Multiple forms of beta-amyloid peptide precursor RNAs in a single cell type

Neurobiology of Aging, Vol. 9, pp. 333-338. ©Pergamon Press plc, 1988. Printed in the U.S.A.

0197-4580/88 $3.00 + .00

Multiple Forms of Beta-Amyloid Peptide Precursor RNAs in a Single Cell Type R. J. D O N N E L L Y , C. G. R A S O O L , R. B A R T U S , 1 S. V I T E K , A. J. B L U M E A N D M. V I T E K ~

Molecular Neurobiology and Geriatrics, Central Nervous System Biological Research Department Medical Research Division, Lederle Laboratories, American Cyanamid Company Pearl River, N Y 10965 R e c e i v e d 1 A p r i l 1988 DONNELLY, R. J., C. G. RASOOL, R. BARTUS, S. VITEK, A. J. BLUME AND M. VITEK. Multipleforms ofbetaamyloid peptide precursor RNAs in a single cell type. NEUROBIOL AGING 9(4) 333-338, 1988.--The longest open reading frames (ORFs) of three different cDNAs ([10, 12, 18, 26], and this report) contain the exact 42 amino acid (aa) sequence of the beta-amyloid peptide (BAP) which is selectively deposited in Alzheimer's diseased (AD) brains. Each of the three cDNAs for the putative amyloid peptide precursor (APP) has been cloned from a different cell type. Using an HL 60 library, we have cloned two of these three APP cDNAs from a single cell type. The sequences of the HL 60 cDNAs are identical to the APP 751 and to the APP 770 forms of APP cDNAs. Northern blots show that oligonueleotide probes drawn from unique regions of the APP 751 and APP 770 cDNAs both hybridize to 4.0 Kilobase (Kb) and to 1.6 Kb APP RNAs from HL 60 cells. In human adult brain, an oligonucleotide probe drawn from the unique region of the APP 751 eDNA hybridizes to a 3.5 Kb APP RNA. However, a DNA probe drawn from the BAP region, which is common to the 695,751, and 770 forms ofAPP cDNAs, hybridizes to 3.5, 3.2 and 1.6 Kb APP RNAs. Taken together, these results sha~' that at least two forms ofAPP RNAs can exist within a single cell type and that the diversity of possible APP RNAs and complexity of their expression may have been underestimated. Thus, in addition to identifying the ceils which produce BAP, a new challenge consists of determining which form of forms of APP RNAs and hence APP proteins are associated with BAP deposition in AD and Down syndrome (DS). Alzheimer's disease Northern analysis

Beta amyloid peptide Diversity

Amyioid peptide precursor RNA

A L Z H E I M E R ' s disease is a degenerative disorder which affects about 10% of the population aged 65 years and older [9], is the leading cause of dementia and is among the leading causes of death in the United States [I 1]. The brains of AD and Down syndrome (DS) patients contain extra-normal numbers of amyloid plaques and cerebrovascular amyloid deposits [9,29] from which a 42 aa beta-amyloid peptide (BAP) has been isolated and sequenced [7,15]. Using a fetal brain library and an oligonucleotide probe drawn from the aa sequence, Kang isolated what appears to be a full length eDNA clone with a 695 aa open reading frame (ORF) for a protein (APP 695) which may be the putative precursor to BAP [10]. Recently, two groups have isolated APP cD N A s with ORFs encoding a 751 aa putative precursor (APP 751) to BAP from a SV 40 transformed fibroblast Jibrary [18] and from an H L 60 library [26]. A third group isolated an APP eDNA with an ORF encoding a 770 aa putative precursor (APP 770) from a human glioblastoma library [12]. The ORFs of APP 751 and APP 770 contain identical 56 aa domains (PI domain) which share structural and functional similarity with the Kunitz domain of serine protease inhibitors [12, 18, 26].

DNA sequencing

ABBREVIATIONS aa AD BAP APP bp Kb

amino acids Alzheimer's disease beta-amyloid amyloid peptide precursor base pairs kilobases

DS FAD DNA mRNA Kd

Down Syndrome familial Alzheimer's disease deoxyribonucleic acid messenger ribonucleic acid Kilodaltons

Using Northern blots, diversity is also found at the R N A level where portions of the APP 695 e D N A hybridize to 3.5, 3.2 and 1.6 Kb APP R N A s from human brain [8, 10, 19, 25, 28, 31]. Though it is tempting to suggest that each of the differently sized species of APP R N A could direct the synthesis of different APP proteins, the data shows that three similar APP eD N A s are individually found in different cell types [10, 12, 18, 26]. In addition to resolving how many form or forms of APP protein are,, associated with BAP deposition, these data have focused our efforts on determin-

1Present address: Cortex Pharmaceuticals, Inc., Coppertree Park, HI0, 151 Kalmus Drive, Costa Mesa, CA 92626. ~Requests for reprints should be addressed to M. Vitek.

333

D O N N E L L Y ET AL.

334 ing whether a single cell type makes a single form of APP RNA. In this paper we report that extensive APP R N A diversity can be found within a single cell type, H L 60. This diversity is shown by isolation and sequencing of e D N A clones for APP 751 and APP 770 from an H L 60 library and by Northern analysis of H L 60 RNA. METHOD

289 i

IM

A. APP-695

VRVPT

B. APP-751

JM

vR

C. APP-770

I"

VR

~ RAP

r,'r

NI

~

NI

Tissue and R N A Preparation Human brains were recovered 12 hours after death, dissected on ice and frozen in liquid nitrogen. RNAs from frozen human brain, fresh rat brain and H L 60 cell pellets were prepared by a modification of the method described by Chirgwin [3]. Briefly, tissue was homogenized in guanidine isothiocyanate, the homogenate overlayed atop a cesium chloride cushion, and RNA recovered by ultracentrifugation. P o l y - A + R N A was isolated using oligo-dT cellulose chromatography as previously described [27]. Some RNAs were purchased from Clonetech (Paio Alto, CA).

Hybridizations and Probes Oligonueleotide probes were synthesized with beta-cyanoethyl phosphoramidite chemistry on an Applied BioSystems 380B D N A synthesizer using protocols from the manufacturer. Probes were purified on polyacrylamide gels as described [28] and 5' end-labeled with gamma-32P-ATP and T4 polynucleotide kinase according to Davis [4]. Lambda D N A was labeled with alpha-32P-dATP by the random priming method of Feinberg and Vogelstein [5]. The plasmid p C L L 701 [28] and SP6 R N A polymerase (Amersham) were used to generate antisense riboprobes labeled with alpha-32P-UTP as described in protocols supplied from Amersham. Riboprobes were hybridized at 45"C in 50% formamide, 5 x S S C ( I × S S C = 0.15 M sodium chloride/0.015 M sodium citrate, pH 7.0), 0.1% sodium dodecyl sulphate (SDS), 10xDenhardt's ( l x D e n h a r d t ' s = 0.02% polyvinylpyrrolidone/0.02% bovine serum albumin/0.02% Ficoll 500), I mg/ml yeast RNA, 10/zg/ml sheared and denatured salmon sperm DNA and washed at 55°C in 0.1 x SSC/0.1% SDS. Hybridizations were performed at 10°C below the Tm for each oligonucleotide probe which was calculated using the equation described in Davis [4]: Tm=16.6 log ( N a + ) + 0.41(%GC) + 81.5 - 675/L - 0.65 F where ( N a + ) is the molar concentration of sodium ion in the hybridization mix, (%GC) is the number of guanosine and cytosine bases divided by the total number of bases in the oligonueleotide multiplied times 100, L is the length of the oligonueleotide in bases and F is the percentage of formamide. Blots were washed at 5°C below the Tm to improve the stringency of hybridization. Plaque lifts and colony lifts were performed as described in Davis [4]. R N A s were fractionated in formaldehyde/ agarose gels and Northern blotted as described in Davis [4]. A lambda gt 11, H L 60 e D N A library was purchased from Cloneteeh (Palo Alto, CA).

DNA Sequencing APP e D N A inserts from recombinant lambda clones were subcloned into the EcoR1 site of pUC 19 [30] by standard techniques [14]. Sanger type D N A sequencing [22] of subclone DNAs was performed using multiple oligonucleotides as described by Strauss [24]. Sequence data was managed with the Intelligenetics (Mountain View, CA) family of computer programs.

PT

~

NI

BAP

FIG. 1. Schematic comparison of APP 695, APP 751 and APP 770 cDNAs. Each box represents the open reading frame (ORF) defined by cDNAs encoding APP proteins. The patterned area labeled BAP refers to the position of the 42 amino acid beta amyloid peptide. Wavy vertical lines refer to the position of the protease inhibitor domain while wavy horizontal lines refer to the carboxy terminal domain of the protein insert. (A) APP 695 protein as proposed by Kang [10]. (B) APP 751 protein as proposed by this paper, Ponte [18] and Tanzi [26]. (C) APP 770 protein as proposed by this paper and Kitaguchi [12]. Numbers following APP refer to the total number of amino acids deduced from the sequence of the longest ORF. Within each box, the relative positions of important amino acids are denoted with single letter codes. Prebe

&95 I Bgl 1

-t I

i

Bam~l!

Pi

Probe

E©oRI

Xn~l

I

I IIIIN

AI"W-SYI Probe

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C Probe

pCLL 701 Probe

AHY-SYI

CTCCAGCGCCCGAGCCGTCCAGGCGGCCAGCAGGAGCAGTGCCAJ~CCGGGCAGCAT

Probe PI

GGCGCTGCACCACACGGCCATGCAGTACI"CI"I"CTG~I~, AAGI"I"GI"I"CCGG~GCCGCC

Probe C

GTTTAACAGG ATCTCGGGCA AGAGGTTCCT

Probe 695

ACTGGCTGCT GTTGTAGGAA CTCGRACCACC~I'CCACA

GGGTAGTCTT GAGTAAACTT CCTCGGGACA

FIG. 2. Map of DNA probes used to define various regions of APP eDNA. A truncated APP 770 eDNA is shown schematically as in Fig. 1. Restriction enzymes cut at the locations shown by the vertical lines below their names. Capped bars indicate the relative sizes and positions of probes which are named. Please note that only Probe 695 is derived from two different portions of the sequence and should correspond to a PIC-less eDNA sequence. 5' to 3' sequence of each oligonucleotide probe is below the diagram.

RESULTS We initially screened a lambda gt 11 library of H L 60 cDNAs with random primed D N A of the plasmid p C L L 701 which corresponds to nucleotide positions 1795 to 2323 of APP 695 e D N A (see Fig. 2). The p C L L 701 positives were rescreened with an oligonueleotide probe (AMY-SY1) corresponding to nucleotide positions 1 to 57 of APP 695 e D N A (Fig. 2) and twelve putative full length APP e D N A clones were selected. EcoR1 restriction analysis of the twelve showed that their inserts contained a 1.0/2.2 Kilobase (Kb) pair (lambda clone L E D 708) or a 1.0/2.1 Kb pair (lambda

M U L T I P L E FORMS O F APP RNAs IN A S I N G L E C E L L TYPE

335

A. LED 708 Insert

tl GTT CGA G V

R

E

AG GTG TGC TCT GAA CAA GCC GAG ACG GGG CCG TGC CGA GCA V C S E Q A E T G P C R A

I 287

I 300 ATG ATC TCC CGC TGG TAC ~ M l S R W Y F

GAT GTG ACT GAA GGG AAG D V T E G K C

GCC Cc-A TTC TTT TAC GGC GGA TGT GGC GGC AAC CGG AAC ARC A P F F ¥ G G C G G g R N N

U TTT GAC ACA GAA GAG TAC TGC ATG GCC GTG TGT GGC AGC GCC A F D T E E ¥ C M A V C G S A I

TT CCT ACA P T

I 340

I 346

S. LED 709 Insed

GTT CGA G V R E 1

AG GTG TGC TCT GAA CAR GCC GAG ACG GGG CCG TGC CGA C-CA AT(; V C S E Q A E T G P C R A M

I 300

287

ATC TCC CGC TGG TAC TTT GAT GTG ACT G,J~% GGG AAG TGT GCC CCA I S R W Y F D V T E G K C A P TTC TTT TAC GGC GGA TGT GGC GGC AAC CGG AAC AAC TTT GAC ACA F F Y G G C G G N R N N F D T GAA GAG TAC TGC ATG GCC GTG TGT GGC AGC GCC ATG TCC CAA AGT E E Y C M A V C G S A H S O S

U TTA CTC AAG ACT ACC CAG GAA CCT CTT GCC CGA GAT CCT GTT A A A C L L K T T Q E P L A R D P V K L

l 360

TT CCT ACA P T

i 365

FIG. 3. Sequence of the LED 708 (A) and the LED 709 (B) nucleotide inserts and their deduced amino acid sequences. Single letter amino acid codes appear below nucleotide triplets encoding them. Position numbers of amino acids are given assuming the first Met as defined by Kang is number 1 [10]. Vertical arrows indicate beginning and end of nucleotide inserts. clone L E D 709) of fragments. All four insert fragments of LED 708 and L E D 709 were subcloned into pUC19 and amplified for D N A sequencing. Although the majority of L E D 708 and L E D 709 nucleotide sequences exactly match the APP 695 c D N A sequence, they also contain additional bases not found in the APP 695 cDNA. These new bases form contiguous sequences which are inserted between nucleotide positions 865 and 866 of the APP 695 c D N A (Fig. 3). L E D 708 contains a 168 bp insert which preserves the reading flame and replaces Val 289 with 57 new aa before resuming with Pro and the remainder of the APP 695 amino acid sequence. L E D 709 contains a 225 bp insert which also preserves the original reading flame in the same fashion by replacing Val 289 with 76 new aa. The sequence of the amino terminal 56 aa (PI domain) of each insert is identical as are the 5' 168 nucleotides of the L E D 708 and the L E D 709 inserts encoding them. In L E D 709, the PL domain (168 bp) is followed by an additional 57 bp (the C domain) which could encode 19 additional amino acids. We refer to these 19 aa as the " C " domain and to the entire 76 aa insert of L E D 709 as the PI + C or PIC domain. With these various inserts the total length of the potential APP protein increases from 695 aa to 751 art (PIodomain) 6r to 770 art (PIC domains). We used Northern blots to determine the presence of various APP R N A s in different R N A samples. As shown in Fig. 2, we used an oligonucleotide derived from the APP 695 nucleotide sequences flanking the inserts (Probe 695), an oligonucleotide derived from the 168 bp insert (Probe PI), an oligonucleotide derived from the unique 57 bp of the L E D 709 insert (Probe C), and a riboprobe derived from the BAP

region to stringently hybridize Northern blots (as shown in Fig. 4). The p C L L 701 riboprobe contains the BAP region which identifies APP RNAs of 5.6, 3.5, 3.2, and 1.6 Kb in human brain poly A + R N A (Fig. 4, Lane A) and APP RNAs of 5.6, 4.0, and 1.6 Kb in poly A + and total H L 60 RNAs (Fig. 4, Lanes B and C). We previously reported [28] that p C L L 701 hybridizes to 3.5, 3.2, and 1.6 Kb APP RNAs from normal adult, AD, and DS human brains as well as mouse and rat brains. Probe PI hybridizes to 5.6 and 3.5 Kb transcripts from human brain poly A + RNAs (Fig. 4, Lane D), to 3.5 and 2.4 Kb transcripts from rat brain poly A + RNAs (Fig. 4, Lane E), and to 4.0 and 1.6 Kb transcripts from poly A + H L 60 RNAs (Fig. 4, Lane F). We can only detect Probe C hybridizing to 4.0 and 1.6 Kb transcripts from H L 60 RNAs. Probe 695 hybridizes to a 5.6 Kb transcript from H L 60 RNA. These results are summarized in Table 1. DISCUSSION

Using c D N A cloning and sequencing, we show that multiple forms of APP RNAs exist within a single cell type, H L 60. Two novel forms of APP cDNAs arise from insertions of 168 (LED 708) and 225 (LED 709), additional base pairs (bp) between nucleotide positions 865 and 866 in the APP 695 cDNA. Since the APP gene is present at one copy per haploid genome [6, 16, 17, 21], these novel forms of APP R N A s .probably result from differential splicing of the primary APP gene transcript. Using the open reading frame defined by the 42 aa BAP, these inserts could replace Val 289 with 57 aa (APP 75 l) or 76 aa (APP 770) protein domains before resuming with Pro and the remainder of the APP 695 aa sequence.

336

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D O N N E L L Y E T AL.

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B÷ B

C÷ C

D÷

D

E

F

G

H

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~!ii~ i~'.:~;i':-. , FIG. 4. Northern blots of various RNAs probed with APP DNA fragments. Formaldehyde/agarose gels were used to fractionate 5 micrograms of poly A+ KNA or 15 micrograms of total RNA which were then Northern blotted [4,28] and stringently hybridized to the probes shown in Fig. 2. pCLL 701 riboprobe was hybridized to human adult brain poly A+ RNA (Lanes A+ and A), to HL 60 poly A+ RNA (Lanes B+ and B), and to total cellular HL 60 RNA (Lanes C+ and C). Two exposures of blots in Lanes A, B, C, and D (where +'s indicate longer exposures) are shown so that minor bands may be more easily visualized. Probe PI was hybridized to human adult brain poly A+ RNA (Lanes D+ and D), rat brain poly A+ RNA (Lane E), and to HL 60 poly A+ RNA (Lane F). Total HL 60 RNA was hybridized to Probe C (Lane G) and to Probe 695 (Lane H). Each Northern blot contained Hind Ill cut lambda DNA size markers which, after hybridization to the APP eDNA fragments, were reprobed with random-primed lambda DNA to visualize the positions of the size standards. The sizes of the positively hybridizing transcripts were calculated based on their mobilites relative to the size standards. Blots have been marked on the right with numbers to indicate the positions of differently sized APP RNAs as follows: 1 for 1.6 Kb species, 2 for 3.2 Kb species, 3 for 3.5 Kb species, 4 for 4.0 Kb species, and 5 for 5.6 Kb species of APP RNA. To facilitate comparisons, each strip has been aligned so that the 3.5 Kb band migrates at the same position. TABLE 1 DISTRIBUTIONOF APP RNA SIZES WHICH HYBRIDIZETO VARIOUSAPP PROBES BY NORTHERN ANALYSIS RNA Source Probe*

Total HL 60 RNA

pCLL701 PI C 695

5.6/4.0/1.6Kb 4.0/1.6 Kb 4.0/1.6 Kb 5.6 Kb

Poly A+ HL 60 RNA 5.6/4.0/1.6Kb 4.0/1.6 Kb ND ND

Human Brain Poly A+ RNA 5.6/3.5/3.2/1.6Kb1" 5.6/3.5 Kb NF ND

Rat Brain Poly A+ RNA 3.5/3.2/1.6 Kbl" 3.5/2.4 Kb NF ND

*See Fig. 2 for localization of probes to the APP eDNA. 1"See Vitek [28] for additional Northern blot data. ND---not determined. NF--not found.

The sequence of the amino terminal 56 aa, that is the PI domain, of each insert is identical as are the 5' 168 bp encoding them. In LED709, these 168 bp are immediately followed by an additional 57 bp which could encode 19 aa or the " C " domain. The sequence of the L E D 708 clone (PI domain only) exactly matches the APP 751 e D N A clones isolated from SV 40 transformed fibroblast [18] and H L 60 [26] libraries. The sequence of the L E D 709 clone (PI + C domains) exactly matches the APP 770 e D N A clone isolated from a human glioblastoma library [ 12]. Unlike these other studies, we find that a single H L 60 library contains both APP 751 and APP 770 forms of eDNA.

We confirmed our e D N A cloning results and potentially broadened the diversity of APP R N A forms by hybridizing probes specific for portions of each APP e D N A to Northern blots. We find that the 5.6, 4.0, and 1.6 Kb RNAs from H L 60 ceils hybridize to p C L L 701 which contains the BAP region and identifies them as APP RNAs. Both 4.0 and 1.6 Kb transcripts hybridize to Probe PI which derives from the PI domains of the longer APP c D N A s and to Probe C which derives from the C domain of APP 770. The 5,6 Kb R N A hybridizes to Probe 695 which derives from regions of the PIC-less APP 695 e D N A flanking the PI and PIC inserts. The hybridization of specific probes to three APP R N A s and

M U L T I P L E FORMS O F APP RNAs IN A S I N G L E C E L L TYPE the cloning of two APP cDNAs suggests that the APP 751, APP 770, and probably the APP 695 forms of R N A are expressed in a single cell type. With respect to human brain APP R N A diversity, p C L L 701 hybridizes to 5.6, 3.5, 3.2, and 1.6 Kb transcripts. Our Probe PI hybridizes to a 3.5 Kb APP R N A which is seen by other groups using different PI specific probes [12, 18, 26] and to a novel 5.6 Kb APP R N A in adult brain. Thus, in addition to the 3.5/3.2 K b doublet seen by many groups, the APP R N A candidates might include a shorter 1.6 Kb species which is also seen by Zain [31] and a longer 5.6 Kb species. Though we fail to detect hybridization with Probe C to adult brain R N A (see also Kitaguchi [12]), cDNA cloning and sequencing shows that fetal [10], normal adult [19], and AD [28,31] brains also contain the APP695 species of RNA. The hybridization of specific probes and cloning of APP cDNAs suggests that at least the APP 695 and APP 751 forms of R N A are expressed in human brain. The qualitative diversity of APP R N A forms highlights the challenge of def'ming which APP protein form or forms are associated with BAP deposition. Using Western blots and antibodies to short peptide sequences predicted to be in three of the putative APP proteins, a doublet of immunoreactive bands is found at 80 kilodaltons (Kd) in adult brain [23]. Using the same approach, a 93 Kd immunoreactire band is only found in mature muscle even though myotubes, myoblasts, and innervated muscle all express APP R N A s [32]. These large protein sizes require APP RNAs in the 2.1 Kb or larger size range to encode them. Since some APP RNAs are just 1.6 Kb in size and the D N A motifs surrounding some internal Met residues favorably compare to K o z a k ' s consensus sequence for initiation of translation [13], APP proteins smaller than 80 Kd are theoretically possible. Even if amino terminal protein sequencing connects one form of APP protein with its proper APP RNA, the muscle data suggest that the presence of APP R N A alone does not necessarily predict the presence of APP protein. The function of an APP protein may not be limited exclusively to being a precursor for BAP. The amino terminal 56 aa (PI domain) of the APP 751 and APP 770 protein inserts is

337

structurally similar to the aa sequence of the Kunitz domain of serine protease inhibitors [12, 18, 26]. Lysates of cells transfected with the PIC-containing APP 770 c D N A inhibited trypsin more than lysates of cells transfected with the PIC-less APP 695 c D N A [12], suggesting that the PI domain functions as a protease inhibitor. However, since the amino acid sequence flanking the active site of an enzyme can alter its activity, the placement o f the C domain (in APP 770) versus the remainder of the APP 695 protein sequence (in APP 75 I) could allosterically modulate the potency as well as the specificity of the Kunitz-like PI domain in different APP proteins. These data suggest that a diversity in APP protein function may also be considered in BAP deposition. Though the function of PI-containing APP proteins as protease inhibitors in vivo remains to be shown, Abraham [1] showed that aiphal-antichymotrypsin is present in amyloid plaques from AD brains. Thus, some type of protease inhibitor activity may be required for BAP deposition. The search for a measurable difference in some aspect of BAP accumulation that could explain its deposition in AD brains must continue. Many explanations are now possible, including a developmental program of subtle changes in BAP accumulation rates which is exerted over numerous decades. In addition to the multiplicity of potential precursors to BAP, multiple genetic loci like those for familial Alzheimer's disease (FAD) [20] and DS [2] are associated with BAP deposition. If BAP deposition causes neuronal cell death, then inhibition of the pathway leading to BAP accumulation remains a tenable way to alter the course of Alzheimer's disease. Alternatively, if BAP accumulation represents a tombstone marker for neuronal degeneration, then greater understanding of its expression may produce insight into the unique pathogenesis of AD and DS. ACKNOWLEDGEMENTS We thank Drs. B. Beer, B. Dubnick and A. Oronsky for their continued support of this research at Lederle Laboratories. Dr. C. G. Rasool is a faculty scholar of the Alzheimer's Disease and Related Disorders Association (Chicago, IL).

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