Download Increased D-amino acid oxidase

1
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.
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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
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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 50M Amplex Red, 0.125 units horse-radish peroxidase, and D-proline
(1-10mM) in a total volume of 10l. 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
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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 Dserine, 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
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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 Dserine 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
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genuine differential involvement of DAO in schizophrenia but not in bipolar disorder, a
possibility that merits further study.
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Supplementary References
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2001; 757: 119-125.
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receptor hypofunction hypothesis of schizophrenia. Arch Gen Psychiatry 2003; 60: 572-576.
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cultured cerebellar granule cells. Neuropharmacology 1989; 28: 447-452.
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Supplementary Table 1. Demographic details of subjects studied
Controls
Schizophrenia
Bipolar disorder
Number
35
35
34
Sex (M,F)
26/9
26/9
18/16
Age (y)
44.2 (1.3)
42.6 (1.4)
45.4 (1.8)
Brain pHa
6.61 (0.04)
6.47 (0.04)
6.43 (0.05)
RNA integrity (RIN)
6.15 (0.34)
6.43 (0.24)
6.21 (0.30)
Post mortem interval (h)
29.4 (2.2)
31.4 (2.6)
37.9 (3.2)
Time to refrigeration (h)
3.59 (0.47)
5.97 (0.72)
9.85 (1.8)
Smoker at death (Y/N/NK)
9/9/17
23/4/8
15/6/13
Onset of illness (y)
-
21.3 (1.0)
25.3 (1.6)
Duration of illness (y)
-
21.3 (1.7)
20.1 (1.7)
Total inpatient time (y)
-
1.23 (0.38)
0.53 (0.24)
Suicide (Y/N)
0/35
7/28
15/19
Psychotic symptoms (Y/N/NK)
0/35
35/35
21/10/2
Antipsychotic exposurec
0
85.0 (17)
10.2 (4.0)
b
Values are mean (SEM).
Y=yes, N=no, NK = not known. h=hours. m=months. y=years.
Different between groups (F2,101=4.174, p=0.018).
a
b
Different between groups (F2,97=7.288, p=0.001).
Lifetime exposure, fluphenazine equivalents (g).
c
See
http://www.stanleyresearch.org/dnn/BrainResearchLaboratoryBrainCollection/
ArrayCollection/ for further details of this brain series.
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Supplementary Table 2. Effects of 14 days’ antipsychotics on DAO activity in rat
cerebellum
Saline
Haloperidol
Clozapine
(1mg/kg/d)
(25mg/kg/d)
DAO Vmax
14.4 (1.3)
12.9 (0.8)
13.2 (0.5)
DAO Km
30.1 (1.6)
29.9 (3.5)
33.9 (7.6)
Values are mean (SEM). N=5 in each group. No differences between groups (all p>0.4).
Supplementary Table 3. DAO and G72/G30 genotype effects on DAO
activity and expression
Genotype
1/1
1/2
2/2
F, p
DAO rs2070587
2.80 (0.80) n=6
4.01 (0.50) n=30
3.98 (0.32) n=67
0.6, 0.56
DAO rs3741775
4.23 (0.56) n=22
3.97 (0.41) n=42
3.69 (0.42) n=39
0.3, 0.74
G72/G30 rs2391191 3.49 (0.62) n=15
4.31 (0.38) n=51
3.56 (0.41) n=37
1.1, 0.34
G72/G30 rs3918342 3.07 (0.50) n=22
4.11 (0.38) n=52
4.23 (0.46) n=29
1.5, 0.22
DAO activity
DAO mRNA
DAO rs2070587
0.62 (0.11) n=6
0.97 (0.13) n=29
0.94 (0.06) n=63
1.1, 0.34
DAO rs3741775
1.00 (0.12) n=22
0.93 (0.09) n=41
0.89 (0.05) n=35
0.3, 0.75
G72/G30 rs2391191 0.84 (0.11) n=14
0.96 (0.08) n=48
0.93 (0.09) n=36
0.2, 0.78
G72/G30 rs3918342 0.92 (0.08) n=20
0.94 (0.08) n=52
0.92 (0.10) n=26
0.1, 0.99
All genotypes are in Hardy-Weinberg equilibrium. In each case, allele 2 is the reported
schizophrenia risk allele.
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Supplementary Figure 1.
A-C: Scatterplots showing the relationship between DAO
activity (circles) and (A) post mortem interval (Pearson R=0.11, p=0.28, n=103), (B)
cerebellar pH (R=-0.06, p=0.52, n=103), or (C) antipsychotic exposure (R=0.01, p=0.93,
n=57). D-F: DAO mRNA (triangles), normalised to the mean of four housekeeping genes,
correlates inversely with: (D) delay from death-to-refrigeration (R=-0.22, p=0.03, n=94),
but not with (E) post mortem interval (R=-0.12, p=0.22, n=98), or (F) cerebellar pH (R=0.05, p=0.64, n=98). DAO mRNA was not related to antipsychotic exposure (R=-0.22,
p=0.21, n=55) or RIN (R=0.05, p=0.63, n=98) (data not shown).
B
A
12.5
DAO activity (Vmax)
DAO activity (Vmax)
12.5
10.0
7.5
5.0
2.5
10.0
7.5
5.0
2.5
0.0
5.8
0.0
0
12
24
36
48
60
72
84
6.0
6.2
C
6.6
6.8
7.0
D
Normalised DAO mRNA
12.5
DAO activity (Vmax)
6.4
Brain pH
Post mortem interval (h)
10.0
7.5
5.0
2.5
0.0
3
2
1
0
0
100
200
300
400
0
Antipsychotic exposure
(Lifetime fluphenazine equivalents, g)
E
20
30 54
F
3
Normalised DAO mRNA
Normalised DAO mRNA
10
Death to refrigeration (h)
2
1
0
0
12
24
36
48
60
Post mortem interval (h)
72
84
3
2
1
0
5.8
6.0
6.2
6.4
6.6
Brain pH
6.8
7.0