Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 2000 International Society for Neurochemistry Protective Effect of the Energy Precursor Creatine Against Toxicity of Glutamate and ␤-Amyloid in Rat Hippocampal Neurons Gregory J. Brewer and *Theo W. Wallimann Departments of Medical Microbiology/Immunology and Neurology, Southern Illinois University School of Medicine, Springfield, Illinois, U.S.A.; and *Institute for Cell Biology, Swiss Federal Institute of Technology, Zurich, Switzerland Abstract: The loss of ATP, which is needed for ionic (Manos et al., 1991; Molloy et al., 1992) and in vivo homeostasis, is an early event in the neurotoxicity of (Hemmer et al., 1994; Kaldis et al., 1996), CK isozyme glutamate and ␤-amyloid (A␤). We hypothesize that cells BB catalyzes the reversible conversion of PCr and ADP supplemented with the precursor creatine make more to ATP and Cr to manage different aspects of high- phosphocreatine (PCr) and create larger energy reserves energy demands in the brain (Hemmer and Wallimann, with consequent neuroprotection against stressors. In serum-free cultures, glutamate at 0.5–1 mM was toxic to 1993; Chen et al., 1995). On the other hand, mitochon- embryonic hippocampal neurons. Creatine at Ͼ0.1 mM drial CK catalyzes the reversible conversion of Cr and greatly reduced glutamate toxicity. Creatine (1 mM) could ATP to ADP and PCr to generate energy reserves in the be added as late as 2 h after glutamate to achieve pro- cytoplasm (Wyss et al., 1992). PCr reserves are depleted tection at 24 h. In association with neurotoxic protection even more rapidly than ATP during ischemia (Passon- by creatine during the first 4 h, PCr levels remained neau and Lowry, 1971) owing to their conversion to ATP constant, and PCr/ATP ratios increased. Morphologi- by CK. Although depletion of ATP is an early event in cally, creatine protected against glutamate-induced den- the neurotoxicity of glutamate (Budd et al., 1997) and dritic pruning. Toxicity in embryonic neurons exposed to ␤-amyloid (A␤) (Mark et al., 1997), PCr levels have not A␤ (25–35) for 48 h was partially prevented by creatine as been reported. Thus, an understanding of PCr levels and well. During the first6hoftreatment with A␤ plus crea- tine, the molar ratio of PCr/ATP in neurons increased their maintenance in the presence of the precursor, Cr, from 15 to 60. Neurons from adult rats were also partially are critical to understanding cellular energetics during protected from a 24-h exposure to A␤ (25–35) by creat- normal function and stress. ine, but protection was reduced in neurons from old Given ample PCr, the CK reaction generates ATP at a animals. These results suggest that fortified energy re- rate 10 times faster than oxidative phosphorylation and serves are able to protect neurons against important 40 times faster than glycolysis (Wallimann et al., 1992). cytotoxic agents. The oral availability of creatine may Because brain ATP levels are typically 3–4 mM and PCr benefit patients with neurodegenerative diseases. levels are ϳ5–6 mM (Erecin´ska and Silver, 1989), we Key Words: Creatine—Phosphocreatine—ATP—Ener- measured the concentrations of these critical energy mol- gy—Glutamate toxicity—␤-Amyloid. ecules, rather than ADP levels, which are maintained J. Neurochem. 74, 1968–1978 (2000). around 0.02 mM by adenylate kinase. Although the rates of many enzymes are regulated by ATP/ADP levels or the energy charge, (ATP ϩ 0.5 ADP)/(ATP ϩ ADP ϩ AMP) (Ball and Atkinson, 1975; Bishop and Atkin- The phosphocreatine (PCr)/creatine (Cr) kinase (CK) son, 1984), the immediate energy available to a mam- system is thought to be physiologically important in malian cell may be better represented by the total high- tissues with high and fluctuating energy requirements, energy pool, composed primarily of ATP and PCr. To like muscle and brain (Wallimann and Hemmer, 1994). quantify the extent of decline or the effects of Cr sup- In these cells, high-energy PCr serves not only as an immediate temporal energy buffer, but also as an energy shuttle from subcellular sites of energy production (mi- Received November 23, 1999; revised manuscript received January tochondria and/or glycolysis) to sites of energy con- 4, 2000; accepted January 5, 2000. sumption (ion pumps and various other ATPases) where Address correspondence and reprint requests to Dr. G. J. Brewer at CK isoenzymes are specifically localized in a compart- Departments of Medical Microbiology/Immunology and Neurology, Southern Illinois University School of Medicine, P.O. Box 19626, mented fashion (Bessman and Geiger, 1981; Wallimann Springfield, IL 62794-9626, U.S.A. E-mail: [email protected] et al., 1992) (Fig. 1). In the cytoplasm and membrane Abbreviations used: A␤, ␤-amyloid; CK, creatine kinase; Cr, crea- compartments of neurons, as well as glial cells in culture tine; MAP2, microtubule-associated protein-2; PCr, phosphocreatine. 1968 NEUROTOXIC PROTECTION BY CREATINE 1969 under stress and Cr supplementation. As a follow-up on a preliminary report (Brewer, 1998b), we describe the protective effects of Cr on neurons in serum-free culture as a model for the excitotoxicity of glutamate and the neurodegenerative action of A␤. Thus, Cr supplementa- tion may turn out to be beneficial for several muscular and neurodegenerative diseases (Guerrero-Ontiveros and Wallimann, 1998). MATERIALS AND METHODS Cell culture Hippocampal neurons were isolated from rat brains at em- bryonic day 18 (Brewer et al., 1993), middle age (9–11 months old), or old age (35–37 months old) (Brewer, 1997). The laboratory animal protocol was approved by the Laboratory Animal Medicine Review Committee. In brief, embryonic neu- rons were isolated by mechanical trituration and then plated at 160 cells/mm2 on glass coverslips precoated with poly-D-lysine in 2% (vol/vol) B27 in Neurobasal medium (Life Technologies, Gaithersburg, MD, U.S.A.) with 25 ␮M glutamate and 0.5 mM glutamine. Every 3–4 days, 50% of the culture medium was exchanged for fresh medium without glutamate. Cultures were incubated at 37°C in 5% CO2/95% O2 for 5–8 days. For adult neurons (Brewer, 1997), hippocampi were sliced at 0.5 mm, digested with papain, and triturated in HibernateA/B27 (Brewer FIG. 1. Relationship of Cr and PCr to ATP in the neuron, drawn and Price, 1996) (Life Technologies). Cells were separated largely from information on rat heart and chick brain. Exogenous from debris on a density gradient of Nycoprep. The neuron- Cr should be readily transported into neurons by a Cr transporter enriched fraction was collected and cultured for 7 days in or synthesized if precursors are available (Dringen et al., 1998). serum-free medium, B27/NeurobasalA with 5 ng/ml fibroblast Under resting levels of ADP, ATP produced in the mitochondria growth factor-2 (Life Technologies) and 0.5 mM glutamine is converted to PCr by mitochondrial CK (CKMi) and released into without glutamate. Adult cells were plated at 320 cells/mm2.Cr the cytoplasm. Under conditions of high Cr levels, respiratory (Sigma) was freshly dissolved every 3 days at Յ200 mM in Vmax can increase by three- to fivefold (Farrell et al., 1972; Saks et al., 1975). In the cytoplasm, the equilibrium constants for brain Neurobasal medium so that dilutions of 1:100 or 1:400 were made directly into cultures. Glutamate (Sigma) was dissolved CK, CKBB (Quest et al., 1990; Boehm et al., 1996,1998), favor conversion of PCr to ATP. ATP is consumed to maintain ion at 25 mM in Hanks’ balanced salt solution, filter-sterilized, and homeostasis, e.g., via the plasma membrane ATPase and most stored at 4°C. Dilutions were made directly into 5–8-day-old importantly the sarcoplasmic–endoplasmic reticulum Ca2ϩ- cultures or into cultures with fresh medium or Locke’s salts 2ϩ ATPase Ca pump (Wallimann and Hemmer, 1994). The result- [154 mM NaCl, 5.6 mM KC1, 2.3 mM CaCl2,1mM MgCl2, 3.6 ing ADP is rapidly consumed in the regeneration of ATP from PCr mM NaHCO3,5mM HEPES (pH 7.2), and 5 mM glucose]. A␤ by CKBB. Note that PCr can be either transported from the soma (25–35) (QCB, Hopkinton, MA, U.S.A.) was dissolved at 2.5 to distant sites or generated at sites distant from the nucleus (N), mM in deionized water and stored at Ϫ70°C until the day of such as at synaptically localized mitochondria. At synapses, PCr can also be used directly as an energy source to load synaptic use. Dilutions of 1:100 were made into fresh culture medium vesicles, especially under conditions of low potassium that for cells cultured for 6–8 days (Brewer, 1998a). Killing of would accompany high levels of synaptic activity (Xu et al., neuron-like cells was determined at the indicated times by live 1996). cell fluorescence of fluorescein from the diacetate and dead cell nuclear fluorescence of propidium iodide (Brewer et al., 1993). In contrast to protease-containing cultures in serum, back- plementation on PCr levels relative to ATP levels, we ground levels of dead cells in serum-free cultures are ϳ40% define the energy reserve status as the ratio PCr/ATP. In and do not change with time (Brewer, 1999). Percent killing was calculated as 100 Ϫ (live neuron-like cells with nonsym- resting skeletal muscle and heart, PCr levels are three metric, branched processes)/(live neuron-like cells ϩ dead times higher than ATP levels (Passonneau and Lowry, cells). Reported treatment values were corrected by subtraction 1971), but in whole brain they are at most twofold higher of the percent killing in control, untreated cultures. Twelve (Gatfield et al., 1966; Goldberg et al., 1966; Ackerman consecutive fields of 0.313 mm2 were counted per condition. et al., 1980; Erecin´ska and Silver, 1989).