1. SIRM Investigation of Metabolic Trafficking between Neurons and Astrocytes Fan, T. W-M. Yuan, P., Lane, A.N., Higashi, R.M. Wang, Y., Hamidi, A., Zhou, R., Xavier Guitart-Navarro, X., Chen, G., Manji, H.K., Kaddurah-Daouk, R. (2010) Stable Isotope Resolved Metabolomic Analysis of Lithium Effects on Glial-Neuronal Interactions. Metabolomics 6, 165 - 179

2. Abstract Despite the long-established therapeutic efficacy of lithium in the treatment of bipolar disorder (BPD), its molecular mechanism of action remains elusive. Newly developed stable isotope- resolved metabolomics (SIRM) is a powerful approach that can be used to elucidate systematically how lithium impacts glial and neuronal metabolic pathways and activities, leading ultimately to deciphering its molecular mechanism of action. The effect of lithium on the metabolism of three different 13 C-labeled precursors ([U- 13 C]-Glucose, 13 C-3-lactate or 13 C-2,3-Alanine) was analyzed in cultured rat astrocytes and neurons by nuclear magnetic resonance (NMR) spectroscopy and gas chromatography mass spectrometry (GC-MS). Using [U- 13 C]-Glucose, lithium was shown to enhance glycolytic activity and part of the Krebs cycle activity in both astrocytes and neurons, particularly the anaplerotic pyruvate carboxylation (PC). The PC pathway was previously thought to be active in astrocytes but absent in neurons. Lithium also stimulated the extracellular release of 13 C labeled-lactate, - alanine (Ala), -citrate, and -glutamine (Gln) by astrocytes. Interrogation of neuronal pathways using 13 C-3-lactate or 13 C-2,3-Ala as tracers indicated a high capacity of neurons to utilize lactate and Ala in the Krebs cycle, particularly in the production of labeled Asp and Glu via PC and normal cycle activity. Prolonged lithium treatment enhanced lactate metabolism via PC but inhibited lactate oxidation via the normal Krebs cycle in neurons. Such lithium modulation of glycolytic, PC and Krebs cycle activity in astrocytes and neurons as well as release of fuel substrates by astrocytes should help replenish Krebs cycle substrates for Glu synthesis while meeting neuronal demands for energy. Further investigations into the molecular regulation of these metabolic traits should provide new insights into the pathophysiology of mood disorders and early diagnostic markers, as well as new target(s) for effective therapies.

3. Summary of Major Findings  Astrocytes cooperate with neurons in Glu-Gln cycling, which sustains glutamatergic neurotransmission  Anaplerotic pyruvate carboxylation (PC) is active in astrocytes and important for Gln synthesis & delivery to neurons  PC is also active in neurons, and contributes to anaplerosis  Psychotropic LiCl affects such trafficking  Primary cell cultures are useful for studying metabolic trafficking between astrocytes and neurons

4. Approach  Treat primary rat cortical neurons and astrocytes with LiCl in the presence of 13 C 6 -glucose, 13 C-3- lactate, or 13 C-2,3-Ala  13 C isotopomer/isotopologue analysis of metabolites using NMR & MS  Reconstruct effects of LiCl on metabolic networks  Hypothesis generation regarding LiCl effects on metabolic regulation

5. 13 C-labeling patterns : Krebs cycle Two turns of the cycle

6. 13 C-labeling pattern a/ Krebs cycle Three turns of the cycle One cycle turn+PC

7. Li effect on neurons: 13 C-3-lactate

8.  Stable isotope-labeled tracers and isotopomer/isotopologue profiling are indispensible for tracing metabolic networks at the atomic level  Integrating NMR with accurate mass based FT-ICR-MS enables systematic profiling of isotopomers and isotopologues for metabolic network reconstruction  SIRM can probe metabolic effects of drug treatments such as Li or pathogenesis  Li+ has wide ranging effects on the metabolism of isolated astrocyets and neurons, and may thereby influence the substrate cycling between these cooperating brain cells Conclusions

9. This work was a collaboration between: The Center for Regulatory Environmental Metabolomics (CREAM) at the University of Louisville, The Mood & Anxiety Disorder Program, National Institute of Mental Health, NIH. Department of Psychiatry, Duke University, Durham, Financial Support: NIH Grant Numbers R24GM078233 (RKD, TF), NCRR P20RR018733, 1R01CA118434-01A2 (TF, ANL, RMH), 3R01CA118434-02S1 (TF, RMH), and National Science Foundation EPSCoR grant # EPS-0447479 (TF, ANL). Acknowledgments