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¹ Ottawa Health Research Institute, Department of Neuroscience, University of Ottawa, Ottawa, Ontario, Canada, K1H 8M5
² Department of Biology, University of Rome "Tor Vergata," 00133, Rome, Italy
³ Institute for Cell Engineering, Department of Neurology and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21287
⁴ Centre National de la Recherche Scientifique, Institut Gustave Roussy, F-948054, Villejuif, France

Address correspondence to Ruth S. Slack, Ottawa Health Research Institute, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario, Canada K1H 8M5. Tel.: (613) 562-5800 ext. 8458. Fax.: (613) 562-5403. E-mail: rslack{at}uottawa.ca

Caspase-independent death mechanisms have been shown to execute apoptosis in many types of neuronal injury. P53 has been identified as a key regulator of neuronal cell death after acute injury such as DNA damage, ischemia, and excitotoxicity. Here, we demonstrate that p53 can induce neuronal cell death via a caspase-mediated process activated by apoptotic activating factor-1 (Apaf1) and via a delayed onset caspase-independent mechanism. In contrast to wild-type cells, Apaf1-deficient neurons exhibit delayed DNA fragmentation and only peripheral chromatin condensation. More importantly, we demonstrate that apoptosis-inducing factor (AIF) is an important factor involved in the regulation of this caspase-independent neuronal cell death. Immunofluorescence studies demonstrate that AIF is released from the mitochondria by a mechanism distinct from that of cytochrome-c in neurons undergoing p53-mediated cell death. The Bcl-2 family regulates this release of AIF and subsequent caspase-independent cell death. In addition, we show that enforced expression of AIF can induce neuronal cell death in a Bax- and caspase-independent manner. Microinjection of neutralizing antibodies against AIF significantly decreased injury-induced neuronal cell death in Apaf1-deficient neurons, indicating its importance in caspase-independent apoptosis. Taken together, our results suggest that AIF may be an important therapeutic target for the treatment of neuronal injury.

Key Words: neurodegeneration; neurons; apoptosis; p53; Bax

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