Characterisation of messenger extracted post-mortem

Journal of Neurology, Neurosurgery, and Psychiatry 1988;51:325-331

Characterisation of messenger RNA extractedpost-mortem from the brains of schizophrenic,depressed and control subjects

C W PERRETT, R M MARCHBANKS, S A WHATLEY

From the Department ofBiochemistry, Institute ofPsychiatry, London, UK

SUMMARY Messenger RNA, obtained from post-mortem brain of 10 schizophrenics, five depressedpatients and 10 control subjects, was characterised with respect to a number of parameters. It wasfound that post-mortem delay was not the major factor in determining RNA yield, size (as deter-mined by cDNA synthesis) and biological activity. Biological activity, as determined by in vitrotranslation in a reticulocyte-lysate system, could be observed using messenger RNA from periods of0 to 84 hours post-mortem. Two-dimensional gel analysis of the newly-synthesised radiolabelledproducts obtained from this material revealed several hundred individual species but no consistentdegradation of any particular species with post-mortem delay. It is suggested, therefore, that pre-

mortem changes are as important as post-mortem changes in determining RNA yield, size andbiological activity. Although no consistent difference could be found between patients and controlsusing any of these parameters, this study confirms that, by isolating messenger RNA from post-mortem human brain, valuable information can be gained on gene expression in psychiatricdisorders.

There is considerable evidence from classical family,twin and adoption studies for a genetic predispositionto many types of mental disorder, including schizo-phrenia and affective disorder. ' 2 The involvement ofspecific genetic elements, and therefore transcrip-tional mechanisms, in the aetiology of affective disor-der has recently been confirmed using molecular gen-etic techniques, with the reported linkage of affectivedisorder to the insulin/Ha-ras- 1 locus on chromo-some 11 in a large Amish pedigree.3 Despite thisfinding, the existence of genetic heterogeneity4 5 anduncertainty about the true values of penetrance forthe predisposing genes confound the ability of DNAlinkage methodology to define the genetic lesions fur-ther. It is therefore important to identify transcribedgenes which can be envisaged to be associated withthose disorders in order to supplement genetic data.One such approach is to compare brain poly(A)-containing messenger RNA populations from normal

Address for reprint requests: Dr C W Perrett, BiochemistryDepartment, Institute of Psychiatry, De Crespigny Park, DenmarkHill, London SE5 8AF, UK.

Received 28 July 1987. Accepted 26 October 1987

and psychiatrically ill patients. With this in mind, wehave studied the effects of post-mortem delay on thebiological activity and size of poly(A)-containingmessenger RNA isolated from schizophrenic,depressed and control brain tissue. Some of theresults shown here have already been presented in apreliminary communication.6

Methods

Brain samplesBrain tissue (10 schizophrenics, five depressed patients and10 control subjects) was obtained from Dr RH Perry(Newcastle-upon-Tyne General Hospital) and Dr G Rey-nolds (MRC Neurochemical Pharmacology Unit, Cam-bridge). Material from both the frontal cortex and caudatenucleus was used. Subjects had died from a variety of causesand post-mortem delay (the time between death and tissuedissection/freezing) varied from 3 to 84 hours. In addition,control tissue (no post-mortem delay) was obtained during atemporal lobe biopsy performed by Mr CE Polkey (Neuro-surgical Unit, Maudsley Hospital).

RNA extractionTotal cellular RNA was extracted from frozen brains essen-tially as described previously,7 using guanidinium thio-cyanate and a caesium chloride centrifugation step.

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Poly(A)-containing messenger RNA was obtained fromtotal cellular RNA by using oligo (dT)-cellulose chro-matography.8 Both total cellular RNA andpoly(A)-containing messenger RNA were estimated byA260 measurements.8

Protein synthesis and analysisPoly(A)-containing messenger RNA (20 pg ml- 1) was usedto direct the synthesis in vitro of proteins in the reticulocyte-lysate system, using ["S] methionine as radiolabelled pre-cursor, as described previously.' Translation products wereanalysed using high-resolution two-dimensional gel electro-phoresis as described by O'Farrell,9 for isoelectric focusing(ISODALT system), and O'Farrell et al,10 for non-equilibrium pH gradient electrophoresis (BASODALT sys-tem). Minor modifications were employed in samplesolubilisation and pH gradient generation.'

Spot intensities on fluorograms were measured by the den-sitometric scanning technique of Quitschke and Schechter, I I

where a value proportional to the volume integral is deter-mined. Intensities were normalised by a comparison withtwo standard spots (see figs 4 and 5) whose intensity did notvary significantly from controls (as measured by Studentt test values; data not shown).

cDNA synthesis and analysiscDNA was synthesised by a modification of the proceduredescribed by Arrand. 2 The incubation mixture (10 p1 totalvolume) contained: 75mM KC1, 3mM MgC 12, 50mMTris-HCI (pH 7 5), 20mM dithiothreitol, 2-23 x 10-3 A260units oligo (dT)12 -18, 12U placental RNAsin, 100 nM eachof dATP, dCTP, dGTP, dTTP, [a-32PJdCTP (0-1 pCi; 420Ci mmol-'), 50pgmlP actinomycin D, 200U murinemoloney leukaemia virus reverse transcriptase and 50ngpoly(A)-containing messenger RNA. After incubation(37°C, 1 h) the cDNA was ethanol-precipitated and washedthree times in 75% v/v ethanol: 25% v/v 0-1 M sodium ace-

Perrett, Marchbanks, Whatleytate. The cDNA was analysed for size in. a denaturingagarose gel (1% w/v agarose in 30mM NaOH, 50mMNaCl, 1 mMEDTA), using alkaline electrophoresis buffer(30mM NaOH ImMEDTA).

Results

RNA recoveryRecovery of both total cellular RNA and poly(A)-containing messenger RNA was variable amongdifferent brain samples but there were no significantdifferences between the groups analysed, as measuredby Student t test values (tables 1 and 2). There was nomarked decrease in total cellular RNA with post-mortem delay (fig 1) and, in addition, age at time ofdeath did not affect the RNA yield (results notshown). Similarly, there was no significant change inthe poly(A)-containing messenger RNA (expressedeither as a percentage of total RNA or in terms of pgg ' brain) either with post-mortem delay or with ageover the range studied (results not shown).

Messenger RNA sizePoly(A)-containing messenger RNA size was esti-mated by reverse transcription into cDNA followedby cDNA sizing (fig 2). Although this underestimatesmessenger RNA size owing to premature terminationby reverse transcriptase, relative size distributions ofsamples can be observed. cDNA sizes showed con-tinuous distributions in the range 200-1400 nucleo-tide residues. There was no significant change in sizewith post-mortem delay over the time periodconsidered.

Table I Comparison of total cellular RNA ofbrain samples

Number of Total cellular RNA Age Post-mortem delayGroup samples (ug/g (SD)) (years (SD)) (hours (SD))

Controls* 6 167 (58) 73 (6.9) 36-5 (27 7)Schizophrenics* 8 184 (74) 72 (6.8) 42-2 (26.1)Depressives* 5 207 (89) 76 (4 5) 29-6 (10.1)Totalt 30 174 (60)

*Comparison of cortex values only.tincluding cortex and caudate values plus fresh biopsy material.

Table 2 Comparison ofpoly(A)-containing messenger RNA ofbrain samples

Poly(A)-containing mRNA

Number of (% of total Post-mortem' delayGroup samples cellular RNA (SD)) Age (years (SD)) (hours (SD))

Controls* 5 3-93 (1-63) 73 (7-5) 43 (254)Schizophrenics* 5 2-75 (1 72) 70 (7-4) 58 5 (17 4)Depressives* 5 2-79 (1-76) 76 (4 5) 29-6 (10-1)Totalt 16 3-19 (1-62)

*Comparison of cortex values only.tlncluding fresh biopsy material.tA similar analysis yielded no significant differences when expressed in terms of pg mRNA/g brain tissue.


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Characterisation of messenger RNA from the brains of schizophrenic, depressed and control subjects

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Biological activity ofmessenger RNAAll samples analysed showed biological activity in thein vitro translation system, although there was con-

* siderable variation between samples (table 3). Studentt test results indicated that there were no significantdifferences between groups (table 3) and, perhaps sur-prisingly, there was no marked reduction in biologicalactivity with post-mortem delay (fig 3).

Translation products were analysed using both* equilibrium (fig 4) and non-equilibrium (fig 5) two-

dimensional gel electrophoresis. Analysis of trans-).2) lation product fluorograms revealed a number of pro-

teins which could be consistently identified and over200 individual radiolabelled species could be detected.No significant amounts of high molecular weight spe-cies (Mr greater than 90000) were present in any ofthe samples analysed. However, there was no consis-tent shift to lower molecular weight species withincreased post-mortem delay, nor any consistent lossof specific individual species (results not shown). Itwas apparent, therefore, that molecular weight distri-

60 80 bution and intensity of specific species was not asimple function of post-mortem factors.

,st-mortem delay in Comparisons of translation product patterns!racted as described between normal, schizophrenic and depressedckets denotes the patients revealed some differences in relative

intensities of specific proteins on some gels (figs 4and 5). These differences are quantified in table 4.However, comparisons of translation productfluorograms from all samples indicated that therewere no consistent differences between either normaland schizophrenic or normal and depressed groups.

3r 26h 30t 34h 60h 72'r t-3xk

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Fig 2 cDNA size estimation. cDNA was synthesisedftompoly(A)-containing messenger RNA and analysed as

described in the Methods section. Key: numbers above lanesrepresent post-mortem delay times; right-hand scalerepresents the migration ofMr markers run on the same gel.

RNA recoveryIn this study, the yield of both total cellular RNA andof poly(A)-containing messenger RNA from differentsamples was variable, although the results were con-

sistent with previously published values.7 13-16 Suchvariation for total RNA is probably not due toimpurity of the sample, since RNA obtained by themethod used here is relatively free from both DNA14and protein (different samples had similar A260:A280ratios; results not shown). Contamination of the sam-

ple cannot be discounted for messenger RNA, how-ever, since messenger RNA isolated from an oli-go(dT) column contains relatively high levels ofribosomal RNA,7 14 although these levels are stillsmall compared with the total variability observedhere. It may be that factors such as tissue dissection,handling and storage play an important part in deter-mining the quantity of RNA recovered. Inclusion oftraces of white matter in the sample would affect rela-tive yield, since RNA levels in white matter are onlyhalf those in grey matter.17

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Fig 1 Change in total RNA yield with po.tissue sampling. RNA was isolated and extin the Methods section. The number in bracorrelation coefficient value.

Discussion

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Table 3 Comparison ofmessenger RNA biological activity ofbrain samples

Number of Biological Post-mortem delayGroup samples activity, Age (years (SD)) (hours (SD))

Controls* 5 3 94 (2 42) 73 (7-5) 43 (25-4)Schizophrenics* 5 4-18 (2 64) 70 (7-4) 58 5 (17 4)Depressives* 5 3-32 (1-10) 76 (4-5) 29-6 (10-1)Totalt 18 3-98 (2-21)

*Comparison of cortex values only.fIncluding cortex and caudate values plus fresh biopsy material.tAs measured by trichloracetic acid precipitation of synthesised proteins (7); values quoted are -fold stimulation over the blank value and (SD).

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Fig 3 Change in biological activity ofmepost-mortem delay in tissue sampling. Biolmeasured by trichloroacetic acid precipitaiproteins.7 Values quoted are -fold stimulatThe number in brackets denotes the correlkvalue.

RNA breakdown, both pre- and p(remains the most obvious determiiboth total RNA and poly(A)-contaRNA. However, there was no corrbetween low RNA yield and either sRNA (as judged by reverse transcritranslation products (see below).

Effects ofpost-mortem delayFrom the results obtained here, arwith others,3 -15 it appears that poin tissue sampling per se does not silRNA recovery. Moreover, and su

mortem delay did not appear significantly to affectmessenger RNA size, as judged by cDNA analysis.Rather, it is possible that pre-mortem changes mayplay a more important role than those post-mortem.In support of this proposal, Perry et al'8 have shownthat, when death follows a prolonged period of severeillness, significant differences can be observed in braintissue pH, as well as some brain amino acid and

* enzyme levels, compared with those values in patientswhere death either occurred suddenly or after aperiod of normal health. Variability in the recovery oftotal cellular RNA in this study may therefore be areflection of individual variations in agonal state.

Biological activitya High-resolution two-dimensional gel electrophoresis

has been used in a number of studies examiningchanges in in vitro translation products using messen-ger RNA isolated from both normal and pathologicalhuman brain.7 1419-22 As with RNA yield, recoveryof biological activity (determined by trichloroacetic

60 80 acid precipitation) was variable between samples.This has been observed by others using post-mortem

XssengerRNA with brain tissue of varying times.'4 In connection withfogical activity was this it was significant that biological activity did nottion ofsynthesised decrease markedly with post-mortem delay. In addi-tion over the blank. tion, size of translation products, as observed on theation coefficient fluorograms, did not show degradation over the post-

mortem period studied. Therefore, whilst RNAdegradation remains the most significant factor insuch analyses, it appears that post-mortem delay

ost-mortem, thus alone does not account for this variability. It is proba-nant of yield of ble that the factors which appear to affect RNAlining messenger recovery, most notably tissue dissection, handling,elation observed storage and agonal state, also affect biological activity;ize of messenger of the messenger RNA.iption) or size of No high molecular weight species were observed in

this study, even after long fluorogram exposure times.However, this did not appear to be due to post-mortem breakdown of messenger RNA, since mes-

id in agreement senger RNA derived from freshly frozen biopsy mate-ost-mortem delay rial produced translation products of similar sizegnificantly affect distribution. Differences in translation product sizesirprisingly, post- observed here, when compared with earlier results,7

Perrett, Marchbanks, Whatley328

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Characterisation of messenger RNA from the brains of schizophrenic, depressed and con

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Fig 4 Two-dimensional ISODALTpolyacrylamide-gel analysis of translation products. In vitro translation and gel analysiswere performed as described in the Methods section. The sample contained 6 x iO' acid-precipitable c.p.m. Key:(a), translation products ofpoly(A)-containing messenger RNA isolatedftom control subjects; (b) translation products ofpoly(A))-containing messenger RNA isolatedftom schizophrenics; (c) translation products ofpoly(A)-containing messengerRNA isolatedfrom depressives; a, b, c, d, spots whosefluorographic intensities changed in a comparison ofsome control andpsychiatric subjects; (*), the standard spotfor the ISODALTsystem, as described in the Methods section; right-hand scalerepresents the migration ofMr markers run on the same gel; numbers above the gel indicate the pH of the ends of theisoelectric-focusing gels. Abbreviations: A, actin; T, tubulin; 14-3-3, brain 14-3-3 protein; BJ, [35SI methionine-bindingproteins of the lysate system.

may reflect the developmental period studied (adultbrain compared with foetal brain), since similar sizedistributions have been observed by other authors inadult post-mortem brain.20 22 In addition, no consis-tent selective loss of individual species was observedwith increased post-mortem delay.The observation that intact messenger RNA, capa-

ble of translating proteins in the in vitro system, was

obtained at post-mortem delays of up to 84 hours issignificant, since such translation is highly dependenton the integrity of the 51-terminal cap structure inbrain messenger RNA.23 This extends the results ofother studies indicating that messenger RNA seemsintact for periods of up to 36 hours post-mortem1 5 21and confirms that the use of messenger RNA samples,extracted post-mortem from human brain, is likely to

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infig 4. Key: e, a spot whosefluorographic intensity changed in a comparison ofsome control andpsychiatric subjects; (*), thestandard spotfor the BASODALTsystem, as described in the Methods section; signs above the gel indicate the electrodepolarityfor the BASODALT separation.

Table 4 Comparison ofspecific spot intensities onfluorograms shown infigs 4 and5

Schizophrenics DepressivesSpot* (%)t (%Jt

a 4 93b 5 100c 0 96d 0 100e 3333 8914-3-3

(basic) 35 3514-3-3

(acidic) 26 26

*Spot designations as in figs 4 and 5.tlntensities were measured and normalised against the standardspots shown in figs 4 and 5. Values are expressed as a percentage ofcontrol intensities.

provide information on expressed genes in neu-

ropsychiatric conditions. Indeed, the data presentedin this study suggest that the state of the tissue pre-mortem may be more important than post-mortemdelays in tissue sampling in determining both recov-ery and biological activity of RNA species. It is possi-ble, however, that important changes occur within 33hours postmortem; we are therefore currently study-ing samples of this time interval.With these considerations in mind, in this study we

found no consistent significant differences onfluorograms between the control and psychiatricgroups which suggests that, at least in the samplesstudied here, changes in abundant messenger RNApopulations are not a feature of the disease state. Fur-

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Characterisation of messenger RNA from the brains ofschizophrenic, depressed and control subjectsther analysis of the rarer populations using cDNAcloning techniques is now under way to obtain a morecomplete picture of these processes.

The support of the Medical Research Council (GrantNo. G8514318N) is gratefully acknowledged.

References

1 Dunner DL. Recent genetic studies of bipolar and unipolardepression. In: Davis JM, Maas JW, eds. The Affective Disor-ders. Washington: American Psychiatric Press, 1983:183-91.

2 Murray RM, Lewis SW, Reveley AM. Towards an aetiologicalclassification of schizophrenia. Lancet 1985;i: 1023-6.

3 Egeland JA, Gerhard DS, Pauls DL, et al. Bipolar affectivedisorders linked to DNA markers on chromosome I1. Nature1987;325:783-7.

4 Baron M, Risch N, Hamburger R, et al. Genetic linkage betweenX-chromosome markers and bipolar affective illness. Nature1987;326:289-92.

5 Hodgkinson S, Sherrington R, Gurling H, et al. Moleculargenetic evidence for heterogeneity in manic depression. Nature1987;325:805-6.

6 Perrett CW, Whatley SA. An analysis of the products of trans-lation in vitro from normal and schizophrenic brain by two-dimensional polyacrylamide-gel electrophoresis. Biochem SocTrans 1987;15:546-7.

7 Whatley SA, Hall C, Davison AN, Lim L. Alterations in therelative amounts of specific mRNA species in the developinghuman brain in Down's Syndrome. Biochem J 1984;220:179-87.

8 Hall C, Lim L. Developmental changes in the composition ofpolyadenylated RNA isolated from free and membrane-boundpolyribosomes of the rat forebrain, analysed by translation invitro. Biochem J 1981;196:327-36.

9 O'Farrell PH. High resolution two-dimensional electrophoresisof proteins. J Biol Chem 1975;250:4007-21.

10 O'Farrell PZ, Goodman HM, O'Farrell PH. High resolution two-dimensional electrophoresis of basic as well as acidic proteins.Cell 1977;12:1133-42.

11 Quitschke W, Schechter N. A noncomputerized scanning methodfor determining relative quantities and synthesis rates on two-dimensional electrophoretic gels. Analyt Biochem 1982;124:231-8.

12 Arrand JE. Preparation of nucleic acid probes. In: Hames BD,Higgins SJ, eds. Nucleic Acid Hybridisation: A PracticalApproach. Oxford: IRL Press, 1985:17-45.

13 Sajdel-Sulkowska EM, Marotta CA. Alzheimer's disease brain:Alterations in RNA levels and in a ribonuclease-inhibitorcomplex. Science 1984;225:947-9.

14 Guillemette JG, Wong L, Crapper McLachlan DR, Lewis PN.Characterization of messenger RNA from the cerebral cortexof control and Alzheimer-afflicted brain. J Neurochem1986;47:987-97.

15 Johnson SA, Morgan DG, Finch CE. Extensive post-mortemstability of RNA from rat and human brain. J Neurosci Res1986;16:267-80.

16 Wood TL, Frantz GD, Menkes JH, Tobin AJ. Regionaldistribution of messenger RNAs in post-mortem human brain.J Neurosci Res 1986;16:31 1-24.

17 Naber D, Dahnke HG. Protein and nucleic acid content in theaging human brain. Neuropathol AppI Neurobiol 1979;5:17-24.

18 Perry EK, Perry RH, Tomlinson BE. The influence of agonalstatus on some neurochemical activities of post mortem humanbrain tissue. Neurosci Lett 1982;29:303-8.

19 Gilbert JM, Brown BA, Strocchi P, Bird ED, Marotta CA. Thepreparation of biologically active messenger RNA fromhuman postmortem brain tissue. J Neurochem 1981;36:976-84.

20 Marotta CA, Brown BA, Strocchi P, Bird ED, Gilbert JM. Invitro synthesis of human brain proteins including tubulin andactin by purified post mortem polysomes. J Neurochem1981;36:966-75.

21 Morrison MR, Griffin WST. The isolation and in vitro translationof undegraded messenger RNAs from human post mortembrain. Analyt Biochem 1981;113:318-24.

22 Sajdel-Sulkowska E, Coughlin JF, Marotta CA. In vitro synthesisof polypeptides of moderately large size by poly(A)-containingmessenger RNA from post mortem human brain and mousebrain. J Neurochem 1983;40:670-80.

23 Murthy MRV. Effects of cap analogue or cap removal on thetranslation of rat brain mRNA in vitro. J Neurochem1982;38:41-5 1.

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Characterisation of messenger extracted post-mortem