A. F. Macq

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 (Aβ), Aβ was not detected in the culture medium of rat astrocytes producing human WtAPP. Because a low β-secretase activity was previously reported in rodent astrocytes, we wondered whether modifications of the APP amino acid sequence at the β-secretase clipping site would modify the astrocytic production of Aβ. Interestingly, rat astrocytes produced high amounts of Aβ 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 β-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 β-secretase activities.

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 Alzheimers disease. Using bacterial expression of several cDNA fragments, the epitope was mapped to an amino acid sequence of APP not investigated before.

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 Alzheimers dementia.

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

Neurofibrillary tangles and senile plaques, two neuropathological markers of Alzheimers 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 Alzheimers 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 Alzheimers disease, different RNA species coding for the ? amyloid peptide precursor arise by alternative splicing of a single transcriptional unit, and use different polyadenylation sites.

CONSTRUCTION OF LAMBDA GT 11 cDNA LIBRARIES FROM POST-MORTEM HUMAN BRAINS

ABSTRACT The lambda gt 11 expression vector has been used in order to construct cDNA libraries from human brain RNA. If brain suffering and post-mortem delay are not too long, RNA was able to produce high molecular weight proteins when translated in a rabbit reticulocyte lysate. The mRNA was transformed into cDNA in order to be cloned in the lambda vector. After in vitro packaging, libraries were obtained, containing between 106 and 2.106 plaques, 92 to 98% of them being recombinants. cDNA libraries from normal cerebral cortex, caudate nucleus, cerebellum, hippocampus and substancia nigra as well as from Alzheimers brain have been constructed.

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 Different Amyloid Peptide Precursors from Brains of Patients with Alzheimer’s Disease

Alzheimer’s disease (AD) is a progressive degenerative disorder of the central nervous system characterized by neuronal loss and by brain lesions including senile plaques and neurofibrillary tangles (Roth et al. 1966; Terry et al. 1981; Whitehouse et al. 1982; Glenner 1983). Recent studies have provided an amino acid sequence for a major polypeptide of the cerebral amyloid found in AD, the amyloid β-protein (Glenner and Wong 1984) or A4 protein (Masters et al. 1985).

Cloning of the Beta Amyloid Peptide Gene in Alzheimer’s Disease

We have isolated and sequenced clones from the brain cDNA libraries derived from three patients with sporadic Alzheimer’s disease (AD). Our results indicate that ADβ-amyloid peptide (AD-BAPP) clones have sequences which do not differ from those found in BAPP cDNAs isolated from a normal fetus and a normal adult brain. We have noted that the sequence of the non-AD partial BAPP cDNA isolated by Goldgaber et al. differs from the sequences of our AD-B APP cDNAs. The inverted dinucleotide would translate into serine instead of tyrosine at position 687 in BAPP and thereby create a putative glycosylation site at the C-terminus of the postulated BAPP. Such a difference might affect the processing of BAPP. However, as only one sequence from non-AD brains differs among the five human BAPP cDNAs now reported, the significance of such heterogeneity is unclear.