Control of Aß release from human neurons by differentiation status and RET signaling.

Few studies have compared the processing of endogenous human amyloid precursor protein (APP) in younger and older neurons. Here, we characterized LUHMES cells as a human model to study Alzheimer's disease-related processes during neuronal maturation and aging. Differentiated LUHMES expressed and spontaneously processed APP via the secretase pathways, and they secreted amyloid ß (Aß) peptide. This was inhibited by cholesterol depletion or secretase inhibition, but not by block of tau phosphorylation. In vitro aged cells increased Aß secretion without upregulation of APP or secretases. We identified the medium constituent glial cell line-derived neurotrophic factor (GDNF) as responsible for this effect. GDNF-triggered Aß release was associated with rapid upregulation of the GDNF coreceptor "rearranged during transfection" (RET). Other direct (neurturin) or indirect (nerve growth factor) RET activators also increased Aß, whereas different neurotrophins were ineffective. Downstream of RET, we found activation of protein kinase B (AKT) to be involved. Accordingly, inhibitors of the AKT regulator phosphatidylinositol-3-kinase completely blocked GDNF-triggered AKT phosphorylation and Aß increase. This suggests that RET signaling affects Aß release from aging neurons.

Requirement of a dopaminergic neuronal phenotype for toxicity of low concentrations of 1-methyl-4-phenylpyridinium to human cells.

LUHMES cells are conditionally-immortalized non-transformed human fetal cells that can be differentiated to acquire a dopaminergic neuron-like phenotype under appropriate growth conditions. After differentiation by GDNF and cyclic adenosine monophosphate, LUHMES were sensitive to 1-methyl-4-phenylpyridinium (MPP(+)) toxicity at < or =5 microM, but resistant to the parental compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The high homogeneity and purity of the cultures allowed the detection of metabolic changes during the degeneration. Cellular ATP dropped in two phases after 24 and 48 h; cellular glutathione (GSH) decreased continuously, paralleled by an increase in lipid peroxidation. These events were accompanied by a time-dependent degeneration of neurites. Block of the dopamine transporter by GBR 12909 or mazindol completely abrogated MPP(+) toxicity. Inhibition of de novo dopamine synthesis by alpha-methyl-l-tyrosine or 3-iodo-l-tyrosine attenuated toxicity, but did not reduce the initial drop in ATP. Inhibition of mixed lineage kinases by CEP1347 completely prevented the MPP(+)-induced loss of viability and intracellular GSH, but failed to attenuate the initial drop of ATP. For the quantitative assessment of neurite degeneration, an automated imaging-based high content screening approach was applied and confirmed the findings made by pharmacological interventions in this study. Our data indicate that inhibition of mitochondrial ATP synthesis is not sufficient to trigger cell death in MPP(+)-treated LUHMES.