide fragments derived from the ? amyloid protein. Human ? amyloid peptide precursor cDNAs have been isolated from normal foetal and adult brain libraries. In peripheral tissue and cultured cells, a novel precursor containing a protease inhibitor domain has been cloned. A cDNA library from the cerebral cortex of a patient with sporadic Alzheimer's disease was constructed and several clones coding for the ? amyloid peptide precursor were isolated cDNAs containing two types of insertion coding for a serine protease inhibitor domain were identified. The use of another polyadenylation site available in the 3?-untranslated region of the mRNA was observed. These results indicate that, in one patient with Alzheimer's disease, different RNA species coding for the ? amyloid peptide precursor arise by alternative splicing of a single transcriptional unit, and use different polyadenylation sites.

A new monoclonal antibody against the anionic domain of the amyloid precursor protein of Alzheimer's disease.

A new mouse monoclonal antibody was raised to a bacterial fusion protein between beta-galactosidase and the extracellular domain of the human amyloid protein precursor (APP). In immunoblotting experiments, this monoclonal antibody labelled the bacterial fusion protein used as an immunogen, the human brain APP, and different full-length APP isoforms expressed by transfected cells. In immunocytochemistry, the monoclonal antibody stained the dystrophic neurites of abundant senile plaques found in the cerebral cortex of patients with Alzheimer's disease. Using bacterial expression of several cDNA fragments, the epitope was mapped to an amino acid sequence of APP not investigated before.

Absence of mutation in the beta-amyloid cDNAs cloned from the brains of three patients with sporadic Alzheimer's disease.

Using an oligonucleotide probe, we isolated cDNA clones corresponding to the precursor of the beta-amyloid peptide (BAP) from brain libraries of 3 patients with sporadic Alzheimer's disease (AD). DNA sequencing showed that the largest cDNA clone encompasses 83% of the open reading frame proposed by Kang et al. to encode the BAP precursor (APP). cDNA clones from each of the 3 AD brain libraries were identical to the sequence of the APP-cDNAs cloned from normal adult human and fetal brain. An antisense-radiolabeled RNA copy of one of the AD clones detected a pattern of 3 gene transcripts measuring 3.5, 3.2 and 1.6 kilobases (kb) in both normal and AD brain RNAs. These data suggest that there are no mutations in or about the 42 amino acid (aa) sequence of BAP and that the accumulation of amyloid consistently found in AD may result from altered post-translational processing of APP.

The amyloid peptide of Alzheimer's disease is not produced by internal initiation of translation generating C-terminal amyloidogenic fragments of its precursor.

The molecular mechanisms of the amyloid peptide (A beta) production from the amyloid precursor protein (APP) remain unclear and it has been suggested that initiation of translation at methionine 596, which immediately precedes the A beta sequence, could generate soluble amyloidogenic fragments. We show that the amyloid peptide is actually produced by expression of the C-terminal 100 residues of the APP, using methionine 596 as an initiation codon. However, the amyloid peptide is no longer detectable when a stop codon is introduced in the APP mRNA, before the A beta coding region. These results strongly suggest that A beta is produced by degradation of APP and not by local translation of its mRNA.

Cloning of the cDNA from normal brain and brain of patients with Alzheimer's disease in the expression vector lambda GT 11.

1. RNA was purified from postmortem human brains, and the poly A+ RNA was isolated by oligo dT cellulose. 2. Double stranded cDNA was synthesized using reverse transcriptase, RNAse H and DNA polymerase. 3. cDNA was cloned in the lambda GT 11 expression vector, and libraries containing between 1 and 2 millions clones were obtained. 92 to 98% of the plaques contained a recombinant phage. 4. Such libraries will allow the molecular characterization of cDNA and corresponding proteins which play a key role in brain functions and in particular which could be involved in the etiology of Alzheimer's dementia.

Missense mutations associated with familial Alzheimer's disease in Sweden lead to the production of the amyloid peptide without internalization of its precursor.

Production of soluble amyloid peptide precursor (APP) and amyloid peptide (A beta) was measured in CHO cells transfected by the wild-type APP 695 cDNA sequence or by the same sequence carrying missense mutations associated with familial Alzheimer's disease in Sweden. Deletion of the C-terminal domain of the protein corresponding to residues 654 to 695 of APP 695 not only inhibited very significantly the internalization of APP at 37 degrees C, but also led to the secretion of an uncleaved APP in the culture medium of CHO cells. This deletion did not affect A beta production from the Swedish APP but was able to inhibit the production of the wild-type APP. These results demonstrate that, in CHO cells, the internalization of the wild-type APP is needed for A beta production, while the production of the amyloid peptide from Swedish APP is independent of the internalization process.

The long term adenoviral expression of the human amyloid precursor protein shows different secretase activities in rat cortical neurons and astrocytes.

Recombinant adenoviruses were used for the expression of human amyloid precursor protein (APP) of Alzheimer's disease in primary cultures of rat cortical neurons and astrocytes. The catabolic pathways of human APP were studied 3 to 4 days after infection, when the equilibrium of APP production was reached. Although the expression of human wild type APP (WtAPP) by rat neurons induced the production of both extracellular and intraneuronal amyloid peptide (Abeta), Abeta was not detected in the culture medium of rat astrocytes producing human WtAPP. Because a low beta-secretase activity was previously reported in rodent astrocytes, we wondered whether modifications of the APP amino acid sequence at the beta-secretase clipping site would modify the astrocytic production of Abeta. Interestingly, rat astrocytes produced high amounts of Abeta after expression of human APP carrying a double amino acid substitution responsible for Alzheimer's disease in a large Swedish family (SwAPP). In both rat cortical neurons and astrocytes, the beta-secretase cleavage of the human SwAPP occurred very early in the secretion process in a cellular compartment in which a different sorting of SwAPP and WtAPP seems unlikely. These results suggest that human WtAPP and SwAPP could be processed by different beta-secretase activities.