Increased D-Amino Acid Oxidase (DAO) Activity and Expression in Schizophrenia but Not Bipolar

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SUPPLEMENTARY MATERIAL

D-Amino acid oxidase (DAO) activity and expression are increased in schizophrenia

PWJ Burnet, SL Eastwood, GC Bristow, BR Godlewska, P Sikka, M Walker, PJ Harrison.

Department of Psychiatry, University of Oxford, Oxford, U.K.

Acknowledgments

Supported by the UK Medical Research Council (Research grant #G0500180) and a Centre Award from the Stanley Medical Research Institute. Tissue provided by the Stanley Medical Research Institute, courtesy of Drs Michael B. Knable, E. Fuller Torrey, Maree J. Webster, and Robert H. Yolken. We thank them for their major contribution to the field through this endeavour. We thank Louise Verrall for her role in our DAO research and Valerie West for secretarial assistance.

Supplementary Introduction

Evidence for DAO involvement in schizophrenia

D-amino acid oxidase (DAO, DAAO) degrades D-amino acids, notably the NMDA receptor modulator D-serine.S1,S2 It is therefore directly implicated in pathophysiological and therapeutic models of schizophrenia based upon NMDA receptor hypofunction,S3-S7 especially following the genetic association of DAO with the disorder,S8 a finding replicated in some though not all studies,S9-S12 such that overall, the genetic candidacy of DAO in schizophrenia remains weak.

Chumakov et alS8 also found association with schizophrenia for a novel gene, G72/G30, which they identified as interacting genetically and biochemically with DAO, and which has also become known as D-amino acid oxidase activator (DAOA). Genetic association of G72/G30 with schizophrenia is supported by a meta-analysis.S10

Supplementary Materials and Methods

Subjects studied

Frozen blocks of cerebellar cortex were provided by the Stanley Medical Research Institute (SMRI). The series, known as the Microarray Collection, comprises 35 controls, 35 subjects with schizophrenia, and 34 with bipolar disorder (one case originally in the latter group has been removed for neuropathological reasons). The demographic details are summarised in Supplementary Table 1. From each block, RNA and DNA were extracted using standard methods.

We also used cerebellar tissue from rats administered haloperidol (1mg/kg/d) or clozapine (25mg/kg/d) i.p. once daily for 14 days, as describedS13 to investigate antipsychotic effects on DAO activity.

DAO activity assay

Fragments of frozen cerebellar tissue (50-100mg) were homogenised in twenty volumes of assay buffer (50mM Na2HPO4, pH 7.4), centrifuged for 1 min at 12,000g, and the supernatants stored at -70oC. The DAO activity assay was performed using the Amplex Red kit (Molecular Probes/Invitrogen, Paisley, UK) as described.S14 Briefly, the supernatants were incubated with 50mM Amplex Red, 0.125 units horse-radish peroxidase, and D-proline (1-10mM) in a total volume of 10ml. Samples were incubated at 37oC for 60min, and the absorbance read at 571nm. The maximum DAO reaction rate (Vmax) and affinity (Km) for D-proline metabolism were calculated from Lineweaver-Burke plots.

Measurement of DAO mRNA

DAO mRNA was measured by qPCR (Applied Biosystems [AB] 7900HT, Warrington, UK) using published primers (0.2 µM final concentration) and SYBR green (AB), and normalised to the geometric mean of four Taqman housekeeping assays (AB: β-2-microglobulin: assay Hs99999907_m1; GAPDH: assay Hs99999905_m1; GUSB: assay Hs99999908_m1; TFRC: assay Hs00951094_m1). Triplicate reactions were performed for all subjects concurrently on a single plate. The mean absolute measures were calculated using a standard curve of serially diluted pooled cDNA and sequence detector software (AB, SDS v2.2.2).

Genotyping

To assess if variation in DAO or in G72/G30 affected DAO expression or activity, we genotyped two SNPs in each gene. The SNPs were selected based on three pragmatic criteria: a) the SNP is amongst those most strongly associated with schizophrenia in each gene, b) the SNP has a high minor allele frequency, and c) the SNP tags a haplotype block.S8,S10 In DAO, we chose rs2070587 (G/T) and rs3741775 (G/T). In G72/G30, we selected rs2391191 (A/G, a coding substitution) and rs3918342 (C/T). Genotyping was performed, in duplicate, using AB Taqman pre-designed genotyping assays in accordance with manufacturer’s instructions (details on request). Genotyping reproducibility was > 99%.

Statistical analysis

All datasets met criteria for normality using the Kolomogorov-Smirnov one-sample test, and parametric tests were therefore applied. Diagnostic and genotype effects were assessed by ANOVA. Potential influences of continuous variables (e.g. age, brain pH, post mortem interval, RIN)S15,S16 were examined using the Pearson coefficient, and any showing significant (p<0.05) correlations were included as covariates. Potential effects of other categorical variables (e.g. sex, suicide) were also explored using ANOVA. Statistical analyses were performed by PJH; the rest of the group remain blinded to the diagnostic code. The data have been deposited with the SMRI.

Supplementary Discussion

What are the functional consequences of enhanced DAO activity?

Many factors other than DAO activity likely influence D-serine availability at the NMDA receptor, including the rate of its synthesis by serine racemase, and its release into and reuptake from the synapse. Alterations in these processes in schizophrenia could counteract – or exacerbate – the effect of enhanced DAO activity. Notably, there does not appear to be a compensatory effect on serine racemase, in that its expression is unaltered in schizophrenia in the cerebellum,S17 with an inconsistent profile in cortical regions;S18,S19 the most relevant parameter would be its enzyme activity, but this has never been reported, and we have failed to get reliable measurements in post mortem brain tissue (unpublished observations). Regarding the status of neuronal and glial D-serine transport in schizophrenia, this is unknown except for our recent finding of a reduced protein abundance of the neuronal transporter Asc-1.S20 Data from a mutant (ddY/DAO-) mouse strain, in which DAO is inactive, support there being a relationship between DAO activity and D-serine levels and NMDA receptor function, as well as a lack of effect upon D-serine synthesis and transport. That is, the mice have increased cerebellar D-serine, increased extracellular D-serine, and enhanced cerebellar NMDA receptor function, but no changes in serine racemase or Asc-1 expression nor in D-serine uptake by cerebellar synaptosomes.S21,S22 Together, these complementary human and mouse findings do not suggest that the increased DAO activity in schizophrenia is compensated for by increases in D-serine synthesis or marked changes in its synaptic transport; as such, a resulting impairment of D-serine modulation of the NMDA receptor remains a plausible interpretation.

Does increased DAO activity (only) affect D-serine?

Since DAO also metabolises other D-amino acids, a further issue to consider is whether D-serine is the only, or even the primary, substrate affected by an increase in cerebellar DAO activity. A functional role for D-serine in cerebellar NMDA receptor modulation has been questioned,S1 and D-serine concentration in the adult cerebellum is very low compared to the forebrain.S23-S26, but see S27 Moreover, it is not clear whether synaptic D-serine is actually decreased in schizophrenia, since brain tissue levels are unchanged,S19,S24,S28 in contrast to the reductions seen in plasmaS29 and CSF.S30 Apart from D-serine, one candidate D-amino acid that may be affected by the DAO elevation is D-alanine, which is present in the cerebellum,S21 is an NMDA receptor modulatorS31 and may also be therapeutically beneficial in schizophrenia.S32 Overall, whilst a primary effect on D-serine, and thence the NMDA receptor, is an attractive interpretation of the DAO increase in schizophrenia, further studies are needed to confirm the biochemical consequences - as well as the cause - of the elevation, and the extent to which the impact is mediated via effects on D-serine and/or the NMDA receptor.

Unaltered DAO activity in bipolar disorder

DAO activity was unaltered in bipolar disorder (Fig. 1A). Given the increase in schizophrenia this is a somewhat unexpected finding, in that most results in the two SMRI brain series show changes that are in the same direction, and often of similar magnitude, in the two diagnostic groups (e.g. refs. S33-S35). There may be residual confounding by a factor that differs between the schizophrenia and bipolar disorder groups and that impacts on DAO activity; the fact that DAO mRNA did not show a similar differential change in the two disorders highlights the need for caution. On the other hand, the result may reflect a genuine differential involvement of DAO in schizophrenia but not in bipolar disorder, a possibility that merits further study.

Supplementary References

S1.Mothet JP, Parent AT, Wolosker H, Brady RO, Linden DJ, Ferris CD et al. D-serine is an endogenous ligand for the glycine site of the N-methyl-D-aspartate receptor. Proc Natl Acad Sci USA 2000; 97: 4926-4931.

S2. Pollegioni L, Piubelli L, Sacchi S, Pilone MS, Molla, G. Physiological functions of D-amino acid oxidases: from yeast to humans. Cell Mol Life Sci 2007; 64: 1373-1394.

S3. Olney JW, Farber NB. Glutamate receptor dysfunction and schizophrenia. Arch Gen Psychiatry 1995; 52: 998-1007.

S4. Tsai GC, Coyle JT. Glutamatergic mechanisms in schizophrenia. Annu Rev Pharmacol Toxicol 2002; 42: 165-179.

S5. Moghaddam B. Bringing order to the glutamate chaos in schizophrenia. Neuron 2003; 40: 881-884.

S6. Harrison PJ, Owen MJ. Genes for schizophrenia? Recent findings and their pathophysiological implications. Lancet 2003; 361: 417-419.

S7. Javitt DC.Glutamate as a therapeutic target in psychiatric disorders. Mol Psychiatry 2004; 9: 984-997.

S8. Chumakov I, Blumenfeld M, Guerrasimenko O, Cavarec L, Palicio M, Abderrahim H, et al. Genetic and physiologic data implicating the new human gene G72 and the gene for D-amino acid oxidase in schizophrenia. Proc Natl Acad Sci USA 2002; 99:13675-13680.

S9. Harrison PJ, Weinberger DR. Schizophrenia genes, gene expression, and neuropathology: on the matter of their convergence. Mol Psychiatry 2005; 10: 40-68.

S10. Detera-Wadleigh SD, McMahon FJ. G72/G30 in schizophrenia and bipolar disorder: Review and meta-analysis. Biol Psychiatry 2006; 60: 106-114.

S11. Wood LS, Pickering EH, Dechairo BM. Significant support for DAO as a schizophrenia susceptibility locus: Examination of five genes putatively associated with schizophrenia. Biol Psychiatry 2007; 61: 1195-1199.

S12. Schumacher J, Jamra RA, Freudenberg J, Becker T, Ohlraum S, Otte AC et al. Examination of G72 and D-amino-acid oxidase as genetic risk factors for schizophrenia and bipolar disorder. Mol Psychiatry 2004; 9: 203-207.

S13. Law AJ, Hutchinson LJ, Burnet PW, Harrison PJ. Antipsychotics increase microtubule-associated protein 2 mRNA but not spinophilin mRNA in rat hippocampus and cortex. J Neurosci Res 2004; 76: 376-382.

S14. Brandish PE, Chiu CS, Schneeweis J, Brandon NJ, Leech CL, Kornienko O, et al. A cell-based ultra-high-throughput screening assay for identifying inhibitors of D-amino acid oxidase. J Biomol Screening 2006; 11: 481-487.

S15. Harrison PJ, Heath PR, Eastwood SL, Burnet PWJ, McDonald B, Pearson RCA: The relative importance of premortem acidosis and postmortem interval for human brain gene expression studies: selective mRNA vulnerability and comparison with their encoded proteins. Neurosci Lett 200: 151-154

S16. Lipska BK, Deep-Soboslay A, Weickert CS, Hyde TM, Martin CE, Herman MM, Kleinman JE: Critical factors in gene expression in post-mortem human brain: focus on studies in schizophrenia. Biol Psychiatry 2006: 650-658.

S17. Verrall L, Walker M, Rawlings N, Benzel I, Kew JNC, Harrison PJ, Burnet PJW. d-Amino acid oxidase and serine racemase in human brain: normal distribution and altered expression in schizophrenia Eur J Neurosci 2007; 26: 1657-1669.

S18. Steffek AE, Haroutunian V, Meador-Woodruff JH. Serine racemase protein expression in cortex and hippocampus in schizophrenia. NeuroReport 2006; 17: 1181-1185.

S19. Bendikov I, Nadri C, Amar S, Panizzutti R, De Miranda J, Wolosker H, Agam G. A CSF and postmortem brain study of D-serine metabolic parameters in schizophrenia. Schiz Res 2007; 90: 41-51.

S20. Burnet PJW, Hutchinson L, von Hesling M, Gilbert E-J, Harrison PJ. Expression of D-serine and glycine transporters (GlyT1, Asc-1 and SNAT2) in the prefrontal cortex and cerebellum in schizophrenia. Schizophr Res (in press)

S21. Morikawa A, Hamase K, Inoue T, Konno R, Niwa A, Zaitsu K. Determination of free D-aspartic acid, D-serine and D-alanine in the brain of mutant mice lacking D-amino-acid oxidase activity. J Chromatogr B 2001; 757: 119-125.

S22. Almond SL, Fradley RL, Armstrong EJ, Heavens RB, Rutter AR, Newman RJ, et al. Behavioral and biochemical characterization of a mutant mouse strain lacking D-amino acid oxidase activity and its implications for schizophrenia. Mol Cell Neurosci 2006: 324-334.

S23. Hashimoto A, Oka T, Nishikawa T. Anatomical distribution and postnatal changes in endogenous free D-aspartate and D-serine in rat brain and periphery. Eur J Neurosci 1995; 7: 1657-1663.

S24. Kumashiro S, Hashimoto A, Nishikawa T. Free D-serine in post-mortem brains and spinal cords of individuals with and without neuropsychiatric diseases. Brain Res 1995; 681: 117-125.

S25. Schell MJ, Molliver ME, Snyder SH. D-Serine, an endogenous synaptic modulator - localization to astrocytes and glutamate-stimulated release. Proc Natl Acad Sci USA 1995; 92: 3948-3952.

S26. Schell MJ, Brady RO, Molliver ME, Snyder SH. D-Serine as a neuromodulator: Regional and developmental localizations in rat brain glia resemble NMDA receptors. J Neurosci 1997; 17: 1604-1615.

S27. Williams SM, Diaz CM, Macnab LT, Sullivan RKP, Pow DV. Immunocytochemical analysis of D-serine distribution in the mammalian brain reveals novel anatomical compartmentalizations in glia and neurons. Glia 2006; 53: 401-411.

S28. Hashimoto K, Sawa A, Iyo M. Increased levels of glutamate in brains from patients with mood disorders. Biol Psychiatry; In Press, doi 10.1016/j.biopsych.2007.03.017.

S29. Hashimoto K, Fukushima T, Shimizu E, Komatsu N, Watanabe H, Shinoda N, et al. Decreased serum levels of D-serine in patients with schizophrenia - Evidence in support of the N-methyl-D-aspartate receptor hypofunction hypothesis of schizophrenia. Arch Gen Psychiatry 2003; 60: 572-576.

S30. Hashimoto K, Engberg G, Shimizu E, Nordin C, Lindstrom LH, Iyo M. Reduced D-serine to total serine ratio in the cerebrospinal fluid of drug naive schizophrenic patients. Prog Neuropsychopharmacol Biol Psychiatry 2005; 29: 767-769.

S31. Wroblewska JT, Fadda E, Mazzetta J, Lazarewicz JW, Costa E. et al. Glycine and D-serine act as positive modulators of signal transduction at N-methyl-D-aspartate sensitive glutamate receptors in cultured cerebellar granule cells. Neuropharmacology 1989; 28: 447-452.

S32. Tsai GE, Yang P, Chang Y-C, Chong M-Y. D-Alanine added to antipsychotics for the treatment of schizophrenia. Biol Psychiatry 2006; 59: 230-234.

S33. Torrey EF, Barci BM, Webster MJ, Bartko JJ, Meador-Woodruff JH, Knable MB. Neurochemical markers for schizophrenia, bipolar disorder, and major depression in postmortem brain. Biol Psychiatry 2005; 57: 252-260.

S34. Xu B, Wratten N, Charych EI, Buyske S, Firestein BL, Brzustowic LM. Increased expression in dorsolateral prefrontal cortex of CAPON in schizophrenia and bipolar disorder. PLoS Med 2005; 2: 999-1007.

S35. Eastwood SL, Harrison PJ. Decreased mRNA expression of Netrin-G1 and Netrin-G2 in the temporal lobe in schizophrenia and bipolar disorder. Neuropsychopharmacology; 2008; 33; 933-945.