AMPA-induced dark cell degeneration of cerebellar Purkinje neurons involves activation of caspases and apparent mitochondrial dysfunction.
Cerebellar Purkinje neurons (PNs) are selectively vulnerable to AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepriopionic acid)-induced delayed neurotoxicity known as dark cell degeneration (DCD) that is expressed as cytoplasmic and nuclear condensation, neuron shrinkage, and failure of physiology. The present study was initiated to determine whether AMPA-receptor-induced DCD in PNs is associated with Bax translocation to the mitochondria, cytochrome C release from the mitochondria, changes in mitochondrial potential, and activation of representative initiator and executor caspases that include caspase-9, caspase-3, and caspase-7. AMPA consistently and rapidly hyperpolarized mitochondria as reflected by an increase in MitoTracker Red CMS Ros fluorescence. Increases in Bax immunoreactivity were quantitatively and temporally variable and Bax failed to localize to mitochondria. Additionally, we observed a marked increase in immunoreactivity of cytochrome C although its release from mitochondria was not apparent. Mitochondrial membrane hyperpolarization and increases in cytochrome C immunoreactivity preceded caspase activation. Immunohistochemical analyses revealed the active form of caspases-3 and -9 were markedly and significantly increased in PNs following 30 microM AMPA, and caspase-9 activation preceded caspase-3. Increases in active caspase-7 immunoreactivity were less frequently encountered in PNs. Thus DCD shares some characteristics of apoptotic programmed cell death, but lacks typical mitochondrial pathophysiology associated with classic apoptosis. These findings suggest that AMPA-induced DCD is a form of active PCD that lies on a spectrum between classical apoptosis and passive necrosis.
Strahlendorf
Jean
J
Department of Physiology, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA. Jean.Strahlendorf@ttuhsc.edu
Box
Cathy
C
Attridge
Jennifer
J
Diertien
Janet
J
Finckbone
VelvetLee
V
Henne
William M
WM
Medina
Margarita S
MS
Miles
Randy
R
Oomman
Sowmini
S
Schneider
Marcia
M
Singh
Hema
H
Veliyaparambil
Madhu
M
Strahlendorf
Howard
H
D000818:Animals; D000831:Animals, Newborn; D020169:Caspases / Q000378:metabolism*; D002452:Cell Count; D002531:Cerebellum / Q000473:pathology; D045304:Cytochromes c / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D066298:In Vitro Techniques; D018613:Microscopy, Confocal / Q000379:methods; D028361:Mitochondrial Diseases / Q000139:chemically induced / Q000378:metabolism*; D009410:Nerve Degeneration / Q000139:chemically induced / Q000201:enzymology / Q000378:metabolism*; D009930:Organic Chemicals; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D011689:Purkinje Cells / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D051028:bcl-2-Associated X Protein
C494828:Bax protein, rat; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C107472:red dye CMXRos; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D045304:Cytochromes c; D003576:Electron Transport Complex IV; D020169:Caspases