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The Journal of Neuroscience, January 1, 1998, 18(1):156-163
Neuroprotective Effects of Creatine and Cyclocreatine in Animal
Models of Huntington's Disease
Russell T.
Matthews1,
Lichuan
Yang1,
Bruce G.
Jenkins1,
Robert J.
Ferrante2,
Bruce R.
Rosen1,
Rima
Kaddurah-Daouk3, and
M. Flint
Beal1
1 Neurochemistry Laboratory, Neurology Service and
Massachusetts General Hospital Nuclear Magnetic Resonance Center,
Department of Radiology, Massachusetts General Hospital and Harvard
Medical School, Boston, Massachusetts 02114, 2 Geriatric
Research Educational and Clinical Center, Bedford Veterans
Administration Medical Center, Department of Neurology and Pathology,
Boston University School of Medicine, Boston, Massachusetts 02115, and
3 The Avicena Group, Inc., Cambridge, Massachusetts 02139
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ABSTRACT |
The gene defect in Huntington's disease (HD) may result in an
impairment of energy metabolism. Malonate and 3-nitropropionic acid
(3-NP) are inhibitors of succinate dehydrogenase that produce energy
depletion and lesions that closely resemble those of HD. Oral
supplementation with creatine or cyclocreatine, which are substrates
for the enzyme creatine kinase, may increase phosphocreatine (PCr) or
phosphocyclocreatine (PCCr) levels and ATP generation and thereby may
exert neuroprotective effects. We found that oral supplementation with
either creatine or cyclocreatine produced significant protection
against malonate lesions, and that creatine but not cyclocreatine
supplementation significantly protected against 3-NP neurotoxicity.
Creatine and cyclocreatine increased brain concentrations of PCr and
PCCr, respectively, and creatine protected against depletions of PCr
and ATP produced by 3-NP. Creatine supplementation protected against
3-NP induced increases in striatal lactate concentrations in
vivo as assessed by 1H magnetic resonance
spectroscopy. Creatine and cyclocreatine protected against
malonate-induced increases in the conversion of salicylate to 2,3- and
2,5-dihydroxybenzoic acid, biochemical markers of hydroxyl radical
generation. Creatine administration protected against 3-NP-induced
increases in 3-nitrotyrosine concentrations, a marker of
peroxynitrite-mediated oxidative injury. Oral supplementation with
creatine or cyclocreatine results in neuroprotective effects in
vivo, which may represent a novel therapeutic strategy for HD
and other neurodegenerative diseases.
Key words:
creatine; ATP; oxidative injury; 3-nitrotyrosine; 3-nitropropionic acid; Huntington
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INTRODUCTION |
There is substantial evidence that
impairment of energy production may play a role in the pathogenesis of
neurodegenerative diseases (Albin and Greenamyre, 1992 ; Beal, 1992).
Impaired energy production may lead to activation of excitatory amino
acid receptors, increases in intracellular calcium, and the generation
of free radicals (Beal, 1995). In Huntington's disease (HD) there is
reduced mitochondrial complex II-III activity in postmortem tissue (Gu et al., 1996; Browne et al., 1997) and increased cerebral lactate concentrations in vivo (Jenkins et al., 1993). Animal models
of Huntington's disease involve defects in energy production. Malonate and 3-nitropropionic acid are, respectively, reversible and
irreversible inhibitors of mitochondrial complex II (succinate
dehydrogenase), which produce striatal lesions similar to those of HD
(Beal et al., 1993b; Greene et al., 1993; Brouillet et al., 1995). The pathogenesis of lesions produced by these compounds involves energy depletion, followed by activation of excitatory amino acid receptors and free radical production (Schulz et al., 1995a,b). The ensuing cell
death involves both apoptosis and necrosis (Pang and Geddes, 1997; Sato
et al., 1997).
Creatine kinase (CK) is a key enzyme involved in regulating energy
metabolism in cells with intermittently high and fluctuating energy
requirements, including the brain (Chen et al., 1995). The enzyme
catalyzes the reversible transfer of the phosphoryl group from
phosphocreatine (PCr) to ADP to generate ATP (for review, see
Wallimann, 1992). Several cytoplasmic and mitochondrial isoforms have
been identified, which along with the substrates creatine and PCr
constitute an intricate cellular energy buffering and transport system,
connecting sites of energy production with sites of energy consumption
(Hemmer and Wallimann, 1993).
The mitochondrial isoform of creatine kinase (Mi-CK) is located at
contact sites between the inner and outer membranes, where it is
associated with porin (Wallimann et al., 1992; Brdiczka et al., 1994).
Mi-CK can convert intramitochondrially produced ATP to PCr directly,
which then gets transported to sites of energy consumption. The
mitochondrial isoform is also coupled to oxidative phosphorylation via
the adenine nucleotide translocator, and a functional coupling between
the isoenzyme and porin has been postulated (Hemmer and Wallimann,
1993). A complex between porin and the adenine nucleotide translocator
appears to play a role in the mitochondrial permeability transition,
which is linked to both apoptotic and necrotic cell death (Beutner et
al., 1997).
If energy impairment plays a critical role in the aforementioned animal
models of Huntington's disease, then compounds that increase the
cerebral energy reserve might be neuroprotective. Both creatine and
cyclocreatine are substrates for mitochondrial creatine kinase and have
been shown to modulate rates of ATP production (Boehm et al., 1996).
Cyclocreatine is the most kinetically active analog of creatine in the
CK reaction, leading to formation of phosphocyclocreatine (PCCr). We
therefore examined whether oral administration of either creatine or
cyclocreatine could exert neuroprotective effects against malonate and
3-nitropropionic acid neurotoxicity. We also examined the ability of
creatine and cyclocreatine to buffer malonate-induced ATP depletions
and whether neuroprotective effects correlated with reduced free
radical generation.
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MATERIALS AND METHODS |
We initially examined whether oral administration of creatine or
its analog cyclocreatine could attenuate malonate lesions. Male Sprague
Dawley rats (Charles River, Cambridge, MA) weighing 300-325 gm were
used. Malonate, 3-nitrotyrosine, creatine, and 2,3- and
2,5-dihydroxybenzoic acid (DHBA) were obtained from Sigma (St. Louis,
MO). Cyclocreatine was supplied by the Avicena Group and was
synthesized as reported previously (Roberts and Walker, 1982). Creatine
was administered orally to rats in their feed at doses of 0.25-3% in
the diet. Cyclocreatine was administered at 0.25-1.0%. Controls
received unsupplemented but otherwise identical diets. The compounds
were administered for 2 weeks before intrastriatal administration of
malonate and then for a further week before sacrifice. Malonate was
dissolved in distilled, deionized water, and the pH was adjusted to 7.4 with 0.1 M HCl. Intrastriatal injections of 1.5 µl of
malonate containing 3 µmol were made with a 10 µl Hamilton syringe
fitted with a 26 gauge blunt-tipped needle, into the left striatum at
the level of the bregma, 2.4 mm lateral to the midline, and 4.5 mm
ventral to the dura as described previously. Animals were killed at
7 d after injection, and the brains were quickly removed and
placed in ice-cold 0.9% saline solution. Brains were sectioned at 2 mm
intervals. Slices were then placed posterior side down in 2%
2,3,5-triphenyltetrazolium chloride (TTC). Slices were stained in the
dark at room temperature for 30 min and then removed and placed in 4%
paraformaldehyde, pH 7.3. Lesions, noted by pale staining, were
evaluated on the posterior surface of each section using a Bioquant 4 system by an experienced histologist blinded to experimental
conditions. These measurements have been validated by comparing them
with measurements obtained on adjacent Nissl-stained sections (Schulz
et al., 1995a).
Creatine or cyclocreatine was administered orally at a dose of 1% in
feed to animals treated with 3-nitropropionic acid (3-NP). Controls
received unsupplemented rat chow. 3-NP was diluted in water and
adjusted to pH 7.4 with NaOH and administered at a dose of 10 mg/kg
intraperitoneally every 12 hr for 9-11 d. Animals became acutely ill
after 9-11 d. Because of variability in the times at which animals
became ill, they were examined clinically 3 hr after the injections,
and when an animal was acutely ill one animal from each group was
killed, regardless of whether it was on a control diet or a
creatine-supplemented diet (Schulz et al., 1995a). Nine to 10 animals
were examined in each group. Animals were killed after showing acute
illness, and striatal lesion volume was assessed by TTC staining as
described above. Four additional animals that received 1% creatine and
four controls were perfused with 4% buffered paraformaldehyde. They
were then cryoprotected, and brains were sectioned at 50 µM intervals on a freezing microtome. Sections were
double-stained using a combined technique for NADPH histochemistry with
Nissl (Schulz et al., 1995a).
We investigated the effects of oral supplementation with 1% creatine
for 2 weeks on increases in striatal lactate concentrations produced
in vivo by 3-nitropropionic acid. Male Sprague Dawley rats
weighing 250-300 gm were administered 3-nitropropionic acid at a dose
of 33 mg/kg intravenously and were imaged 2.5-3.0 hr later. Seven
control and six creatine-fed animals were examined. Nuclear magnetic
resonance (NMR) imaging experiments were performed at 4.7 tesla on a
General Electric Omega chemical shift imager (CSI) with a 35 mm bird
cage. Lactate signal intensity was measured using a rapid
two-dimensional (w,y) water-suppressed CSI
sequence with a preparatory inversion pulse for lipid suppression based on the difference in T1 values between brain metabolites and lipids with repetition time of 2200 msec, inversion time of 208 msec, echo
time of 272 msec, NA of 8, and 16 phase-encode steps (Jenkins et al.,
1996). This sequence allows spatial localization in one dimension of
the important brain metabolites lactate, N-acetylaspartate (NAA), glutamate, glutamine, creatine, and choline. Lactate
measurements from the acute lesions and NAA signal intensity were
measured by extracting spectra from the two-dimensional files
corresponding to the lines that project along the superior-inferior
axis in the rat brain from coronal slices. Because NAA is unaltered
during the first few hours after 3-NP lesioning, lactate signal
intensity of the lines projected through the striatum was measured and
normalized to the NAA signal intensity in the unaffected regions of the
brain by direct integration of the extracted spectra. The NAA
concentration was assumed to be 7 mM. The effects of
feeding with 1% cyclocreatine on brain PCr and ATP levels were
examined using 31P magnetic resonance spectroscopy at
baseline and 1, 1.5, 2, and 6 weeks compared with normal controls
(Koroshetz et al., 1997).
To investigate the effects of 3-NP on energy metabolites further, we
administered 1% creatine in the diet and 1% cyclocreatine compared
with a normal diet. In the group fed with creatine we also examined the
effect of 3-NP compared with a control diet. Eight to 10 animals were
examined in each group. Animals were treated with 3-NP at a dose of 10 mg/kg intraperitoneally every 12 hr until they developed symptoms, and
then they were killed 2 hr later by the freeze-clamp technique in pairs
(either a control or a creatine-fed animal depending on which initially
became ill). Creatine, lactate, PCr, inosine monophosphate (IMP), AMP,
nicotinamide adenine dinucleotide (NAD), GDP, ADP, and ATP were
measured using a modification of published procedures (Bernocchi et
al., 1994).
The apparatus consisted of a Perkin-Elmer (Norwalk, CT) gradient HPLC
pump, a Waters Associates (Milford, MA) 409 multiple UV wavelength
detector, and a Shimadzu 501 integrator. Frozen striatal tissue was
dissected on a freezing cold plate ( 20°C) and placed in 0.4 M perchloric acid (10 µl/mg wet weight), homogenized, and
centrifuged. The supernatant was neutralized with 25 µl of 2 M K2CO3 added to 200 µl of the
supernatant and recentrifuged. Supernatants were then stored at
80°C until injected. Standards were prepared in 0.4 M
perchloric acid at concentrations of 10 µM for creatine,
cyclocreatine, PCr, and IMP, 5 µM for NAD, ADP, and ATP,
and 200 µM for lactate (based on tissue concentrations). Samples were separated on a 15 cm 3 µM Nikko Bioscience
C18 HPLC column (ESA, Inc., Chelmsford, MA) at a flow rate of 1 ml/min using a gradient. Buffer A was 25 mM
Na2HPO4 with 100 mg/l tetrabutylammonium (TBA),
pH 5.5, whereas buffer B was 200 mM
NaH2PO4 with 100 mg/l TBA, pH 4.0, and 10%
acetonitrile. The gradient was 100% buffer A for 0-5 min, 100%
buffer A to 100% buffer B for 5-25 min, and 100% buffer B for 25-34
min. Samples were monitored at 214 nm for 0-9 min, 260 nm for 9-25
min, and 214 nm for 25-34 min. The retention times were (in min):
creatine, 1.8; cyclocreatine, 2.0; lactate, 3.1; PCr (PCCr),
5.4; IMP, 13.8; GDP, 17.2; AMP, 18.4; NAD, 19.0; ADP-GTP,
21.0; and ATP, 23.4. The ADP and GTP peaks did not resolve, and the
combined peak was calculated using the ADP standard. All standards were
linear over a 100-fold concentration range.
The salicylate hydroxyl radical-trapping method was used for measuring
levels of hydroxyl radicals in striatal tissue after malonate
injections. Eight animals in each group were fed either a normal diet,
a 1% creatine-supplemented diet, or a 1% cyclocreatine-supplemented diet for 2 weeks before intrastriatal malonate, as described above. Animals were injected with 200 mg/kg salicylate intraperitoneally just
before the malonate injections and were killed 1 hr later. The striata
were then dissected rapidly from a 2-mm-thick slice and placed in 0.25 ml of chilled 0.1 M perchloric acid. Samples were
subsequently sonicated, frozen rapidly and thawed, and centrifuged twice. An aliquot of supernatant was analyzed by HPLC with 16-electrode electrochemical detection (Beal et al., 1990). Salicylate, 2,3- and
2,5-DHBA, tyrosine, and 3-nitrotyrosine were measured electrochemically by oxidation at 840, 240, 120, 600, and 840 mV, respectively, with
retention times of 20.5, 9.4, 6.3, 10.5, and 18.2 min, respectively. The data were expressed as the ratio of 2,3- and 2,5-DHBA to salicylate to normalize the DHBA concentrations for differing brain concentrations of salicylate. Similarly, 3-nitrotyrosine levels were normalized to
tyrosine levels. We also examined the effects of 1% creatine supplementation for 2 weeks on 3-NP-induced increases in
3-nitrotyrosine levels. Male Sprague Dawley rats were treated with 3-NP
at a dose of 20 mg/kg intraperitoneally and then killed at 3 hr. Ten
animals were examined in each group. The striata were dissected and
placed in chilled 0.1 M perchloric acid. 3-Nitrotyrosine
and tyrosine concentrations were measured by HPLC with electrochemical
detection as above.
Statistical comparisons were made by unpaired Student's t
test or by one-way ANOVA followed by Fisher's protected least
significant difference test for post hoc comparisons.
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RESULTS |
In initial pilot experiments we found that oral supplementation
with both creatine and cyclocreatine protected against striatal malonate lesions. We then examined a dose-response curve for
neuroprotection of both creatine and cyclocreatine against
malonate-induced striatal lesions. As shown in Figure
1, increasing doses of creatine from 0.25 to 3% administered for 2 weeks in the diet exerted dose-dependent neuroprotective effects against malonate-induced striatal lesions. Significant protection occurred with doses of 1 and 2% in the diet.
There was less protection at 3% creatine, suggesting that a U-shaped
dose response may occur with higher doses. Administration of
cyclocreatine resulted in dose-dependent neuroprotective effects, which
were significant at a dose of 1% cyclocreatine.
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Figure 1.
Effects of increasing doses of creatine
(CRT) and cyclocreatine (Cyclo) on striatal
lesions produced by 3 µmol of malonate. *p < 0.05; **p < 0.01 (ANOVA). Eight to 10 animals were
used in each group.
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We examined the effects of both creatine and cyclocreatine on subacute
3-NP neurotoxicity. Dietary supplementation with 1% creatine for 2 weeks resulted in a significant 83% reduction in lesion volume
produced by 3-NP (Fig. 2). In contrast,
animals treated with cyclocreatine became ill earlier and appeared to have an exacerbation of toxicity (data not shown). This striking neuroprotection was histopathologically confirmed using Nissl stains
(Fig. 3). Lesions could not be detected
in the creatine-fed animals, whereas the mean lesion volumes in the
3-NP-treated animals on control diets were 19.7 ± 5.1 mm3.
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Figure 2.
Effects of 1% creatine on lesions produced by
subacute systemic administration of 3-NP. ***p < 0.001. Ten animals were used in each group.
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Figure 3.
Nissl and NADPH-diaphorase double-stained coronal
brain sections at the level of the striatum and anterior commissure
from a creatine-fed rat (A, C) and a non-creatine-fed
rat (B, D). Rats prefed 1% creatine with subsequent
administration of 3-nitropropionic acid resulted in no demonstrable
striatal lesions (A) or neuronal loss or
alteration (C). In contrast, treatment with
3-nitroproprionic acid alone resulted in bilateral striatal lesions
within the caudate-putamen (arrows)
(B) with marked neuronal loss and relative
preservation of NADPH-diaphorase neurons (dark-stained
neurons) (D). Note the ventricular
enlargement attributable to striatal atrophy in the 3-nitroproprionic
acid-treated rat.
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We investigated the mechanism of neuroprotection of creatine against
malonate and 3-NP-induced neurotoxicity. We examined whether creatine
can increase brain energy reserves and can prevent depletions of
high-energy phosphate compounds induced by 3-NP. We measured creatine,
lactate, PCr, GDP, AMP, NAD, ADP-GTP, and ATP levels after oral
administration of 1% creatine (column 2) or 1% cyclocreatine (column
3) for 2 weeks compared with an unsupplemented diet (control) (Table
1). The ADP-GTP peak did not resolve the two compounds and therefore reflects a combination of both. Creatine administration produced a significant increase in striatal PCr levels.
Cyclocreatine resulted in significant reductions in creatine, which has
been noted previously, presumably because of its own buildup in the
brain. Cyclocreatine was phosphorylated efficiently by creatine kinase
to yield PCCr, which builds up to levels almost 20-fold greater than
PCr levels. Administration of 3-NP in controls (column 4) produced a
significant decrease in striatal concentrations of creatine, PCr, GDP,
AMP, NAD, ADP-GTP, and ATP, whereas there was a significant increase
in lactate. The reductions in Cr, PCr, GDP, AMP, NAD, ADP-GTP, and ATP
and the increase in lactate were attenuated significantly in animals
fed for 2 weeks with 1% creatine (column 5).
We also investigated the effects of creatine feeding on increases in
striatal lactate concentrations induced by 3-NP in vivo using 1H magnetic resonance spectroscopy. Animals were fed
with 1% creatine for 2 weeks before intravenous administration of 3-NP
at a dose of 33 mg/kg. We adopted this route of administration because
it resulted in more reproducible increases in lactate. As shown in Figure 4, there was a significant
reduction in lactate/NAA ratios in animals fed with creatine.
Representative chemical shift spectra are shown in Figure
5. The effects of feeding 1%
cyclocreatine on PCr and ATP levels were assessed by phosphorus
magnetic resonance spectroscopy. Relative concentrations were
calculated compared with external standards. We examined six animals
fed with cyclocreatine and nine controls. Animals fed with
cyclocreatine showed increases in ATP levels from 0.076 ± 0.009 at baseline to 0.115 ± 0.098 at 2-6 weeks
(p < 0.05) and increases in PCr and PCCr from
0.057 ± .008 at baseline to 0.137 ± 0.098 at 2-6 weeks
(p < 0.05).
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Figure 4.
Striatal lactate/NAA ratios in seven control and
six creatine-fed animals 2.5-3.0 hr after intravenous administration
of 3-NP. *p < 0.05.
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Figure 5.
Representative proton chemical shift spectra of a
control and a creatine-fed rat after intravenous administration of
3-NP. Lactate is reduced, and creatine is increased in the creatine-fed rat.
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A consequence of energy impairment produced by either malonate or 3-NP
is the generation of free radicals, which appear to play a role in cell
death, because both free radical scavengers and nitric oxide synthase
inhibitors can attenuate the toxicity produced by either compound
significantly (Greenamyre et al., 1994; Schulz et al., 1995a,b, 1996).
In the present experiments we investigated the effects of oral
administration of either 1% creatine or 1% cyclocreatine for 2 weeks
on malonate-induced increases in DHBA/salicylate and
3-nitrotyrosine/tyrosine. Animals were killed 1 hr after the malonate
injection. As shown in Figure 6, malonate
produced significant increases in both 2,3- and 2,5-DHBA/salicylate, which were significantly attenuated in animals fed with either creatine
or cyclocreatine. A significant increase in 3-nitrotyrosine/tyrosine was found in control animals, but smaller increases in
3-nitrotyrosine/tyrosine in the creatine- and cyclocreatine-fed animals
did not reach significance. We also examined the effects of 1%
creatine feeding for 2 weeks on increases in 3-nitrotyrosine/tyrosine
levels produced by 20 mg/kg 3-NP at 3 hr after administration. As shown
in Figure 7, increases in
3-nitrotyrosine/tyrosine were significantly decreased in the
creatine-fed animals.
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Figure 6.
Production of 2,3- and 2,5-DHBA and
3-nitrotyrosine after intrastriatal injection of malonate in controls
(Cont), animals fed with creatine, and those fed with
cyclocreatine (Cyclo). **p < 0.01 compared with uninjected side; #p < 0.05 compared with DHBA elevation in controls. Eight animals were used
in each group.
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Figure 7.
3-Nitrotyrosine levels after systemic
administration of 3-NP to controls and creatine-fed animals.
**p < 0.01 compared with saline-injected controls;
##p < 0.01 compared with 3-NP-injected
animals on a normal diet. Ten animals were used in each group.
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DISCUSSION |
There is substantial evidence that a secondary consequence of the
gene defect in HD may be an impairment of energy metabolism. HD
patients show weight loss despite a normal or increased caloric intake
(O'Brien et al., 1990). Furthermore, we showed increased lactate
concentrations in both the striatum and cerebral cortex of HD patients,
as assessed using 1H magnetic resonance spectroscopy
(Jenkins et al., 1993). We recently found a reduced PCr/Pi ratio in
resting gastrocnemius muscle of HD patients (Koroshetz et al., 1997).
In postmortem brain tissue there is decreased complex II-III activity
in HD basal ganglia (Gu et al., 1996; Browne et al., 1997).
We and others found that intrastriatal injections of the reversible
succinate dehydrogenase inhibitor malonate or systemic administration
of the irreversible succinate dehydrogenase inhibitor 3-nitropropionic
acid results in striatal lesions that closely mimic HD neuropathology
(Beal et al., 1993a,b; Greene et al., 1993; Brouillet et al., 1995).
There is sparing of striatal afferents and striatal NADPH-diaphorase
interneurons, with a depletion of striatal projection neurons. In
primates systemic administration of 3-NP produces the characteristic
histopathological features of HD, as well as both a choreiform movement
disorders and frontal-type cognitive deficits, which are typical
clinical manifestations of HD (Brouillet et al., 1995; Palfi et al.,
1996).
Both malonate and 3-NP produce energy defects in vivo,
followed by activation of excitatory amino acid receptors and the
generation of free radicals (Beal et al., 1993a,b; Greene et al., 1993;
Greenamyre et al., 1994; Schulz et al., 1995a,b). If the initiating
step in the pathological cascade is a depletion of cellular energy stores, then agents that can buffer cellular energy stores may be
neuroprotective. The brain isoform of creatine kinase, along with the
mitochondrial isoform and the substances Cr and PCr, constitute a
system that seems to be critical in regulating energy homeostasis in
the brain and other organs with high and fluctuating energy demands
(Hemmer and Wallimann, 1993). The mitochondrial isoform is part of a
complex of proteins that form an efficient, tightly coupled multienzyme
energy channel, which generates and transports energy in the form of
PCr, from the mitochondrial matrix to the cytoplasm. Creatine is an
excellent stimulant for mitochondrial respiration, resulting in the
generation of PCr (Kernec et al., 1996; O'Gorman et al., 1996).
Substantial evidence supports a direct functional coupling of creatine
kinase with Na+/K+ ATPase,
neurotransmitter release, and in maintenance of membrane potentials and
restoration of ion gradients before and after depolarization (Dunant et
al., 1988; Hemmer and Wallimann, 1993). High-energy turnover and high
creatine kinase concentrations have been found in those regions of the
brain that are rich in synaptic connections, e.g., molecular layer of
the cerebellum, glomerular structures of the granule layer, and the
hippocampus (Kaldis et al., 1996). An important role of creatine kinase
in the adult brain is supported by in vivo
31P NMR transfer measurements showing the
pseudo-first-order rate constant of the creatine kinase reaction (in
the direction of ATP synthesis) (Corbett and Laptook, 1994). Creatine
kinase flux correlates with brain activity, as measured by EEG, as well
as the amount of 2-deoxyglucose phosphate found in the brain (Sauter and Rudin, 1993).
A novel strategy to improve brain energy stores is therefore to
administer either creatine or an analog such as cyclocreatine, which
leads to high-energy phosphagens and which potentially could sustain
ATP production by the creatine kinase reaction. Previous studies in
both the heart and skeletal muscle showed that cyclocreatine administration resulted in increased tissue levels of
cyclocreatine and phosphocyclocreatine, delayed depletion of ATP levels
under ischemic conditions, and delayed onset of ischemia-induced rigor (Griffiths and Walker, 1976; Annesley and Walker, 1980; Roberts and
Walker, 1982; Turner and Walker, 1987; Elgebaly et al., 1994). Creatine
had no effect on ischemia-induced ATP depletion in the heart, but it
did protect against ATP depletion produced by arterial hypertension
(Turner and Walker, 1985; Osbakken et al., 1992; Constantin-Teodosiu et
al., 1995). In hippocampal slices creatine supplementation increased
PCr levels, delayed synaptic failure, and ameliorated neuronal damage
produced by anoxia (Whittingham and Lipton, 1981; Carter et al., 1995).
Cyclocreatine administration in vivo increased brain
concentrations of PCCr and appeared to buffer ATP stores (Woznicki and
Walker, 1980).
In the present study we found that oral administration of both
creatine and cyclocreatine produced dose-dependent neuroprotective effects against malonate lesions. The protection with creatine was
diminished at the highest dose level, suggesting that there may be a
U-shaped dose response at higher doses. We observed similar effects
with MPTP-induced dopamine depletions (M. F. Beal et al., unpublished data). The best neuroprotection was that seen after subacute administration of 3-NP. A significant 83% reduction in lesion
volume was observed in animals fed 1% creatine.
The mechanism of neuroprotection involves protection against depletions
of both PCr and ATP. We found that administration of either creatine or
cyclocreatine results in increased brain concentrations of PCr or PCCr,
respectively. There was a trend toward increased ATP, as determined
biochemically and by magnetic resonance spectroscopy. Increases in ATP
levels are somewhat unexpected, because brain ATP levels are thought to
be regulated tightly (Erecinska and Silver, 1989). Administration of
3-NP produced significant decreases in creatine, PCr, GDP, AMP, NAD,
ADP-GTP, and ATP. These energy metabolites are also decreased by
cerebral ischemia (Lazzarino et al., 1992) and by 3-NP in
vitro (Erecinska and Nelson, 1994) and in vivo
(Brouillet et al., 1993; Tsai et al., 1997). Creatine administration
significantly protected against the decreases. Furthermore, creatine
administration protected against 3-NP-induced increases of lactate, as
assessed by 1H magnetic resonance spectroscopy in
vivo. These findings therefore provide the first in
vivo data that creatine administration can increase brain
high-energy phosphate compounds and can protect against energy
compromise produced by mitochondrial toxins. Decreases in NAD and ATP
may be a consequence of both impaired mitochondrial function as well as
activation of poly-ADP-ribose polymerase, which plays a role in
neuronal cell death in vitro (Zhang et al., 1994). The
ability of PCr to stimulate synaptic glutamate uptake and thereby to
reduce extracellular glutamate may also play a role in the
neuroprotective effects of creatine and cyclocreatine (Xu et al.,
1996).
A secondary consequence of energy impairment is increased intracellular
concentrations of calcium attributable to both impaired mitochondrial
calcium buffering and activation of voltage-dependent NMDA excitatory
amino acid receptors (Beal, 1992, 1995). This leads to increased free
radical production by mitochondria as well as activation of neuronal
nitric oxide synthase, which is calmodulin-dependent. This can lead to
the generation of peroxynitrite, formed by the interaction of
O·2 with NO·. Peroxynitrite can
oxidize a intracellular molecules by a "hydroxyl radical"-like
activity, and it also can lead to nitration of tyrosines (Beckman et
al., 1990, 1992). We showed previously that both malonate and 3-NP
result in increases in both hydroxyl radical activity, as well as
3-nitrotyrosine (Schulz et al., 1995a,b). Furthermore, both free
radical scavengers and nitric oxide synthase inhibitors can attenuate
both malonate and 3-NP neurotoxicity (Greenamyre et al., 1994; Schulz
et al., 1995a,b, 1996).
Creatine kinase appears to be coupled directly or indirectly to
energetic processes required for calcium homeostasis (Wallimann et al.,
1992; Steeghs et al., 1997). Creatine pretreatment delayed increases in
intracellular calcium produced by 3-NP in cortical and striatal
astrocytes in vitro (Deshpande et al., 1997). Administration of creatine or cyclocreatine therefore may improve intracellular calcium buffering and may prevent free radical production by
mitochondria. In the present experiments we found that administration
of both creatine and cyclocreatine significantly attenuated
malonate-induced increases in 2,3- and 2,5-DHBA/salicylate, markers of
hydroxyl radical generation (Floyd et al., 1984). Increased
3-nitrotyrosine levels after malonate were also attenuated. Oral
administration of creatine reduced 3-NP-induced increases in
3-nitrotyrosine significantly. These findings therefore suggest that
improved energy buffering can act upstream to attenuate free radical
generation, which is associated with cell death produced by
mitochondrial toxins.
The present studies demonstrate that oral administration of
either creatine or cyclocreatine can buffer cellular ATP concentrations and can attenuate cell death in animal models that mimic the
neuropathological and clinical phenotype of HD. By attenuating ATP
depletion these compounds appear to prevent a pathological cascade,
which leads to free radical generation and eventual cell death.
Creatine administration is well tolerated in man and may have benefits
in pathological conditions (Balsom et al., 1994; Dawson et al., 1995).
Long-term administration to patients with gyrate atrophy of the choroid and retina prevented visual field constriction and resulted in improvement of muscle biopsy findings (Sipila et al., 1981). A pediatric patient with creatine deficiency in brain accompanied by an
extrapyramidal movement disorder showed partial restoration of cerebral
creatine concentrations and clinical improvement with oral creatine
administration (Stockler et al., 1994, 1996). Creatine administration
resulted in improvement in a patient with the mitochondrial disorder
mitochondrial encephalopathy lactic acidosis and strokes (Hagenfeldt et
al., 1994). The present findings suggest that treatment with creatine
or its analogs might be a novel therapeutic strategy to slow or halt
neurodegeneration in HD. Similar strategies might be effective in other
neurodegenerative diseases in which defects in energy metabolism are
implicated (Beal, 1992).
| |
FOOTNOTES |
Received Aug. 12, 1997; revised Sept. 30, 1997; accepted Oct. 23, 1997.
This work was supported by National Institutes of Health Grants
NS35255, NS37102 (R.J.F.), NS32365, NS31579, NS16367, and NS35255
(M.F.B.). The secretarial assistance of Sharon Melanson is gratefully
acknowledged.
Correspondence should be addressed to Dr. M. Flint Beal, Neurology
Service, WRN 408, Massachusetts General Hospital, 32 Fruit Street,
Boston, MA 02114.
| |
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Top
Abstract
Introduction
Compound via MeSH
