Cell death

Neuronal population

Target

1.2. Key molecules regulating neuronal apoptosis during development

The breakthrough for understanding the mechanism of neuronal cell death induced by trophic factor deprivation came from the studies in the nematode Caenorhabditis elegans. During C. elegans development, of 1,090 somatic cells (of which 302 are neurons and 56 are glial cells), 131 undergo programmed cell death. The death of these 131 cells is predetermined by their genetic lineages and occurs at predictable times during the development of each individual worm. Thus programmed cell death in C. elegans is a form of cellular suicide. In contrast, there is no evidence to indicate that the death of mammalian neurons occurring during the period when they are establishing synaptic connection is predetermined. As discussed before, the verdict regarding which neuron will die and which will live is reached through a competition process during which neurons compete for establishing the correct synaptic connection and trophic factor availability. Therefore, mammalian neurons that die during the period of establishing synaptic connection are not predetermined by their lineage; rather, they are induced as a result of lacking trophic factor support.

The central components of the programmed cell death machinery in C. elegans are three CED proteins: CED-3, CED-4, and CED-9. In this cellular suicide machinery, CED-9 functions as an inhibitor of apoptosis by preventing CED-4 from interacting with CED- 3, whereas CED-4 is a proapoptotic adaptor molecule

required for the activation of CED- 3, a cysteine protease responsible for the execution of cell death program.

In mammals, the homologs of CED-9,

CED-4, and CED-3 are members of the Bcl-2 family, Apaf-1/NOD-like receptor family, and caspase family, respectively. The demonstration that neuronal cell death induced by the lack of trophic factors can be prevented by overexpression of Bcl-2 or by expression of a virally encoded caspase inhibitor crmA provided the first insights into the role of apoptosis in regulating neuronal cell death. Thus, surprisingly, although mammalian neuronal cell death induced by trophic factor deprivation occurs through a stochastic process of competition, they are regulated by a cellular suicide machinery that is very likely to share the same evolutionary origin as that of programmed cell

death in C. elegans that is predetermined by their cell lineages during development. Next we review the mechanisms by which neuronal cell death is regulated and executed.

1.2.1. Roles of caspases and Apaf-1 in neuronal cell death

Multiple virally encoded caspase inhibitors, such as crmA and p35, provided useful tools for demonstrating the roles of apoptosis in a variety of cellular systems, including neurons. The expression of crmA (a caspase inhibitor encoded by cowpox virus) in chicken dorsal root ganglion (DRG) neurons or p35 (a caspase inhibitor encoded by baculovirus) in rat sympathetic neurons inhibits apoptosis on NGF deprivation in culture. Peptide-based chemical inhibitors derived from the preferred caspase cleavage sites in their substrates also block neuronal cell death in vitro. Newborn DRG neurons from mutant caspase-1 (C285G) transgenic mice and caspase-1–/– mice are resistant to trophic factor withdrawal-induced apoptosis in culture. However, it has been challenging to demonstrate the precise roles of caspases in regulating neuronal cell death during the period of establishing synaptic connection in vivo because until now, no caspase-deficient mice have shown a significant defect in the elimination of developing postmitotic neurons while establishing synaptic connections in vivo, despite the establishment of almost all caspase mutant mice.

Genetic analysis of caspase-deficient mice demonstrated the roles of caspase-3 and caspase-9 in mediating apoptosis of mitotically active neural progenitor cells or immature neurons in the forebrain during early developmental stages before the formation of synaptic connections. Caspase-3–/– mice in mixed 129/SvJ and C57BL/6 background die perinatally with a variety of hyperplasia and disorganized cell deployment in the brain, similar to that of caspase-9–/– mice. Ectopic cell masses appear in the cerebral cortex, hippocampus, and striatum, whereas the incidence of pyknosis, a prominent feature of apoptosis during normal neurogenesis in the periventricular zone, is significantly reduced in caspase- 3–/– and caspase-9–/– mice. Certain mutant phenotypes of caspase-deficient mice, however, can have a strong dependency on genetic background (e.g., caspase-3–/– mice are viable and developmentally normal in C57BL/6 background). The interaction of genetic background with a specific caspase deficiency might be a subject of interest for future studies.

The death of newborn neural precursor cells in the periventricular zone as those impaired in caspase-3–/– and caspase-9–/– mice, however, occurs before the time of establishing synaptic connection because neurons are born in the periventricular zone and the neuronal connections are only made after their migration to the appropriate layers of the brains. The regulation of neuronal cell death in population in vivo before the formation of synaptic connections may differ from the well-known target-dependent type of naturally occurring neuronal death that occurs when synaptic connections are being formed. Interestingly, in contrast to the striking perturbations in the morphology observed in the more rostral, mitotically active regions of the brain of caspase-3–/– and caspase-9–/– embryos, the spinal cord, brainstem, and peripheral ganglia in these caspase mutant mice appear completely normal at both embryonic and postnatal stages. Developing postmitotic neurons at these regions ultimately undergo normal number of neuronal loss, despite the temporal delay in cell death. Thus, although the studies using in vitro model of trophic factor–dependent neuronal cell death predict an important role of caspases in apoptosis, there is a general lack of evidence for the involvement of any specific caspase in postmitotic neuronal death in vivo. There might be a number of reasons to explain this. First of all, it is possible that the constitutive loss of a caspase in the germline might lead to upregulated expression of other caspases in the mutant background, which might compensate for the loss of a single caspase. This has been demonstrated for caspase- 3–/–, caspase-7–/–, and caspase-9–/– mice. However, a

compensatory expression of other caspases makes it difficult to explain the lack of obvious deficiency in the death of all neurons since the majority of late-stage caspase-3–/–; caspase-7–/– double knockout embryos, which lack both major downstream caspases required for the execution of apoptosis, did not show abnormality in brain morphology. It will be interesting to examine whether conditional caspase mutant mice that are specifically deficient for a caspase in neuronal lineage or in a temporally regulated manner show a defect in neuronal cell death induced by trophic factor deprivation. On the other hand, it is possible that in caspase-deficient mice, additional caspase-independent cell death mechanism is activated to compensate for the loss of caspase deficiency. In fact, although many populations of developing postmitotic neurons are able to exhibit normal amount of cell death in the absence of either caspase-3 or caspase-9, the morphology of these degenerating neurons differs from the more typical apoptotic cell death, and the kinetics of their degeneration is delayed. Ultrastructural analysis of degenerating spinal cord neurons from E14.5 caspase-3–/– embryos revealed the presence of extensive cytoplasmic vacuoles that are not usually detected in apoptotic cells and that are seldom observed in dying neurons of control embryos. A delay in the degeneration of caspase-deficient neurons suggests that caspase-mediated neuronal cell death is more efficient than caspase-independent cell death.

Similar to caspase-9–/– mice, regardless of genetic background, apaf-1–/– mice also display a massive overgrowth of cells in the brain (exencephaly) that was attributed in part to the reduced cell death of both immature neurons and dividing neuronal precursor cells and that is consistent with the role of apaf-1 being an activator of caspase-9. In mature neurons, however, Apaf-1 was found to be dispensable for neuronal cell death caused by the lack of trophic signaling input from TrkA deficiency or synaptic activity from Munc-18 deficiency in vivo. Many populations of postmitotic, “targetdependent” neurons, including spinal and cranial motor neurons, spinal interneurons, DRG sensory neurons, and sympathetic neurons in the SCG, undergo a quantitatively normal amount of cell death in the absence of apaf-1, although caspase-3 activation is blocked. The degenerating apaf-1–/– neurons show numerous vacuoles atypical for apoptosis, suggesting the activation of a back-up cell death mechanism in developing neurons when caspase activation is inhibited. Thus it is possible that in postmitotic mature neurons that are deficient for caspase activation mechanism, the lack of trophic factor support might trigger an active form of cell death mediated through a caspase-independent mechanism.

1.2.2. Role of Bcl-2 family members in neuronal cell death

The Bcl-2 family includes both antiapoptotic and proapoptotic proteins that contain one or more Bcl- 2 homology (BH) domains. Bcl-2 and Bcl-xl are two major antiapoptotic members of the Bcl-2 family. Overexpression of Bcl-2 prevents apoptosis of sympathetic neurons induced by NGF deprivation in culture. Transgenic mice expressing Bcl-2 in the nervous system show reduced neuronal cell death during developmental hypertrophy of the brain and increased numbers of facial motor neurons and retinal ganglion cells. It is interesting, however, to compare the neuronal phenotypes of the mice over-expressing Bcl-2 with that of caspase-9–/– and apaf-1–/– mice: although they all show reductions in neuronal cell death and increases in neuronal cell numbers, over-expression of Bcl-2 in the brains results in larger brains with more neurons but otherwise normal brain morphology, whereas caspase-9 deficiency or apaf-1 deficiency leads to severe defects in brain morphogenesis. Given the brain morphology of Bcl-2 transgenic mice, one might argue that a simple reduction in neuronal cell death is not sufficient to alter brain morphogenesis. By the same reasoning, it is possible that caspase-9 and apaf-1 have additional functions unrelated to regulation of apoptosis, a possibility that should be examined in the future.

The expression of Bcl-2 is high in the central nervous system during development and downregulated after birth; however, the expression of Bcl-2 is retained in neurons of the peripheral nervous system throughout life. Although the prenatal development of the nervous system in Bcl-2–/– mice is normal, there is a subsequent loss of motor, sensory, and sympathetic neurons after birth, suggesting that Bcl-2 is crucial for the maintenance of specific populations of neurons during the early postnatal period.

The normal development of nervous system in Bcl- 2–/– mice might be attributed to the redundancy in the functions served by other members of the Bcl-2 family. Bcl-xl appears to be a good candidate because it is also expressed in the developing brain. Unlike Bcl-2, whose expression decreases after birth, Bcl-xl expression is retained in the neurons of the adult central nervous system. Bcl-xl–/– mice die around embryonic day 13, with extensive apoptotic cell death in postmitotic differentiating neurons of the developing brain, spinal cord, and dorsal root ganglia. Striking deficiency of neuronal survival in Bcl-xl–/– mice indicates its pivotal role in maintaining neuronal survival.

Mcl-1 is another important antiapoptotic Bcl-2 family member. Mcl-1–/– mice die very early in embryonic development around embryonic day 3.5, which is the most severe phenotype among all the known mutant mice in the members of antiapoptotic Bcl-2 family. Robust expression of Mcl-1 is present in both proliferating neuronal progenitor cells and postmitotic neurons during brain development. Mcl-1 deficiency, especially in neuronal lineage, results in the apoptotic death of both Nestin+ neural progenitors and Tuj1+ newly committed neurons. During the development of the nervous system, proapoptotic Bax and bid are expressed in neural precursor cells within the ventricular zone, with the expression of Bax peaking at E12 to E15, corresponding to the period of early neurogenesis when widespread apoptosis of neural precursors occurs in the Mcl-1 conditional mutants. Although both Mcl-1 and Bcl-xl have been shown to block Baxand Bak-mediated apoptosis, Bcl-xl is expressed at very low levels in the neural precursor populations, and apoptosis is observed in more mature neuronal populations in the Bcl-xl–/– mutant mice at E12.5. Therefore, Mcl-1 plays an important role in regulating the survival of neurons during the transition from the progenitor to the postmitotic period.

Antiapoptotic members of the Bcl-2 family act as antagonists for the proapoptotic members of Bcl-2 family. Bax is a key member of proapoptotic Bcl-2 family in regulating neuronal apoptosis. Wild-type neonatal sympathetic superior cervical ganglion neurons and facial motor neurons express Bax mRNA at a time when these neuronal populations are susceptible to growth factor deprivation in vivo. Deletion of Bax results in profound effects on the survival of many kinds of neurons. Bax–/– neonatal sympathetic neurons and facial motor neurons survive nerve growth factor deprivation in culture and disconnection from their targets by axotomy in vivo, respectively. Bax–/– mice have increased neuronal numbers in the superior cervical ganglia and facial nuclei. Thus the activation of Bax may be a critical event for neuronal cell death induced by trophic factor withdrawal, as well as injury. Further studies examined the fate of the excess rescued neurons in postnatal Bax–/– mice during muscle target innervation and revealed that although initially all of the motor neurons, including those rescued by Bax deletion, are able to project to and innervate targets, only a subpopulation can grow and retain target contacts postnatally. Treatment with exogenous trophic factor can reverse their atrophy and promote regrowth of the axons of the excess surviving motor neurons, suggesting that even after their developmental role in