Establishment of cholinergic neuron-like cell lines with differential vulnerability to nitrosative stress.
Cholinergic cell lines were established by fusion of embryonic day 17 wild-type neurons from rat basal forebrain (BF) and upper brainstem (BS) with N18tg neuroblastoma cells. Isolated clones expressed choline acetyltransferase (ChAT) and neuronal nitric oxide synthase (nNOS) activities that were increased upon differentiation with retinoic acid. Clones from the BF expressed high levels of the tyrosine kinase type A (TrkA) receptor expression and activation of the mitogen-activated kinase ERK2 upon treatment with nerve growth factor. Like wild-type cholinergic populations, the six clones studied were variably resistant to nitric oxide (NO) excess from addition of S-nitroso-N-acetyl-D, L-penicillamine (SNAP). Of these, the BS2 clone exhibited resistance like in vivo BS cholinergic neurons, while the MS10 clone mimicked in vivo BF vulnerability. Apoptosis in response to NO excess was preceded by increases in mitochondrial responses bax/bcl-2 ratios, but cytochrome C was not released. Mitochondrial levels of apoptosis initiating factor (AIF) were either unchanged or increased, and only in MS clones was endonuclease G (EndoG) released. Microarray data indicated the existence of endoplasmic reticular (ER) stress and caspase-4 and caspase-12 were involved in the pathway to DNA fragmentation. The array data also indicated a survival role for mdm2, and its blockade rendered vulnerable the brainstem survivor clone BS2. Akt and ERK1/2 pathways were activated in response to NO and their blockade increased DNA fragmentation. Blockade of GSK-3 alpha/beta, a downstream target of Akt, reduced SNAP toxicity and this was more prominent in basal forebrain clones. We have identified two cholinergic cell lines useful for molecular studies of cholinergic vulnerability. We hypothesize that, in cholinergic neurons, control of ER stress signaling may be a major factor in differential vulnerability.
Personett
David A
DA
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, 4500 San Pablo Road, Jacksonville, Fl 32224, USA.
Williams
Katrina
K
Baskerville
Karen A
KA
McKinney
Michael
M
D000818:Animals; D001921:Brain / Q000166:cytology; D002454:Cell Differentiation / Q000502:physiology*; D002459:Cell Fusion / Q000379:methods; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002795:Choline O-Acetyltransferase / Q000378:metabolism*; D053938:DNA Fragmentation / Q000187:drug effects; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D046228:Microarray Analysis / Q000379:methods; D020932:Nerve Growth Factor / Q000494:pharmacology; D009447:Neuroblastoma / Q000503:physiopathology; D009474:Neurons / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism*; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013312:Stress, Physiological / Q000139:chemically induced / Q000378:metabolism*; D013997:Time Factors
D004791:Enzyme Inhibitors; D020030:Nitric Oxide Donors; C110881:S-nitro-N-acetylpenicillamine; D020932:Nerve Growth Factor; D052248:Nitric Oxide Synthase Type I; D002795:Choline O-Acetyltransferase; D010396:Penicillamine