Rapid subcellular redistribution of Bax precedes caspase-3 and endonuclease activation during excitotoxic neuronal apoptosis in rat brain.
Neuronal apoptosis is induced prominently in the newborn rodent brain by glutamate receptor excitotoxicity and related insults, including trauma and hypoxia-ischemia. However, the molecular mechanisms of this neurodegeneration are unclear. We tested the hypothesis that changes in the subcellular distribution of the proapoptotic protein Bax precede the activation of downstream apoptosis-effector mechanisms such as caspase-3 cleavage and endonuclease activation during the progression of excitotoxic neuronal apoptosis in the striatum of newborn rat. Kainic acid (4 nmol) was injected into striatum of anesthetized 7-day-old rats, and the animals were killed at 2, 6, 12, and 24 h postinsult. Controls were age-matched, vehicle-injected, or naive rats. Counts of ultrastructurally confirmed striatal neuron apoptosis in brain sections were highest at 24 h. Striatal tissue was microdissected and fractionated into cytosolic, mitochondrial-, and nuclear-enriched compartments. Immunoblots showed that Bax translocates from the cytosol fraction to the mitochondrial fraction, with maximal translocation by 2 h in the absence of changes in mitochondrial accumulation. Cleaved caspase-3 levels increase progressively in both cytosolic and mitochondrial fractions between 6 and 24 h. Cleaved caspase-3 accumulates in apoptotic striatal neurons as shown by immunolocalization. Internucleosomal fragmentation of DNA coincides with caspase-3 cleavage. We conclude that rapid translocation of Bax to mitochondria precedes caspase-3 and endonuclease activation during excitotoxic neuronal apoptosis in newborn rat brain and that initiation of this death cascade occurs within 2 h after glutamate receptor activation.
Department of Anesthesiology & Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196, USA.
Josephine
J
Lok
Lee J
LJ
Martin
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001921:Brain / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D004720:Endonucleases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology*; D005260:Female; D007608:Kainic Acid / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D011247:Pregnancy; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D051028:bcl-2-Associated X Protein
C494828:Bax protein, rat; D018690:Excitatory Amino Acid Agonists; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D004720:Endonucleases; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D007608:Kainic Acid