Neuroprotective Effects of Creatine and Cyclocreatine in Animal Models of Huntington’S Disease
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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. Matthews,1 Lichuan Yang,1 Bruce G. Jenkins,1 Robert J. Ferrante,2 Bruce R. Rosen,1 Rima Kaddurah-Daouk3 and M. Flint Beal1 1Neurochemistry Laboratory, Neurology Service and Massachusetts General Hospital Nuclear Magnetic Resonance Center, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, 2Geriatric Research Educational and Clinical Center, Bedford Veterans Administration Medical Center, Department of Neurology and Pathology, Boston University School of Medicine, Boston, Massachusetts 02115, and 3The Avicena Group, Inc., Cambridge, Massachusetts 02139 The gene defect in Huntington’s disease (HD) may result in produced by 3-NP.Creatine supplementation protected against an impairment of energy metabolism. Malonate and 3-NP induced increases in striatal lactate concentrations in vivo 3-nitropropionic acid (3-NP) are inhibitors of succinate dehy- as assessed by 1H magnetic resonance spectroscopy. Creatine drogenase that produce energy depletion and lesions that and cyclocreatine protected against malonate-induced in- closely resemble those of HD. Oral supplementation with cre- creases in the conversion of salicylate to 2,3- and 2,5- atine or cyclocreatine, which are substrates for the enzyme dihydroxybenzoic acid, biochemical markers of hydroxyl radical creatine kinase, may increase phosphocreatine (PCr) or phos- generation. Creatine administration protected against 3-NP- phocyclocreatine (PCCr) levels and ATP generation and thereby induced increases in 3-nitrotyrosine concentrations, a marker may exert neuroprotective effects. We found that oral supple- of peroxynitrite-mediated oxidative injury. Oral supplementa- mentation with either creatine or cyclocreatine produced sig- tion with creatine or cyclocreatine results in neuroprotective nificant protection against malonate lesions, and that creatine effects in vivo, which may represent a novel therapeutic strat- but not cyclocreatine supplementation significantly protected egy for HD and other neurodegenerative diseases. against 3-NP neurotoxicity. Creatine and cyclocreatine in- creased brain concentrations of PCr and PCCr, respectively, Key words: creatine; ATP; oxidative injury; 3-nitrotyrosine; and creatine protected against depletions of PCr and ATP 3-nitropropionic acid; Huntington There is substantial evidence that impairment of energy produc- ing energy requirements, including the brain (Chen et al., 1995). tion may play a role in the pathogenesis of neurodegenerative The enzyme catalyzes the reversible transfer of the phosphoryl diseases (Albin and Greenamyre, 1992; Beal, 1992). Impaired group from phosphocreatine (PCr) to ADP to generate ATP (for energy production may lead to activation of excitatory amino acid review, see Wallimann, 1992). Several cytoplasmic and mitochon- receptors, increases in intracellular calcium, and the generation of drial isoforms have been identified, which along with the sub- free radicals (Beal, 1995). In Huntington’s disease (HD) there is strates creatine and PCr constitute an intricate cellular energy reduced mitochondrial complex II–III activity in postmortem buffering and transport system, connecting sites of energy pro- tissue (Gu et al., 1996; Browne et al., 1997) and increased cerebral duction with sites of energy consumption (Hemmer and Walli- lactate concentrations in vivo (Jenkins et al., 1993). Animal mod- mann, 1993). els of Huntington’s disease involve defects in energy production. The mitochondrial isoform of creatine kinase (Mi-CK) is lo- Malonate and 3-nitropropionic acid are, respectively, reversible cated at contact sites between the inner and outer membranes, and irreversible inhibitors of mitochondrial complex II (succinate where it is associated with porin (Wallimann et al., 1992; Brdiczka dehydrogenase), which produce striatal lesions similar to those of et al., 1994). Mi-CK can convert intramitochondrially produced HD (Beal et al., 1993b; Greene et al., 1993; Brouillet et al., 1995). ATP to PCr directly, which then gets transported to sites of The pathogenesis of lesions produced by these compounds in- energy consumption. The mitochondrial isoform is also coupled volves energy depletion, followed by activation of excitatory to oxidative phosphorylation via the adenine nucleotide translo- amino acid receptors and free radical production (Schulz et al., cator, and a functional coupling between the isoenzyme and 1995a,b). The ensuing cell death involves both apoptosis and porin has been postulated (Hemmer and Wallimann, 1993). A necrosis (Pang and Geddes, 1997; Sato et al., 1997). complex between porin and the adenine nucleotide translocator Creatine kinase (CK) is a key enzyme involved in regulating appears to play a role in the mitochondrial permeability transi- energy metabolism in cells with intermittently high and fluctuat- tion, which is linked to both apoptotic and necrotic cell death Received Aug. 12, 1997; revised Sept. 30, 1997; accepted Oct. 23, 1997. (Beutner et al., 1997). This work was supported by National Institutes of Health Grants NS35255, If energy impairment plays a critical role in the aforementioned NS37102 (R.J.F.), NS32365, NS31579, NS16367, and NS35255 (M.F.B.). The secre- animal models of Huntington’s disease, then compounds that tarial assistance of Sharon Melanson is gratefully acknowledged. increase the cerebral energy reserve might be neuroprotective. Correspondence should be addressed to Dr. M. Flint Beal, Neurology Service, WRN 408, Massachusetts General Hospital, 32 Fruit Street, Boston, MA 02114. Both creatine and cyclocreatine are substrates for mitochondrial Copyright © 1997 Society for Neuroscience 0270-6474/97/180156-08$05.00/0 creatine kinase and have been shown to modulate rates of ATP Matthews et al. • Neuroprotection of Creatine in Huntington’s Disease Models J. Neurosci., January 1, 1998, 18(1):156–163 157 production (Boehm et al., 1996). Cyclocreatine is the most kinet- lines that project along the superior–inferior axis in the rat brain from ically active analog of creatine in the CK reaction, leading to coronal slices. Because NAA is unaltered during the first few hours after formation of phosphocyclocreatine (PCCr). We therefore exam- 3-NP lesioning, lactate signal intensity of the lines projected through the striatum was measured and normalized to the NAA signal intensity in ined whether oral administration of either creatine or cyclocrea- the unaffected regions of the brain by direct integration of the extracted tine could exert neuroprotective effects against malonate and spectra. The NAA concentration was assumed to be 7 mM. The effects of 3-nitropropionic acid neurotoxicity. We also examined the ability feeding with 1% cyclocreatine on brain PCr and ATP levels were exam- of creatine and cyclocreatine to buffer malonate-induced ATP ined using 31P magnetic resonance spectroscopy at baseline and 1, 1.5, 2, depletions and whether neuroprotective effects correlated with and 6 weeks compared with normal controls (Koroshetz et al., 1997). To investigate the effects of 3-NP on energy metabolites further, we reduced free radical generation. administered 1% creatine in the diet and 1% cyclocreatine compared with a normal diet. In the group fed with creatine we also examined the MATERIALS AND METHODS effect of 3-NP compared with a control diet. Eight to 10 animals were We initially examined whether oral administration of creatine or its examined in each group. Animals were treated with 3-NP at a dose of 10 analog cyclocreatine could attenuate malonate lesions. Male Sprague mg/kg intraperitoneally every 12 hr until they developed symptoms, and Dawley rats (Charles River, Cambridge, MA) weighing 300–325 gm then they were killed 2 hr later by the freeze-clamp technique in pairs were used. Malonate, 3-nitrotyrosine, creatine, and 2,3- and 2,5- (either a control or a creatine-fed animal depending on which initially dihydroxybenzoic acid (DHBA) were obtained from Sigma (St. Louis, became ill). Creatine, lactate, PCr, inosine monophosphate (IMP), MO). Cyclocreatine was supplied by the Avicena Group and was synthe- AMP, nicotinamide adenine dinucleotide (NAD), GDP, ADP, and ATP sized as reported previously (Roberts and Walker, 1982). Creatine was were measured using a modification of published procedures (Bernocchi administered orally to rats in their feed at doses of 0.25–3% in the diet. et al., 1994). Cyclocreatine was administered at 0.25–1.0%. Controls received un- The apparatus consisted of a Perkin-Elmer (Norwalk, CT) gradient supplemented but otherwise identical diets. The compounds were ad- HPLC pump, a Waters Associates (Milford, MA) 409 multiple UV ministered for 2 weeks before intrastriatal administration of malonate wavelength detector, and a Shimadzu 501 integrator. Frozen striatal and then for a further week before sacrifice. Malonate was dissolved in tissue was dissected on a freezing cold plate (220°C) and placed in 0.4 M distilled, deionized water, and the pH was adjusted to 7.4 with 0.1 M HCl. perchloric acid (10 ml/mg wet weight), homogenized, and centrifuged. Intrastriatal injections of 1.5 ml of malonate containing 3 mmol were m m The supernatant was neutralized with 25 lof2M K2CO3 added to 200 made with a 10 l Hamilton syringe fitted with a 26 gauge blunt-tipped ml of the supernatant and recentrifuged. Supernatants were then stored needle, into