The presenilin 1 deltaE9 mutation gives enhanced basal phospholipase C activity and a resultant increase in intracellular calcium concentrations.
We studied effects of the familial Alzheimer's disease presenilin 1 (PS1) exon 9 deletion (PS1-DeltaE9) mutation on basal and carbachol-stimulated phosphoinositide (PI) hydrolysis and intracellular Ca(2+) concentrations ([Ca(2+)](i)) in human SH-SY5Y neuroblastoma cells. We demonstrate that PS1-DeltaE9 cells have an enhanced basal PI hydrolysis and [Ca(2+)](i) as compared with both wild type PS1 (PS1-WT) and nontransfected (NT) cells. Both were reversed by the phospholipase C (PLC) inhibitor neomycin. The PS1-DeltaE9-related high basal [Ca(2+)](i) was also reversed by xestospongin C confirming that this effect was inositol trisphosphate receptor-mediated. Carbachol gave a greater stimulation of [Ca(2+)](i) in PS1-DeltaE9 cells that took longer to return to basal as compared with responses seen in NT and PS1-WT cells. This long tail-off effect seen in PS1-DeltaE9 cells after carbachol stimulation was reversed by xestospongin C and dantrolene, suggesting that it was mediated by inositol trisphosphate receptor and ryanodine receptor amplification of Ca(2+). Ruthenium red only reduced carbachol peak elevations of [Ca(2+)](i) in NT and PS1-WT cells and not in PS1-DeltaE9 cells. No significant between cell type differences were seen for basal and carbachol-stimulated [Ca(2+)](i) with either ryanodine or the endoplasmic reticulum Ca(2+) ATPase inhibitor cyclopiazonic acid. Immunostaining experiments revealed that for all the cell types PS1 is present at the plasma membrane and co-localizes with N-cadherin, a component of the cell-cell adhesion complex. Immunoblotting of cell extracts for PLC-beta1 showed that, compared with NT and PS1-WT cells, the PS1-DeltaE9 transfectants gave a relative increase in levels of the calpain generated N-terminal fragment (100 kDa) over full-length (150 kDa) PLC-beta1. Our results suggest that the PS1-DeltaE9 mutation causes upstream changes in PI signaling with enhanced basal PLC activity as a primary effect that leads to a higher [Ca(2+)](i). This may provide a novel mechanism by which the PS1-DeltaE9 mutation sensitizes cells to apoptotic stimuli and enhanced amyloid beta generation.
Section of Experimental Geriatrics, Karolinska Institutet, Neurotec, Kliniskt Forskningscentrum (KFC), 141 86 Huddinge, Sweden. angel.cedazo-minguez@neurotec.ki.se
Angel
A
Cedazo-Minguez
Bogdan O
BO
Popescu
Maria
M
Ankarcrona
Takeshi
T
Nishimura
Richard F
RF
Cowburn
D016229:Amyloid beta-Peptides / Q000378:metabolism; D017209:Apoptosis; D015820:Cadherins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004396:Coloring Agents / Q000494:pharmacology; D003620:Dantrolene / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005091:Exons; D006801:Humans; D006868:Hydrolysis; D015151:Immunoblotting; D007150:Immunohistochemistry; D007211:Indoles / Q000494:pharmacology; D047028:Macrocyclic Compounds; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D018613:Microscopy, Confocal; D009125:Muscle Relaxants, Central / Q000494:pharmacology; D009154:Mutation; D009355:Neomycin / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D053764:Presenilin-1; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012430:Ruthenium Red / Q000494:pharmacology; D012433:Ryanodine / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D016229:Amyloid beta-Peptides; D015820:Cadherins; D004396:Coloring Agents; D004791:Enzyme Inhibitors; D007211:Indoles; D047028:Macrocyclic Compounds; D008565:Membrane Proteins; D009125:Muscle Relaxants, Central; D010080:Oxazoles; C508853:PSEN1 protein, human; D053764:Presenilin-1; C108644:xestospongin A; D012430:Ruthenium Red; D012433:Ryanodine; D002217:Carbachol; D010738:Type C Phospholipases; D003620:Dantrolene; D009355:Neomycin; D002118:Calcium; C000543:cyclopiazonic acid