NEUROBIOLOGY
`
`Caspase-1 and -3 are sequentially activated in motor
`neuron death in Cu,Zn superoxide dismutase-
`mediated familial amyotrophic lateral sclerosis
`
`Piera Pasinelli*†, Megan K. Houseweart†‡, Robert H. Brown, Jr.*§, and Don W. Cleveland‡§
`
`*Massachusetts General Hospital-East, Charlestown, MA 02129; and ‡Ludwig Institute for Cancer Research University of California at San Diego,
`La Jolla, CA 92093
`
`Familial amyotrophic lateral sclerosis-linked mutations in copper-zinc
`superoxide dismutase cause motor neuron death through one or
`more acquired toxic properties. An early event in the mechanism of
`toxicity from such mutants is now demonstrated to be activation of
`caspase-1. Neuronal death, however, follows only after months of
`chronic caspase-1 activation concomitantly with activation of the
`executioner caspase-3 as the final step in the toxic cascade. Thus, a
`common toxicity of mutant SOD1 is a sequential activation of at least
`two caspases, caspase-1 that acts slowly as a chronic initiator and
`caspase-3 acting as the final effector of cell death.
`
`Edited by Jeremy Nathans, Johns Hopkins University School of Medicine, Baltimore, MD, and approved September 22, 2000 (received for review July 3, 2000)
`caspase inhibitor zVAD-fmk (z-benzyloxycarbonyl, fmk 5 flu-
`oromethyl ketone) delays disease onset.
`Altered expression of some members of the Bcl-2 family has
`been found in affected regions of symptomatic G93A mice (18).
`In human ALS tissues, Bcl-2 and BAX mRNA levels were
`generally decreased and increased respectively (19, 20). Further,
`in spinal cords of transgenic ALS mice carrying the G37R and
`G85R mutations, caspase-1 is proteolytically processed (21),
`although how this relates to the timing of disease onset or
`progression is unknown. Additionally, in differentiated mouse
`neuroblastoma N2a cells, all three SOD1 mutants examined
`provoked caspase-1 activation and release of active IL-1b fol-
`lowing oxidative challenge (21).
`Despite this evidence, doubt about an apoptotic cell death
`mechanism in the G93A mice has emerged (22). Furthermore,
`the slow cell death process in ALS mouse models, where
`pathology occurs months before motor neuron loss, contrasts
`strikingly with the very rapid caspase-dependent cell suicide
`pathways in other contexts. We now demonstrate that lines of
`mice that develop ALS-like disease by expressing any of three
`SOD1 mutants (including G93A) activate caspase-1 as an early
`event in the motor neuron death. This is temporally followed by
`activation of caspase-3 specifically in affected regions simulta-
`neously with the appearance of apoptotic neurons and astro-
`cytes. These findings indicate that a toxic cascade common to
`ALS-mutant SOD1 proteins is the sequential activation of at
`least two caspases, caspase-1 that acts slowly as a chronic
`initiator, and caspase-3 acting as the final effector of cell death.
`
`Amyotrophic lateral sclerosis (ALS) is a paralytic disorder
`
`caused by selective death of motor neurons. From 2 to 3% of
`cases are caused by mutations in the gene encoding the free radical
`scavenging enzyme, Cu,Zn superoxide dismutase (SOD1) (1).
`More than 70 different mutations in SOD1 are known. That mutant
`SOD1 triggers the disease through one or more toxic properties (2)
`is suggested by the dominant inheritance pattern in familial ALS
`and the observation that SOD1-null mice do not develop motor
`neuron disease (3). Moreover, transgenic mice expressing three
`different familial ALS-linked mutants in SOD1 develop motor
`neuron disease despite elevated (4, 5) or unchanged (6) SOD1
`activity, whereas neither onset age nor rapidity of disease progres-
`sion correlates with SOD1 activity in patients (7).
`One possible element in SOD1-mediated neuronal death is
`apoptosis, or programmed cell death, in which the cell activates a
`preprogrammed, intracellular suicide machinery. The central com-
`ponents in this pathway are caspases, cysteine proteases with
`aspartate specificity (8). These proteases are translated as inactive
`pro-enzymes that are activated after cleavage at specific aspartate
`residues. Once activated, they cleave other selected intracellular
`targets including caspases,
`leading to an amplified cell death
`cascade.
`Caspases are divided into two major subgroups: upstream (ini-
`tiator) caspases that initiate the proteolytic cascade and down-
`stream (effector) caspases, such as caspase-3, that kill the cell by
`cleaving specific intracellular targets (9). After a death stimulus,
`upstream caspases are directly recruited by ligand-bound death
`receptors (e.g., fas, tumor necrosis factor receptor) or, as in the case
`of caspase-9, activated intracellularly by a caspase-activating factor
`(9). These events trigger activation of downstream caspases, pro-
`cessing of the effector caspases and cell death.
`For neuronal cells in the high oxygen environment of cell culture,
`mutant SOD1 protein is pro-apoptotic after withdrawal of trophic
`support (10–12) in contrast to native wild-type (wt) SOD1, which
`is anti-apoptotic (10, 13, 14). In mice that develop motor neuron
`disease from expression of one ALS-linked SOD1 mutant, post-
`natal elevation of the anti-apoptotic gene Bcl-2 delays onset of
`motor neuron disease and prolongs survival without affecting
`disease duration (15). Expression of a mutant caspase-1 that
`dominantly inhibits caspase-1 activity also slows disease progression
`(16). Recently, Li et al. (17) demonstrated activation of caspases-1
`and -3 in the G93A mice and showed that treatment with the broad
`
`Materials and Methods
`N2a Cell Culture. N2a cell cultures were grown as described (21).
`At 6 days after differentiation, cells were incubated with xan-
`thineyxanthine-oxidase (XyXO; 100 mM-10 milliunitsyml) as
`described (21). When Ac-YVAD-CMK and Ac-YVAD-CHO
`(Bachem; CMK 5 chloromethylketone, CHO 5 aldehyde de-
`rivative) were used, the cells were preincubated for 1 h with the
`inhibitor before XyXO addition.
`
`Western Immunoblots. N2a cells or tissues from transgenic mice
`were lysed in Hepes buffer (pH 7.6) containing 40 mM KCl, 5
`
`This paper was submitted directly (Track II) to the PNAS office.
`
`Abbreviations: ALS, amyotrophic lateral sclerosis; SOD1, CU,Zn superoxide dismutase; wt,
`wild-type; GFAP, glial fibrillary acidic protein; hSOD1, human SOD1; fmk, fluoromethyl
`ketone; CMK, chloromethyl ketone; CHO, aldehyde derivative; AMC, 7-amino-4-methyl-
`coumarin.
`†P.P. and M.K.H. contributed equally to this work.
`§To whom reprint request should be addressed. E-mail: dcleveland@ucsd.edu or
`brown@helix.mgh.harvard.edu.
`
`The publication costs of this article were defrayed in part by page charge payment. This
`article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
`§1734 solely to indicate this fact.
`Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073ypnas.240305897.
`Article and publication date are at www.pnas.orgycgiydoiy10.1073ypnas.240305897
`
`PNAS u December 5, 2000 u vol. 97 u no. 25 u 13901–13906
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`a Fluo-star BMG fluorometer (excitation and emission wave-
`lengths of 380 nm and 420 nm).
`
`Immunofluorescence and Immunohistochemistry. Mice were tran-
`scardially perfused and postfixed overnight with 4% paraformal-
`dehyde in 0.1 M sodium phosphate (pH 7.6). Paraffin-embedded
`spinal cord sections were deparaffinzed and incubated with the
`anti-activated caspase-3 (CM1), anti-glial fibrillary acidic protein
`(GFAP; Research Diagnostics, Flanders, NJ), or the anti-
`nonphosphorylated neurofilament (SMI32, Sternberger Mono-
`clonals, Baltimore, MD) antibodies. The peroxidase-antiperoxidase
`method (Vector Laboratories) was used to visualize immunoreac-
`tivity. For double immunofluorescence, deparaffinized spinal cord
`sections were incubated in primary antibodies and incubated with
`either FITC or Texas Red conjugated secondary antisera. DAPI
`was used to stain nuclei. Deconvolution microscopy was performed
`using Applied Precision DELTA VISION digital deconvolution
`software.
`
`Tissue Preparation for Axon Counting and Electron Microscopy. Mice
`were perfused and postfixed overnight in 0.1 M sodium phosphate
`(pH 7.6). Samples were treated with 2% osmium tetroxide, washed,
`dehydrated, and embedded in Epon-araldite resin. Thick sections
`(0.75 mm) for light microscopy were stained with toluidine blue.
`Axons were counted from the L5 ventrals root of three to five mice
`from each genotype and age. Thin sections (70 nm) for electron
`microscopy were stained with uranyl acetateylead citrate.
`Results
`Caspase-1 Activation Is a Very Early Event in Mutant SOD1 Toxicity. To
`extend our earlier evidence for caspase-1 cleavage in spinal cords
`of SOD1 mutant mice at endstage disease (21), we sought to
`determine the timing of caspase-1 activation by assaying for
`cleavage of this caspase in G37R (line 42) and G85R (line 148)
`mice at different stages of disease. Immunoblotting revealed
`early appearance of the caspase-1 p20 active fragment in spinal
`cord extracts of both lines of mice. In the G37R mice (Fig. 1 a
`and c), the fragment was seen by 2 months, approximately 3–4
`months before disease onset. In the G85R mice (Fig. 1b), this
`fragment appeared as early as 1 month, ’10–11 months before
`
`Caspase-1 cleavage appears early in the lifespan of G37R- and
`Fig. 1.
`G85R-transgenic mice. Immunoblots of spinal cord extracts from G37R (a) and
`G85R (b) mice probed with a polyclonal antibody against both the inactive
`(p45) pro-enzyme and the active (p20) caspase-1. In mice over-expressing the
`wt human SOD1 (hSOD1) protein, the antibody only recognized the p45
`inactive and the p30 intermediate form. (c) Caspase-1 cleavage in G37R mice
`detected with the polyclonal antibody used for the cell culture experiments.
`wt indicates mice overexpressing wt hSOD1.
`
`mM MgCl2, 1% sodium lauryl sulfate salt (SDS), 1 mM EGTA,
`and 1 mM EDTA with protease inhibitors. Proteins (30 mgylane)
`were electrophoresed and blotted to poly(vinyldene difluoride)
`membrane. Blots were probed with anti-human SOD1 (Calbio-
`chem), rabbit anti-caspase-1 active fragment (Santa Cruz or
`Biosource), or a rabbit anti-activated caspase-3 p20 (CM1;
`IDUN Pharmaceuticals, La Jolla, CA) antibodies and visualized
`by using chemilumenescence detection (Amersham).
`
`Caspase Activity Assay. N2a cells were lysed in buffer containing
`10 mM TriszHCl, 10 mM NaH2PO4yNaHPO4 (pH 7.5), 130 mM
`NaCl, 1% Triton X-100, and 10 mM NaPPi with protease
`inhibitors. Equal amounts of lysates were incubated for 1 h at
`37°C with 200 ml of Hepes buffer with either Ac-YVAD-AMC
`or Ac-DEVD-AMC (PharMingen; AMC 5 7-amino-4-
`methylcoumarin) to measure caspase-1 and -3, respectively.
`Fuorescence of the free AMC fluorophore was measured using
`
`Caspase-3 is activated late in mutant SOD1-transgenic mouse spinal cord and cortex but not in cerebellum or sciatic nerve. Immunoblots of spinal cord
`Fig. 2.
`and cortex extracts from G37R (a and b) and G85R (e and f ) mice probed with the CM1 antibody. No immunostaining is detected early in life. Staining for the
`p18 fragment appears at 4 months in the G37R mice and at 10 months in the G85R mice but not in littermate nontransgenic controls (wt). Activation of caspase-3
`is tissue-specific; no active fragment appears in cerebellum or sciatic nerve of either G37R (c and d) or G85R (g and h) mice. Each gel was loaded with 30 mgylane.
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`Caspase-3 immunoreactivity appears ’2 months before onset and
`Fig. 3.
`increases with age in the spinal cord anterior horn of all mutant SOD1 mouse
`lines tested. Immunostaining for active caspase-3 (brown precipitate) in G37R
`(a and b) and G85R (c and d) mice and their nontransgenic littermate controls.
`(b and d) Increased magnification of a and c (end stage). (e) Active caspase-3
`immunoreactivity in G93A mouse, nontransgenic littermate control, and wt
`hSOD1-expressing mouse spinal cord. Black arrows indicate active caspase-3-
`positive inclusions. White arrows indicate caspase-3-positive motor neurons.
`Arrowheads indicate vacuoles. (Bars 5 25 mm.)
`
`was not uniform throughout the neuronal cytoplasm but rather was
`characterized by a mixture of diffuse cytoplasmic staining in a few
`neurons as well as positive inclusions in others andyor surrounding
`debris as seen in the G37R and G85R mice. Overall, the temporal
`pattern of specific caspase-3 activation detected in all three of the
`SOD1 mouse models of ALS matched the timing of motor neuron
`loss and phenotypic onset.
`
`Caspase-1 Activation Precedes and Caspase-3 Activation Coincides
`with Loss of Large Motor Axons in Mice with Different SOD1 Muta-
`tions. To determine how the timing of caspase-1 and- 3 activation
`relates to the death of motor neurons, L5 motor axons were
`counted from SOD1 mutant mice at several times. This analysis,
`in combination with immunoblotting and immunocytochemistry
`assays, revealed that the onset of motor axon loss occurred much
`later than the appearance of activated caspase-1 and was coin-
`cident with or just after the earliest detected activated caspase-3.
`For G85R mice that developed clinical weakness only in the last
`2–4 weeks of life, loss of large motor axons could not be detected
`before 12 months of age (Fig. 4a). By contrast, caspase-1
`activation was found by immunoblotting as early as 1 month (Fig.
`1b), and caspase-3 activation was detectable at 10 months [by
`immunohistochemistry and immunoblot (Figs. 3c and 2e)].
`The G37R mice experienced a significant loss of large motor
`axons by 4 months, 2 months after caspase-1 activation and
`concomitant with caspase-3 activation (Fig. 4b). Mice with the
`G93A mutation first exhibited caspase-3 activation at 6 months,
`whereas a significant loss of large motor axons was not evident
`
`clinical disease and before neuronal death could be detected in
`spinal cord sections or motor roots (6). In both lines of mice, the
`caspase-1 cleavage product was present throughout the disease,
`whereas no active fragment was detected in mice overexpressing
`wt hSOD1 by approximately sixfold (Fig. 1c), even late in life (15
`months, Fig. 1b). Thus, caspase-1 activation is abundant long
`before neuronal death andyor phenotypic onset.
`
`Caspase-3 Is Activated in Motor Neurons Late in the Course of Mouse
`ALS. That the timing of caspase-1 activation is not closely correlated
`with the phenotypic onset of disease in SOD1 mutant mice sug-
`gested that either caspase-1 activation is not sufficient to induce
`motor neuron cell death or that within motor neurons chronic
`caspase-1 activation represents only an initial phase of a cell death
`response whose kinetics are much slower than that observed in the
`typical developmental or in vitro cell culture models. To test
`whether cell death arose from sequential activation of an initiator
`caspase (caspase-1) ultimately triggering activation of the execu-
`tioner caspase-3, extracts from spinal cords of G37R, G85R, and
`littermate control mice were immunoblotted for caspase-3 activa-
`tion by using the CM1 antibody specific for the p20-p18 active
`fragment (23–25). Activation of caspase-3 was detected in the spinal
`cords and brain cortices of both mouse lines ’2 months before the
`onset of hindlimb weakness (Fig. 2 a, b, e, and f). In the G37R mice
`(Fig. 2 a and b), activated caspase-3 first appeared at 4 months,
`increasing in abundance by 6 months, concomitant with the onset
`of clinical disease. In the G85R mice for which no pathology is
`observable before 10 months (6), the active caspase-3 fragment was
`not detected on immunoblots before 10 months but was observable
`at later ages (Fig. 2 e and f) when weakness began. No active
`fragments were seen in the nontransgenic littermate controls at any
`age. This activation of caspase-3 was selective for regions under-
`going neurodegeneration: the p18 fragment was detected in spinal
`cord and cortex (Fig. 2 a, b, e, and f) but not cerebellum or sciatic
`nerve (Fig. 2 c, d, g, and h).
`Immunocytochemistry with the CM1 antibody defined the iden-
`tities of cells activating caspase-3 in mutant G37R and G85R mouse
`spinal cords. At 2 months, G37R mutant spinal cords contained
`vacuoles but no detectable activated caspase-3 (Fig. 3a). By 4
`months of age, caspase-3 immunoreactivity was present as small
`dots, at a time that degenerating axons were visible by examination
`of motor roots (’2 months before the onset of weakness). By 6
`months of age, when most motor neurons have been lost and the
`mice are clinically affected, caspase-3 activation was greatly in-
`creased in remaining neurons and in small cells with caspase-3-
`positive, round inclusions. In addition, caspase-3-positive debris not
`associated with cells became more abundant (Fig. 3b), probably
`representing degenerating cells or phagocytic cells that have en-
`gulfed cell remnants. Similarly, G85R mutant spinal cords displayed
`no activated caspase-3 through 8 months, but by 10 months (’2
`months before onset of clinical disease), several small caspase-3-
`positive bodies were observed (Fig. 3c). By endstage, more acti-
`vated caspase-3-positive debris was present and the few surviving
`neurons contained cleaved caspase-3 (Fig. 3d). Activated caspase-3
`was not detected in nontransgenic littermate control mice from any
`line or in age-matched mice overexpressing wt hSOD1 (Fig. 3 a, c,
`and e).
`To further test the generality of caspase-3 activation just before
`disease onset, spinal cords from mice that develop motor neuron
`disease from another ALS-linked SOD1 mutation G93A (low
`expressing line; ref. 26) also were examined with the anti-activated
`caspase-3 antibody. We could not confirm an earlier report of
`appearance of caspase-3 activation diffusely within the cytoplasm of
`most motor neurons before phenotypic onset in a G93A line with
`earlier disease onset (17). Rather, caspase-3 activation was first
`visible in a few motor neurons at 6 months of age, contemporaneous
`with phenotypic onset, and the proportion of motor neurons stained
`for caspase-3 increased by 10 months (Fig. 3e). Activated caspase-3
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`Caspase-1 and caspase-3 activation precedes loss of large motor axons
`Fig. 4.
`and the appearance of apoptotic morphology in all mutant SOD1 mouse lines
`tested. Axon counts from the L5 motor root of the spinal cord for G85R (a), G37R
`(b), and G93A (c) mice (dotted lines) and their littermate nontransgenic controls
`(solid lines) at various ages. The appearance of activated caspase-1 in immunoblot
`is indicated by the leftmost arrowhead in a and b. The appearance of activated
`caspase-3 in immunoblot is indicated by solid triangles. The appearance of
`activated caspase-3 in immunohistochemistry is indicated by open triangles in a,
`b, and c. Axon counts are averages from three to five animals of each genotype
`and age. Horizontal brackets indicate the age of disease onset (DO). Error bars are
`the SD of the data. (d and e) Electron microscopy images show apoptotic changes
`within the ventral horn of 8-mo-old G93A-mutant mice. Arrows indicate con-
`densed chromatin within nuclei. Vacuoles are marked with a V. Filaments within
`neurons are indicated with an F. (Bars in d and e 5 2 mm.)
`
`until 8 months (Fig. 4c). These data are consistent with a
`sequence involving early activation of caspase-1 followed much
`later by caspase-3 activation that occurs just before or coincident
`with significant axon loss and clinical disease.
`Electron microscopy of tissues demonstrated that caspase-3
`activation was accompanied by ultrastructural evidence of apopto-
`sis. The anterior horn region of spinal cords from G85R, G37R, and
`G93A mice all contained cells with classic apoptotic features
`(shown in Fig. 4 d and e for G93A) including chromatin conden-
`sation (arrows) and cell shrinkage with relative preservation of
`subcellular organelles (27). Chromatin condensation was evident in
`cells at least as early as 10 months in G85R mice, 4 months in G37R,
`and 8 months in G93A mice, ages when caspase-3 is activated and
`motor axons begin to die in the respective lines. Nontransgenic
`littermate control mice did not contain observable apoptotic cells.
`
`Activated Caspase-3 Appears Within Neurons and Glia of SOD1 Mutant
`Mouse Spinal Cords. To determine the identity of cells containing
`activated caspase-3, double-immunofluorescence was used to stain
`simultaneously for the active fragment of caspase-3 and markers of
`either neurons (anti-neurofilament) or astrocytes (GFAP). This
`revealed that in the anterior horn region of G85R (Fig. 5a) and
`
`Fig. 5. Activated caspase-3 appears within the neuronal and glial inclusions
`present in mutant SOD1 mouse spinal cords. Double immunofluorescence of
`spinal cord anterior horn using CM1 antibody (Casp 3 in the Left panels) and
`either GFAP antibody to stain astrocytes, ubiquitin antibody (UBIQ) to stain
`inclusions, or an antibody recognizing nonphosphorylated neurofilaments (NF)
`to visualize neurons. Right panels depict the merged image from the first two
`channels with DAPI to show the position of nuclei. Activated caspase-3-positive
`inclusions (arrowheads) within astrocytes (A) andyor extracellular debris (*) are
`common features within mutant G85R (a) and G93A-mutant (b) spinal cords. (c)
`Activated caspase-3 colocalizes with ubiquitin-positive inclusions in G93A-
`mutant mice. Activated caspase-3 immunoreactivity is present within motor
`neurons (MN) (d) and motor neuron inclusions (e) (arrow) of mutant G85R mice.
`(f) Activated caspase-3 is absent from 10-mo-old wt hSOD1-transgenic mice
`whereas astrocytic GFAP staining is normal. (Bar 5 12.5 mm.)
`
`G93A mutant mice (Fig. 5b), activated caspase-3 was present within
`inclusions circumscribed by GFAP, demonstrating either that the
`inclusion is within glial cells (28) or that glial processes envelop
`extracellular inclusions. These round inclusions (Fig. 5c) were
`identical to the Lewy-body-like, ubiquitin- and SOD1-positive
`inclusions described previously both in G85R (6, 28) and G93A
`mice (26) and in some examples of SOD1-mutant mediated human
`ALS (28, 35). Because electron microscopy of these earlier exam-
`ples has demonstrated inclusions clearly within astrocyte cell bodies
`in mice and humans (6, 35), we infer that at least some of these
`caspase-3 aggregates must be within dying astroctyes. Several of the
`few motor neurons remaining at endstage disease contained acti-
`vated caspase-3 (Fig. 5 d and e). The caspase-3 staining appeared
`in a punctate pattern within the cell body and proximal processes
`of motor neurons and stained neuronal inclusions intensely (Fig.
`5e). A significant proportion of activated caspase-3 was found in
`smaller, irregular patches that did not colocalize with GFAP or
`anti-neurofilament (Fig. 5 a, b, c, and e). This immunoreactivity did
`not appear to be associated with any cell and may represent
`extracellular aggregates or debris within phagocytic cells. For G37R
`mice, which lack prominent Lewy body-like astrocytic inclusions,
`caspase-3 activation solely localized within neurons and debris (not
`shown). In spinal cords of nontransgenic littermate controls or in
`mice expressing wt hSOD1 (Fig. 5f ), activated caspase-3 was not
`detected.
`Thus, from the timing of appearance of active caspase-3 and
`its known properties as an executioner protease (9), activation of
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`Fig. 6. Caspase-1 is activated early in differentiated mouse neuroblastoma N2a
`cells. Immunoblots showing caspase-1 activation in differentiated G37R (a) or
`G85R (b) positive N2a exposed to XyXO for 0, 1, 3, or 4 h. (c) Immunoblot of lysates
`from wt hSOD1-expressing cells (left) or G85R-positive cells (right) 4 h after XyXO
`treatment. (d) YVAD-AMC cleavage was measured as described and is reported
`relative to the cleavage induced by lysates of untreated (time 0) wt hSOD1-
`positive cultures. Data are the mean 6 SD of three independent experiments for
`the G85R-positive cells and of two independent experiments assayed in duplicate
`for the G37R and wt hSOD1 expressing cells. Asterisks (*, P , 0.05; **, P , 0.01)
`indicate significant differences between groups. (e) Immunoblot showing levels
`of wt and mutant SODs in N2a cell lines by using an antibody that recognizes
`human and mouse SOD1 equally. The hSOD1 protein migrates more slowly than
`the mouse, only the G85R mutant comigrates with the mouse SOD1. (f) Caspase-3
`activity recorded as DEVD-AMC cleavage by cell lysates relative to the cleavage
`induced by lysates of untreated wt hSOD1-positive cells. Data are the mean 6 SD
`of three independent experiments. Asterisks (*, P , 0.05) indicate a statistical
`difference between groups. (g) Percentage of cells that stained positive for
`activated caspase-3 at different times after XyXO treatment. (h) YVAD-AMC
`cleavage and DEVD-AMC cleavage measured in lysates from G85R cells treated
`with XyXO in the presence of caspase-1-and-3 inhibitors. Data are the mean 6 SD
`of six replicates. Fluorescence emitted from the fluorogenic substrate is expressed
`relative to that emitted from untreated cells.
`
`Caspase-3 activation occurs after caspase-1 activation both in vitro
`and in vivo. In all three lines of SOD1 mutant expressing mice,
`caspase-3 activation appears at or just before the onset of motor
`axon loss and the appearance of apoptotic morphology. This
`sequential caspase activation only occurs in regions affected by
`neurodegeneration in ALS. This proteolytic cascade is shared by
`SOD1 mutants that provoke quite diverse pathologies in the various
`ALS mouse models. Li et al. (17), reported that both caspase-1 and
`-3 are activated in one line of ALS transgenic mice and that
`intrathecal administration of the pan-caspase inhibitor zVAD-fmk
`prolongs the life of these mice by ’25%. Our data extend those
`findings in four important directions.
`
`caspase-3 appears to be the final step in the toxicity of familial
`ALS mutants of SOD1.
`
`An inVitroTemporal Cascade of Caspase-1 and -3 from Mutant SOD1.
`Caspase-1 has previously been shown to be activated in neuroblas-
`toma cells expressing human mutant but not wt hSOD1 following
`differentiation into neurons induced by serum withdrawal and an
`additional oxidative stress from treatment with XyXO (21). To test
`if neuronal death arose from a cascade of caspase-1 activation
`followed by caspase-3 activation, the activities of both were deter-
`mined in a set of N2a cells stably expressing wt hSOD1 (three times
`the endogenous mouse SOD1 level) or any of three mutants (G37R,
`G41D, or G85R) to levels equivalent to mouse SOD1 (Fig. 6e).
`Every hour for 4 h, cells were harvested and caspase-1 activation
`detected by protein immunoblot using the antibody recognizing the
`p20 active fragment of caspase-1. Activation was seen within 1 h
`after XyXO treatment in G37R-expressing cells (Fig. 6a), whereas
`in the G85R expressing cells, the active p20 fragment was chron-
`ically present even without XyXO treatment (Fig. 6b). In a third line
`of cells expressing the ALS related G41D mutation, the cleaved p20
`fragment also was detected before XyXO treatment and increased
`within 1 h (not shown). By contrast, cells expressing the wt hSOD1
`protein did not show evidence of the p20 product even after 4 h of
`XyXO treatment (Fig. 6c).
`Use of the substrate Ac-YVAD-AMC, which is preferentially
`cleaved by caspase-1 or caspase-1-like proteases (25) and which
`yields a fluorescent cleavage product, confirmed that the early and
`rapid appearance of the caspase-1 cleavage product was accompa-
`nied by a corresponding increase in caspase-1 like activity (Fig. 6d).
`For the G85R mutant, an elevated level was detected even without
`XyXO treatment. As early as 1 h after treatment with XyXO, there
`was a significant increase in caspase-1 activity in both mutant
`expressing cells with maximal increase detected by 3 h of XyXO
`treatment, whereas no changes in caspase-1 activity were detected
`in wt hSOD1-expressing cells (Fig. 6d).
`Caspase-3-like activity in the G85R-expressing cells was moni-
`tored with the caspase-3-like protease specific Ac-DEVD-AMC
`fluorogenic substrate. Significant increases in caspase-3 activity
`were detected by 3 h of XyXO treatment, 2 h after the increase in
`caspase-1 activation was first observed in the previous experiment
`(Fig. 6f). The caspase-3 inhibitor Ac-DEVD-CHO completely
`blocked the XyXO-mediated DEVDase activity (not shown). This
`was confirmed by immunocytochemistry: despite chronic, partial
`caspase-1 activation (Fig. 6 b and d), the proportion of G85R-
`expressing N2a cells with detectable activated caspase-3 escalated
`markedly after imposition of oxidative stress (Fig. 6g). Counting the
`number of cells containing activated caspase-3 revealed a sizable
`loss of cells over the 4-h period after XyXO treatment, with ’50%
`of those cells remaining contained the active caspase-3 fragment
`(Fig. 6g). Similar results were obtained for the G37R and G41D-
`expressing cells. In contrast, activated caspase-3 was always absent
`in the wt hSOD1-expressing cells.
`The selective caspase-3 inhibitor Ac-DEVD-CHO (100 mM)
`yielded almost complete blockage of caspase-3 activity after impo-
`sition of oxidative stress, with no effect on caspase-1 (Fig. 6h). The
`caspase-1 and caspase-1-like protease inhibitor Ac-YVAD-CMK
`blocked activation of both caspases, as expected (Fig. 6h). These
`results confirm that caspase-1 activation is independent of
`caspase-3 activity, whereas the sequential order of caspase activa-
`tion suggests that mutant SOD1-dependent increases in caspase-3
`activity depend on previous activation of caspase-1.
`
`Discussion
`This study provides evidence for a sequential cascade of caspase
`activation in mutant SOD1-mediated ALS both in an in vitro cell
`culture model and in transgenic mouse models of ALS. In mutant
`SOD1 mice, caspase-1 is activated early, months before neuronal
`death, and in vitro, activation occurs soon after oxidative stress.
`
`Pasinelli et al.
`
`PNAS u December 5, 2000 u vol. 97 u no. 25 u 13905
`
`Page 5 of 6
`
`Fluidigm
`Exhibit 1014
`
`

`
`First, whereas Li et al. (17) did not resolve the timing of activation
`of the two caspases, we have clearly established that caspase-1 is
`activated months before activation of caspase-3. The protracted
`time course of caspase-1 activity, essentially throughout the adult
`life of these mice, raises several hypotheses concerning mechanisms
`of caspase-1-dependent motor neuron toxicity. These include a
`possible interplay between inflammation and apoptosis, and a role
`for caspase-1 as an early mediator of cell death, because caspase-1
`is responsible for the activation of executioner caspases during the
`progression of apoptosis. Each mechanism can now be assessed.
`Second, we also have documented that the sequential activation
`of caspase-1 and -3 is induced by oxidative stress in a mutant
`SOD1-dependent manner in vitro. Within a time course that is
`compressed in vitro, this strikingly recapitulates the activation
`pattern in the mouse spinal cord. These observations suggest that
`specific upstream caspases activate other downstream effector
`caspase family members within a preexisting apoptotic caspase
`signaling pathway.
`Third, the close temporal relationship between caspase-3
`activation and cell death that we demonstrate in vivo and in vitro
`firmly argues that this downstream executioner caspase mediates
`the death of motor neurons in ALS. Indeed, this is supported by
`prior evidence that caspase-3 is one of the key effector caspases
`in mammalian apoptosis. Caspase-3-deficient mice have distinc-
`tive developmental abnormalities, such as brain enlargement,
`that arise because neuronal apoptotic death is defective in the
`absence of caspase-3 (29). Unlike the other major effector
`caspase, caspase-7 (30), caspase-3 is prominently expressed in
`multiple regions of the brain (25).
`Fourth, caspase-3 activation is especially prominent within
`astrocytes of mutant G85R and G93A mice. In light of the
`dependence of motor neurons on astrocytes to prevent excito-
`toxicity by rapid recovery of synaptic glutamate by the EAAT2
`glutamate transporter (31, 32), it is possible that astrocytes are
`themselves direct targets for SOD1-mediated toxicity and that
`the resulting impairment of astrocyte function hastens disease
`progression. It may be relevant that astrogliosis is common in
`ALS spinal cord and that glial inclusions containing wt and
`mutant SOD1 are found in G93A mice (28), G85R mice (6), and
`some examples of SOD1-mediated human disease.
`How might caspase-1 participate in this disease? In several
`systems, caspase-1 is essential in inflammation andyor in patholog-
`ical apoptotic death, in the latter instances acting as both activator
`and ef