In <I>C. Elegans</I>, Aspartyl Proteases and Calpain, Not Caspases, Play the Grim Reaper
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The proteolytic degradation that accompanies neurodegeneration in animals may be mediated not by apoptotic proteases, but by a group of aspartyl and calcium-dependent proteases, at least in the roundworm, researchers report in today's Nature.
First author Popi Syntichaki and colleagues working in Nektarious Tavernarakis's lab at the Institute of Molecular Biology and Biotechnology, Heraklion, Greece, came to this conclusion after probing the proteolytic pathways that accompany neurodegeneration in the nematode C. elegans. In these animals, hyperactivity of the ion channel protein mec-4 leads to the death of touch receptor neurons in a manner that resembles the excitotoxic destruction of mammalian neurons by neurotransmitters such as glutamate.
Syntichaki et al. found that in animals with congenital loss of aspartyl protease activity, mec-4 induction of neurodegeneration failed. The authors also showed that the aspartyl protease inhibitor, pepstatin A, protected the six touch receptor neurons from mec-4, as did starvation, which also reduces aspartyl protease activity.
The authors extended these observations to neurons that degenerate in response to other toxic genes, such as mutants of degenerin. In all cases, reduction in aspartyl protease activity was protective, indicating that these enzymes are general mediators of neuronal cell death. But which protease is the culprit? To answer this question the authors systematically suppressed the expression of each C. elegans aspartyl proteases. Of the six in the genome, two, asp-3 and asp-4, were found to be necessary for neurodegeneration, while silencing of a third, asp-1, partly protected against cell death.
Syntichaki et al. then looked at what might trigger activation of these grim reapers. Knowing that rising intracellular calcium and calcium-dependent cysteine proteases, such as calpain, have been implicated in cell death in mammals and flatworms, the authors revisited their mec-4 necrosis model, but this time they administered the calpain inhibitor MDL-28170 to some of the animals. In these worms the number of dying neurons was reduced by as much as twofold. Again, capitalizing on the tractability of the flatworm model and the availability of the genome sequence, the authors were able to narrow the candidate pool for cell death mediators down to two out of 17 calpain homologs, clp-1 and clp-5 (also known as Tra-3).
All told, these experiments seem to link calcium and neurodegeneration through calpain and aspartyl proteases. However, as the authors mention, they never achieved complete blockage of neurodegeneration by RNAi-mediated silencing of these enzymes, which hints that other pathways may also be involved.—Tom Fagan
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Further Reading
Primary Papers
- Syntichaki P, Xu K, Driscoll M, Tavernarakis N. Specific aspartyl and calpain proteases are required for neurodegeneration in C. elegans. Nature. 2002 Oct 31;419(6910):939-44. PubMed.
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Picower Institute of MIT
This intriguing paper uses a well-executed genetic approach to demonstrate the significance of the calcium-dependent cysteine protease calpains and the cathepsin-like aspartyl proteases in neurodegeneration. It has been postulated that, in mammalian systems, calpains and lysosomal enzymes such as cathepsins play a role in acute (such as ischemic brain damage) and chronic (such as Alzheimer's disease) neurodegeneration. However, due to the complexity of the mammalian nervous system and the lack of suitable pharmacologic inhibitors, it has been difficult to unambiguously demonstrate the importance of these enzymes in neurodegeneration. This study not only shows that these enzymes play an essential role in neuronal death, but also offers a cascade of events initiating from Ca influx to activation of calpains and the aspartyl proteases.
This study also suggests that caspases are responsible for apoptosis during development, whereas calpains and the aspartyl proteases are more responsible for neurodegeneration in C. elegans. The picture may not be as black and white in mammals. There is a large body of literature describing the involvement of caspases in a number of neurodegenerative diseases. This underscores the challenge we are facing in combating neurodegenerative diseases such as Alzheimer's in that we need to deal with multiple pathways to save neurons from dying.
New York University School of Medicine/Nathan Kline Institute
In their careful genetic analysis in C. elegans, Syntichaki and colleagues present a compelling case that calpains and aspartyl proteases are essential participants in a form of neurodegeneration that occurs independently of CED-3 and several related proteases important to programmed cell death in the nematode. The study provides the most direct evidence to date that activation of these two proteolytic systems can execute death of mature neurons in situ, and offers a valuable window into the still-murky realm of "necrotic" cell death.
Although the genetic perturbations were not intended to model a specific human disease, the cellular pathways involved and the outcomes invite interesting comparisons to findings in Alzheimer’s disease. In AD, calpain activation and high expression of certain lysosomal proteases, including the aspartyl protease cathepsin D, develop very early in neurons in vulnerable areas and become robust as the neurons begin to show other signs of metabolic compromise. Growing evidence links the actions of these two proteolytic systems in the Alzheimer brain to cytoskeletal and APP-related pathobiology as well as to neurodegeneration (reviewed in Ann.NY.Acad.Sci, 2000). In the C. elegans model, neurodegeneration was elicited by inducing the release of calcium from endoplasmic reticulum stores. By somewhat different mechanisms, mutant forms of presenilin that cause familial AD potentiate agonist-induced release of calcium from the ER of neurons in transgenic mice. Although neuronal death has not been seen in these mice, the sensitivity to excitotoxin-induced neurodegeneration is increased. Other factors important to AD development—aging, oxidative stress, and Aβ toxicity—also promote calcium dysregulation as well as activate calpain and cathepsin D. The nematode findings should stimulate research on how these proteolytic pathways are regulated in the brain and behave in mammalian models of human neurodegenerative disease.
The Syntichaki et al. results also touch on an emerging theme within cell death circles that the different protease systems of the cell interact extensively. This cross-talk has contributed to the present confusing picture in aging-related neurodegenerative disease of which protease systems mediate neuronal cell death. Given the extent of these interactions, it would not be surprising if some caspase activation could also be detected late in the demise of neurons in the worm model, even though its role is minor. Until more specific protease inhibitors are developed, the genetic approach taken by Syntichaki et al. holds the greatest promise for sorting out which activated proteases in a dying cell are essential to the particular degenerative process and are, perhaps, viable therapeutic targets. The prospect that multiple protease systems contribute to neurodegeneration does pose a challenge for therapy development. At the same time, the substantial rescue seen in this study even when only one of the implicated proteases was blocked provides a ray of hope that targeting the pathological activation of "utility" proteases, a situation often considered too catastrophic or end-stage to rescue, may yet have a place in the treatment of AD and related disorders.
References:
Nixon RA. A "protease activation cascade" in the pathogenesis of Alzheimer's disease. Ann N Y Acad Sci. 2000;924:117-31. PubMed.
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