Neuroscience Letters, 132 (1991) 109-112 © 1991 Elsevier Scientific Publishers Ireland Ltd. All fights reserved 0304-3940/91/$ 03.50 ADONIS 030439409100624E

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NSL 08140

Alzheimer's disease: fl-amyloid precursor protein mRNA expression in mononuclear blood cells John S. Allen 1, Greer M. M u r p h y Jr. 1, Lawrence F. Eng 2, Karen E. Stultz 1, Helen D. Davies 1, Lesley B. Pickford 2 and Jared R. Tinklenberg ~ Departments of 1Psychiatry and Behavioral Sciences and 2Pathology, Stanford University School of Medicine, Palo Alto VeteransAffairs Medical Center, Palo Alto, CA 94304 (U.S.A.) (Received 9 July 1991; Accepted 23 July 1991)

Key words: Alzheimer's disease; Monocyte; Lymphocyte; fl-Amyloid precursor protein; Polymerase chain reaction fl-Amyloid precursor protein (flAPP) mRNA was examined in peripheral mononuclear blood cells (PMBCs) in Alzheimer's disease, Down's syndrome and control subjects. Total RNA from PMBCs was reverse transcribed and then amplified using the polymerase chain reaction (PCR). The 3 major//APP transcripts were expressed in PMBCs from all subjects. These results suggest that PMBCs could be a circulating source for abnormal amyloid deposition in the brain and in peripheral tissues.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by abnormal deposition of the fl amyloid protein (flAP) in plaques and cerebral blood vessels, flAP is a small polypeptide of 39-42 residues, probably derived from a larger, membrane-bound fl-amyloid precursor protein (flAPP). In human brain there are mRNAs coding for 3 major flAPP isoforms [11,13,18,23]. flAPP-770 and -751 contain a Kunitz serine protease inhibitor (KPI) domain, and are highly homologous to cell-secreted protease nexin II [14]. flAPP-695 lacks the KPI domain, as does the rarer flAPP-714 [6,12]. AD can be diagnosed with certainty only by examination of cerebral tissue. However, the search for peripheral markers has been extensive [7]. Recently flAPP has been targeted, since flAPP mRNAs are found in a variety of extraneural tissues [6,12,22]. A hematogenous source for abnormal amyloid deposits in AD is possible because at least some forms of flAPP are present in human serum, plasma and platelets [17,20,24], and because flAP is often found in the cerebral vasculature. Amplification with polymerase chain reaction (PCR) of cDNA synthesized by reverse transcription (RT) is a powerful tool in the analysis of mRNA expression [25]. Although RT-PCR has been used to study flAPP mRNA levels in brain and some peripheral tissues [6,12],

Correspondence: G. Murphy, Psychiatry Service (116A3), Palo Alto Veterans Affairs Medical Center, Palo Alto, CA 94304, U.S.A.

the formed elements of the blood have not been examined with this technique. We report on the expression of the 3 prinicpal flAPP mRNA transcripts in peripheral mononuclear blood cells (PMBCs; monocytes and lymphocytes) in AD, Down's Syndrome (DS) and control subjects. Monocytes are important because developmentally they probably give rise to microglia [16], which are often seen in proximity to flAPP deposits in AD brain [4]. Lymphocytes are found more frequently in AD brain tissue than in controls [8], and hence could also be involved in amyloidosis. Subjects included six patients with AD, one with DS and four controls (Table I). All AD subjects met NINCDS criteria for probable AD. On the basis of global deterioration scale (GDS) scores [19], two AD subjects were designated as moderate, three as moderately severe and one as severe. The DS subjects was karyotypically trisomy 21, but showed no signs of cognitive decline. All subjects were without significant medical problems, except the DS subject who suffered from a congenital heart defect. All subjects were also free of acute or chronic infectious disease. Complete blood counts were obtained on all the AD and DS subjects, and all had normal leukocyte counts and normal lymphocyte and monocyte differentials. PMBCs were isolated by centrifugation at 500 g with ficoll and sodium diatrizoate, following the method described in B6yum [1] (Histopaque-1077, Sigma Diagnostics). From each subject 20 ml of blood were collected in 3 sodium heparinized tubes; this quantity of blood

110 TABLE I AD, Alzheimer'sdisease; DS, Down's syndrome. Fig. 1 lane

Age

Sex

Diagnosis

GDS

Degree impairment

I 2 3 4 5 6 7 8 9 10 Southern (Fig. 2A)

73 76 74 81 78 81 19 26 71 66 37

F M F F F M M F F F M

AD AD AD AD AD AD DS Control Control Control Control

5 6.5 5 5 4 4

moderatelysevere severe moderatelysevere moderatelysevere moderate moderate not impaired not impaired not impaired not impaired not impaired

typically yielded 5-10 x 10 7 mononuclear cells. Immediately following isolation of the PMBC pellet, total R N A was extracted using the guanidinium thiocyanatephenol-chloroform method of Chomczynski and Sacchi [3]. The final R N A pellet was dissolved in 20-50 #! of 1 mM EDTA, pH 8, and stored at - 70°C. The concentration (typically 3-5 #g/#l) and purity (OD260/OD280 = 1.72.0) of the RNA sample were measured in a spectrophotometer. The structural integrity of the total RNA samples was confirmed by electrophoresis on a 1% formaldehyde-agarose gel (data not shown). First strand cDNA synthesis was performed with the cDNA Cycle Kit (Invitrogen). For each reaction, 3 #g of RNA in a total volume of 8 #1 was used. RNA secondary structures were denatured by the addition of 2/A of 100 mM M e H g O H and incubation at room temperature for 5 min. Following the addition of a reducing agent (2.5 #1 of 0.7 M fl-mercaptoethanol) and chilling on ice, 1 #1 of random primer (1.0 #g/#l) was added, and the mix was incubated at 65°C for 2 rain and chilled on ice. The volume was brought up to 20 #l by the addition of l #l of placental RNase inhibitor, 4 #l of 5 × RT buffer, l #l of 25 mM dNTPs, and 0.5 #l of AMV reverse transcriptase (10 units/#l). The mixture was incubated for 1 h at 42°C, and then heated to 95°C for 3 min. After chilling on ice, 0.5 #1 o f A M V RT was again added, and the incubation repeated. In a typical reaction, 4/A of the R T / c D N A mixture was added directly to the PCR reaction mixture. Oligonucleotide primers originally designed by Golde et al. [6] were used which simultaneously amplified cDNA sequences coding for flAPP-695 (87 bp), flAPP714 (144 bp), flAPP-751 (255 bp) and flAPP-770 (312 bp). The sequence of the forward primer was: 5'-CACC A C A G A G T C T G T G G A A G - 3 ' ; the reverse primer had

the sequence: 5 ' - A G G T G T C T C G A G A T A C T T G T - 3 ' . For PCR, 4 #1 (one-fifth) of the R T / c D N A mixture was added to 5 #1 10 x PCR buffer (Perkin Elmer Cetus, containing 100 mM Tris-HCl, 500 mM KCI, 15 mM MgC12, and 0.01% (w/v) gelatin), 2.5 #1 of 10 mM dNTPs, 1 #1 of each primer (0.3 #g/#l), and 0.8 #1 (4 units) of Taq polymerase (Perkin Elmer Cetus). Sterile water was added to bring the volume to 50 #1. Thirty-five cycles of amplification (1 min at 95°C, 1 min at 60°C) were followed by a single extension incubation of 7 min at 72°C. All amplifications were carried out using a programmable DNA Thermal Cycler (Perkin Elmer Cetus). After amplification, the PCR products were precipitated by adding 2 vols. of 100% ethanol in the presence of 0.1 M NaC1 to the entire PCR reaction volume; the mixture was chilled for at least one hour at - 70°C. After centrifugation, the resulting DNA pellet was resuspended in 10 #1 of TE buffer and run on a 2% agarose gel. Amplified c D N A was visualized with ethidium bromide. To confirm the identity of flAPP-695, -751 and -770 cDNA, some gels were Southern blotted with 10 x SSC onto a nylon filter (Bio Trace RP, Gelman Scientific). Individual lanes were cut from the filter, and hybridized to 35S-labelled riboprobes complementary to a sequence in the KPI domain (nucleotides 1062-109l [18] - - 'insert probe') and to regions flanking the inserted KPI domain (nucleotides 975-989 and nucleotides 1158-1171 - 'junction probe'). The insert probe detects flAPP-770 and -751, while the junction probe specifically detects flAPP-695. After hybridization, the filter lanes were washed twice in 1 x SSC for 30 rain at 65°C, twice in 0.2 x SSC at 65°C for 30 min, and exposed to X-ray film for 24-48 h. The results of reverse transcription and amplification of flAPP mRNAs from PMBCs are shown in Fig. 1. For

111

BAPP-770 8APP-751

CH0-751

8APP-895 ~

5-AD

6

7 DS

8

.............. 9 10 Control

Fig. I. flAPP cDNAs amplifiedfrom PMBC total RNA using RTPCR. Informationon subjects givenin Table I. Sizesof the amplified fragmentsare givenin the text. 2%agarosegel,ethidiumbromidestaining. all subjects, the 3 major transcripts were present; in 3 subjects (lanes 6, 9 and 10), additional weak bands may indicate the presence of flAPP-714. In Fig. 2, Southern blotting and riboprobe hybridization showed specific binding of the insert probe to cDNA for flAPP-770 and -751 and of the junction probe to flAPP-695 cDNA. In Fig. 3, cDNAs derived by RT-PCR from total RNA isolated from Chinese hamster ovary (CHO) cell lines transfected to overexpress either human BAPP-751 or human flAPP-695 transcripts are shown alongside a typical PMBC result. Relative positions of the signal in the 3 lanes showed that the flAPP-751 and -695 transcripts were expressed in PMBCs. No direct identification of the flAPP-714 transcript was done. However, the position of the band relative to flAPP-695 (87 bp) indicates that it is the correct length for the flAPP-714 cDNA (144 bp). Samples run as controls for contamination either without total R N A or without RT showed no flAPP cDNA signal. Replications on different days using the same total RNA samples yielded identical PCR results (data not shown). These results demonstrate that PMBCs from AD, DS

Insert Probe

[SAPP-770/751]

Junction Probe

[SAPP-695] Fig. 2. Autoradiograph demonstrating specific binding of insert and junction probes for flAPP-770/751 and flAPP-695, respectively, after Southern blotting and hybridization. Subject described in Table I.

lira

W

CH0-695 AD Fig. 3. Position of cDNA bands from PMBC RNA, relative to that from total RNA from CHO cells transfectedwith flAPP-751 and -695. PMBCs from same AD subjects as in lane 4, Fig. 1.2% agarose gel, ethidium bromidestaining.

and control subjects of a variety of ages contain the 3 major flAPP mRNAs. The presence of flAPP message in these cells suggests they could be an important circulating source of flAPP. In AD flAPP is thought to undergo abnormal proteolysis to form flAP [5]. flAP is found not only in the brain, but in the cerebral vasculature and in some peripheral tissues [9]. The origin of the flAPP giving rise to peripheral flAP deposits has not been identified. Lymphocytes, as well as monocyte-derived macrophages, often migrate outside the vascular compartment and could be a source of flAPP in peripheral tissues. PMBCs could also contribute to the flAPP which has been found in plasma and serum [17,20], which in turn might give rise to flAP in vessel walls. We have detected cell-surface flAPP with immunofluoresence on monocytes and lymphocytes (unpublished observations), and in a preliminary report another group has described flAPP in lymphocyte membrane extracts [21]. If PMBCs are a circulating source of flAPP, then the presence of all 3 transcripts in these cells may be important. Some reports have indicated that abnormalities in the relative amounts of the 3 alternatively spliced flAPP transcripts could contribute to the formation of flAP in AD brain [6,10,15]. In the present study all RT-PCR reactions started with the same amount of total RNA, but Fig. 1 demonstrates that there was considerable intersubject variation in the overall amount of PCR product, as well as in the relative amounts of product resulting from the 3 individual transcripts. This may indicate that subjects differed in overall BAPP m R N A expression, and in the ratios of the 3 splicing variants. However, the precise starting quantities of the flAPP mRNAs cannot be determined from the present data because PCR amplification may vary for different sized cDNAs [2]. In summary, these results demonstrate that AD, DS and control PMBCs show the 3 major flAPP transcripts. PMBCs and their derivatives are found in the vascular

112

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