S282

Symposium: Apoptosis

pi&q THE ROLE OF CASPASE-12 IN MEDIATING AN ER-SPECIFIC APOPTOSIS PATHWAY AND ABETA PROTEIN NEUROTOXIC- ITY

Jun),ing Yuan, Toshiyuki Nakagawn, Harvard Medical School, Boston, MA

Apoptosis is a genetically regulated cellular suicide mechanism that plays an important role in regulating normal development and tissue homeostasis as well as pathogen&s of diseases. Apoptotic signals can target different compartments of a cell: death receptors located on cytoplasmic membrane, DNA damage to nuclei, free radical insult of mitochondria, and unfolded protein response directed at endoplasmic reticulum (ER stress). Mammalian caspase family of cyst&e proteases, homologues of C. elegans cell death gene product Ced-3, are critical mediators of apoptosis signal transduction and execution. Fourteen members of mammalian caspase family have been identified. Some of the caspase family members. such as caspase-8, mediate membrane-targeted apoptotic signals for death receptors. Other caspases, such as caspase-9. mediate apoptotic rignal downstream from mitochondrial damage. We have identified a novel ER-specific apoptosis pathway mediated by caspase-12. We demonstrated that caspase-I2 is localized specifically in ER and activated by ER stress apoptotic signals which include disruption of ER calcium homeostasia and accumulation of excers proteins in ER, but not by membrane- or mitochondrial targeted apoptotic signal. Caspase-12-/- cella are resistant specifically to ER stress apoptoris signals. Furthermore, caspase-12-1. cortical neurons are resistant to cell death induced by amyloid beta peptide. We conclude that caspase-I2 mediates an ER specific apoptosir pathway that plays an important role in amyloid beta protein neurotoxicity.

p?iq BCL-2 FAMILY PROTEINS IN NEURONAL DISEASE

Jun M. Hurdwck, The Johns Hopkins Univervihj School of Public Health, Baltimore.

MD

Caspases cleave key substrates within the cell to facilitate apoptosis, often converting anti-apoptotic factors into pro apoptotic factors. During VUIIS infections and other death stimuli, Bcl-2 and Bcl-XL are cleaved by caspase-3 in the loop domain during apoptosis, converting these proteins from anti-apoptotic into pro-apoptotic proteins (Cheng et al. Science 278, 1966, 1997; Clem Chrm, 274:21155. 1999). Furthermore, the cleavage products kills cells by a caspase-dependent mechanism involving the induction of channel activity in mitochondria. These events presumably function to ttp the balance and amplify the death pathway. In the absence of thia conversion from anti- to pro-apoptotic function as pro-apoptotic proteins (Lewis et al. Nature Medicme 5:832, 1999). In addition to B&2 family members, other factors including neurolog- ical disease genes can be converted from anti- to pro-apoptotic factors.

pig SYNAPTOSIS

Greg Michael Cole. VA Greatrr 01 and UCLA, North Hills, CA

Despite evidence of pro-apoptotic pathway activation and neuronal nuclear DNA strand breaks in AD. end-stage nuclear apoptotic event5 are rare suggesting only partial actwatlon of apoptotic pathways may occur in neurites at a level rufficient to cause local aynapae and arbor loss, but insufficient to activate nuclear destruction. Trophic factor IOFS, oxidatwe damage to mitochondria or AD accumulation could initiate cytochrome C release and focal synaptic caspase activation followed by flipping of phosphatidylserine and rapid neurite phagocytosis by glia. Results with fractin and other probes are consistent with focal synaptic mitochondrial alterations leading to activation of pro-apoptotic pathways in neurites of AD and APP transgenic mice. Because this “falling off’ of arbor shares apoptotic mechanisms without neuron loss, the term “synaptosis” has been proposed to describe it. Synaptosis may play a role m synapse loss in development and neurodegenerative conditions where synapse loss and regression of dendritic arbor occur prior to, and in excess of actual neuron IOSS.

piq MITOCHONDRIAL STATUS OF CASPASE INHIBITOR-SAVED NEURONS: ROLE OF MEMBRANE POTENTIAL IN COMMIT- MENT(S) TO DIE.

Eugene M Johnson Jr., Mohanish Deshmukh, Washington UniversiQ School of Medicine, St. Lours, MO

Trophic factor deprived neurons undergo a Bax-dependent, macromolecular synthe- G-dependent, apoptosis associated with mitochondrial cytochrome c release and caspase activation. The mitochondrial translocation of Bax is a critical step m the process. The release of cytochrome c in some models of apoptosis has been associated with activation of the permeability transition pore (PTP). an incompletely understood phenomenon associated with loss of mitochondrial membrane potential. We set out to determine: I) was loss of cytochrome c in trophic factor-deprived neurons associated with loss of mitochondtial membrane potential: 2) for how long would caspase inhibition prevent neuronal commitment to die; and 3) what events might be associated with caspase-independent death. Nerve growth factor (NGF) deprivation of sympathetic neurons led to a loss of mitochondrial cytochrome c. However, this loss of cytochrome c occurred prior to the loss of mitochondrial membrane potential, Indicating that PTP was not involved in this process. Cell death could be markedly retarded by pharmacological inhibition of caspases or deletion of caspase 9; however these neurons ultimately beame committed to die (i.e., were no longer savable by readdition of NGF). The time course of this committment was indistinguishable with the time course of loss of mitochondrial membrane potential. NGF-deprived neurons from Bax knockout mice neither released cytochrome c, nor lost their mitochondrial membrane potential. These data indicate that whereas release of cytochrome c causes caspase activation and commitment to death in normal neurons, this commitment to death is delayed up to the point of mitochondrial membrane potential loss in caspase inhibitor-treated neurons. Thus caspase inhibitors delay death and provide a window of time (subsequent to cytochrome c release and prior to loss of mitochondtial potential) in which re-establishment of trophic environment is compatible with the long-term survival of the cell. (Supported by NIA and NINDS)

MECHANISMS OF CELL DEATH IN ALZHEIMER’S DISEASE: ROLE OF PRESENILIN AND MITOCHONDRIAL FUNCTION.

Maria Ankarcrona, Camilla Skagermark, Kjell Hulrenby. Brngr Winblud, Kurolinska

Instituter, HUDDINGE Sweden

The mechanisms of neuronal death in Alzheimer’s disease (AD) are not fully understood. Degeneration of neurite, and lobs of connectivity is likely to be responsible for the early neuronal dysfunction m AD. Such cells may survive as “ghost cells” which are alive but not functionable and eventually these cells will be cleared off by phagocytosis. Other possible mechanisms for cell death in AD include necrosis and apoptosis, where cells elther die from an acute damage or are degraded after activation of the striktly controlled apoptotic programme. All these forms of cell death probably occur, maybe at different time-points, during the progression of AD. We are investigating the role of presenilins and mitochondrial function for the onset and progression of cell death. Presenilins (PSI and PS2) are mutated in most cases of autosomal dominant inherited forms of early onset AD and such mutations sensitize cells to apoptotic stimuli in vitro. The onset of apoptosis is associated with loss of mitochondrial membrane potential and opening of transition pores in the mitochon- drial membrane. Opening of such megapores lead to release of death factors and caspase activation. Previous studies show that presenilins are localized to subcellular compartments and particulary to ER-membranes. Here we show for the first time that PSI is also localized to mitochondrial membranes. Homogenized rat brains were wbcellulary fractionated into nuclear, microsomal and mitochondrial fractions by differential centrifugation on a sucrose gradient. Using immunoblot analysis we detected PSI in the crude nuclear fraction, microsomal fraction as well as in the mitochondrial fraction. Immunoelectron microscope studies show in detail that PSI is localized to mttochondrial inner membranes. The function of PSI localized to mitochondrial membranes is presently unknown. It may be speculated that PSI is part of or regulates the megapores opened during permeability transition occuring early in apoptosis. PSI mutations may make cellp more vulnerable to apoptotic stimuli due to dysfunction of this protein at the mitochondrial level. These possibilities are under current investigation in our laboratory.

pq SYNAPTIC APOPTOTIC AND ANTI-APOPTOTIC SIGNALING IN AD AND SUCCESSFUL BRAIN AGING

M. P. Mattson, National Institute on Aging, Baltimore. MD

Dysfunction and degeneration of synapses are fundamental to the pathogen&s of AD and related disorders. We have been studying signaling cascades that occur locally in pre- and postsynaptic terminals that may either contribute to the neurodegenerative process in AD. or protect therefrom. Environmental (e.g., exposure to amyloid