Processing of Amyloid Precursor Protein in Human Primary Neuron and Astrocyte Cultures

Abstract

Increased production of amyloid /3 peptide (A/3) is highly suspected to play a major role in Alzheimer's disease (AD) pathogenesis. Because A/I deposits in AD senile plaques appear uniquely in the brain and are fairly restricted to humans, we assessed amyloid precursor protein (APP) metabolism in primary cultures of the cell types associated with AD senile plaques: neurons, astrocytes, and microglia. We find that neurons secrete 40% of newly synthesized APP, whereas glia secrete only 10%. Neuronal and astrocytic APP processing generates five C-terminal fragments similar to those observed in human adult brain, of which the most amyloidogenic higher-molecular-weight fragments are more abundant. The level of amyloidogenic 4-kDa A/I exceeds that of nonamyloidogenic 3-kDa A/I in both neurons and astrocytes. In contrast, microglia make more of the smallest C-terminal fragment and no detectable A/I. We conclude that human neurons and astrocytes generate higher levels of amyloidogenic fragments than microglia and favor amyloidogenic processing compared with previously studied culture systems. Therefore, we propose that the higher amyloidogenic processing of APP in neurons and astrocytes, combined with the extended lifespan of individuals, likely promotes AD pathology in aging humans.

Role of Endoplasmic Reticulum, Endosomal–Lysosomal Compartments, and Microtubules in Amyloid Precursor Protein Metabolism of Human Neurons

“…It is therefore conceivable that human neurons are also different from these cell types with respect to APP metabolism. We note that each of these models was transfected or infected to produce abnormally high levels of APP, whereas human primary neurons already produce high constitutive levels of APP,,, (LeBlanc et al, 1997). It is conceivable that infection, transfection, or high abnormal expression of APP affects the integrity of the endoplasmic reticulum and Golgi in these cells.…”

“…We have shown that APP processing in these human neurons is highly amyloidogenic compared with rodent primary neurons (LeBlanc et al, 1997). AP1-4, is 10 times more abundant than AP,-,, in human primary neurons, but both species are made in human neurons (LeBlanc et al, 1997). The sequence of human APP is partly responsible for the higher amyloidogenic processing (De Strooper et al, 1995).…”

“…In transfected cell lines, inhibition of lysosomal enzymes with leupeptin increases the level of intracellular CTFs, indicating that these are degraded in the lysosome Haass et al, 1992a,b). In human brain and primary cultures of neurons, five distinct CTFs are produced LeBlanc, 1995;LeBlanc et al, 1997), but the site of their turnover has never been assessed. The size and immunoprecipitation profile of the human CTFs indicate that the two largest CTFs, C l and C2, both contain the entire AP region, that C3 and C4 contain the AP17-24 epitope, and that C5 does not immunoprecipitate with anti-AP antisera (LeBlanc, 1995;LeBlanc et al, 1997LeBlanc et al, , 1998.…”

“…We have established a human fetal primary neuron culture model system to address issues of cell and species type-specific APP metabolism (LeBlanc, 1995). We have shown that APP processing in these human neurons is highly amyloidogenic compared with rodent primary neurons (LeBlanc et al, 1997). AP1-4, is 10 times more abundant than AP,-,, in human primary neurons, but both species are made in human neurons (LeBlanc et al, 1997).…”

Cellular prion protein inhibits proapoptotic Bax conformational change in human neurons and in breast carcinoma MCF-7 cells

“…27,62 Hippocampal HW3-5 and HpL3-4 cell lines established from PrP þ / þ and PrP À/À mice, respectively, were a kind gift from Dr. T Onodera (University of Tokyo, Japan) and were serum-deprived as described previously. …”