Carmit Nadri

A CSF and postmortem brain study of D-serine metabolic parameters in schizophrenia

Clinical trials demonstrated that D-serine administration improves schizophrenia symptoms, raising the possibility that altered levels of endogenous D-serine may contribute to the N-methyl D-aspartate receptor hypofunction thought to play a role in the disease. We hypothesized that cerebro-spinal fluid (CSF) D-serine levels are decreased in the patients due to reduced synthesis and/or increased degradation in brain. We now monitored amino acid levels in CSF from 12 schizophrenia patients vs. 12 controls and in postmortem parietal-cortex from 15 control subjects and 15 each of schizophrenia, major-depression and bipolar patients. In addition, we monitored postmortem brain serine racemase and D-amino acid oxidase protein levels by Western-blot analysis. We found a 25% decrease in D-serine levels and D/L-serine ratio in CSF of schizophrenia patients, while parietal-cortex D-serine was unaltered. Levels of L-serine, L-glutamine and L-glutamate were unaffected. Frontal-cortex (39%) and hippocampal (21%) serine racemase protein levels and hippocampal serine racemase/D-amino acid oxidase ratio (34%) were reduced. Hippocampal D-amino-acid-oxidase protein levels significantly correlated with duration of illness (r=0.6, p=0.019) but not age. D-amino acid oxidase levels in patients with DOI>20 years were 77% significantly higher than in the other patients and controls. Our results suggest that reduced brain serine racemase and elevated D-amino acid oxidase protein levels may contribute to the lower CSF D-serine levels in schizophrenia.

GSK-3 parameters in postmortem frontal cortex and hippocampus of schizophrenic patients

The protein kinase glycogen synthase kinase-3 (GSK-3) is highly abundant in brain and involved in signal transduction cascades, particularly during neurodevelopment. We have previously found reduced GSK-3beta mRNA levels, protein levels and GSK-3 total (alpha+beta isoforms) activity in postmortem frontal cortex of schizophrenic patients in the Stanley Medical Research Institutes Brain Collection. To verify and extend these findings, GSK-3 parameters were now measured in the frontal cortex (BA9) and hippocampus obtained from the Rebecca L. Cooper Research Laboratories postmortem brain collection. Fifteen pairs of schizophrenic patients and matched control subjects have been studied. No significant differences in GSK-3alpha and GSK-3beta mRNA levels, GSK-3beta protein levels or total GSK-3 (alpha+beta) activity were found in the frontal cortex of the two diagnostic groups. Hippocampal GSK-3alpha and GSK-3beta mRNA levels were significantly lower (22% and 28%, respectively) in the tissue from the schizophrenic patients compared with the normal controls. Hippocampal GSK-3beta protein levels in the schizophrenic patients were 24% significantly lower than control values only after omission of three outlier subjects. Hippocampal total GSK-3 (alpha+beta) activity in the patients was 31% lower in the schizophrenic patients vs. control subjects. This difference was marginally significant. While our previous data on GSK-3beta in postmortem brain and the recent report that there is impaired AKT1-GSK-3beta signaling in schizophrenia suggest that changes in pathways involving protein kinases such as AKT1 and GSK-3beta in schizophrenia are complex, our present data do not provide strong evidence in support of the involvement of GSK-3beta in schizophrenia. Therefore, further investigation in a greater number of brain samples is warranted to better clarify the possible role of this enzyme in the pathophysiology of schizophrenia.

Low GSK-3β in schizophrenia as a consequence of neurodevelopmental insult

Glycogen synthase kinase-3 (GSK-3) is a protein kinase highly abundant in brain and involved in signal transduction cascades, particularly neurodevelopment. Its activity and protein levels have been reported to be over 40% lower in postmortem frontal cortex of schizophrenic patients. GSK-3beta in occipital cortex of schizophrenic patients was not reduced, suggesting regional specificity. There was no reduction in GSK-3beta protein levels in fresh and immortalized lymphocytes and both GSK-3 activity and GSK-3beta mRNA levels in fresh lymphocytes from schizophrenic patients. In the schizophrenia-related neonatal ventral hippocampal lesion rat model, we measured GSK-3beta protein levels and GSK-3 activity in the frontal cortex. GSK-3beta protein levels in lesioned rats were significantly lower than in sham rats, favoring perinatal insult as a cause of low GSK-3beta in schizophrenia. Taken together, these studies suggest that low GSK-3 in postmortem brain of schizophrenic patients is a late consequence of perinatal neurodevelopmental insult in schizophrenia. In rats, acute or chronic cold restraint stress did not change GSK-3beta protein levels. Chronic treatment of rats with lithium, valproate, haloperidol or clozapine did not change rat cortical GSK-3beta protein levels ex vivo, supporting the concept that low GSK-3beta in schizophrenia is not secondary to stress or drug treatment. Our initial findings of low GSK-3beta protein levels in postmortem brain have been replicated by another group. Our own group has found additionally that GSK-3beta mRNA levels were 40% lower in postmortem dorsolateral prefrontal cortex (DLPFC) of schizophrenic patients, supporting our previous findings. Further studies will be aimed at determining whether nonspecific neonatal damage or only specific factors cause low GSK-3 as a late effect. We plan to study whether low GSK-3beta activity is associated with biochemical effects such as elevated beta-catenin levels.

Glycogen synthase kinase (GSK)-3β levels and activity in a neurodevelopmental rat model of schizophrenia

We have previously reported reduced GSK-3beta protein levels and GSK-3 total (alpha + beta isoforms) activity in postmortem frontal cortex of schizophrenic patients. We now studied whether GSK-3beta is altered in the frontal cortex of rats with the neonatal excitotoxic hippocampal lesion used as a model of schizophrenia. Rats were infused with ibotenic acid (or artificial CSF in controls) bilaterally into the ventral hippocampus (VH) at postnatal day 7, then killed at postnatal day 35 (pre-puberty) or 56 (post-puberty). GSK-3beta protein levels were reduced in the frontal cortex of the lesioned rats as compared to sham animals; post-hoc comparisons revealed that the reduction was statistically significant at a pre-pubertal age. Total GSK-3 (alpha + beta) activity was not different between lesioned and sham rats at any age. These results demonstrate that reduced frontal cortical GSK-3beta levels may occur as a result of neonatal hippocampal damage and suggest that this animal model may be utilized to study the mechanism of GSK-3 reduction in schizophrenia, a disorder in which postmortem changes in GSK-3 were found.

Lack of effect of mood stabilizers or neuroleptics on GSK-3β protein levels and GSK-3 activity

Glycogen synthase kinase (GSK)-3 protein levels and GSK-3 activity were previously found to be over 40% reduced in the post-mortem prefrontal cortex of schizophrenic patients. Lithium and valproate have been reported to selectively inhibit GSK-3. We hypothesized that in-vivo administration of lithium and valproate would result in up-regulation of GSK-3 protein levels and GSK-3 activity. The present study aimed to evaluate the possible involvement of neuroleptic treatment in the decrease of GSK-3 in schizophrenia. Rat frontal cortex GSK-3 protein levels and GSK-3 activity were measured following administration of therapeutic doses of lithium or valproate for 11 d, or of haloperidol, chlorpromazine or clozapine for 21 d. None of the drugs induced a change in GSK-3 protein levels. All the drugs except chlorpromazine (which was not tested) did not affect GSK-3 activity. This suggests that GSK-3 inhibition by lithium or valproate does not induce regulation of protein levels or activity and that the reduction in GSK-3 protein levels and GSK-3 activity in the post-mortem prefrontal cortex of schizophrenic patients is not neuroleptic-treatment related.

GSK-3 parameters in lymphocytes of schizophrenic patients

Glycogen synthase kinase-3 (GSK-3) is a highly conserved serine/threonine protein kinase that is involved in the signal transduction cascades of multiple cellular processes. GSK-3 has two isoforms, designated alpha and beta. GSK-3beta protein levels and GSK-3 enzyme activity have been reported to be reduced by over 40% in postmortem frontal cortex of schizophrenic patients. GSK-3 is also present in peripheral tissue such as lymphocytes. In this study we aimed to find whether the reduction in brain GSK-3beta measures is reflected in peripheral tissue of schizophrenic patients. Fresh lymphocytes from schizophrenic patients showed no difference in GSK-3 alpha and GSK-3beta mRNA levels, GSK-3beta protein levels, or total GSK-3 (alpha+beta) enzyme activity compared with findings in control subjects. In addition, lymphocyte-derived cell lines from schizophrenic patients did not differ in their GSK-3beta protein levels from levels in normal control subjects. The results rule out the use of lymphocyte GSK-3 as a marker for central GSK-3 abnormalities in schizophrenia.

Oxygen restriction of neonate rats elevates neuregulin-1α isoform levels: Possible relationship to schizophrenia

Neuregulin-1 (NRG-1), a replicated gene in schizophrenia-association studies, exhibits six mRNA-types and two types of the EGF-like domain, alpha and beta. The beta-isoform was extensively studied, less is known about the extent and specific localization of adult brain NRG-1alpha. NRG-1alpha protein levels were reported reduced in postmortem prefrontal-cortex of schizophrenia patients. NRG-1 type I mRNA levels were found higher in postmortem brain in schizophrenia. In an attempt to decipher between a genetic or environmental involvement in the differences in NRG-1 levels in postmortem brain in schizophrenia, and since obstetric complications were suggested non-genetic risk-factors of schizophrenia, we studied the effect of perinatal hypoxia in rats on brain NRG-1alpha protein levels. Seven-day-old rats were exposed to hypoxia versus air. Frontal-cortex levels of NRG-1alpha isoform were quantified at adulthood by Western blotting. Frontal-cortex NRG-1alpha was 32% elevated in hypoxia-exposed rats. The data support the role of non-genetic factors, e.g. oxygen restriction, in the expression of genes associated with schizophrenia.

Genetic correlational analysis of glycogen synthase kinase-3beta and prepulse inhibition in inbred mice

In humans, GSK‐3β activity is diminished in schizophrenic patients as is prepulse inhibition of the startle response (PPI). We performed a genetic correlational analysis between published PPI values and frontal cortex GSK‐3 activity analyzed in our laboratory in 10 inbred mouse strains. This methodology could indicate relevant parameters for study in an animal model. Indeed, we obtained significant correlations between the enzymes activity and PPI measured by two different methods. This may indicate that investigation of the genetics of GSK‐3β regulation holds promise for understanding some of the biochemical underpinnings of schizophrenia.

Elevated urinary ADAM12 protein levels in lithium-treated bipolar patients

SummaryADAM (A Disintegrin And Metalloprotease)12 is a member of a family of integral membrane and secreted glycoproteins. ADAM12 has recently been detected in urine. In the present study we measured ADAM12 protein levels in urine from bipolar patients vs. healthy controls.Nineteen bipolar patients and 22 matched-control subjects were studied. Urine samples were concentrated and Western-blot analysis used to determine ADAM12 protein levels.The 92 kDa form of urine ADAM12 protein levels were highly elevated in Li-treated bipolar patients compared with normal controls. The 68 kDa form of urine ADAM12 protein levels did not differ. Future experiments are needed to explore a potential link between ADAM12 protein level elevation and lithium response.