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Superoxide dismutase in familial amyotrophic lateral sclerosis: models for gain of function
Commentary Superoxide dismutase in familial amyotrophic lateral sclerosis: models for gain of function Robert H Brown Jr Massachusetts
Current
Genetics
and biochemistry
dismutase in familial
Opinion
General
in Neurobiology
of superoxide
amyotrophic
Hospital,
lateral
sclerosis Arnyotrophic lateral sclerosis (ALS) is a lethal, paralytic disorder caused by death of motor neurons in adult human brain, brainstem and spinal cord (reviewed in [ 1.21). The disease often begins focally and disseminates in a pattern suggesting spread among contiguous pools of motor neurons. About 10% of all ALS cases arise as a dominantly inherited trait (f&ilial ALS, FALS), which is clinically indistinguishable from sporadic ALS (SALS) [Xl. Approximately 20’%~ of FALS cases are associated with mutations in the gene encoding cytosolic Cu/Zn superoxide dismutase 1 (SODl) [4]. More than 35 mutations affecting SOD1 have been identified, specifically, in patients with FALS (summarized in [.5]). The SOD 1 enzyme functions as a honlodimer, with each protein consisting of 153 amino acids. It has a positively charged active channel that electrostatically attracts the superoxide anion (Oa*-) into the active site to react with copper, thereby converting (‘dismutating’) it to hydrogen peroxIde (HzO?). Normally, hydrogen peroxide exits the channel into free solution where it, in turn, is converted to water (HzO) by catalase or glutathione peroxidase [h] (Fig. 1). That SOD1 thus plays a pivotal role in free radical homeostasis has prompted the hypothesis that motor neuron degeneration in FALS may be triggered by reactive oxygen species. Recent studies have examined the efGcts of SOD1 mutations on the biochemical properties of the enzyme. In brain homogenates, SOD1 activity is reduced in FALS patients with SOD1 mutations, but not in SALS patients or individuals with FALS not associated with SOD1 mutations [7]. An analysis using quantitative irr bird hybridization in six SALS patients concluded that levels of SOD1 mRNA were significantly increased in motor
Boston,
1995,
USA
5:841-846
neurons; similar increases were not seen in Purkinje cells [8]. Several reports have also now documented a loss of SOD1 activity in lymphoblasts and red blood cells selectively in FALS patients with SOD? mutations [Y-13]. The latter is likely to reflect diminished stability of the mutant enzyme in this non-nucleated cell type. At least two reports describe reductions in SOD1 activity levels iu the spinal fluid of ALS patients (presumably SALS patients) [14,15]. Several studies have characterized molecular properties of some of the mutant SOD1 molecules. When expressed transiently in COS cells, different FALS-related SOD 1 mutations varied considerably in enzyme activity. For example, the activity of the Ala++Val (A3V) mutant was reduced by about 50%, whereas Gly37-+Arg (G37R) activity was normal [lh]. The half-lives of the mutant proteins were reduced, most markedly in those with the least residual SOD1 dismutation activity. Thus, the mutations render the molecule less stable while variably reducing activity levels. Because it flmctions as a honmdimer, the mutant SOD1 molecule might be expected to impair function of the enzyme when dimerized with wild-type protein. However, two inactive mutant SOD1 proteins, Gly85+Arg (G85R) and Gly41 +Asp (G-llD), failed to diminish the activity or half-life of the wild-type molecule 1171 when these proteins Lvere expressed simultaneously in COS cells. On the other hand, a recent analysis of the activity of mutant SOD 1 in Drm~~~~/~i/arr~clarrc~~clrtcrdoes suggest that, in that organism, some SOD 1 mutations (not necessarily related to FALS) may act as donlinant-Ilcgatives [18]. Few data exist to test the hypothesis that there is oxidative stress or toxicity in tissues of FALS patients. Levels of protein carbonyl groups, use&l markers of oxidntive injury to proteins, are elevated in brain [7] and spinal cord [19] of SALS but not FALS patients. By contrast, selected markers of oxidative stress are not elevated in the spinal fluid of ALS patients [20].
Abbreviations ALS-amyotrophic
lateral sclerosis;
NMDA-N-methyl-o-aspartate;
AMPAd-amino-3-hydroxy-5.methyl-l-isoxazole NO-nitric
0 Current
oxide; SALS-sporadic
Biology
proprionic
ALS; SODl-Cu/Zn
Ltd ISSN 0959-4388
acid; FALS-familial
superoxide
dismutase
ALS; 1.
841
842
Commentary
Fig.
1.
SOD1
mutations,
and neuronal neurotransmitter, with
SOD7
excltotoxicity
cell death. The excitatory glutamate, may interdct
mutations
to cause neuro-
nal cell death. Glutamate termined
by neuronal
cytic uptake, as GLT-1.
levels are de-
release and astro-
through
transporters
Glutamate toxicity
by the elevations
in cytosolic- calcium
(Ca2+) that follow
binding
of glutamate
to its receptors on the neuronal Similar Neuron
such
is trIggered
elevations
mediated by antibodies channels, channels
surfare.
in Ca2+ may also be (1~) against Ca’+
such as voltage-sensitive
Ca?+
(VSCC). In turn, Ca2+ activates
nitric oxide synthetase (NOS) to form nitric oxide xanthine
(NO).
Ca?+ can also converl
dchydrogenase
thine oxidase (X0), tion of O,‘cytosolic
to xaI,-
from xanthinc.
The elevated
Ca2+ levels may also generate
02*-through A2 (PLA,).
activation of phosphollpd\c Elevated CaJ+ may be toxtc
to mitochondria,
releasing both OH* and
02’-.
SOD1
Normally,
(onverts
0,‘~
to
hydrogen peroxide (H,O,),
which i\ thrn
converted to water (H,C)
by glutathionc~
peroxidase
or
conditions,
(I,‘-
to iorm
Novel SOD1 **
(XDH)
leading to the iorrna-
c-atalase. Llndcr
peroxynitrite
(ONOO~.
the inset are possible of the mutant SOD1 which are iurther
nomlal
can combincx with NO l.lstcd
111
novel, toxic efic(.t\ moles ule (SOD1 “),
described
111Flgurct .!
and the text. The events deplc-ted 111tht\ figure
may be self-reiniorclng,
and may
thus continue after the inc-iting, upstream stimuli
(such
a\ high injuries
tar structures
or moIcc&5
brane, rnitochondria) tosolic
1
Cytotoxicity
are injured,
4
Moreover,
rc’a[ttv(, ohy-
If mitoc hondr/.l
suh\equent energy dcpk~tlon,
c-ellutar degradation
Surface membrane, Mitochandria
may accentuate the scnsltlvity H,02
1 Cell death ) 2995 Current Opinion in Neurobrology
SODl-related
(c1.g ccll mc’n)
further in< rca\c’cv-
Neurofilaments, Axonal transport
toxic stimuli.
ALS does not arise from loss of
SOD1 function The foregoing analyses can be interpreted to argue that tttotor muron death in FALS is a consequence of loss of SOD1 function. Although studies of neurons in cell culture clearly demonstrate that diminished scavenging of the superoxide anion can precipitate apoptotic neuronal death [21-2X], at least five argunients f&or the alternate possibility, natttely, that the mutant SOD1 tnolecule has one or more novel, adverse functions that are ultimately lethal for the motor neuron. First,
tially
and de~~~)l,~r~r,~t~or~
In dddition,
are lipid diiiuse
I~cl\i
to critic dl subs clltr-
CaL+ and influx
gcn species.
w
glutamate
cease. Thus,
soluble
to C’Y(lttr
both NO ,intl and (an potc,n
a(ro\\ the < c’ll m(,mt)r,1n<+
to interact with
constituc~nt\of
ad~a((,nt
cells and strut ture\, thcbrchy propaKatlng the cytotoxlc arrows,
proces\. KA. kalnatcx. Grey
postulated toxI(
pathway\.
FALS associated with SO111 tnutntiotts is ittheritcd as a dominant rather than a recessive trait. Sccottd, thcrc is no correlation between the levels of loss of fktcttott and clinical tnarkcrs of severity, including age ofomct ot duration ofdiscasc 191. Third, no clear null tttutattotts itt SO111 have yet bcctt detected. One tttutatiott itttroducc\ a stop codon, which is located toward the 3 crud of the coding sequence, but has not bun s11ow11 to diminish levels of expressed SOI) 1 protcttt 1241. l-ourth, in yeast lacking the SODI gene (mf I-). two tttutattts (GlyW+Ala [G93A] and LeulOO+Gly [I_1OOG]) rc’t.titt enough SOD1 activity to rcscuc the yca\t from OX~~CII
Superoxide
and paraquat sensitivity, whereas a third mutant, G8SR, lacks SOD1 activity and fails to rescue sodl- yeast. Yet, despite marked variation in residual superoxide anion disrnutation activity, all three mutations trigger motor neurm death in FALS [25]. Fifth, three groups have now documented that over-expression of SOD1 protein with mutations associated with FALS produces a lethal, at 3-5 months paralytic disorder in mice, beginning
dismutase
in familial
amyotrophic
SOD1
lateral
sclerosis Brown Jr
in FALS Functional consequence
Model
of age [26-281. These animals have elevated levels of SOD1 activity, strongly arguing that the disorder does not arise from insufficient SOD1 activity. Equivalent over-expression of the wild-type enzyme does not cause motor neuron pathology.
Cu/Zn toxicity
7-Y Mechanisms
for cell death in FALS
These observations imply that one or more distinctive cytotoxic functions are associated with the mutant SOD1 molecule in FALS. Information accumulating on this crucial subject allows speculation on at least five hypotheses (Fig. 2). Central to most is the implicit argument that the mutations alter the folding of the SOD1 molecule, reducing its stability and relaxing the configuration of the active channel or site. This argument is intuitively appealing because it may explain why 35 different mutations can produce the same altered function and clinical phenotype.
Nitration
release of copper
tyrosines
Hydroxyl
radical
H (4
\
(e)
Accelerated
of
critical
/
Apoptosis
Protein
aggregation
and zinc
Experiments in yeast and bacteria indicate that the do not bind metals normally. In mutant proteins sodl- yeast, copper and zinc binding by G93A was indistinguishable from normal, whereas that of G85R was markedly labile, raising the possibility that one aspect of the physicochemistry of the SOD1 mutation is altered affinity for copper and/or zinc [25] (Fig. 2). Analogously, in SOD l-deficient Es&cricllia m/i, the mutant His46+Arg (H46R) protein showed almost no SOD1 activity, failed to restore resistance to paraquat, and had a reduced affinity of the mutant SOD1 protein for copper [29]. Elevated levels of copper and zinc may be directly toxic (Fig. 2b): copper can participate in potentially harmful redox reactions; zinc may intoxicate neurons, possibly by interacting with NMDA and AMF’A receptors [30,31].
~~
Fig. 2. Models of gain of function for mutant SOD1 molecules in FALS. (a) In the normal SOD1 molecule, copper Ku) is located in the active site at the end of a positively charged active channel whose dimensions allow only entry of superoxide anion (0-O) and some small molecules such as phosphate or azide. Zinc (Zn) assists in maintaining the structure of the enzyme. (b) In this model, the mutations relax and open the overall conformation of the enzyme, releasing Zn and Cu, which are potentially toxic. In (c), the mutations effectively open up the molecule, allowing access of atypical substrates (e.g. peroxynitrite, as illustrated). In (d), mutations reposition hydrogen peroxide with respect to Cu, enhancing the iormation of hydroxyl radicals. These may interact with SOD1 itseli or diffuse into the cytosol where they can affect redox status, potentially triggering expression of transcriptional factors (e.g. c-jun, fos family proteins) involved in initiation of programmed cell death. In (e), it is speculated that the major eifect of the mutations is to diminish SOD1 stability so severely that the molecule forms toxic precipitates.
Tyrosine nitration
A second hypothesis invokes accelerated nitration of critical tyrosine residues (Fig. 2~). As shown in Figure 1, the superoxide anion normally can combine with nitric oxide (NO) to f&n peroxynitrite (ONOO). This process may be enhanced as SOD1 activity falls and levels of superoxide anion increase. By itself, NO can act as a nitrogen douor, nitrating tyrosine residues. A more important route to nitration may be through
either the normal or the mutant SOD 1 niolccule, which can catalyze nitration by accepting peroxynitrite as a substrate, forming a nitronium ion with enhanced ability to nitrate tyrosines [X2]. By relaxing the structure of the SOD1 molecule, the SOD1 mutations may enhance this nitration process [33]. One prediction of this hypothesis is that levels of nitrotyrosine in ALS neural tissues should
843
844
Commentary
be elevated. A feature of this model, also sununarized in Figure 1, is that the nitration process can be enhanced by exposure of neurons to glutamate. This neurotranslllittcr can augment free cytosolic calcium levels and thereby enhance generation of reactive oxygen species, such as NO and superoxide anion.
Hydroxyl
radical
formation
Under certain circumstances, hydrogen peroxide can interact with copper in the active site ofwild-type SOD 1 to form hydroxyl radicals (OH’) (341 (Fig. 2d). The hydroxyl radicals may react it1 siflr with SOD1 itself, thereby inactivating it, or diffilsuse out into the cytosol to react with other targets. Hydroxyl radicals formed in the wild-type active channel can be trapped by anionic scavengers that bind to the charged channel. Iu normal circumstances, the charge profile on the channel and the local rate of production of hydrogen peroxide (0.1 mM nlin-1) preclude generation of significant hydroxyl radicals [34]. It is conceivable, however, that mutations that perturb the structure of SOD1 might alter the relationship of hydrogen peroxide to copper or retard the egress of hydrogen peroxide, thereby augmenting hydroxyl radical generation within the SOD1 active site.
Apoptosis
A fourth hypothesis is that mutant SOD1 proteins promote apoptosis (Fig. 2d). This is prompted by the report that in immortalized rat nigral neural cells, the A4V mutant triggers apoptosis, whereas wild-type SOD1 is protective [35]. Whether the ability of the mutant protein to cause apoptosis operates through one of the above mechanisms (enhanced copper or zinc release; increased protein nitration or hydroxyl radicals) or a novel property is not known. In this context, it is striking that apoptosis induced in sympathetic to withdrawal of nerve growth neurons in response factor involves early (within three hours) generation of superoxide anion. The interval to onset of apoptosis is inversely proportional to the levels of superoxide anion, perhaps because it serves as a signalling element early in apoptosis in neurons [22]. One potential set of targets for superoxide-anion-mediated signalling are c-jun and Fos family proteins; levels of these redox-sensitive transcriptional factors are up-regulated following withdrawal of nerve growth factor in the superior cervical ganglion sympathetic neurons [36].
in protein aggregates within motor sporadic and &lilial ALS [37-391.
Overview
neurons
in both
and future questions
Figure 1 outlines a possible cascade of events lc~dulg to motor neuron death in ALS. It ascribe? a central role for glutamate (e.g. LAO]), cytosolic calcium I-1l--1.3] and SODI, and designates possible altered fimctiollf ot SOD1. These are proposed to have adverse cff&th OII targets such as ncurofilaments (e.g. [JJ]) and procmcs such as axonal transport [as]. This scheme is intclldcd to provide a framework for testing hypotheses and ral\illg critical questions. Firstly, above ‘111, the outstalldill~ problem is the mechanism of cell death in nc‘umls expressing mutant forms of SODl. Secondly, is there unequivocal evidence that the disease entail\ osidativc toxicity to any type of uiolecular specie\ or cellul.lr constituent? Despite the continuing enttiusidsui f0r thik hypothesis, it remains to be determined \vhcther free radical metabolisnl is significantly perturbcsd ill ally fimll of ALS and, ifit is, what the primary targets oiosldntivc il?jury are. Thirdly. do dowmtreanl steps in the dc.lth necrosis or an alternate death cascade entail apoptosir, paradignl? This question will best be addrmed through analyses of brain and spinal cord h-om ALS patient\. and ALS transgenic mice for markers of apoptmis and perhaps for expression of death gene\ illq)licnted in mammalian apoptosis (e.g. interleukirlI fi-converting enzyme [G] or Yanla/CPP32b [~7,#]). Fourthly, do the pathogenetic mechanisms proposed fi)r FALS explain SALS? At what points do these convcrqc? And, as a corollary, will this infi,rmation point to nc‘\\ candidate genes for the 80’%, of FALS not arising ti-otll SOl)l mutations? And, finally, what are the therapeutic implications of this type of model?
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I
Urown
Massachusetts
13th Street,
Jr, Day Neuron~uscul~r I
Current
Genetics
and biochemistry
dismutase in familial
Opinion
General
in Neurobiology
of superoxide
amyotrophic
Hospital,
lateral
sclerosis Arnyotrophic lateral sclerosis (ALS) is a lethal, paralytic disorder caused by death of motor neurons in adult human brain, brainstem and spinal cord (reviewed in [ 1.21). The disease often begins focally and disseminates in a pattern suggesting spread among contiguous pools of motor neurons. About 10% of all ALS cases arise as a dominantly inherited trait (f&ilial ALS, FALS), which is clinically indistinguishable from sporadic ALS (SALS) [Xl. Approximately 20’%~ of FALS cases are associated with mutations in the gene encoding cytosolic Cu/Zn superoxide dismutase 1 (SODl) [4]. More than 35 mutations affecting SOD1 have been identified, specifically, in patients with FALS (summarized in [.5]). The SOD 1 enzyme functions as a honlodimer, with each protein consisting of 153 amino acids. It has a positively charged active channel that electrostatically attracts the superoxide anion (Oa*-) into the active site to react with copper, thereby converting (‘dismutating’) it to hydrogen peroxIde (HzO?). Normally, hydrogen peroxide exits the channel into free solution where it, in turn, is converted to water (HzO) by catalase or glutathione peroxidase [h] (Fig. 1). That SOD1 thus plays a pivotal role in free radical homeostasis has prompted the hypothesis that motor neuron degeneration in FALS may be triggered by reactive oxygen species. Recent studies have examined the efGcts of SOD1 mutations on the biochemical properties of the enzyme. In brain homogenates, SOD1 activity is reduced in FALS patients with SOD1 mutations, but not in SALS patients or individuals with FALS not associated with SOD1 mutations [7]. An analysis using quantitative irr bird hybridization in six SALS patients concluded that levels of SOD1 mRNA were significantly increased in motor
Boston,
1995,
USA
5:841-846
neurons; similar increases were not seen in Purkinje cells [8]. Several reports have also now documented a loss of SOD1 activity in lymphoblasts and red blood cells selectively in FALS patients with SOD? mutations [Y-13]. The latter is likely to reflect diminished stability of the mutant enzyme in this non-nucleated cell type. At least two reports describe reductions in SOD1 activity levels iu the spinal fluid of ALS patients (presumably SALS patients) [14,15]. Several studies have characterized molecular properties of some of the mutant SOD1 molecules. When expressed transiently in COS cells, different FALS-related SOD 1 mutations varied considerably in enzyme activity. For example, the activity of the Ala++Val (A3V) mutant was reduced by about 50%, whereas Gly37-+Arg (G37R) activity was normal [lh]. The half-lives of the mutant proteins were reduced, most markedly in those with the least residual SOD1 dismutation activity. Thus, the mutations render the molecule less stable while variably reducing activity levels. Because it flmctions as a honmdimer, the mutant SOD1 molecule might be expected to impair function of the enzyme when dimerized with wild-type protein. However, two inactive mutant SOD1 proteins, Gly85+Arg (G85R) and Gly41 +Asp (G-llD), failed to diminish the activity or half-life of the wild-type molecule 1171 when these proteins Lvere expressed simultaneously in COS cells. On the other hand, a recent analysis of the activity of mutant SOD 1 in Drm~~~~/~i/arr~clarrc~~clrtcrdoes suggest that, in that organism, some SOD 1 mutations (not necessarily related to FALS) may act as donlinant-Ilcgatives [18]. Few data exist to test the hypothesis that there is oxidative stress or toxicity in tissues of FALS patients. Levels of protein carbonyl groups, use&l markers of oxidntive injury to proteins, are elevated in brain [7] and spinal cord [19] of SALS but not FALS patients. By contrast, selected markers of oxidative stress are not elevated in the spinal fluid of ALS patients [20].
Abbreviations ALS-amyotrophic
lateral sclerosis;
NMDA-N-methyl-o-aspartate;
AMPAd-amino-3-hydroxy-5.methyl-l-isoxazole NO-nitric
0 Current
oxide; SALS-sporadic
Biology
proprionic
ALS; SODl-Cu/Zn
Ltd ISSN 0959-4388
acid; FALS-familial
superoxide
dismutase
ALS; 1.
841
842
Commentary
Fig.
1.
SOD1
mutations,
and neuronal neurotransmitter, with
SOD7
excltotoxicity
cell death. The excitatory glutamate, may interdct
mutations
to cause neuro-
nal cell death. Glutamate termined
by neuronal
cytic uptake, as GLT-1.
levels are de-
release and astro-
through
transporters
Glutamate toxicity
by the elevations
in cytosolic- calcium
(Ca2+) that follow
binding
of glutamate
to its receptors on the neuronal Similar Neuron
such
is trIggered
elevations
mediated by antibodies channels, channels
surfare.
in Ca2+ may also be (1~) against Ca’+
such as voltage-sensitive
Ca?+
(VSCC). In turn, Ca2+ activates
nitric oxide synthetase (NOS) to form nitric oxide xanthine
(NO).
Ca?+ can also converl
dchydrogenase
thine oxidase (X0), tion of O,‘cytosolic
to xaI,-
from xanthinc.
The elevated
Ca2+ levels may also generate
02*-through A2 (PLA,).
activation of phosphollpd\c Elevated CaJ+ may be toxtc
to mitochondria,
releasing both OH* and
02’-.
SOD1
Normally,
(onverts
0,‘~
to
hydrogen peroxide (H,O,),
which i\ thrn
converted to water (H,C)
by glutathionc~
peroxidase
or
conditions,
(I,‘-
to iorm
Novel SOD1 **
(XDH)
leading to the iorrna-
c-atalase. Llndcr
peroxynitrite
(ONOO~.
the inset are possible of the mutant SOD1 which are iurther
nomlal
can combincx with NO l.lstcd
111
novel, toxic efic(.t\ moles ule (SOD1 “),
described
111Flgurct .!
and the text. The events deplc-ted 111tht\ figure
may be self-reiniorclng,
and may
thus continue after the inc-iting, upstream stimuli
(such
a\ high injuries
tar structures
or moIcc&5
brane, rnitochondria) tosolic
1
Cytotoxicity
are injured,
4
Moreover,
rc’a[ttv(, ohy-
If mitoc hondr/.l
suh\equent energy dcpk~tlon,
c-ellutar degradation
Surface membrane, Mitochandria
may accentuate the scnsltlvity H,02
1 Cell death ) 2995 Current Opinion in Neurobrology
SODl-related
(c1.g ccll mc’n)
further in< rca\c’cv-
Neurofilaments, Axonal transport
toxic stimuli.
ALS does not arise from loss of
SOD1 function The foregoing analyses can be interpreted to argue that tttotor muron death in FALS is a consequence of loss of SOD1 function. Although studies of neurons in cell culture clearly demonstrate that diminished scavenging of the superoxide anion can precipitate apoptotic neuronal death [21-2X], at least five argunients f&or the alternate possibility, natttely, that the mutant SOD1 tnolecule has one or more novel, adverse functions that are ultimately lethal for the motor neuron. First,
tially
and de~~~)l,~r~r,~t~or~
In dddition,
are lipid diiiuse
I~cl\i
to critic dl subs clltr-
CaL+ and influx
gcn species.
w
glutamate
cease. Thus,
soluble
to C’Y(lttr
both NO ,intl and (an potc,n
a(ro\\ the < c’ll m(,mt)r,1n<+
to interact with
constituc~nt\of
ad~a((,nt
cells and strut ture\, thcbrchy propaKatlng the cytotoxlc arrows,
proces\. KA. kalnatcx. Grey
postulated toxI(
pathway\.
FALS associated with SO111 tnutntiotts is ittheritcd as a dominant rather than a recessive trait. Sccottd, thcrc is no correlation between the levels of loss of fktcttott and clinical tnarkcrs of severity, including age ofomct ot duration ofdiscasc 191. Third, no clear null tttutattotts itt SO111 have yet bcctt detected. One tttutatiott itttroducc\ a stop codon, which is located toward the 3 crud of the coding sequence, but has not bun s11ow11 to diminish levels of expressed SOI) 1 protcttt 1241. l-ourth, in yeast lacking the SODI gene (mf I-). two tttutattts (GlyW+Ala [G93A] and LeulOO+Gly [I_1OOG]) rc’t.titt enough SOD1 activity to rcscuc the yca\t from OX~~CII
Superoxide
and paraquat sensitivity, whereas a third mutant, G8SR, lacks SOD1 activity and fails to rescue sodl- yeast. Yet, despite marked variation in residual superoxide anion disrnutation activity, all three mutations trigger motor neurm death in FALS [25]. Fifth, three groups have now documented that over-expression of SOD1 protein with mutations associated with FALS produces a lethal, at 3-5 months paralytic disorder in mice, beginning
dismutase
in familial
amyotrophic
SOD1
lateral
sclerosis Brown Jr
in FALS Functional consequence
Model
of age [26-281. These animals have elevated levels of SOD1 activity, strongly arguing that the disorder does not arise from insufficient SOD1 activity. Equivalent over-expression of the wild-type enzyme does not cause motor neuron pathology.
Cu/Zn toxicity
7-Y Mechanisms
for cell death in FALS
These observations imply that one or more distinctive cytotoxic functions are associated with the mutant SOD1 molecule in FALS. Information accumulating on this crucial subject allows speculation on at least five hypotheses (Fig. 2). Central to most is the implicit argument that the mutations alter the folding of the SOD1 molecule, reducing its stability and relaxing the configuration of the active channel or site. This argument is intuitively appealing because it may explain why 35 different mutations can produce the same altered function and clinical phenotype.
Nitration
release of copper
tyrosines
Hydroxyl
radical
H (4
\
(e)
Accelerated
of
critical
/
Apoptosis
Protein
aggregation
and zinc
Experiments in yeast and bacteria indicate that the do not bind metals normally. In mutant proteins sodl- yeast, copper and zinc binding by G93A was indistinguishable from normal, whereas that of G85R was markedly labile, raising the possibility that one aspect of the physicochemistry of the SOD1 mutation is altered affinity for copper and/or zinc [25] (Fig. 2). Analogously, in SOD l-deficient Es&cricllia m/i, the mutant His46+Arg (H46R) protein showed almost no SOD1 activity, failed to restore resistance to paraquat, and had a reduced affinity of the mutant SOD1 protein for copper [29]. Elevated levels of copper and zinc may be directly toxic (Fig. 2b): copper can participate in potentially harmful redox reactions; zinc may intoxicate neurons, possibly by interacting with NMDA and AMF’A receptors [30,31].
~~
Fig. 2. Models of gain of function for mutant SOD1 molecules in FALS. (a) In the normal SOD1 molecule, copper Ku) is located in the active site at the end of a positively charged active channel whose dimensions allow only entry of superoxide anion (0-O) and some small molecules such as phosphate or azide. Zinc (Zn) assists in maintaining the structure of the enzyme. (b) In this model, the mutations relax and open the overall conformation of the enzyme, releasing Zn and Cu, which are potentially toxic. In (c), the mutations effectively open up the molecule, allowing access of atypical substrates (e.g. peroxynitrite, as illustrated). In (d), mutations reposition hydrogen peroxide with respect to Cu, enhancing the iormation of hydroxyl radicals. These may interact with SOD1 itseli or diffuse into the cytosol where they can affect redox status, potentially triggering expression of transcriptional factors (e.g. c-jun, fos family proteins) involved in initiation of programmed cell death. In (e), it is speculated that the major eifect of the mutations is to diminish SOD1 stability so severely that the molecule forms toxic precipitates.
Tyrosine nitration
A second hypothesis invokes accelerated nitration of critical tyrosine residues (Fig. 2~). As shown in Figure 1, the superoxide anion normally can combine with nitric oxide (NO) to f&n peroxynitrite (ONOO). This process may be enhanced as SOD1 activity falls and levels of superoxide anion increase. By itself, NO can act as a nitrogen douor, nitrating tyrosine residues. A more important route to nitration may be through
either the normal or the mutant SOD 1 niolccule, which can catalyze nitration by accepting peroxynitrite as a substrate, forming a nitronium ion with enhanced ability to nitrate tyrosines [X2]. By relaxing the structure of the SOD1 molecule, the SOD1 mutations may enhance this nitration process [33]. One prediction of this hypothesis is that levels of nitrotyrosine in ALS neural tissues should
843
844
Commentary
be elevated. A feature of this model, also sununarized in Figure 1, is that the nitration process can be enhanced by exposure of neurons to glutamate. This neurotranslllittcr can augment free cytosolic calcium levels and thereby enhance generation of reactive oxygen species, such as NO and superoxide anion.
Hydroxyl
radical
formation
Under certain circumstances, hydrogen peroxide can interact with copper in the active site ofwild-type SOD 1 to form hydroxyl radicals (OH’) (341 (Fig. 2d). The hydroxyl radicals may react it1 siflr with SOD1 itself, thereby inactivating it, or diffilsuse out into the cytosol to react with other targets. Hydroxyl radicals formed in the wild-type active channel can be trapped by anionic scavengers that bind to the charged channel. Iu normal circumstances, the charge profile on the channel and the local rate of production of hydrogen peroxide (0.1 mM nlin-1) preclude generation of significant hydroxyl radicals [34]. It is conceivable, however, that mutations that perturb the structure of SOD1 might alter the relationship of hydrogen peroxide to copper or retard the egress of hydrogen peroxide, thereby augmenting hydroxyl radical generation within the SOD1 active site.
Apoptosis
A fourth hypothesis is that mutant SOD1 proteins promote apoptosis (Fig. 2d). This is prompted by the report that in immortalized rat nigral neural cells, the A4V mutant triggers apoptosis, whereas wild-type SOD1 is protective [35]. Whether the ability of the mutant protein to cause apoptosis operates through one of the above mechanisms (enhanced copper or zinc release; increased protein nitration or hydroxyl radicals) or a novel property is not known. In this context, it is striking that apoptosis induced in sympathetic to withdrawal of nerve growth neurons in response factor involves early (within three hours) generation of superoxide anion. The interval to onset of apoptosis is inversely proportional to the levels of superoxide anion, perhaps because it serves as a signalling element early in apoptosis in neurons [22]. One potential set of targets for superoxide-anion-mediated signalling are c-jun and Fos family proteins; levels of these redox-sensitive transcriptional factors are up-regulated following withdrawal of nerve growth factor in the superior cervical ganglion sympathetic neurons [36].
in protein aggregates within motor sporadic and &lilial ALS [37-391.
Overview
neurons
in both
and future questions
Figure 1 outlines a possible cascade of events lc~dulg to motor neuron death in ALS. It ascribe? a central role for glutamate (e.g. LAO]), cytosolic calcium I-1l--1.3] and SODI, and designates possible altered fimctiollf ot SOD1. These are proposed to have adverse cff&th OII targets such as ncurofilaments (e.g. [JJ]) and procmcs such as axonal transport [as]. This scheme is intclldcd to provide a framework for testing hypotheses and ral\illg critical questions. Firstly, above ‘111, the outstalldill~ problem is the mechanism of cell death in nc‘umls expressing mutant forms of SODl. Secondly, is there unequivocal evidence that the disease entail\ osidativc toxicity to any type of uiolecular specie\ or cellul.lr constituent? Despite the continuing enttiusidsui f0r thik hypothesis, it remains to be determined \vhcther free radical metabolisnl is significantly perturbcsd ill ally fimll of ALS and, ifit is, what the primary targets oiosldntivc il?jury are. Thirdly. do dowmtreanl steps in the dc.lth necrosis or an alternate death cascade entail apoptosir, paradignl? This question will best be addrmed through analyses of brain and spinal cord h-om ALS patient\. and ALS transgenic mice for markers of apoptmis and perhaps for expression of death gene\ illq)licnted in mammalian apoptosis (e.g. interleukirlI fi-converting enzyme [G] or Yanla/CPP32b [~7,#]). Fourthly, do the pathogenetic mechanisms proposed fi)r FALS explain SALS? At what points do these convcrqc? And, as a corollary, will this infi,rmation point to nc‘\\ candidate genes for the 80’%, of FALS not arising ti-otll SOl)l mutations? And, finally, what are the therapeutic implications of this type of model?
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