Programmed cell death in amyotrophic lateral sclerosis (original) (raw)

Caspases are members of a distinct family of cysteine proteases that share the ability to cleave their substrates after specific aspartic acid residues and that are present in cells as inactive zymogens, called procaspases. So far, 14 different mammalian caspases have been identified that differ in primary sequence and substrate specificity. An instrumental role for caspases in ALS neurodegen-eration is supported by the demonstration that the irreversible broad-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(_O_-methyl)-fluoromethylketone attenuates mutant SOD1–mediated cell death in transfected PC-12 cells (29) and in transgenic SOD1G93A mice (57).

All of the identified caspases are grouped based on their function. One group includes caspases -1, -4, -5, -11, -12, and -14, which are now believed to play a primary role in cytokine maturation. Among these, in ALS, the lion’s share of attention until now has been given to caspase-1, the key enzyme responsible for the activation of IL-1. Procaspase-1 is highly expressed in spinal cord motor neurons, and its activation in the spinal cord of transgenic mutant SOD1 mice coincides with the development of the glial response and with the very beginning of the loss of motor neurons (33, 34, 57, 63, 72). Despite caspase-1’s likely indirect role in PCD, chronic inhibition of caspase-1 by a dominant negative mutant of the enzyme has been proven effective in prolonging the life of transgenic SOD1G93A mice (73). So far, the status of the other members of the caspase-1 subfamily in ALS is unknown. Some preliminary investigations show that caspase-12, which is known to be activated following ER stress (74), is expressed in motor neurons of nontransgenic mice, and even more so in those of symptomatic transgenic SOD1G93A mice (C. Guégan et al., unpublished observations). In symptomatic transgenic SOD1G93A mice, most of the motor neurons immunopositive for caspase-12 appear condensed, shrunken, and vacuolized. Although more work on caspase-12 remains to be done in this model of ALS, our preliminary data argue that sick cells are the site of an ER stress whose occurrence could well contribute to the overall cascade of deleterious events that ultimately underlies the demise of spinal cord motor neurons in the mutant SOD1 model.

By contrast, caspases -2, -3, -6, -7, -8, -9, and -10 have been implicated in apoptosis per se, although their roles can be further divided into “initiator” and “effector.”

Initiator caspases include procaspases -2, -8, -9, and -10, all of which have long prodomains and protein-protein interaction motifs, such as the death-effector domain and the caspase-activation and -recruitment domain, that contribute to the transduction of various signals into proteolytic activity. Procaspase-8 is activated after ligation of certain cell surface receptors, such as the TNF receptors. Interestingly, while significant glial response and production of IL-1β occur early in transgenic mutant SOD1 mice (see above), activation of procaspase-8, like induction of TNF-α (56), is only detected in spinal cords near the end stage (65). This suggests that, in this ALS model, the TNF/caspase-8 machinery may be a late contributor to the degenerative process. Caspase-2 is another initiator of PCD whose activation occurs in the spinal cord of affected transgenic mutant SOD1 mice (S. Vukosavic et al., unpublished observations). Yet ablation of caspase-2 in transgenic SOD1G93A mice has been reported to be of no consequence to the expression of the disease (75), indicating that whatever the role of caspase-2 is in ALS, it is dispensable. A third caspase initiator is caspase-9, whose role is pivotal in the so-called mitochondria-dependent PCD pathway (53). Here, after a death stimulus, released mitochondrial cytochrome c interacts in the cytosol with apoptotic protease-activating factor-1 (Apaf-1) in the presence of dATP, which stimulates the processing of procaspase-9 into its active form, which in turn can activate the downstream executioner caspases (see below). Evidence of prominent recruitment of this mitochondrial pathway has been documented in spinal cord specimens of both ALS patients and transgenic SOD1G93A mice (63). In that study, it is shown that, while cytochrome c is confined to the mitochondria in cells in the control samples, it is diffusely distributed in the cytosol in several of the spared cells, especially neurons, in the pathological samples. It is also demonstrated, at least in transgenic mutant SOD1G93A mice, that the mitochondrial cytochrome c translocation to the cytosol occurs at the same time as the cytosolic Bax translocation to the mitochondria and activation of procaspase-9, and before activation of downstream caspase executioners such as procaspase-3 and procaspase-7 (Figure 1, b–d). Because caspase-9 is thought to be so critical in many cell-death settings, it is very likely that the observed translocation of cytochrome c and activation of procaspase-9 in ALS represent significant pathological events. Consistent with this view is the finding that prevention of mitochondrial cytochrome c release lengthens the lifespan of transgenic SOD1G93A mice (76).

Effector caspases include procaspases -3, -6, and -7, all of which have short prodomains and lack intrinsic enzymatic activity. However, upon their cleavage, which is triggered by, for example, initiator caspases, effector caspases acquire the capacity to cleave a large number of intracellular substrates, which probably results in the eventual death of the cell. Consistent with this scenario, it has been reported that key effector caspases such as caspase-3 and caspase-7 (see Figure 1d) are indeed activated in spinal cords of transgenic mutant SOD1 mice in a time-dependent manner that parallels the time course of the neurodegenerative process (33, 34); activation of procaspase-3 has also been observed in spinal cord samples from ALS patients (38). Yet current data on the sequence of events in the PCD cascade indicate that, once effector caspases have been activated, the cell death process, at least in certain pathological settings, has reached a point of no return. This would suggest that, in these specific conditions, the death commitment point is situated upstream of these caspases, and, consequently, interventions aimed at inhibiting these downstream caspases may fail to provide any real neuroprotective benefit (77). Whether this applies to the demise of motor neurons in ALS remains to be determined.