Molecular Mechanisms of Developmental Neuronal Apoptosis

Studies of the nematode Caenorhabditis elegans first elucidated the presence of a genetically controlled suicide cascade that determines apoptotic cell death. Three apoptosis regulatory genes have been identified in this species in which 131 of 1,090 somatic cells undergo developmental apoptosis: ced-3 and ced-4 act in a proapoptotic way, whereas ced-9 prevents apoptotic cell death (Horvitz, 1999). In biochemical studies, it has been shown that these three proteins function as a complex in which the antiapoptotic ced-9 normally suppresses ced-4 dependent activation of ced-3. The mammalian homologues of ced-3, ced-4, and ced-9 were cloned and it became clear that these genes were core components of the mammalian apoptosis machinery as well (for review, see Bahr, 2000). The mammalian homologues of ced-3 belong to a family of proteases called caspases, which consists of at least 14 members (Thornberry and Lazebnik, 1998). During cell death, caspases are converted from their proenzymatic forms into activated proteases in a cascade-like order and thereby mediate signaling and execution of apoptosis (Fig. 1).

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Figure I The mitochondrial pathway of apoptosis. Following an apoptotic trigger, cytochrome c (cyt c) is released from the mitochondrium. Cyt c liberation into the cytosol results in formation of the apoptosome (in combination with Apaf1 and pro-caspase 9), which is cleaved in the presence of ATP or dATP. This triggers the downstream caspase cascade, in which cas-pase-3 plays a major role in the execution of cell death. (Adapted from Bahr, 2000.)

On the basis of the peptide-sequence preference of their substrates, the caspase family can be divided into three subgroups: the Interleukin-ip-converting enzyme protease family (caspases 1, 4, 5, and 11-14), the ced-3 subfamily (caspases 3 and 6-10), and a third subfamily containing only caspase-2. Studies on knock-out mice revealed the individual in vivo relevance of each caspase. Whereas the pheno-type of caspase-1-knock-out mice, for example, does not indicate any disturbances in developmental neuronal apop-tosis, mice deficient in caspase-3 develop phenotypical changes characterized by an excessive number of neurons in the CNS and retina (Nicholson and Thornberry, 1997).

Ced-4 shares homology with the mammalian apoptosis regulator Apoptosis protease activated factor-1 (Apaf-1) (Zou et al., 1997), which can associate with several death proteases, including caspase-4, -8, and -9. Together with cytochrome c released from the mitochondria, complex formation of Apaf-1 and pro-caspase-9 builds the "apopto-some" as shown in Fig. 1, a trigger complex for the activation of the further downstream caspase cascade (for review, see Bahr, 2000). The phenotype of Apaf-1-knock-out mice shows similarities to caspase-3 and -9 knock-outs characterized by reduced developmental apoptosis in the CNS and consecutive brain, as well as craniofacial malformations (Cecconi et al., 1998).

Ced-9 shows sequence and structural similarities to the mammalian proto-oncogene Bcl-2 (Hengartner and Horvitz, 1994). The growing Bcl-2 family consists of at least 15 Bcl-2 homologues, which function either as antagonists (e.g., Bcl-2, Bcl-XL, or Bcl-w) or agonists (Bax, Bcl-Xs, BAD) of apoptotic cell death (for review, see Bahr, 2000). Parts of the Bcl-2 family members are located at the outer mitochondrial membrane where they can protect or disrupt membrane integrity. Whereas Bcl-2 and Bcl-XL prevent cytochrome c release from the mitochondrium, conformational changes of Bax lead to release of cytochrome c and consecutive formation of the apoptosome. This mitochondrial pathway of apoptosis is located upstream from caspase activation and triggers the further execution of cell death (Fig. 1). Studies on Bcl-2 knock-out mice revealed that this member of the Bcl-2 family is not essential for developmental neuronal cell death: At birth, the number of motoneurons, sensory, or sympathetic neurons in these mice was not significantly changed when compared to wild-type animals (Michaelidis et al., 1996). However, substantial degeneration of central and peripheral neurons occurred during the early postnatal period. In the retina, Bcl-2 knock-outs showed a loss of 29% of their RGC axons until postnatal day 15 indicating a physiological role of Bcl-2 for the maintenance of neurons soon after the period of naturally occurring cell death (Cellerino et al., 1999). In contrast, mice deficient for Bcl-XL die around embryonic day 13 because of a massive increase of developmental apoptosis in the hematopoietic and the nervous system (Motoyama et al., 1995). Knock-out mice of the death-promoting Bcl-2 family member Bax show increased numbers of motor and sympathetic neurons, as well as increased developmental survival of RGCs (Deckwerth et al., 1996; Mosinger Ogilvie et al., 1998).

II. Lesion-Induced Apoptosis: Apoptotic Neuronal Cell Death in Models of Experimental Autoimmune Encephalomyelitis

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