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Efficient three-drug cocktail for disease induced by mutant superoxide dismutase.

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ORIGINAL ARTICLES
Efficient Three-Drug Cocktail for Disease
Induced by Mutant Superoxide Dismutase
Jasna Kriz, MD, PhD, Geneviève Gowing, BSc, and Jean-Pierre Julien, PhD
There is currently no effective pharmacological treatment for amyotrophic lateral sclerosis (ALS). Because evidence suggests that multiple pathways may contribute to ALS pathogenesis, we tested in a mouse model of ALS (SOD1G37R mice)
a combination approach consisting of three drugs for distinct targets in the complex pathway to neuronal death: minocycline, an antimicrobial agent that inhibits microglial activation, riluzole, a glutamate antagonist, and nimodipine, a
voltage-gated calcium channel blocker. The efficacy of this three-drug cocktail was remarkable when administered in the
diet from late presymptomatic stage (8 –9 months). It delayed the onset of disease, slowed the loss of muscle strength,
and increased the average longevity of SOD1G37R mice by 6 weeks. The protective effect of the treatment was corroborated by the reduced immunodetection signals for markers of gliosis and neurodegeneration in the spinal cord of
SOD1G37R mice. These results indicate that such three-drug combination may represent an effective strategy for ALS
treatment.
Ann Neurol 2003;53:429 – 436
Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disease characterized by progressive
muscle weakness and atrophy followed by eventual paralysis, leading to death within 2 to 5 years after diagnosis.1–3 The disease occurs in both sporadic and familial forms with highly similar clinical courses. In the
past decade, much effort has been focused to elucidate
the mechanisms of disease induced by missense mutations in the gene coding for superoxide dismutase 1
(SOD1) that are responsible for approximately 25% of
familial ALS.4,5 Yet, the toxicity of SOD1 mutants is
not fully understood.
Transgenic mice overexpressing various SOD1 mutants linked to familial ALS develop progressive motor
neuron disease with many pathological features found
in both familial and sporadic ALS cases.6 – 8 The current view is that the motor neuron death induced by
mutant SOD1 is complex and may involve multiple
pathways including formation of protein aggregates,
proteosome dysfunction, axonal transport defects, oxidative damage, mitochondrial defects, alterations in
calcium homeostasis, caspase activation, and changes in
levels of Bcl-2 members.1–3,9 Moreover, excitotoxicity
due to astrocyte dysfunction and proinflammatory
molecules released from activated microglia are other
factors that might contribute to propagation of the
neurodegenerative process.10 –13
So far, the various pharmacological agents that have
From the Centre for Research in Neurosciences, McGill University,
Research Institute of the McGill University Health Centre, Montréal, Québec H3G 1A4, Canada.
Received Aug 21, 2002, and in revised form Nov 20. Accepted for
publication Nov 21, 2002.
been tested in ALS mice have produced only modest
benefits.1–3 Recently, three different groups including
ours reported the beneficial effects of minocycline in
ALS mice expressing mutant SOD1.14 –16 Because
much evidence suggests that multiple pathways may
contribute to ALS pathogenesis, in addition to minocycline, we designed a drug combination approach
aimed at different targets in the complex pathway to
neuronal death. This drug cocktail consisted of minocycline, an antibiotic that also inhibits microglial activation, riluzole, a glutamate antagonist, and nimodipine, a voltage-gated calcium channel blocker. Here, we
report that such three-drug cocktail is remarkably effective to delay onset of disease and mortality in a
mouse model of ALS.
Materials and Methods
Generation of SOD1G37R Mice
Transgenic mice overexpressing SOD1G37R by approximately
fivefold (line 29)7,17 were enriched in C57BL/6 background.
Only mice heterozygous for the SOD1G37R transgene were
used for our study. All mice were genotyped by Southern
blotting. The use of animals and all surgical procedures were
conducted according to the Guide of Care and Use of Experimental Animals of the Canadian Council of Animal
Care.
Address correspondence to Dr Julien, Research Institute of the
McGill University Health Centre, 1650 Cedar Avenue, Montréal,
Québec H3G 1A4, Canada. E-mail: jean-pierre.julien@mcgill.ca
© 2003 Wiley-Liss, Inc.
429
Three-drug Therapy Protocol
The SOD1G37R mice were housed at the standard temperature (21°C) and in a light-controlled environment with ad
libitum access to the food and water. The study was conducted using littermates. The mouse littermates were fed a
regular rodent food (Harlan Teklad, Houston, TX) and were
randomly divided into three-drug therapy–treated and control groups, including wild-type littermates. At the age of 8
to 9 months, SOD1G37R mice from the experimental groups
were administered the triple medicated diet TD 01146 (Harlan, Teklad), containing 1,000mg/kg of minocycline,
500mg/kg of riluzole, and 500mg/kg of nimodipine. All
three compounds were purchased from Sigma (Oakville, Ontario, Canada). Daily intake of food in mice was 12 to
18gm/100gm of body weight. Calculated loss of compounds
during the food processing was 10 to 15%. Thus, we estimate that the mice received daily approximately 80 to
100mg/kg of minocycline and 30 to 40mg/kg for each nimodipine and riluzole. Onset of the clinical disease was determined by measurement of motor strength and by the hind
limb contraction when mice were suspended by their tail.14
They were killed when they started to lie on the side and to
express difficulties in grooming, as described previously.14,18
To confirm the effects of the three-drug therapy, we conducted two independent experiments with different sets of
SOD1G37R mouse littermates. The therapy was initiated at
the late presymptomatic stage of disease (8 –9-month-old
mice).
Muscle Strength Test
With minor adjustments, the muscle strength test was conducted as previously described.14 The mice were allowed to
grab vertically oriented wire (approximately 2mm in diameter) with the loop at the lower end. For more consistent
measurements, the wire was maintained in the vertically oriented circular motion (circle radius, approximately 15cm at
35r.p.m.).
Immunohistochemistry
Mice were killed by intraperitoneal injection of chlorale hydrate, perfused with 16gm/L sodium cacodylate buffer (pH
7.4) followed by fixative (3% glutaraldehyde in sodium cacodylate buffer). Immunohistochemical studies were performed as previously described.14,17 Incubation with the primary antibodies anti-Cdk5 (C-8, 1:1,000; Santa Cruz
Biotechnology, Santa Cruz, CA), anti–glial fibrillary acidic
protein monoclonal antibody (anti-GFAP 1:200 dilution;
Sigma), anti–mouse Mac2 rat monoclonal antibody (TIB166) distributed by ATCC (1:500 dilution; Manassas, VA),
and anti–p-p38 and anti-cleaved caspase-3 rabbit polyclonal
antibody (1:500 dilution; New England Biolab, Missisagua,
Ontario, Canada) was performed overnight at room temperature in phosphate-buffered saline/bovine serum albumin.
The labeling was developed using a vector ABC kit (Vector
Laboratories, Burlington, Ontario, Canada) and Sigma-fast
tablets (Sigma). Tissue sections for the axonal counting were
prepared for embedding in Epon as described previously.17
Western Blots
The mice were killed by overdose of chloral hydrate (intraperitoneally). Immediately after, total protein extracts were
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obtained from L4 –L5 spinal cord sections by homogenization in sodium dodecyl sulfate–urea (0.5% sodium dodecyl
sulfate, 8M urea in 7.4 phosphate buffer) with a cocktail of
protease inhibitors (phenylmethyl sulfonyl fluoride 2mM,
leupeptin 2mg/ml, pepstatin 1mg/ml, and 1 mg/ml aprotinin). The protein was measured using a detergent compatible
(DC) protein assay (BioRad, Hercules, CA). The proteins
were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and detected using monoclonal primary antibodies
against anti-GFAP (1:2,000; Sigma), anti-actin (C-4;
1:5,000; Boehringer-Manheim, Indianapolis, IN), and anti–
p-p38, rabbit polyclonal antibody (anti–p-p38, Thr180/
Tyr182 1:500 dilution; New England Biolab). The Western
blots were revealed using the Renaissance chemiluminescence
kit (NEN Life Science, Boston, MA).
Data Analysis
Data are expressed as a mean ⫾ standard error. Statistical
significance was assessed by two-tailed Student’s t test ( p ⱕ
0.05).
Results
Three-drug Cocktail Increases the Life Span of
SOD1G37R Mice
Mouse littermates heterozygous for the SOD1G37R
transgene (line 29) were fed a regular rodent food
(Harlan Teklad). At late presymptomatic stage (8 and
9 months), the mouse littermates were randomly divided into two groups, the three-drug therapy–treated
and control groups. The three drugs (minocycline, riluzole, and nimodipine) were delivered as a dietary
supplement in a special custom-made rodent diet. Figure 1A shows the survival curves of the drug cocktail–
treated SOD1G37R mice and control SOD1G37R mice
fed on regular diet. As compared with control littermates, the average life span of the drug-treated
SOD1G37R mice was increased by 6 weeks (54.1 ⫾
0.9; n ⫽ 10 vs 48.0 ⫾ 0.6; n ⫽ 10, p ⱕ 0.05; Table).
As shown in Figure 1B, the distribution of mortality
for the tested mice showed almost no overlapping between the two tested groups. For control SOD1G37R
mice, the mortality peak was at 47 to 48 weeks,
whereas the mortality distribution for the drug-treated
mice was more equally spread between 52 to 58 weeks
(see Fig 1B).
Three-drug Cocktail Delays the Onset of Disease and
Muscle Strength Decline in SOD1G37R Mice
To determine the effects of the three-drug therapy on
disease onset and progression in SOD1G37R mice, we
conducted a muscle strength assay. This assay is based
on the time that mice are able to grip a vertical circulating wire. Unlike normal mice, the SOD1G37R mice
exhibited an age-dependent decline in hanging time
(Fig 2). The onset of disease in SOD1G37R mice was
characterized by a rapid decline in muscle strength (at
treatment significantly delayed the onset of muscle
weakness and improved the motor performance of the
SOD1G37R mice throughout the tested period (see Fig
2 and Table).
Effective Protection against the Loss of Motor Axons
in SOD1G37R Mice
To assess whether the three-drug therapy delayed degeneration of motor neurons, we counted the total
number of motor axons in L4 and L5 ventral roots
from treated SOD1G37R mice (n ⫽ 3 or 4) and control
SOD1G37R littermates (n ⫽ 4) at early symptomatic
stage of disease (44 weeks) and at late stage of disease
(48 weeks) (Fig 3A and B). At early stage of disease,
motor axons from treated SOD1G37R mice were
mostly spared unlike axons from control SOD1G37R
littermates (see Fig 3A). For instance, at 44 weeks, the
L5 ventral roots from control SOD1G37R mice had
390 ⫾ 23 remaining axons, whereas those from drugtreated mice had 823 ⫾ 41 axons, which is not significantly different from control values (911 ⫾ 36). A
similar pattern was observed at the level of L4 ventral
roots (713 ⫾ 20 for drug-treated vs 352 ⫾ 20 for control SOD1G37R mice). At 48 weeks, the numbers of
remaining axons in the L4 and L5 ventral roots from
drug-treated SOD1G37R mice were 637 ⫾ 112 and
685 ⫾ 115, respectively. Thus, whereas some axonal
loss was evident at 48 weeks, most motor axons were
still present in the drug-treated SOD1G37R mice.
Fig 1. The three-drug therapy increases the life span of
SOD1G37R mice. (A) Survival curves of control SOD1G37R
mice and drug-treated SOD1G37R mice (n ⫽ 10). The survival probability of transgenic mice is plotted as a function of
the age in weeks. Three-drug treatment starting at late
presymptomatic stage of disease increased the average life span
of SOD1G37R mice by 6 weeks. (triangles) Three-drug therapy; (squares) SOD1G37R. (B) Mortality distribution of drugtreated versus control SOD1G37R mice according to their age
in weeks. (striped bars) SOD1G37R; (filled bars) three-drug
therapy.
the age of 43– 44 weeks), followed by a slower declining stage (46 – 47 weeks) that progressed to a stage of
complete hind limb paralysis. Note that the three-drug
Reduced Cdk5 Mislocalization and Capsase-3
Activation
Recent studies demonstrated the involvement of
caspase-3 activation in ALS pathogenesis.1,9,19 Activation of caspase-3 occurs late in the course of disease
and it is associated with the loss of large motor neurons.9 Two other pathological hallmarks of degenerating neurons in SOD1G37R mice are the mislocalization
of Cdk517 and the nuclear localization of Cdk420).
Cdk5 is normally targeted to the cell membrane by its
activator p35. However, in SOD1G37R mice, Cdk5 is
mostly detected in the cytoplasm of motor neurons.17
To examine whether the three-drug therapy attenuated
the signals for markers of neurodegeneration in the spinal cord sections of SOD1G37R mice, we conducted
immunohistochemistry with anti–Cdk5, anti–Cdk4,
and anti–caspase-3 antibodies.
Table. Three-Drug Therapy Delays the Onset of Disease and Increase Longevity of SOD1G37R Mice (mean ⫾ SEM)
SOD1G37R Mice
Three-drug therapy (n ⫽ 10)
Control (n ⫽ 10)
a
Muscle Weakness (weeks)
Onset of Paralysis (weeks)
End-Stage Paralysis (weeks)
47.8 ⫾ 0.95a
43.0 ⫾ 0.92
49.6 ⫾ 1.06a
45.4 ⫾ 0.59
54.1 ⫾ 0.98*
48.0 ⫾ 0.62
Significantly different from control ( p ⱕ 0.05).
SEM ⫽ standard error of the mean.
Kriz et al: Three-Drug Therapy for ALS
431
nocycline exerted additional effects on glial cell activation in SOD1G37R, we examined by immunohistochemistry and Western blotting expression of Mac-2 and
phosphorylated form of p38 MAPK (p-p38), which are
markers of microglial activation, and GFAP as a marker
for astrogliosis. At early symptomatic stage (44 weeks),
the spinal cord sections of age-matched normal mice
and drug-treated SOD1G37R mice were almost completely devoid of Mac-2 immunoreactivity (Fig 5). In
contrast, the spinal cord of control SOD1G37R mice
showed a robust Mac-2 immunoreactivity. The Mac2–immunoreactive cells showed morphology typical
of activated microglia/macrophages (irregular shape,
short processes; see Fig 5a, panel C). A similar pattern
of immunoreactivity was observed with antibodies
Fig 2. The three-drug combination improved muscle strength
and delayed disease onset. Muscle strength was assessed by the
time that mice were able to grip a circulating wire. Each
point represents mean ⫾ SEM, Significant difference in comparison of drug-treated versus control SOD1G37R mice (p ⱕ
0.05 by two-tailed Student’s t test). The number of mice in
each group was as follows: normal mice, n ⫽ 6; drug-treated
SOD1G37R mice and control SOD1G37R mice, n ⫽ 8.
Whereas the spinal motor neurons of control
SOD1G37R mice exhibited robust cytoplasmic immunoreactivities for Cdk5 and Cdk4 at 10 months (Fig
4G, H), very low levels of cytoplasmic Cdk5 and Cdk4
immunoreactivity was detected in spinal cord sections
of 10-month-old SOD1G37R mice under drug treatment (see Fig 4D, E). At the age of 11 months, cytoplasmic immunoreactivities for Cdk5 and Cdk4 were
detected in spinal motor neurons of drug-treated
SOD1G37R mice but at reduced levels as compared
with control SOD1G37R mice (see Fig 4J, K, M, N).
Antibodies against activated form of caspase-3
yielded a weak cytoplasmic immunostaining in several
spinal motor neurons of drug-treated SOD1G37R mice
at 10 months (see Fig 4F), indicating that caspase-3
activation preceded axonal degeneration. Again, much
stronger caspase-3 immunoreactivity was detected in
motor neurons of 10-month-old control SOD1G37R
littermates. We conclude that beneficial effects of the
three-drug treatment were associated with reduced signals for markers of neurodegeneration.
Three-Drug Therapy Attenuates Astrocytosis and
Microglial Activation
Astrocytosis and microgliosis are nonneuronal events
that are likely to contribute to the neurodegenerative
processes in ALS.11–13 Recently, we showed that minocycline alone attenuates microglial activation but not astrocytosis in SOD1G37R mice.14 To determine whether
inclusion of nimodipine and riluzole together with mi-
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Fig 3. The three-drug therapy delayed axonal loss in L4 and
L5 ventral roots of SOD1G37R mice. The histograms show the
total number of axons in L4 and L5 ventral roots of normal
mice (wt), drug-treated (G37R-tr), and control SOD1G37R
(G37R) mice at the age of 10 months (A) and 11 months
(B). The counts were determined using ventral root sections of
three to four animals for each group. (asterisks) Significant
difference for the comparison of drug-treated or control
SOD1G37R littermates versus wild-type mice/ p ⱕ 0.05 by
two-tailed Student’s t test.
Fig 4. The three-drug treatment attenuates deregulation of Cdks (Cdk5 and Cdk4) and caspase-3 activation. The micrographs show
the immunoreactivities of Cdk5, Cdk4, and activated caspase-3 in the spinal cord of WT mice (A–C), of drug-treated SOD1 G37R
mice (D–F) and of control SOD1G37R littermates (G–I) at 10 months as well as of drug-treated SOD1G37R mice (J–L) and of
control SOD1G37R littermates (M–O) at 11 months. Bar ⫽ 100␮m.
against p-p38. Control SOD1G37R mice yielded a
strong p-p38 immunoreactivity in the white and gray
matter (predominantly ventral horns) of the spinal
cord (see Fig. 5a, panel F). The p-p38 signal was considerably attenuated by the three-drug treatment
showing the similar level of immunoreactivity as in
normal mice (see Fig 5a, panels D, E). This was further confirmed by Western blotting. At age of 10
months, spinal cord extracts from normal mice or
from drug-treated SOD1G37R mice showed very low
levels of p-p38 as compared with control SOD1G37R
mice (see Fig 5b). During disease progression, the lev-
els of p-p38 in spinal cord extracts gradually increased
in drug-treated SOD1G37R mice.
Unlike minocycline alone,14 the presence of riluzole and nimodipine in our three-drug cocktail
markedly attenuated GFAP immunoreactivity in spinal cord sections of SOD1G37R mice at 44 week (see
Fig 5a, panels G, H). This was further confirmed by
the weak GFAP immunostaining on Western blot of
spinal cord extracts from drug-treated SOD1G37R
mice as compared with control SOD1G37R littermates
(see Fig 5c). Note, however, that the three-drug therapy was unable to completely abolish astrogliosis as
Kriz et al: Three-Drug Therapy for ALS
433
Fig 5. (a) The three-drug therapy attenuates microglial activation and astrogliosis in the spinal cord of SOD1G37R mice. Micrographs show the Mac-2, p-p38, and GFAP immunoreactivities in spinal cords of 10-month-old wild-type mice (A, D, G), drugtreated SOD1G37R mice (B, E, H), and control SOD1G37R littermates (C, F, I). Bar ⫽ 100␮m. At 10 months of age, lower levels
of p-p38 (b) and GFAP (c) were detected by immunoblotting of spinal cord extracts from drug-treated SOD1G37R mice as compared with control SOD1G37R mice. WT ⫽ wild type; GFAP ⫽ glial fibrillary acidic protein.
reflected by increased GFAP levels at later stages of
disease (see Fig 5c).
Discussion
Here, we report for the first time to our knowledge a
new and effective pharmacological approach to slow
ALS pathogenesis caused by mutant SOD1. This treatment is based on the combination of three drugs, minocycline, riluzole, and nimodipine, aiming at distinct
targets in the disease process. Remarkably, the three-
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drug cocktail delayed the onset of disease by 4 weeks
and increased the average longevity of SOD1G37R mice
by 6 weeks. Our analysis confirmed the striking neuroprotective effect of the three-drug cocktail. At 11
months, which corresponds to end-stage paralysis for
control SOD1G37R mice, the motor axons in drugtreated SOD1G37R littermates were still largely spared
(see Fig 3). In addition, the three-drug therapy attenuated microgliosis and reactive astrocytosis and reduced
markers of neurodegeneration such as deregulation of
Cdk5 and Cdk4 as well as caspase-3 activation (see Fig
4).
Minocycline, a semisynthetic tetracycline derivative
that effectively crosses the blood–brain barrier, is extensively used in humans with relatively few side effects.21
Several reports showed that minocycline can exhibit biological effects completely distinct from its antimicrobial action.22,23 Minocycline exerted neuroprotective
effects in experimental brain ischemia and brain injury
and also in mice models of Huntington’s and Parkinson’s disease.24 –28 It has been suggested that minocycline exerts neuroprotective effects by preventing microglial activation, reducing the induction of caspase-1
and by inhibiting cytochrome c release from mitochondria.14 –16,22–28 In addition, minocycline inhibits matrix metalloproteases, nitric oxide synthases, protein tyrosin nitration, cyclooxygenase-2, and prostaglandin E2
production and also may confer neuroprotection
through inhibition of excitotoxin-induced microglial
activation.22,23,29
We showed previously that minocycline attenuated
microglial activation and extended the longevity of
SOD1G37R mice by 3 weeks. Similar results were obtained by other groups in testing the effects of minocycline in ALS mice expressing another SOD1 mutant
(G93A).14 –16 Although minocycline can reduce microgliosis, it had no effect on astrogliosis in animal models
of stroke24,25 or of ALS.14 The efficacy of the cocktail
described here may be related in part to synergistic effects of the three drugs and to the ability to attenuate
both microgliosis and astrogliosis. Yet, it remains unclear how the addition of riluzole and nimodipine to
minocycline may alter astrogliosis, and certainly further
studies with combinations of two drugs may be needed
to elucidate the separate beneficial effects of nimodipine and riluzole.
The precise mechanism of action of riluzole has not
been fully elucidated. It appears to involve interference
with excitatory amino acid in the central nervous system, possibly through inhibition of glutamic acid release, blockade or inactivation of sodium channels,
and/or activation of G-protein coupled transduction
pathways.3,18,30,31 In mice models of ALS, treatment
with riluzole alone extended survival for only 13 to 15
days.18 Nimodipine is the L-type voltage-gated calcium
channel blocker with preferential effects on the central
nervous system.32 Although, in a small clinical trial nimodipine alone was not effective in slowing the progression of disease, its pharmacological properties may
be of potential benefit in a drug cocktail.33 Some previous studies suggested altered calcium currents and increased intracellular calcium in ALS pathogenesis.34 –36
In PC12 cells, treatment with nimodipine prevented
massive Ca2⫹ influx through voltage-gated calcium
channels induced by membrane depolarization, a phenomenon associated with mitochondrial disruption and
followed by cell apoptosis and/or necrosis.37 It also exhibits neuroprotective effects in ischemia/hypoxiainduced neuronal damage, and it posses certain anticonvulsant effects against kainic acid–induced
seizures.38,39 Recently, nimodipine was shown to promote regeneration and functional recovery after intracranial facial nerve crush.40
Previous attempts to alleviate SOD1-mediated disease with pharmacological approaches in transgenic
mouse models have produced only modest benefits. Vitamin E, gabapentin, and salicylate had no effect on
survival of SOD1G93A mice.18,41 More benefits were
provided in SOD1G93A mice by the intracerebroventricular administration of zVAD-fmk, a broad caspase
inhibitor,19 janus 3 inhibitor,42 RPR119990,43 and,
with creatine, a compound believed to improve mitochondrial function.44 The three-drug cocktail described
here represents one of the most efficient pharmacological treatment in a mouse model of ALS. By conferring
neuroprotection together with attenuation of microgliosis and reactive astrocytosis, this three-drug therapy
may represent a novel and effective treatment for ALS
and possibly other neurodegenerative diseases.
This work was supported by the Canadian Institutes of Health Research (CIHR; 42 503), the Neuromuscular Research Partnership
(ALS Canada, Muscular Dystrophy Association of Canada, and
CIHR), the Center for ALS Research at Johns Hopkins, and a
CIHR Senior Investigator Award (J.-P.J.).
The technical help of P. Hince and D. Houle is gratefully acknowledged. We are grateful to Drs D. L. Price and D. W. Cleveland for
the kind gift of SOD1G37R mice.
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