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can be broadly divided into two groups:initiator caspases (such as caspase-8 and cas-pase-9) whose main function is to activatedownstream caspases, and executor caspases(such as caspases-3, -6 and -7), which areresponsible for dismantling cellular pro-teins. The two main apoptotic pathways —the death receptor and mitochondrial path-ways — are activated by caspase-8 and cas-pase-9, respectively, both of which are foundin the cytoplasm (Fig. 1). Caspase-8 isrecruited to a death-inducing signallingcomplex only when death receptors such as

The philosophical muse proclaiming that“death is the essential condition of life”1

has a firm footing in biology, for pro-grammed cell death (apoptosis) is obligatoryfor normal development of multicellularorganisms2. But apoptosis is a double-edgedsword, and deregulated cell death is impli-cated in a growing number of clinical disor-ders3. So, the apoptotic process needs to betightly regulated. One way in which this isdone is by physically segregating the differ-ent components of the apoptotic machinery— only when the death switch is trippedare the tools of execution brought togetherin the cytosol and the suicide programmeactivated.

The two main compartments known tobe involved in such segregation are the plas-ma membrane, where both death and sur-vival receptors reside, and the mitochon-drion, which is home to several proteins thatregulate apoptosis. Now, reporting on page98 of this issue, Yuan and colleagues4 pointto the endoplasmic reticulum as a third sub-cellular compartment implicated in apop-totic execution. Furthermore, they provideevidence linking activation of a hithertoobscure apoptotic enzyme, caspase-12, toAlzheimer’s disease.

Famous mainly for the synthesis and pro-cessing of secreted proteins and the storageof intracellular calcium, the endoplasmicreticulum has already provided us with cluesas to its alter ego. First, both pro- and anti-apoptotic members of the Bcl-2 family arelocated in intracellular membranes includ-ing the nuclear envelope, the outer mito-chondrial membrane and the endoplasmicreticulum5. And second, cytosolic Ca2& hasbeen implicated as a pro-apoptotic secondmessenger6 involved in both triggeringapoptosis and in regulating death-specificenzymes.

Among the most prominent of thesedeath-specific enzymes is a family of cys-teine-dependent aspartate-specific prote-ases known as the caspases7. These enzymes

8. Alroy, J. in Extinctions in Near Time: Causes, Contexts, and

Consequences (ed. MacPhee, R. D. E.) 105–143

(Kluwer/Plenum, New York, 1999).

9. Williams, P. H. in Conservation in a Changing World (eds Mace,

G. M., Balmford, A. & Ginsberg, J. R.) 211–249 (Cambridge

Univ. Press, 1998).

10.Mittermeier, R. A., Myers, N., Robles Gil, P. & Goettsch

Mittermeier, C. Hotspots (CEMEX, Mexico City, 1999).

11.Brooks, T. M., Pimm, S. L. & Collar, N. J. Conserv. Biol. 11,

382–394 (1997).

12.Pimm, S. L. & Brooks, T. M. in Nature and Human Society: The

Quest for a Sustainable World (ed. Raven, P.) 38–54 (Natl Acad.

Press, Washington DC, 1999).

Fas or the tumour-necrosis factor receptorare oligomerized after binding of specific lig-ands. In contrast, caspase-9 is activated whencytochrome c is released into the cytoplasmfrom the space between the inner and outermitochondrial membranes.

Yuan and co-workers4 now show thatanother caspase, caspase-12, localizes not tothe cytosol but to the endoplasmic reticu-lum. Caspase-12 is specifically involved inthe apoptosis that results from stress in theendoplasmic reticulum. Treatment withcompounds such as brefeldin A (whichinhibits transport from the endoplasmicreticulum to the Golgi body) or tunicamycin(which inhibits N-glycosylation in the endo-plasmic reticulum) triggers activation of cas-pase-12. But the strongest activation is seenin response to thapsigargin, which disruptsintracellular Ca2& homeostasis, or to theCa2& ionophore A23187. Apoptosis trig-gered through pathways that do not involvethe endoplasmic reticulum, such as serumdeprivation or Fas activation, do not result inactivation of caspase-12.

To study the localization of caspase-12,Yuan and colleagues used a specific caspase-12 antibody in immunoblotting experi-ments and detected bands in a fraction ofbrain extract that also contained an endo-plasmic-reticulum-specific protein calledTRAPa. Immunocytochemistry thenrevealed the perinuclear distribution of cas-

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1. Curnutt, J. L., Pimm, S. L. & Maurer, B. A. Oikos 76, 131–144

(1996).

2. Lomolino, M. V. & Channell, R. J. Mamm. 76, 335–347

(1995).

3. Lomolino, M. V. & Channell, R. Conserv. Biol. 12, 481–484

(1998).

4. Channell, R. & Lomolino, M. V. Nature 403, 84–86

(2000).

5. Gaston, K. J. Ecography 17, 198–205 (1994).

6. http://www.creo.org

7. Olson, S. L. & James, H. F. in Quaternary Extinctions: A

Prehistoric Revolution (eds Martin, P. S. & Klein, R. G.) 768–780

(Univ. Arizona Press, Tucson, 1984).

Apoptosis

Caspases find a new place to hideHuseyin Mehmet

Figure 1 Three distinct apoptotic signalling pathways. a, When the mitochondrion receivesappropriate apoptotic cues, or is irreversibly damaged, pro-apoptotic molecules such as cytochrome care released into the cytosol. Together with ATP, cytochrome c forms a complex with Apaf-1 andprocaspase-9, which is released in an active form. b, Oligomerization of death receptors (by specificdeath ligands) recruits adaptor molecules involved in activation of caspase-8. The active caspase isformed from two procaspase-8 molecules. c, After stress to the endoplasmic reticulum, including therelease of Ca2& from intracellular stores, caspase-12 is activated. Activated initiator caspases, such ascaspase-8 and caspase-9, activate executioner caspases, including caspase-3. Active caspase-3 cleavesthe b-amyloid precursor protein (APP), resulting in increased production of amyloid b-peptide (Ab)which can feed back into caspase-3 activation and execute apoptosis in a caspase-12-dependentmanner. The question marks denote possible, but unconfirmed, pathways.

Apoptotic trigger

Death ligand Endoplasmic reticulumstress

Death receptor Ca2+

?

??

?

Adaptor molecule

Cytochrome cApaf-1

Procaspase-9(inactive)

Procaspase-8(inactive)

Procaspase-3(inactive)

Procaspase-12(inactive)

Caspase-3(active)

Caspase-12(active)

Caspase-8(active)

Caspase-9(active)

APP Aβ

a b c

Apoptosis

© 2000 Macmillan Magazines Ltd

pase-12, suggesting that it is situated in eitherthe mitochondria or the endoplasmic reticu-lum. The co-localization of caspase-12 withseveral proteins found in the endoplasmicreticulum — grp78, presenilin-2, the b-amyloid precursor protein (APP) and greenfluorescent protein targeted to the endoplas-mic reticulum — confirmed that caspase-12resides in the endoplasmic reticulum.

It seems, then, that the main function ofcaspase-12 is to facilitate apoptosis in cellsirreversibly damaged by stress signals fromthe endoplasmic reticulum. Further evi-dence of this comes from Yuan and col-leagues’ studies of transgenic mice that lackintact caspase-12 protein. Thymocytes fromthese mice die in response to the mitochon-dria-dependent death signal dexametha-sone, and hepatocytes die after injection ofFas-agonist antibodies, indicating that cas-pase-12 is not required for apoptosis initiat-ed in the mitochondria or plasma mem-brane. In contrast, apoptosis of the renaltubular epithelium, which is mediated byendoplasmic reticulum stress, dependsentirely on caspase-12 activity.

But this is not the end of the story. Theendoplasmic reticulum, along with the as-sociated presenilins and APP and the acc-umulation of Ca2&, are all implicated inAlzheimer’s disease8. Apoptosis has alsobeen linked to Alzheimer’s disease, with APPidentified as a specific substrate for caspase-3(ref. 9), and Yuan and colleagues’ work nowsupports this idea. They found that primarycortical neurons isolated from their caspase-12-deficient mice are resistant to apoptosisinduced by the cleavage product of APP,the amyloid-b peptide (Ab). To excludethe possibility that the mutant mice lack asub-population of Ab-sensitive neurons, theauthors used an antisense approach toswitch off caspase-12 in normal cells, andfound that these cells were also protectedagainst Ab toxicity.

Putting these data together we can envis-age a model in which APP is cleaved by cas-pase-3, releasing Ab which, in turn, activatesdownstream caspases including caspase-3(ref. 9) and, possibly, caspase-12 to amplifythe effects. Although this needs furtherinvestigation, it does explain why, despite thehigh speed of apoptosis, Alzheimer’s diseasedevelops slowly over a period of years. Per-haps the presenilins and APP are innocentbystanders in neuronal cells that are trig-gered to undergo apoptosis after irreparablestress damage. The gradual accumulation ofAbcould eventually reach critical levels, trig-gering neuronal death through a self-perpet-uating circle of APP cleavage, Ab productionand caspase-12-dependent apoptosis.

Like many important insights, Yuan andcolleagues’ experiments raise more ques-tions than they answer (Fig. 1). For example,what are the physiological regulators of cas-pase-12? Although increases in the cytosolic

concentration of Ca2& are enough to activatecaspase-12, the protein intermediates in thisprocess are not known. What are the down-stream targets of caspase-12? Because cas-pase-3 is activated by Ab, caspase-12 may bethe missing link between APP cleavage, acti-vation of caspase-3 and neuronal apoptosis.Where do Bcl-2 family proteins fit in? Theirfunction is closely linked with caspase activi-ty, and their localization in the endoplasmicreticulum suggests that they are involved inregulating caspase-12-mediated death.

If the identification of caspase-12 in theendoplasmic-reticulum stress pathway fol-lows recent trends, we can expect there to beredundancy in the system. Indeed, the obser-vations4 that such apoptosis is not complete-ly inhibited in embryonic fibroblasts fromthe caspase-12-deficient mice suggests thatthere are caspase-12-independent pathwaysin the endoplasmic reticulum. But what setscaspase-12 apart from other apoptotic pro-teases is that it really does seem to be restrict-ed to stress responses in the endoplasmicreticulum.

This observation has clear clinical ramifi-cations. Caspases are central to both normalprogrammed cell death and injury-depen-dent apoptosis, so any therapy that manipu-lates caspase activity must take into accountthe possible effects on tissue homeostasis.In this regard, though, caspase-12 seems to

have a strong advantage as a target over othercaspases. In contrast to other caspase knock-outs10, caspase-12-deficient mice have nonoticeable developmental or behaviouraldefects, and have a normal incidence oftumours. So, caspase-12 is probably notessential in normal developmental death ortumorigenesis. And if activation of caspase-12 does turn out to be confined to only anarrow band of cellular stress signals, it willbe a promising potential target for treatingneurodegenerative diseases and cancer withfew side effects. ■

Huseyin Mehmet is in the Weston Laboratory,Division of Paediatrics, Obstetrics and Gynaecology,Imperial College of Science, Technology andMedicine, Hammersmith Hospital, Du Cane Road,London W12 0NN, UK.e-mail: h.mehmet@ic.ac.uk

1. Gilman, C. P. The Living of Charlotte Perkins Gilman: An

Autobiography (Appleton-Century, New York, 1935).

2. Jacobson, M. D., Weil, M. & Raff, M. C. Cell 88, 347–354

(1997).

3. Thompson, C. B. Science 267, 1456–1462 (1995).

4. Nakagawa, T. et al. Nature 403, 98–103 (2000).

5. Krajewski, S. et al. Cancer Res. 53, 4701–4714 (1993).

6. Nicotera, P. & Orrenius, S. Cell Calcium 23, 173–180

(1998).

7. Wolf, B. B. & Green, D. R. J. Biol. Chem. 274, 20049–20052

(1999).

8. Mattson, M. P., Guo, Q., Furukawa, K. & Pedersen,W. A.

J. Neurochem. 70, 1–14 (1998).

9. Gervais, F. G. et al. Cell 97, 395–406 (1999).

10.Zheng, T. S., Hunot, S., Kuida, K. & Flavell, R. A. Cell Death

Differ. 6, 1043–1053 (1999).

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30 NATURE | VOL 403 | 6 JANUARY 2000 | www.nature.com

Over the past 30 years, astronomers haveaccumulated an impressive body ofevidence suggesting that there is more

to our Galaxy than meets the eye. Themotions of the stars we see betray the exis-tence of a much larger gravitational pull thanwe can infer from the mutual interactions ofthe stars themselves. In fact as much as 90%of our Galaxy may be invisible to observers.This unseen mass has been dubbed ‘darkmatter’ and is distributed roughly spherical-ly throughout the Galaxy — mainly withinthe faint Galactic halo that surrounds theluminous Galactic disk. But the nature of thisdark matter remains a complete mystery. Onpage 57 of this issue, Hodgkin et al.1 present asmall but vital piece of evidence supportingthe notion that old dead stars may be animportant constituent of dark matter.

Dark-matter theories fall into two broadclasses. The first postulates that dark matteris not truly dark and resides in some kindof faint stellar object. Favoured candidatesinclude brown dwarfs (an object about one-hundredth the mass of our Sun, too small tobecome a real star) and white dwarfs (a dead

star, the remnant of a star more massive thanour own, which lived fast and died young).

The second class of theory is more specu-lative but has profound consequences if cor-rect. In this class, the dark-matter candidatesare various kinds of small elementary par-ticles left over from the Big Bang. Such ma-terial was produced when the Universe wasmuch hotter and denser, and would form thegravitational background of all subsequentevolution of stars and galaxies. These parti-cles are much smaller than stars, but alsomore numerous, so that occasionally theywill pass through the Earth and eventually,we hope, pass through subtle experimentsdesigned to detect them. But until theseexperiments bear fruit, most progress on thedark-matter front will continue to focus onfaint stellar objects.

In the past few years, gravitationalmicrolensing experiments have opened anew avenue of investigation into this prob-lem. These experiments monitored aroundten million stars in the Magellanic Clouds(our nearest galactic neighbours), in thehope of detecting the occasional brightening

Astronomy

A bright future for dark matterBrad Hansen