A mitochondrial DNA sequence variant associated with schizophrenia and oxidative stress.

We have previously reported a changed mitochondrial (mt) gene expression in brain from patients with schizophrenia [Schizophr. Res. 14 (1995) 203]; now, we describe the distribution in the mtDNA from lymphocytes of a heteroplasmic sequence variation that was originally found in the mtDNA from the postmortem brain of a patient with schizophrenia. The variant is m.12027T>C and results in the change from isoleucine to threonine at position 423 of the ND4 subunit of NADH-ubiquinone reductase. Using a PCR-RFLP method, we have determined the heteroplasmy as the ratio of variant to total (variant ratio) at m.12027 in 184 controls and 181 patients with schizophrenia as well as 24 postmortem brain samples. The distribution of variants is bimodal having peaks at variant ratios of 0.262 and 0.732. The variant-rich fraction is very significantly associated with schizophrenia in males (47%), while there is only 18% in control males. There are significantly more variant-rich control females (36%) than control males (18%), suggesting that the female population is less sensitive to the presence of a variant in terms of liability to schizophrenia. In variant-rich samples from postmortem brain originating from both sexes, there is an increased superoxide production, suggesting that the variation contributes to oxidative stress. Antioxidant glycosides, such as quercetin rutoside, quench the superoxide production without (in contrast to neuroleptic drugs) interfering with the electron transfer activity of the reductase.

Circadian regulation of prion protein messenger RNA in the rat forebrain: a widespread and synchronous rhythm.

Although the expression of the normal prion protein in the host is critical to the development of transmissible spongiform encephalopathies, the physiological role of this protein and the processes regulating its expression remain obscure. We now report that the messenger RNA for the prion protein is regulated in the rat brain in a marked circadian manner not only in the suprachiasmatic nuclei, the principal site for the generation of mammalian circadian rhythms, but also in other forebrain regions. The data show a remarkable consistency in the concurrence of a single peak of prion protein messenger RNA at each of the sites early in the animal's phase of increased locomotor activity; behavioural arousal does not, however, appear to affect this expression. We believe this to be the first study demonstrating that the expression of prion protein messenger RNA can change over a relatively short period in vivo. The results are discussed with reference to the range of recently discovered "clock-related" transcripts which also have widespread tissue expression; these include the messenger RNAs for D-box binding protein and thyroid embryonic factor, transcription factors which bind to the prion protein promoter.

Superoxide, neuroleptics and the ubiquinone and cytochrome b5 reductases in brain and lymphocytes from normals and schizophrenic patients.

The effects of the neuroleptic flupenthixol on the expression of the genes coding for the mitochondrial ubiquinone and cytochrome b5 reductases have been studied because of the importance of these enzymes in energy metabolism, oxidative stress and also because similar but oppositely directed changes have been previously observed in the cerebral cortex from schizophrenics. The neuroleptic flupenthixol reduces the expression in rats of the gene coding for NADH-cytochrome b5 reductase as measured by in situ hybridisation and its enzymic manifestation. Flupenthixol also reduces the enzymic activity of the mitochondrial NADH-ubiquinone reductase, and it has been previously shown that mRNA from the mitochondrially coded parts of the enzyme are reduced by the drug. Both the cis- and therapeutically less active trans-flupenthixol were found to produce these changes in rats. Post-mortem brain tissue from schizophrenics who have received neuroleptic medication have reduced levels of both reductases as measured enzymically, Lymphocyte samples from schizophrenics also have reduced levels of both reductases compared with normals. The superoxide anion O2- is the principle agent of oxidative stress and both the cytochrome b5 and the ubiquinone reductase enzymes were semi-purified from sheep liver and shown to produce appreciable amounts of superoxide. Superoxide production is reduced in brain homogenates from rats treated with flupenthixol. Its production is also reduced in brain tissue and lymphocytes from schizophrenics receiving neuroleptic medication. We conclude that neuroleptic medication reduces the expression of both the ubiquinone and cytochrome b5 reductase and among the effects of this reduction is a decrease in the production of neurotoxic superoxide.

Phosmet induces up-regulation of surface levels of the cellular prion protein.

Chronic (2 day) exposure of human neuroblastoma cells to the organophosphate pesticide phosmet induced a marked concentration-dependent increase in the levels of PrP present on the cell surface as assessed by biotin labelling and immunoprecipitation. Levels of both phospholipase C (PIPLC)-releasable and non-releasable forms of PrP were increased on the plasma membrane. These increases appear to be due to post-transcriptional mechanisms, since PrP mRNA levels as assessed by Northern blotting were unaffected by phosmet treatment. These data raise the possibility that phosmet exposure could increase the susceptibility to the prion agent by altering the levels of accessible PrP.

Mitochondrial involvement in schizophrenia and other functional psychoses.

Gene expression has been studied in post-mortem frontal cortex samples from patients who had suffered from schizophrenia and depressive illness. mRNA was extracted and characterised by translation and separation of the products by 2D gel electrophoresis. Post-mortem artefacts and the agonal experience did not affect the size distribution or amount of specific translation products. Four expression products were specifically reduced in samples from schizophrenics compared with normals. The expression of six products was altered in affective disorder, one in common with schizophrenia, two the same as in schizophrenia but increased. cDNA libraries were produced from the mRNA samples and 5 clones present at abnormal levels in schizophrenia identified by differential screening, isolated and sequenced. All the sequences encode mitochondrial transcripts; four encode mitochondrial rRNA and one the amino acid sequence of cytochrome oxidase sub-unit II. Increased cytochrome oxidase transcripts were found in a further set of mRNA extracts from schizophrenic patients including two who had not received neuroleptic medication. The effects of neuroleptic administration as exemplified by alpha-flupenthixol compared with the ineffective beta-flupenthixol were studied in experimental animals. It was found that 13 out of 28 clones whose levels were altered were mitochondrial in origin including rRNA, COX I & II and the NADH-Q reductase. Those encoding respiratory enzymes were at abnormally low levels as a result of alpha-flupenthixol administration. Measurements of the enzymic activity of cytochrome c oxidase in post-mortem frontal cortex of schizophrenics did not indicate any differences in overall activity but there was a decreased sensitivity to azide that was abolished by neuroleptics. Studies on NADH-cytochrome c reductase showed that schizophrenics whether medicated or not had a reduced rotenone sensitive activity that was compensated for by increased rotenone insensitive activity. We conclude that changes in mitochondrial gene expression are involved in schizophrenia and probably other functional psychoses.

Effects of sex steroids on protein synthesis in cultured human lymphocytes.

In cultured human lymphocytes, oestrogen and progesterone at concentrations found in plasma during the normal menstrual cycle, significantly increase the incorporation of [35S] methionine into protein and, in addition, both hormones significantly alter the relative synthesis of certain proteins. At concentrations found in plasma during pregnancy, some changes are augmented while others are reversed. These specific sex-steroid-induced changes in protein synthesis provide possible peripheral biological markers of hormone action which may be tested for their association with predisposition to, and/or onset of, conditions such as postpartum psychiatric illness.

Regulation of intracellular free calcium levels by the cellular prion protein.

A rat brain synaptosomal model was used to investigate the possible role of the cellular prion protein (PrP) in the regulation of intracellular free calcium levels ([Ca2+i). Treatment of synaptosomes with bacterially derived recombinant human PrP in the range 20-100 micrograms ml-1 resulted in dose-dependent elevations of [Ca2+]i. These increases were dependent on extracellular calcium and were inhibited by gadolinium chloride, a potent blocker of voltage-sensitive calcium channels. Conversely, when calcium channels were activated by synaptosomal depolarization, treatment with monoclonal antibody to PrP in the range 200-320 ng IgG ml-1 resulted in a dose-dependent reduction of [Ca2+i, which was blocked by competition with PrP preparations. These results indicate that PrP is associated with regulation of intracellular free calcium levels through an interaction with voltage-sensitive calcium channels.

Genetic linkage analysis of schizophrenia using chromosome 11q13-24 markers in Israeli pedigrees.

It is generally agreed that there is a genetic component in the etiology of schizophrenia which may be tested by the application of linkage analysis to multiply-affected families. One genetic region of interest is the long arm of chromosome 11 because of previously reported associations of genetic variation in this region with schizophrenia, and because of the fact that it contains the locus for the dopamine D2 receptor gene. In this study we have examined the segregation of schizophrenia with microsatellite dinucleotide repeat DNA markers along chromosome 11q in 5 Israeli families multiply-affected for schizophrenia. The hypothesis of linkage under genetic homogeneity of causation was tested under a number of genetic models. Linkage analysis provided no evidence for significant causal mutations within the region bounded by INT and D11S420 on chromosome 11q. It is still possible, however, that a gene of major effect exists in this region, either with low penetrance or with heterogeneity.

A study of altered gene expression in frontal cortex from schizophrenic patients using differential screening.

In order to examine the molecular basis of schizophrenia we have employed a sequential differential hybridisation protocol to isolate mRNAs whose abundances are altered in schizophrenic compared to normal frontal cortex. Five cDNAs present at abnormal levels in the schizophrenic brain have been isolated by this method. The sequences were identified on the basis of homologies in the EMBL and Genbank databases. All the sequences encode mitochondrial transcripts; one encodes part of the 12s rRNA, three encode parts of the 16s rRNA region of the mitochondrial genome whilst the fourth encodes part of the amino acid sequence of cytochrome oxidase sub-unit II. It was established that mitochondrial sequences were not over-represented in the library and that this could therefore not account for the isolation of five mitochondrial transcripts by this procedure. Increased levels of cytochrome oxidase mRNA were detected in a further set of extracts from the frontal cortex of eight schizophrenic patients and five controls. The amount of mt-DNA was measured in these samples but there was no difference between schizophrenic and control. These results indicate a possible abnormality of mitochondrial function in the schizophrenic frontal cortex.

Prion protein immunocytochemistry helps to establish the true incidence of prion diseases.

Creutzfeldt-Jakob disease (CJD) and Gerstmann-Strüssler-Scheinker disease (GSSD) are transmissible spongiform encephalopathies or prion diseases affecting man. It has been reported that prion diseases may occur without the histological hallmarks of spongiform encephalopathies: vacuolation of the cerebral grey matter, neuronal loss and astrocytosis. These cases without characteristic neuropathology may go undiagnosed and consequently the true incidence of transmissible dementias is likely to have been under-estimated. Immunocytochemistry using antibodies to prion protein gives positive staining of these cases, albeit the pattern of immunostaining differs from that seen in typical forms. Accumulation of prion protein is a molecular hallmark of prion diseases, and thus a reproducible, speedy and cost-efficient immunocytochemical screening of unusual dementias may help to establish the true incidence of prion diseases.

Analysis of relative mRNA levels and protein patterns in brains of rat strains bred for differing levels of emotionality.

mRNA and protein populations were studied in the brains of Maudsley reactive (MR) and Maudsley nonreactive (MNR) rat strains, which exhibit differing levels of emotionality. Translational analysis of forebrain mRNA indicated that the relative levels of two translation products (42 kDA, pI 5.0; 30 kDa, pI 5.8) were increased in the MR compared to the MNR strain. In addition, a charge-shift variant of a 36 kDa protein was present in the MR strain. Analysis of brain protein patterns indicated that a protein of 39 kDa, pI 5.0, was found to be more abundant in MR compared with MNR strains in both frontal cortex and hippocampus and the relative level of one protein (40 kDa, pI 5.8) was decreased in the frontal cortex.

Changes in brain gene expression in schizophrenic and depressed patients.

Poly(A+) mRNA was extracted from the post-mortem brain of schizophrenics (9 subjects), unipolar depressives (5 subjects) and controls (10 subjects) and used to direct the in vitro translation of radiolabelled protein in a cell-free reticulocyte-lysate system. Protein species were analysed on two-dimensional gels. Over 200 products were detected and, from these, 74 well-resolved species were chosen for further analysis. The optical density of each product was quantified by image analysis and normalised with respect to overall gel intensity. It was found that 7 novel, uncharacterised protein species, ranging from molecular weights (Mr) 17 kDa to 38 kDa and apparent isoelectric points (pI) 5.7-7.1, changed significantly in intensity in the psychotic groups compared to controls. One species changed only in the schizophrenia group (Mr = 26 kDa, pI = 5.8, 18% of control intensity) and 3 changed only in the depressive group (Mr = 38 kDa, pI = 6.2, 540% of control; Mr = 34 kDa, pI = 6.2, 6% of control; Mr = 17 kDa, pI = 5.7, 238% of control). Three further protein species were common to both psychotic groups (one species decreased in both schizophrenia and depression, Mr = 33 kDa, pI = 5.8; two species showed opposing intensity changes, decreasing in schizophrenia and increasing in depression, Mr = 35 kDa, pI = 7.1; Mr = 23 kDa, pI = 6.1). None of these changes was a function of post-mortem delay or mode of death. It is quite likely that such protein species reflect the abundance of specific mRNAs and target gene systems associated with the disease state.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in relative levels of specific brain mRNA species associated with schizophrenia and depression.

Total cellular polyadenylated RNA (poly(A)+ RNA, mRNA) was prepared after guanidinium thiocyanate extraction of frozen brain tissue from age-matched controls and patients suffering from schizophrenia and unipolar depression. These mRNA populations were analysed by in vitro translation followed by two-dimensional gel analysis. Data were obtained from fluorograms derived from 10 different schizophrenic patients, 10 different controls and 5 different depressive patients. The relative concentrations of mRNA species coding for 4 translation products (33 kDa, pI 5.8; 26 kDa, pI 5.8; 35 kDa, pI 7.1; 23 kDa, pI 6.1) were significantly reduced in schizophrenia compared to controls when determined by computerised image analysis of the fluorograms. In the case of depression, the relative concentrations of mRNA species coding for 6 translation products were significantly altered, 4 being increased (38 kDa, pI 6.2, 17 kDa, pI 5.7, 35 kDa, pI 7.1; 23 kDa, pI 6.1) and two decreased (34 kDa, pI 6.2; 33 kDa, pI 5.8). Three translation products were altered in both schizophrenia and depression, one (33 kDa, pI 5.8) being altered according to the same trend, a decrease relative to controls, but two (35 kDa, pI 7.1; 23 kDa, pI 6.1) being altered differently in schizophrenia (reduced) and depression (increased). The effects of post mortem delay, mode of death and drug treatment on mRNA composition were also examined and found not to affect the levels of these translation products significantly. The significance of these changes will be discussed in relation to their relevance of biological mechanisms in the psychoses.

An estimation of the sensitivity of in vitro translation using two-dimensional gel analysis.

Poly (A+ mRNA species, isolated from 100-day-old rat brain, were analysed by in vitro translation and two-dimensional gel electrophoresis. The synthesis of selected protein species was compared to actin on the basis of [35S]methionine incorporation. The estimated molar abundance of translation products varied from abundant species at 0.78% of the total to several are species, detectable below the 0.02% level. If these synthesis rates reflect the abundance of particular mRNAs in the mixture, this sensitivity limit compares well with accepted values using differential cDNA screening techniques. This analysis provides evidence that in vitro translation methodology is able to detect rarer mRNA species than is usually expected--these include similar abundance classes to library screening procedures.

A specific effect of antipsychotic drugs on protein synthesis in human lymphomononuclear cells.

We have studied the effects of psychotropic drugs on patterns of protein synthesis in human lymphomononuclear cells by two-dimensional gel electrophoretic analysis. Drugs effective in treatment of schizophrenia specifically increased the relative synthesis of a 30-kDa polypeptide in cultured human lymphomononuclear cells whereas dopamine (DA) or psychoactive drugs lacking antipsychotic properties did not. The effect was stereospecific with respect to the clinically active and inactive isomers of flupenthixol. Synthesis of the 30-kDa polypeptide appears therefore to be correlated with antipsychotic properties but not with DA receptor binding. It is possible that such effects may be associated with the clinically beneficial effect of antipsychotic drugs in the brain.

The genetics of Alzheimer's disease--where does it lead?

The identification and study of genetic systems with potential involvement in the cellular changes associated with Alzheimer's disease may help us to understand the complex pathways from primary genetic lesion to the final disorder.