Mary A. Walker

d-Amino acid oxidase and serine racemase in human brain: normal distribution and altered expression in schizophrenia.

The N‐methyl‐d‐aspartate receptor co‐agonist d‐serine is synthesized by serine racemase and degraded by d‐amino acid oxidase. Both d‐serine and its metabolizing enzymes are implicated in N‐methyl‐d‐aspartate receptor hypofunction thought to occur in schizophrenia. We studied d‐amino acid oxidase and serine racemase immunohistochemically in several brain regions and compared their immunoreactivity and their mRNA levels in the cerebellum and dorsolateral prefrontal cortex in schizophrenia. d‐Amino acid oxidase immunoreactivity was abundant in glia, especially Bergmann glia, of the cerebellum, whereas in prefrontal cortex, hippocampus and substantia nigra, it was predominantly neuronal. Serine racemase was principally glial in all regions examined and demonstrated prominent white matter staining. In schizophrenia, d‐amino acid oxidase mRNA was increased in the cerebellum, and as a trend for protein. Serine racemase was increased in schizophrenia in the dorsolateral prefrontal cortex but not in cerebellum, while serine racemase mRNA was unchanged in both regions. Administration of haloperidol to rats did not significantly affect serine racemase or d‐amino acid oxidase levels. These findings establish the major cell types wherein serine racemase and d‐amino acid oxidase are expressed in human brain and provide some support for aberrant d‐serine metabolism in schizophrenia. However, they raise further questions as to the roles of d‐amino acid oxidase and serine racemase in both physiological and pathophysiological processes in the brain.

A postmortem study of the mediodorsal nucleus of the thalamus in schizophrenia

Four studies have reported that the mediodorsal nucleus of the thalamus (MD) is smaller and contains fewer neurons in schizophrenia. The MD is a key node in a circuit proposed to be dysfunctional in the disorder. However, one study did not find a MD volume loss in schizophrenia, and all the studies to date are relatively small. Given the importance of establishing unequivocally the presence of MD pathology, we have carried out a study of the volume and number of neurons in the left and right MD in 21 patients with schizophrenia and 27 healthy comparison subjects. We also measured the size of MD neurons, and estimated total thalamic volume. We found no difference in the volume of the MD, the number of MD neurons, or the size of MD neurons in either hemisphere in schizophrenia. Neither was total thalamic volume altered. There are no obvious methodological or clinical factors to explain our failure to replicate the finding of MD involvement in schizophrenia. Hence our negative observations, in the largest sample yet investigated, cast doubt on the robustness and/or the generalisability of MD neuropathology in schizophrenia.

The size and fiber composition of the anterior commissure with respect to gender and schizophrenia

BACKGROUND In light of evidence for deviations in asymmetry and alterations in the anatomy of the corpus callosum in schizophrenia, this study examined the anterior commissure in post mortem brains (n = 14 female control patients, 15 male control patients, 11 female schizophrenic patients, 15 male schizophrenic patients). METHODS Measures were made of the cross-sectional area of the anterior commissure in the midsagittal plane. In addition, the fiber density and fiber number were measured in a subset of cases (n = 10 female control subjects, 10 male control subjects, 8 female schizophrenic patients, 9 male schizophrenic patients), using the Palmgren silver stain and stereological methods. RESULTS In control subjects, fiber numbers were greater (p = .024) in women than men. In schizophrenia, the cross-sectional area was unaffected, but for fiber density there was a significant gender x diagnosis interaction (p = .026), corresponding to a reduction in female, but not male patients with schizophrenia. CONCLUSIONS The reduction in density of fibers in the anterior commissure is consistent with an alteration of interhemispheric connectivity in schizophrenia, but the restriction of the finding to women emphasizes the relevance of gender to understanding the nature of the hemispheric interaction.

D-amino acid oxidase activity and expression are increased in schizophrenia

D-amino acid oxidase (DAO, DAAO) metabolises the NMDA receptor (NMDAR) modulator D-serine.1 Enhanced DAO activity is thus a potential cause of reduced D-serine and thence impaired NMDAR functioning in schizophrenia,2 and an explanation for the genetic contribution that DAO may make to the disorder.3,4 Here we report that DAO activity, and expression, are increased in the cerebellum in schizophrenia, but not in bipolar disorder, and are not related to SNPs in DAO or its putative activator gene G72/G30.3 We measured DAO enzyme activity, and DAO mRNA by qRT-PCR, using established methods, in cerebellar tissue from subjects with schizophrenia, bipolar disorder, and controls, from the Stanley Microarray Collection (Supplementary Table 1). We genotyped subjects for two schizophrenia-associated tag SNPs in DAO and G72/G30. We used cerebellar tissue from rats administered haloperidol or clozapine for 14 days to investigate antipsychotic effects on DAO activity. For demographic and methodological details, see Supplementary Materials. As shown in Fig. 1A, DAO activity differed between groups (F2,100=4.79, p=0.010), being increased in schizophrenia compared to controls (+37%, p=0.027) and compared to bipolar disorder (+57%, p=0.004). The latter groups did not differ (p=0.45). DAO activity was not correlated with post mortem interval, brain pH, or antipsychotic exposure (Supplementary Fig. 1) or age (R=0.03, p=0.79). DAO activity was unrelated to alcohol or substance misuse history (using a five-point scale), smoking, sex, or suicide. DAO activity increased with duration of schizophrenia (R=0.34, p=0.05, n=35). Within the bipolar disorder group, DAO activity did not vary according to a history of psychotic symptoms. DAO activity correlated with DAO mRNA (R=0.43, p=0.0001, n=97). DAO affinity showed no group differences (Fig. 1B). DAO activity in rat cerebellum was unaffected by antipsychotics (Supplementary Table 2). Figure 1 Cerebellar DAO activity and expression in 35 control subjects (CON, white bars), 35 with schizophrenia (SCZ, black bars), and 34 with bipolar disorder (BPD, shaded bars). A) DAO activity (Vmax, nmol D-proline/min/mg protein) is higher in subjects with ... Normalised DAO mRNA differed between groups (F2,88=3.84, p=0.025), being increased compared to controls in schizophrenia (p=0.01) and bipolar disorder (p=0.04; Fig. 1C). DAO mRNA was not related to antipsychotic exposure, post mortem interval, brain pH, or RIN, but was affected by death-to-refrigeration interval (Supplementary Fig. 1). Neither DAO activity nor DAO mRNA were influenced by the DAO or G72/G30 SNPs (Supplementary Table 3), apart from a trend for higher DAO activity in allele 2 carriers of G72/G30 rs3918342 (p=0.08 vs 1/1 homozygotes). There were no genotype-by-diagnosis interactions. Our data show an elevation of DAO activity in schizophrenia accompanied by increased gene expression. The activity increase confirms the results of a pilot study,5 and the elevated DAO mRNA replicates findings in a separate cohort.6 There were no correlations of DAO activity or expression with medication exposure, and no effect of haloperidol or clozapine on DAO activity in rat brain. Other confounders (e.g. smoking) did not have a demonstrable influence either. Hence, within the constraints of a post mortem study, our findings appear related to the diagnosis of schizophrenia. We focused on the cerebellum because DAO is abundant and active therein; it will be important to ascertain whether DAO activity is affected elsewhere in schizophrenia, but problematic since forebrain DAO activity is extremely low, despite robust expression. Parenthetically, this discrepancy is complemented by a difference in the cellular localization of DAO: it is glial in the cerebellum, but mainly neuronal in the cerebral cortex.6 Elevated DAO activity will presumably enhance metabolism of D-serine and, other things being equal, contribute to a reduced synaptic availability of D-serine, potentially impairing NMDAR function.1 As such, our findings support the hypothesized involvement of DAO, and thence D-serine, in the NMDAR hypofunction of schizophrenia and its potential therapeutic amelioration.2,7,8 Clearly any such conclusions are tentative, since many other factors likely also influence D-serine availability, e.g. its synthesis by serine racemase,6,9 and its release and reuptake.10 Alterations in these processes could counteract – or exacerbate – enhanced DAO activity. Moreover, since DAO also metabolises other D-amino acids,1 D-serine is not the only substrate that might be affected by an increase in DAO activity. For example, D-alanine is present in the cerebellum, is an NMDAR modulator, and may be therapeutically beneficial in schizophrenia.7 Overall, whilst a primary effect on D-serine, and thence NMDARs, is an attractive interpretation of the DAO increase in schizophrenia, further studies are needed to confirm the biochemical consequences, as well as the causes, of the elevation. (See Supplementary Materials for further discussion). As the DAO and G72/G30 SNPs did not influence DAO activity or expression, our data provide no evidence that they are functional (nor indexing SNPs that are), nor have we identified a mechanism whereby variation in these genes might confer schizophrenia susceptibility. There may be other SNPs that are relevant in this respect; alternatively, the DAO SNPs might operate at other places and times, whilst G72/G30 SNPs could act via a DAO-independent pathway. In summary, DAO activity and expression are increased in schizophrenia, but not related to DAO or G72/G30 genotype. Any pathophysiological consequences of increased DAO activity seem most likely to impact on D-serine metabolism and thence NMDAR function, but this remains to be established.

Early parental deprivation in the marmoset monkey produces long-term changes in hippocampal expression of genes involved in synaptic plasticity and implicated in mood disorder.

In mood disorder, early stressors including parental separation are vulnerability factors, and hippocampal involvement is prominent. In common marmoset monkeys, daily parental deprivation during infancy produces a prodepressive state of increased basal activity and reactivity in stress systems and mild anhedonia that persists at least to adolescence. Here we examined the expression of eight genes, each implicated in neural plasticity and in the pathophysiology of mood disorder, in the hippocampus of these same adolescent marmosets, relative to their normally reared sibling controls. We also measured hippocampal volume. Early deprivation led to decreases in hippocampal growth-associated protein-43 (GAP-43) mRNA, serotonin 1A receptor (5-HT1AR) mRNA and binding ([3H]WAY100635), and to increased vesicular GABA transporter mRNA. Brain-derived neurotrophic factor (BDNF), synaptophysin, vesicular glutamate transporter 1 (VGluT1), microtubule-associated protein-2, and spinophilin transcripts were unchanged. There were some correlations with in vivo biochemical and behavioral indices, including VGluT1 mRNA with reward-seeking behavior, and serotonin 1A receptor mRNA with CSF cortisol. Early deprivation did not affect hippocampal volume. We conclude that early deprivation in a nonhuman primate, in the absence of subsequent stressors, has a long-term effect on the hippocampal expression of genes implicated in synaptic function and plasticity. The reductions in GAP-43 and serotonin 1A receptor expressions are comparable with findings in mood disorder, supporting the possibility that the latter reflect an early developmental contribution to disease vulnerability. Equally, the negative results suggest that other features of mood disorder, such as decreased hippocampal volume and BDNF expression, are related to different aspects of the pathophysiological process.

Reduced density of calbindin-immunoreactive interneurons in the planum temporale in schizophrenia

Reduced density of calbindin-containing interneurons in the prefrontal cortex in schizophrenia has been reported (Beasley et al 2002; Biol Psych 52:708-715). Calbindin is a calcium-binding protein (CBP) present in a subpopulation of GABAergic neurons restricted mainly to layer II of the cortex. A paraffin-embedded, 10-mum-thick section from the planum temporale (PT) of each hemisphere was prepared from 12 patients with schizophrenia and 12 controls. Calbindin-containing cells were stained using an antibody (D-28K). Counting frames were superimposed to sample within layer II of the PT. A bilateral reduction (20%) in calbindin cell density was found in patients (controlling for fixation time). Furthermore, mean calbindin cell cross-sectional area was increased in female patients and reduced in male patients. Reduced CBP expression (reducing the excitability of interneurons) or reduced number of CBP-containing cells may cause disinhibition of pyramidal cells. The majority of calbindin-containing cells in the mature brain are double-bouquet cells with vertically oriented dendrites and axon bundles. By exercising inhibitory modulation of pyramidal cells in a columnar arrangement, they make possible cohesive vertical inhibition of minicolumns. Loss of columnar inhibition may result in reduced minicolumnar segregation and altered cell size may reflect altered minicolumn size.

Reduced neuron density, enlarged minicolumn spacing and altered ageing effects in fusiform cortex in schizophrenia

Structural and functional MRI studies report reduced volume and activation of the fusiform gyrus in schizophrenia. The fusiform cortex is involved in object naming and face recognition. Neuron cell size, shape and density, glial cell density and minicolumn spacing in layers III and V of the fusiform cortex were assessed following systematic random sampling from 13 controls and 11 schizophrenic patients. Pyramidal cell density was reduced in schizophrenia. Non-pyramidal cell density was reduced in layer III of the left hemisphere in schizophrenia, mostly in females. Non-pyramidal cells were larger in schizophrenia. Glial cell density was unaltered. Fusiform minicolumn spacing was asymmetrically wider in the right hemisphere of normal control subjects. Minicolumns were less dense in schizophrenia, particularly in the left hemisphere of females and the right hemisphere of males. Reduced neuron density in the fusiform cortex in schizophrenia contributes to evidence of functional-anatomical abnormalities from neuroimaging and neuropathology studies. Anatomical sex differences in schizophrenia may relate to anatomical and cognitive sex differences associated with fusiform cortex in the normal population. Wider minicolumn spacing is consistent with reduced cell density and is linked to altered ageing in schizophrenia.

The DISC1 Ser704Cys substitution affects centrosomal localization of its binding partner PCM1 in glia in human brain

Disrupted-in-schizophrenia 1 (DISC1) has been genetically associated with schizophrenia, and with brain phenotypes including grey matter volume and working memory performance. However, the molecular and cellular basis for these associations remains to be elucidated. One potential mechanism may be via an altered interaction of DISC1 with its binding partners. In this context, we previously demonstrated that one DISC1 variant, Leu607Phe, influenced the extent of centrosomal localization of pericentriolar material 1 (PCM1) in SH-SY5Y cells. The current study extends this work to human brain, and includes another DISC1 coding variant, Ser704Cys. Using immunohistochemistry, we first characterized the distribution of PCM1 in human superior temporal gyrus (STG). PCM1 immunoreactivity was localized to the centrosome in glia, but not in neurons, which showed widespread immunoreactivity. We quantified centrosomal PCM1 immunoreactivity in STG glia of 81 controls and 67 subjects with schizophrenia, genotyped for the two polymorphisms. Centrosomal PCM1 immunoreactive area was smaller in Cys704 carriers than in Ser704 homozygotes, with a similar trend in Phe607 homozygotes compared with Leu607 carriers, replicating the finding in SH-SY5Y cells. No differences were seen between controls and subjects with schizophrenia. These findings confirm in vivo that DISC1 coding variants modulate centrosomal PCM1 localization, highlight a role for DISC1 in glial function and provide a possible cellular mechanism contributing to the association of these DISC1 variants with psychiatric phenotypes. Whether this influence of DISC1 genotype extends to other centrosomal proteins and DISC1 binding partners remains to be determined.

Metabotropic glutamate receptor 3 (mGlu3; mGluR3; GRM3) in schizophrenia: Antibody characterisation and a semi-quantitative western blot study

Background Metabotropic glutamate receptor 3 (mGlu3, mGluR3), encoded by GRM3, is a risk gene for schizophrenia and a therapeutic target. It is unclear whether expression of the receptor is altered in the disorder or related to GRM3 risk genotype. Antibodies used to date to assess mGlu3 in schizophrenia have not been well validated. Objective To characterise six commercially available anti-mGlu3 antibodies for use in human brain, and then conduct a semi-quantitative study of mGlu3 immunoreactivity in schizophrenia. Methods Antibodies tested using Grm3−/− and Grm2−/−/3−/− mice and transfected HEK293T/17 cells. Western blotting on membrane protein isolated from superior temporal cortex of 70 patients with schizophrenia and 87 healthy comparison subjects, genotyped for GRM3 SNP rs10234440. Results One (out of six) anti-mGlu3 antibodies was fully validated, a C-terminal antibody which detected monomeric (~ 100 kDa) and dimeric (~ 200 kDa) mGlu3. A second, N-terminal, antibody detected the 200 kDa band but also produced non-specific bands. Using the C-terminal antibody for western blotting in human brain, mGlu3 immunoreactivity was found to decline with age, and was affected by pH and post mortem interval. There were no differences in monomeric or dimeric mGlu3 immunoreactivity in schizophrenia or in relation to GRM3 genotype. The antibody was not suitable for immunohistochemistry. Interpretation These data highlight the value of knockout mouse tissue for antibody validation, and the need for careful antibody characterisation. The schizophrenia data show that involvement of GRM3 in the disorder and its genetic risk architecture is not reflected in total membrane mGlu3 immunoreactivity in superior temporal cortex.

Altered hippocampal gene expression and structure in transgenic mice overexpressing neuregulin 1 ( Nrg1 ) type I

Transgenic mice overexpressing the type I isoform of neuregulin 1 (Nrg1; NRG1) have alterations in hippocampal gamma oscillations and an age-emergent deficit in hippocampus-dependent spatial working memory. Here, we examined the molecular and morphological correlates of these findings. Microarrays showed over 100 hippocampal transcripts differentially expressed in Nrg1tg-type I mice, with enrichment of genes related to neuromodulation and, in older mice, of genes involved in inflammation and immunity. Nrg1tg-type I mice had an enlarged hippocampus with a widened dentate gyrus. The results show that Nrg1 type I impacts on hippocampal gene expression and structure in a multifaceted and partly age-related way, complementing the evidence implicating Nrg1 signaling in aspects of hippocampal function. The findings are also relevant to the possible role of NRG1 signaling in the pathophysiology of schizophrenia or other disorders affecting this brain region.