Molecular Psychiatry (1997) 2, 451–456  1997 Stockton Press All rights reserved 1359–4184/97 $12.00

PROGRESS Changes in brain expression in psychiatric illness: mRNA differential display provides some clues TP Flanigan and RA Leslie

Oxford University SmithKline Beecham Centre for Applied Neuropsychobiology, Department of Clinical Pharmacology, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE, UK

The 20th century has witnessed a progressive increase in our understanding of brain struc- ture, organisation and function and now includes knowledge at the macromolecular and ionic levels. Investigations of such diverse functions as cognition, memory and mood, performed mainly in whole animal studies, are now advancing rapidly with the application of modern molecular biological techniques. In this article we consider the contribution of mRNA differen- tial display to the analysis of altered in vitro and in vivo. The role that this technique may play in the identification of involved in the aetiology of psychiatric dis- orders and their treatment is discussed. Keywords: depression; anxiety; schizophrenia; addiction; neuroplasticity; neuroadaptation; central nervous system

Modern molecular biological techniques have started and noradrenaline in the treatment of such disorders. to contribute greatly to our understanding of the links While studies such as these have highlighted the pri- between gene expression, protein function and behav- mary site of action of some psychiatric drugs, the iour. Genes controlling brain function are now being mechanisms by which they achieve their therapeutic identified and sequenced, and many of their protein effects are still obscure. In large part, this is due to our products, responsible for such events as neuro- very rudimentary understanding of normal brain func- transmitter-receptor interactions, second and third tion, particularly with regard to cognition, memory or messenger function and ion channel modulation, are modulation of mood. A common phenomenon, involv- being identified. Many of these molecules and their ing a considerable lag phase between the onset of psy- complex interactions provide various measures of chotropic drug therapy and clinical effect, is also diffi- specificity in communication amongst neurons. This cult to understand, and has suggested to many workers developing knowledge of normal brain function is a role for neuroadaptive responses in the mode of helping in studies of neurological disorders such as action of these treatments. Such neuroadaptive Alzheimer’s, Huntington’s and Parkinson’s diseases, responses would probably involve not only pharmaco- where some specific neurochemical defects have been kinetic considerations, but also changes in the level of shown to be associated with the illnesses. The psychi- gene expression in the brain, resulting in alterations in atric disorders, on the other hand, may appear to be levels of mRNA as well as their encoded proteins. less accessible to investigation at the molecular level; A growing body of experimental data has shown that however, novel molecular approaches being used to external modulation of neurotransmission leads to investigate gene expression in the nervous system are changes in gene expression in the brain. For example, changing this perception. expression of the immediate-early gene c-fos is altered by drugs that interfere with cholinergic,1 dopaminergic,2 glutamatergic3 and serotonergic4 transmission in rat Gene expression, CNS disorders and drugs brain. It has been shown that activation of glutamate Investigations of psychotropic drug interactions with receptors by kainic acid is associated with alterations normal or diseased brain have yielded useful insights in expression of mRNAs encoding products of several into potential neurochemical deficits underlying psy- immediate-early genes as well as differentiation and chiatric illness. For example, the success of sero- growth response proteins, heat shock proteins and syn- tonergic and adrenergic compounds in the alleviation apse-related proteins.5 This study also identified many of depressive illness has indicated a role for serotonin unknown genes whose expression was altered. Thus, it seems likely that ‘normal’ as well as altered neuro- transmission contributes to an ongoing array of neuroad- Correspondence: Dr TP Flanigan, Oxford University SmithKline aptive responses, some of which involve changes in the Beecham Centre for Applied Neuropsychobiology, Department of Clinical Pharmacology, Radcliffe Infirmary, Woodstock Rd, expression of known, as well as novel, genes. A better Oxford OX2 6HE, UK. E-mail: flaniganȰworf.molbiol.ox.ac.uk understanding of this neuroplasticity will provide Received 27 January 1997; revised and accepted 7 April 1997 further clues into normal and diseased brain function, Changes in brain gene expression TP Flanigan and RA Leslie 452 and offer potential new targets for drug development to iety of molecular procedures including sub-cloning and treat neurological and psychiatric illness. sequencing, RNase protection assays, Northern and Recent studies of genetic abnormalities in psychi- reverse Northern blotting, in situ hybridisation and atric illnesses such as schizophrenia, anxiety and RACE (rapid amplification of cDNA ends). These pro- depression have suggested the involvement of several cedures can provide information on the nature of the genetic factors, although any roles these elements play differential expression of the band, its nucleic acid com- in neurochemical mechanisms producing the disease position, and the cellular localisation of the transcript, states remain a mystery. Studies linking specific genes and can provide access to the gene encoding the mRNA. or gene loci to such diseases provide candidate genes While DD is becoming the method of choice in the to aid investigations of the aetiology of these illnesses. identification of differential expression of mRNA from In polygenetic illnesses with large environmental different sources, the technique does suffer from several components, which is suspected to be the case with drawbacks9 including a higher than expected number of most psychiatric disorders, a broader strategy than this false positive (and negative) bands, and the fact that it will almost certainly be required to arrive at a complete initially targets the 3Ј (untranslated) region of mRNA, understanding of these disease processes. Some new which generally makes gene identification more diffi- molecular strategies are providing ways to attack these cult. Several precautions can be taken in experimental questions. One of these, the technique of subtractive design, however, to help validate the DD procedure. hybridisation, can reveal differences between cDNA To reduce the contribution of bands derived from gen- libraries derived, for example, from diseased vs normal omic DNA (which can often contaminate RNA tissue or cells. This technique has been adopted to preparations) RNA samples can be digested with DNase identify tissue-specific genes which might be involved prior to RT-PCR. Control reactions, in which reverse in the causative mechanisms of schizophrenia.6 transcriptase or RNA is omitted from the RT-PCR, can Because of some serious limitations of this technique also be performed to assess the contribution from however, alternative approaches have been developed sources other than RNA. Wherever possible it is always to allow, for example, comparison of more than two sensible to validate the DD procedure by initially tar- mRNA sources at one time, to enable biologists to work geting a specific RNA whose expression is known to be with smaller amounts of source mRNA, and to reduce altered (from previous studies using candidate gene the time and cost of such experiments. approaches) by the model system under investigation. In addition, variability within the DD runs can be reduced by performing duplicate or multiple reactions Differential display of mRNA and its applications and analysing the products ‘side-by-side’ on the gels. ‘mRNA differential display’ (RT-PCR Differential Dis- This will permit further analysis to be performed only play or DD), first described by Liang and Pardee,7 pro- on bands whose expression is altered in a consistent vides a potentially simple and efficient method for the and reproducible fashion. identification of known and novel genes that are differ- Other aspects of experimental design can also often entially expressed in biological systems, and offers sev- contribute to validation of the DD procedure and sub- eral distinct advantages over subtractive hybridisation. sequent identification of differentially expressed genes. The procedure requires lower amounts of source In studies where the identification of tissue (or cell)- material, permits comparison of mRNA samples from specific gene expression is the ultimate goal, some mea- multiple sources, gives direct access to the nucleic acid sure of validity may be achieved by comparing the target sequence information, and is less technically tissue (cell) type with a battery of different tissue (cell) demanding, thus increasing the likelihood of identifying types. In situations where an external stimulus is differentially expressed genes. DD is a reverse tran- expected to alter gene expression, performing the analy- scriptase-linked polymerase chain reaction (RT-PCR) sis at different time points after the original stimulus approach, which subfractionates transcripts in RT reac- will identify candidate bands whose altered expression tions using primers anchored (‘anchored primer’) at the is time-dependent. Alternatively in the analysis of mod- polyadenylate region of the 3Ј-end of the mRNAs. Par- els in which several stimuli will elicit similar changes, tial specificity is conferred by specific pairs of nucleo- such multiple conditions may be included in the experi- tides at the 3Ј-end of the primers, designed to direct the ment to compare with appropriate controls. In this way, RT reactions. The products of these reactions are further bands can be identified which are differentially regu- fractionated by subsequent PCRs, conducted in the pres- lated in common by these stimuli relative to controls. enceofthesameanchoredprimerandasecondshort These approaches will reduce the incidence of false ‘arbitrary primer’, whose sequence is chosen such that it positives as well as improve validation of the differen- hybridises to almost all cDNAs, but at variable distances tial display technique. upstream from the anchored primer. Low-stringency Despite these considerations the high incidences of amplification, in the presence of radiolabelled nucleo- false positive results which have sometimes been tides, yields labelled PCR products that can then be sep- reported10,11 suggest that the technique itself is suscep- arated according to size on conventional polyacrylam- tible to inherent variation in experimental conditions. ide sequencing gels, and subsequently visualised by The amplification procedure used may serve to exagger- autoradiography.7,8 Differentially expressed bands can ate this variation. This problem may also be com- be recovered directly from the gels and utilised in a var- pounded by the degree of mispriming exhibited by the Changes in brain gene expression TP Flanigan and RA Leslie 453 oligonucleotides employed. Hence, certain signals may cancer31 cells have been identified using this strategy. derive from ‘spurious’ priming occurring in the PCRs Known members of the zinc finger gene family were due to the low annealing temperature used. The latter also detected in these studies. Donohoe et al,28 also used problem has partly been addressed by the introduction a ‘targeted differential display’ to identify a FGF- of ‘hot start PCR’ and more recently by the use of longer inducible kinase (Fnk) which was differentially primers12 and ‘touchdown’ PCR13 where initial PCR expressed during the NIH-3T3 cell cycle. Fnk appears cycles are performed at higher annealing temperatures, to be an immediate-early gene and is a member of the with this temperature gradually being decreased as PCR polo subfamily of serine/threonine protein kinases. cycle number increases until the low temperature is Thus, targeted DD may prove to be a useful tool in the achieved. In this way the contribution of spurious PCR identification of novel members of known gene families, products to the final profile is minimised.14 whose expression may be altered in response to a var- Other improvements, such as altering the design of iety of stimuli. primers12 have been suggested to reduce some of the Clearly DD has been most successfully employed, so drawbacks of the procedure. Furthermore, a sister tech- far, in identifying differentially expressed genes in nique, known as mRNA arbitrarily primed PCR finger- homogeneous cell lines or in tissues that exhibit limited printing (RAP), has been adapted for such studies15,16 cellular heterogeneity. Possibly the greatest challenge to and should improve sensitivity and detectability in the the ability of DD to detect altered gene expression will analysis of differential expression of mRNA. With RAP, be in the investigation of neuroadaptation in the adult the RT-PCRs are performed using only a single arbitrary mammalian brain. Some progress has already been primer that is thought to confer more reliable priming, made, however. It is well documented that in animal and improve the likelihood that the amplified targets models of stroke, induced either by mechanical lesions will represent protein-coding regions of differentially or other types of oxygen deprivation, induction of the expressed mRNAs. immediate-early gene c-fos occurs prior to hippocampal These new technologies have been used to identify cell death (apoptosis), suggesting that altered gene differential gene expression in a range of systems and expression underlies at least some of the brain’s have, for example, identified a variety of genes with response to stroke. Wang et al,32 used DD to implicate oncogenic or tumour suppressor potential in tissues or a previously identified protein, adrenomedullin, in the cell lines derived from of breast,17–19 ovary,20,21 22 23 24 25 response of rat cortex to ischaemia induced by liver, stomach, bone and brain. The success of DD occlusion of the middle cerebral artery. This is an in identifying potentially interesting genes in important study which indicates unequivocally that alt- research can be attributed, in part, to the relatively ered gene expression in adult mammalian brain can be homogeneous nature of the cells and tissues involved. detected by DD and shows that neuroplastic changes It has been estimated that, of the 100 000 or so genes induced by other stimuli, such as pharmacological in the , only about 15 000 of these are agents, may also be amenable to DD analysis. expressed in any given cell.7 Hence, the more homo- Some other recent studies reinforce these obser- geneous the tissue or cell source of mRNA being com- vations. Acute psychomotor stimulants such as cocaine pared, the more likely it is that differences in expression have been shown to elevate expression of c-fos and jun will be detectable. This is well illustrated in the work 33 34 from Jeffrey Winkles’ lab, which has identified genes B in rat striatum and cerebellum, which is consistent with the suggestion that neuroadaptation underlies the involved in regulation of the cell cycle in NIH-3T3 cells 35 following treatment with FGF-1.26–28 behavioural responses to such drugs. Further support- Fortunately, in situations where more heterogeneous ing evidence for this hypothesis has been provided by cell populations have been analysed, DD has also DD which was used to demonstrate that acute cocaine proved successful. In particular, work by Joseph et al,29 and amphetamine administration elevated mRNA levels indicated that analysis of developmentally regulated for a previously unidentified gene in rat striatum, but 36 geneexpressionintheCNScouldsuccessfullyemploy not hippocampus or cerebellum. Thus, DD can be DD. These workers identified a transcript (‘neuronatin’) used successfully to identify genes whose expression is whichisexpressedathighlevelsinneonatal,butnot altered in adult mammalian brain, in a regionally spe- adult rat brain. Downregulation of this mRNA may be cific fashion, in response to drug administration. involved in nerve terminal differentiation in the brain. DD may also prove useful in the identification of Interestingly, neuronatin mRNA was expressed in genes involved in regulation of behaviour. Melanin-con- human foetal but not adult brain, suggesting a develop- centrating has been identified by DD as a can- mental role for this gene in human brain. didate gene that may participate in the hypothalamic An interesting development in DD is the use of ‘tar- regulation of body weight in obese rats.37 This study geted’ primers that are designed to uncover hitherto identified five other candidate genes expressed in the unknown members of families of expressed sequences. CNS that could also be involved in regulation of feeding In this approach the primers used are designed to target behaviour. It is, as yet, unclear whether regulation of regions of mRNAs that bear greatest homology between this gene is involved in feeding behaviour and obesity subfamily members. Novel members of the family of in humans. mRNAs encoding proteins bearing zinc finger domains In summary, DD has been shown to be useful in the from cultures of mouse fibroblast30 and human ovarian identification of candidate genes that are providing new Changes in brain gene expression TP Flanigan and RA Leslie 454 knowledge of physiological, pharmacological and in the rat C6 glioma cell line.38 This is particularly behavioural aspects of CNS function. intriguing because lithium is known to affect second messenger pathways in cellular systems,39 including effects on cAMP and phosphatidyl inositol metabolism. Psychiatric disease: studies of human brain mRNA CNPase II is thought to play a role in myelination, and Since DD is an mRNA-based technique, the availability may also be involved in neuronal growth and repair38 of good quality RNA is a prerequisite in the generation so lithium may well be involved in some of these other of meaningful data. Therefore, whether this technique mechanisms during its role as a psychotropic drug. In will prove to be useful in studies of the role of altered any case, this study suggests that lithium is involved gene expression in diseased human brain, from which with some neuroplasticity, at least in vitro, and ident- it is usually very difficult to extract high quality mRNA, ifies four candidate genes whose altered expression may remains to be determined. Several important factors will be involved in the mode of action of lithium in vivo. have to be taken into consideration when designing a Whether any of these genes truly are involved in the DD of human brain mRNA. Isolation of diseased human therapeutic action of lithium awaits further study. brain mRNA is necessarily compromised by degradation In a similar way to that indicated in the above study, which may be due both to the length of the patient’s DD of mRNA from experimental tissues treated with agonal state, and the post-mortem delay in obtaining the other psychotropic drugs should uncover genes whose tissue. Any differences between diseased and normal expression is altered in response to these treatments. brains in post-mortem delay, and other causes of mRNA Employment of a candidate gene approach, using degradation between samples, would exacerbate the mRNA samples obtained from human brain, will then incidence of false positive or negative signals. Other help to determine the significance of these transcripts potential difficulties include any variability within nor- in the aetiology of the relevant disease. The possible mal healthy human brains as well as any differences in applications of this type of study are many. For sampling procedures during brain dissections. Hence, example, neuroleptic drugs such as haloperidol are sus- any determination of whether differences in expression pected of inducing a range of neuroadaptive responses, of particular transcripts between control and disease including structural changes40 and alterations in samples is truly disease-related will probably be a com- expression of c-fos,41 neuropeptides42 and other pro- plex task. Another issue that will usually have to be teins.43 Synaptophysin mRNA44 and protein45 have been considered is the variability in drug history of tissue shown to be decreased in the medial temporal lobe of donors, since such variability may generate false posi- schizophrenics, which supports a role for altered synap- tives (or negatives) itself. tic activity as a pathological marker in this disease. DD Some of these problems may be minimised by careful of mRNA from brains of animals treated with neurolept- selection of cases, pooling of brain samples, increasing ics should uncover genes involved in the mechanism of the numbers of samples of healthy and diseased brains action of these drugs, and may help in the understand- to be compared, etc, but it is likely that the problems ing of the role of altered gene expression and neuroplas- associated with differences in drug histories (both medi- ticity in schizophrenia. cal and ‘recreational’ use) and other variables will mean Just as for schizophrenia, little is known of the role that generation of extensive analyses of human brain of gene expression in clinical depression or its treat- tissue by DD will be very time consuming. Nonetheless, ment. It seems likely from some recent studies, how- such studies may be worth pursuing; for example, ever, that altered gene expression is involved at least in analysis of brain mRNA from clinically depressed suic- the mechanisms underlying chronic administration of ide victims may provide clues as to the role of altered antidepressant drugs. Chronic (but not acute) adminis- gene expression in affective disorder. Another possi- tration of several antidepressants elevates the levels of bility is that a better understanding of drug addiction the cyclic AMP response element binding protein may arise from DD of mRNA from brains of people who (CREB) and its corresponding mRNA46 as well as brain- die under the influence of addictive drugs of abuse such derived neurotrophic factor (BDNF) and its trkB recep- as cocaine and amphetamine. tor mRNA,47 in rat hippocampus. Neither CREB, BDNF Another approach that is currently being used to nor trkB expression was altered by several other types study alterations of gene expression in psychiatry, will of psychotropic drugs, such as haloperidol or cocaine, probably find wider application in the short term; that suggesting that the antidepressant effects on this gene is, analysis of altered gene expression in mRNA from expression were specific. CREB is a nuclear transcrip- experimental animal brain. Animal brain tissue or cul- tion factor which probably influences the level of tured cells that have been treated with psychotropic expression of a range of ‘downstream’ genes. Similarly, drugs can be studied with DD to provide candidate altered BDNF and trkB expression would have effects genes, novel as well as known, whose expression can on genes controlling neuronal growth and maintenance. then be investigated in other experimental paradigms. Hence, chronic administration of antidepressants is Indeed, the drug lithium, effective in the treatment of likely to influence expression of genes encoding novel, bipolar affective disease, has recently been demon- as well as known, proteins in the brain. DD of mRNA strated by DD to alter the expression of at least three could play a significant role in the identification of these novel mRNAs, as well as the transcript for the enzyme genes and help to unravel their role in the mode of 2Ј,3Ј-cyclic nucleotide 3Ј phosphodiesterase (CNPase II) action of antidepressant drugs. Whether the effects of Changes in brain gene expression TP Flanigan and RA Leslie 455 antidepressants on CREB, BDNF and trkB gene mRNA samples derived from diseased human brain expression are relevant to disorders of mood in man tissues, the use of this technique (or its successors) in remains to be determined. However, it is interesting to animal and cell culture models should reveal known note that altered gene expression has been demonstrated and novel candidate genes for investigation in studies in brains from schizophrenics and unipolar affective of the aetiology of psychiatric illness. This approach disorder sufferers, relative to controls.48 In this study should permit an evaluation of the contribution of alt- proteins were identified that were altered in a disease- ered gene expression in the mechanism of action of psy- specific manner and vice-versa. Whether this altered chotropic drugs and may contribute to the design and gene expression reflects the disease state, or drug his- use of more effective and safer drugs in the treatment tory, or neither in these individuals is unknown; never- of psychiatric diseases. theless, further characterisation of these genes and the Notwithstanding its current popularity, it is imposs- influence of psychotropic drugs on their expression in ible to say how long DD will continue to be the tech- the brain may help our understanding of their role in nique of choice to search for and identify differentially psychiatric illness. expressed genes in specific tissues. Already techno- logies such as SAGE (serial analysis of gene Differential display and gene linkage analysis expression)52 and cDNA microarray techniques53 are providing novel ways to study differential gene The use of DD of mRNA from animal or cell culture expression. Until the entire complement of expressed models in the provision of candidate genes for study in sequences in the human genome is known, however, human psychiatric disorder may be combined with these new technologies must be regarded as simply other technologies to yield even more information about more sophisticated candidate gene approaches. the genetics of disease states. For example, several ner- vous system disorders are thought to be associated with complex genetic patterns in which specific chromo- Acknowledgements somes or gene loci appear to segregate with specific The authors would like to thank Andreja Volenec for pathologies in humans or behavioural patterns in lab- her critical reading of the manuscript. oratory animals used as models of psychiatric dis- eases.49 If it proves to be the case, it is likely that map- ping the candidate genes, identified by DD, to these References chromosomal loci will give researchers more confidence 1 Barone P, Morelli M, Cicarelli G, Cozzolino A, DeJoanna G, Cam- that the genes in question are relevant to the aetiology panella G, DiChiarra G. Expression of c-fos protein in experimental of the disease under investigation rather than just epilepsy induced by pilocarpine. Synapse 1993; 14: 1–9. ‘housekeeping genes’ or ‘coincidental hits’ from the DD 2 Dragunow M, Robertson GS, Faull RL, Robertson HA, Jansen K. 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