Superoxide, Neuroleptics and the Ubiquinone and Cytochrome B5
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Molecular Psychiatry (1998) 3, 227–237 1998 Stockton Press All rights reserved 1359–4184/98 $12.00 ORIGINAL RESEARCH ARTICLE Superoxide, neuroleptics and the ubiquinone and cytochrome b5 reductases in brain and lymphocytes from normals and schizophrenic patients SA Whatley1, D Curti2, F Das Gupta1, IN Ferrier3, S Jones1, C Taylor4 and RM Marchbanks1,5 1Department of Neuroscience, Institute of Psychiatry, Denmark Hill, London SE5 8AF, UK; 2Istituto di Farmacologia, Universita di Pavia, 11 Piazza Botta, 27100 Pavia, Italy; 3MRC Neurochemical Pathology Unit, Newcastle General Hospital, NE4 6BE, UK; 4Department of Forensic Psychiatry, Institute of Psychiatry, Denmark Hill, London SE5 8AF, UK The effects of the neuroleptic flupenthixol on the expression of the genes coding for the mito- chondrial ubiquinone and cytochrome b5 reductases have been studied because of the impor- tance 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 enzy- mic 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. Keywords: oxidative stress; flupenthixol; psychosis; Complex I; mitochondria; gene expression; in situ hybridisation Introduction a disjunction between the timescale of biological effect and therapeutic efficacy.1 Schizophrenia is characterised by delusional experi- Liability to schizophrenia is partially inherited. Cal- ences and thought disorder with a lifetime morbidity culations from twin and family studies indicate that of the order of 1%. Studies on the biological basis of approximately 60–70% of the risk is inherited. A fairly the psychoses have turned up many sometimes contra- intensive study of those parts of the human genome dictory clues but the substantive aetiological theories that code for dopamine receptors and related enzymes result from the effects of drugs which afford some mea- has not so far revealed a linkage that is reproducible sure of symptomatic relief. Hence the ‘dopaminergic’ under extended scrutiny.2,3 A linkage study covering hypothesis of schizophrenia because neuroleptic medi- the whole nuclear coded genome at an average 9 cM cations are found to affect various parts of the dopa- intervals has revealed about 25 loci linked to schizo- mine neurotransmitter system. Despite intensive inves- phrenia4 with probabilities greater than P Ͻ 0.05, but tigation the ‘dopamine’ hypothesis has not been found there does not appear to be any functional connection to be particularly predictive and in particular there is between these loci. An alternative approach is to try and find out which Correspondence: SA Whatley, Dept of Neuroscience, Institute of genes are expressed differently in patients suffering Psychiatry, Denmark Hill, London SE5 8AF, UK. E-mail from psychosis. We characterised the mRNA from post- ϽspbcsawȰiop.bpmf.ac.ukϾ mortem brain of schizophrenics and normals by 2-D 5Current address: Wessex Human Genetics Institute, Duthie translational analysis to establish that it was not so Building (Mailpoint 808), Southampton General Hospital, SO16 5–7 6YD, UK degraded as to confound the whole approach. The Received 21 July 1997; revised 19 November 1997; accepted 10 mRNA populations in post-mortem brain of schizo- December 1997 phrenics were then compared to normals by differen- Superoxide, neuroleptics and reductases in schizophrenia SA Whatley et al 228 tial screening, a protocol that allows the identification samples 24 were from the MRC Brain Bank, Newcastle of genes whose relative transcript levels are altered General Hospital and a further three schizophrenic and between two tissue types.8 Five clones were selected three normal samples were provided from the Institute as being differentially expressed in schizophrenics and of Psychiatry brain bank. The sample set is described were then found to be derived from mitochondrial in Table 1. Because of varying tissue availability the (mt)DNA; four of these were for ribosomal RNA but an same samples were not always used in each study. upregulated clone coding for cytochrome oxidase sub- Diagnosis of the patients was carried out by means of unit II was also found.9 DSM-III. Death was at average age of 65.7 × 16 years Neuroleptic medication is known to cause changes (normals) and 70.6 ± 10 years (schizophrenics) and was in gene expression in brain,10 and in parallel studies it from various causes. The post-mortem delay ranged was found that cis(␣)-flupenthixol by comparison with from 3 to 84 hours with averages of 25.6 ± 13 (normals) its therapeutically inactive isomer trans()-fluphen- and 69.4 ± 3.3 (schizophrenics) and 41.2 ± 16.8 thixol caused the differential expression in rat brain of (unmedicated schizophrenics). Because of the rather 29 clones, 14 of which were of mitochondrial origin (S higher post-mortem delay for the schizophrenic cohort Jones, personal communication and Whatley et al11). a sub-group with shorter post-mortem delay (39.2 ± 6.7 Predominant among the mitochondrial sequences were n = 5) was identified and the results of enzyme assays those coding for cytochrome oxidase subunits I and II and superoxide production calculated separately. In no and the NADH-ubiquinone reductase, all of which case was there a significant difference between the were downregulated by cis-flupenthixol. Expression of results from the short and long post-mortem delay sub- the nuclear gene coding for NADH-cytochrome b5 groups. reductase was also found to be downregulated by cis- flupenthixol. Human lymphocytes These were obtained from nor- There is therefore an apparent involvement of the mals and male patients admitted to the hospital wing processes of mitochondrial oxidative energy metab- of Broadmoor. Lymphocytes were isolated from an 8- olism in the aetiology of schizophrenia and an effect ml whole blood sample by the technique of Boyum.13 of neuroleptic medication on the same processes. An Diagnosis of these patients was carried out by means additional issue is that both the ubiquinone and the b5 of DSM-III and the sample set is described in Table 2. reductase enzymes are flavoproteins that while All tissue was stored at −70°C until use. mediating the transfer of electrons from NAD(P)H are known to produce small amounts of superoxide which Enzyme preparations NADH ubiquinone and cyto- has been invoked as an aggravating or even causal fac- chrome b5 reductases and cytochrome b5 itself were tor in neurotoxicity and mental illness.12 To study this, made from 66 g fresh sheep liver following the general a simple method of measuring superoxide production procedure described by Yang and Cederbaum.14 The has been devised and applied to the brain samples. tissue was homogenised in 600 ml 0.25 M sucrose buff- In this investigation we seek to relate the amounts of ered with 10 mM Tris-Cl pH 7.4 and 1.0 mM EDTA. the reductases in brain and lymphocyte tissues from The nuclear fraction was removed as a pellet after cen- schizophrenics, the extent to which any changes might trifugation twice at 500 g for 10 min. Mitochondria be due to neuroleptic medication and the role both were sedimented at 11 000 g for 16 min and washed play in the production of toxic superoxide. twice with 20 volumes sucrose buffer. The mitochon- Preliminary studies11 of the activity of the two drial fraction was frozen in aliquots and used without reductase enzymes suggested that in the brains of further treatment except for dilution to 0.85 g protein schizophrenic patients either the respiratory chain l−1 as a source of NADH-ubiquinone reductase. Each (ubiquinone) reductase activity was decreased with a microgram of protein in the preparation was derived compensating increase in the outer membrane (b5) from 0.26 mg tissue. reductase or that there was a change in the properties Polyethylene glycol (PEG average molecular wt of ubiquinone reductase in schizophrenics such that it 6000) was added to the post-mitochondrial supernatant is less sensitive to rotenone. In order to distinguish and to a final concentration of 8.5% and spun at 13 000 g measure the two reductases correctly in small amounts for 20 min. The microsomal pellet was washed twice of tissue we have investigated the properties of the with 60 ml PEG-sucrose. The washed pellet was resus- semi-purified enzymes and applied improved methods pended in 100 ml (ie approximately 8 mg protein ml−1) so that rotenone sensitivity of the ubiquinone of 0.1 M Tris-acetate pH 8.1 containing 1 mM EDTA, reductase from schizophrenics can be independently 1 mM phenylmethyl sulphonyl fluoride and 2% final monitored to resolve this question. concentration Triton X-100 and stirred for 1 h.