PA4-Amyloid Protein Precursor mRNA Isoforms without Exon 15 Are ...

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8 are well known to be alternatively spliced, APP mRNA isoforms without exon 15 were only recently identified in leukocytes and rat brain microglial cells and ...
THE JOURNALOF BIOIOCICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology,Inc.

Vol. 269,No. 2,Issue of January 14,pp. 1510-1517,

1994 Printed in U.S.A.

PA4-Amyloid Protein Precursor mRNA Isoforms without Exon 15 Are Ubiquitously Expressed in Rat Tissues Including Brain, but Not in Neurons* (Received for publication, May 21, 1993, and in revised form, August 24, 1993)

Rupert SandbrinkSB, Colin L. Mastersn, and Konrad BeyreutherS From the $Center for Molecular Biology Heidelberg (ZMBH), University of Heidelberg, Im Neuenheimer Feld 282, 0-69120 Heidelberg, Germany and the Wepartment of Pathology, University of Melbourne, Parkville, Victoria 3052, Australia

The PA4-amyloidprotein precursor (APP), the source of the PA4-amyloid deposits found in Alzheimer brains, constitutes afamilyoftransmembrane glycoproteins generated by alternative splicing. While exon 7 and exon 8 are well known to be alternatively spliced, APP mRNA isoforms without exon 15 were only recently identified in leukocytes and rat brain microglial cells and therefore denoted as L-APP mRNAs. In order to perform a detailed analysis of individual L-APP mRNA isoforms in perfused rat tissues, we developed a quantitative polymerase chain reaction assay from reverse transcribed RNA allowing us to analyze the alternatively spliced region in between exon 6 and 16. In all peripheral tissues examined, L-APP mRNA isoforms were detected comprising between 25% (skeletal muscle) and about 70% (aorta, pancreas) of total APP transcripts. All four possible APP mRNA isoforms without exon 15 were shown L-APP733, L-APP696, and to exist, i.e. L-APP752, L-APP677. L-APP expression in the central nervous system (“4% of total APP mRNA) was then studied in more detail by analyzing different brain regions and tissues and primary cultured brain cells. The only cell type which was shown not to express L-APP mRNA to a detectable level is the neuronal cell. Ubiquitous expression of APP mRNAs lacking exon 15 except for neurons indicates an important function in non-neuronal cells and is especially remarkable since neurons are the primarily affected cells inAlzheimer’s disease.

10). However, the major part of APP molecules undergoes proteolytic cleavage within the PA4 region to give rise to soluble, non-amyloidogenic secretory products (11, 12). Analysis of APP mRNA expression revealed that APP canbe detected in almost all tissues examined (10, 13-16) as well as cultured cells. The biological activity of APP is still notunderstood. Several putative functions have been ascribed, including participation in cell-cell and cell-matrix adhesion (171, heparin-binding (21), growth promoting activities (18-201, and an excitoprotective role in neurons (53). Other suggestions comprise proteinase inhibition (13, 28, 29, 541, synaptic function (22), and receptor activity (8, 23). APP constitutes a family of different isoforms that are generated by alternative splicing of the 19 exons encoded by the APP gene (24-26). These different APP isoforms are named according to their length in aminoacids. APP695 mRNA lacking exon 7 and 8 is the most abundant APP transcript in the brain (13,14,16,271.As major primary translationproducts in peripheralorgansthe APP770 and APP751 isoforms were found (13,28,29), which represent thefull-length and theexon 8 lacking isoform, respectively. On RNA levels the APP714 mRNAisoform, solely lacking exon 7, was alsodetected (16,271. Exon 7 codes for a 57-amino-acid domain with considerable homology to a serine protease inhibitorof the Kunitz-type (13, 28, 29). Exon 8 encodes for a 19-amino-acid domain with homology to the MRCOX-2 antigen (30). Two other rare transcripts of the APP gene have also been identified. Bothencode C terminallytruncated, non-amyloidogenic forms and were therefore denoted as APRP563 (for “amyloid precursor-related protein”) and APRP365 mRNAs (31, 39). Since altered APP Alzheimer’s disease (AD)’is neuropathologically characterprocessing required for amyloid deposition in AD could very ized by senile amyloid plaques, neurofibrillary tangles, and well be influenced by altered expression of the various APP cerebrovascular amyloidosis (1,2). The PA4-amyloid protein is mRNAs, the alternative splicing of exon 7 and 8 in AD and the major proteinaceous component of amyloid deposits in AD aging hasbeen studied by many groups (14,16,32-40).Unforbrains. It is derived by an only partially characterized metatunately, the results of these experiments are inconsistent, and bolic pathway (3-5) from a larger transmembraneglycoprotein, as yet no consensus has been reached regarding the disease therefore denoted as PA4-amyloid protein precursor (APP)(6status and thelevels of specific mRNAs for APP. Recently, a third alternatively used splice site in the APP * This work was supportedthrough SFB 317 and 258 of the Deutsche gene was identified, involving the 54-bp large exon 15 (41). This Forschungsgemeinschaft, the Bundesminister fur Forschung und Technologie of Germany, the Metropolitan Life Foundation, the Fonds der exon codes for 18 amino acids which precede the PA4 region of Chemischen Industrie, the Forschungsschwerpunkt Baden-WiirttemAPP by 16 aminoacids. It was thereforesuggested that alterberg (to K. B.), and the National Health and Medical Research Council of Australia, theVictorian Health Promotion Foundation,and the Alu- native splicing of exon 15 may influence the pathway leading to minium Development CorporationofAustralia (to C. L. M.). Thecosts of the liberationof the PA4 subunit. TheAPP transcripts excludpublication of this article were defrayedin part by the paymentof page ing exon 15 were first discovered in peripheral leukocytes and charges. This article must therefore be hereby marked“aduertisement” microglial cells, and are therefore denoted as leukocyte-derived in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 0 To whom correspondence shouldbe addressed: Center for Molecular APP (L-APP) mRNAs (41, 42). On the protein level, L-APPs Biology Heidelberg (ZMBH), University of Heidelberg, Im Neuenheimer were found to be also expressed in astrocyte-enriched cultures Feld 282, D-69120 Heidelberg, Germany. Tel.: +49-6221-566848;Fax: (43). Since a regulated alternative splicing of exon 15 in re+49-6221665891. sponse t o immunogenic stimuli was demonstrated, it wasproThe abbreviations used are: AD, Alzheimer’sdisease;APP,PA4posed that L-APPs play a role in immunocompetent cell activity amyloid protein precursor; APRP, PAC-amyloid precursor related pro(43). tein; L-APP, leukocyte-derived APP; PCR, polymerase chain reaction; An exact analysis of the tissue-specific regulation of L-APP RT-PCR, PCR of reverse transcribed R N A , bp, base paifis).

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Ubiquitous Expression of L-APPmRNAs mRNA expression has not yet been performed. Therefore, we developed a quantitative polymerase chain reaction assay from reverse transcribed RNA (RT-PCR), allowing us to analyze the whole region between exon 6 and 16 ofAPP transcripts (24-26). This strategy enabled us to distinguish between APP splice isoforms generated by alternative splicing of exons 7,8, and 15. To answer the open question of L-APP mRNA expression in vivo, we then applied this method to RNA from perfused rat tissues. Expression of L-APP mRNA was studied in detail in tissues of the peripheral system and compared to L-APP mRNA expression in different braintissuesandprimarycultured brain cells. In all tissues examined, including the centralnervous system, L-APP mRNA isoforms were detected, but not in primary cultured neurons. We therefore presume that high levels of APP mRNA expression by neurons in vivopaired with the inability to alternatively splice exon 15 are the reasonsfor the characteristic,low portion of L-APP transcripts detected in brain, and thisconstellation is noteworthy in regard toAD as a brain-specific disorder.

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PCR productswith sr@41/nr1970: APP 770

1153 bp

L-APP 752

1099 bp

APP 751

1096 bp

L-APP 733

1042 bp

APP 714

985 bp

L-APP 696

931 bp

APP 695

928 bp

L-APP 677

874 bp

FIG.1.RT-PCR assay to detect alternative spliced APP mRNA isoforms. The exon structure of the full-length APP transcript is shown, indicating alternative splicing of exon 7, 8, and 15. Bottom, the series of PCR employed in this study is depicted. The following primer MATERIALSANDMETHODS pairs were used: PCR A, sr037/ar664; PCR B , sx169/ar664; PCR C, sx1200/ax1810; PCR D, sr841/ax1810;PCRE , sx1200/ar1970; andPCR Animals and Tissue Preparation-Unless otherwise noted, tissues by use of sr841/ or 87months old, which were obtained F, sr841/ar1970. Below this, amplified cDNAfragments were prepared from Wistar rats, from the animal facility of the Centerfor Molecular Biology, Heidelberg. ar1970 asPCR primers are illustratedfor all eightpossible APP mRNA Animals were sacrificed by C 0 2 inhalation and immediately transcar- splice isoforms. BglI and FspI restriction digestion sites are indicated dially perfused with ice-cold phosphate-buffered saline. Only well per- above. pA4, the pA4-amyloid encoding sequence (gray), while Tm and coding for the transmembrane part of fused animals were used for tissue dissection.removed The tissues were striped burs indicate the sequence APP. quickly frozen in liquid nitrogen. Cell Preparation and Cell Culture Conditions-More than 95% pure neuronal cultures were obtained when septal cells of E18 rat brain were 480. Reaction conditions wereas following: 200 nM of sense primer, 200 of radioactive labelingof PCR prodplated on poly-DL-ornithine-coated dishes and kept in serum-free me- nM of antisense primer (in the case dium together with 2PM cytosine-arabinoside for 8 days (44). A rather ucts, 50 nM of [33P-lar1970and 150 nM of unlabeled ar1970), 0.2 mM mixed culture of neurons, astrocytes, and some microglial cells was dNTP, and 2.5 units of Taq polymerase (Boehringer Mannheim) with obtained by plating thecells on poly-ornithine-coated dishes and culti- buffer conditions from the Taq polymerase supplier. Amplification was vating them in serum-containing medium (5% fetal calf serum, GIBCO/ performed with a "hot start": 2 min a t 95 "C without Taq polymerase, BRL) without cytosine arabinoside for 8 days. Astrocytes (more than then, at 75 "C, addition of Taq; cycle conditions were 94 "C for 1 min, of 95% pure, glial fibrillary acidic protein-positive) were obtained by cul- 55 "C for 1min, 72"C for 2.5 min (2-s extensiodcycle, final extension 35 cycles were 10 rnin). For PCR analysis without labeling the products, turing rat postnatal (P6) cerebellar cells onto uncoated dishes for 2 performed; for radioactively labeled PCR products, usually 24 cycles weeks, shaking them for several hours, and subsequently cultivating adhering cells in serum-containing medium without cytosine arabino- were performed. Only the blood and the pancreas sample were amplified using 26 cycles in order to get a comparably strong signal. For side for more than 6 weeks (modified from Ref. 45). analysis of PCR amplification as a function of the cycle number, the Isolation of RNA-Total RNA was prepared as described (46). For final extension step was omitted. RNA extraction of brain vessels (A. basilaris and circulus arteriosus Analysis ofPCR Products-PCR products (between 4 to 20 pl) were Willisi) 2 pg of tRNA was addedas a carrier. Yield and quality of RNA analyzed either on 1.5% agarose gels by ethidium bromide staining or preparations were determined by spectrophotometry (47). PCR Primers andAPP cDNA Encoding Plasmids-The designation by denaturing polyacrylamidegel electrophoresis (4.0%) and autoradiography as described (47). Restriction digestionof unlabeled PCR prodof PCR primers reflects the position of the primer in regardto the rat cDNA APP sequence including exon 7 and 8 (25, 26). The following ucts before electrophoresis was performed with FspI (New England primers were used:srO37 (20 bp): 5'-TGGACGG?TCGGGCTCTGGA-3' Biolabs), while radioactively labeled products were digested with BglI of individualbands,gels (nucleotides 37-56); sx169 (22bp): 5'-TCAGGGACCAAAACCTG- (Boehringer Mannheim). For quantitation were analyzedon a phosphorimager (Molecular Dynamics) by overnight CAITG-3' (nucleotides 169-190); sr841 (22bp): 5"GAGTCTGTGGAGGAGGTAGTCC-3' (nucleotides 841-862); sx1200(22 bp): 5'-CAAG- exposure; in each case a corresponding background value was subtracted. The figures shown were prepared from corresponding autoraCACCGAGAGAGAATGTCC-3' (nucleotides 1200-1221); sr1995(24 diograms (Kodak X-OmatA F t ) with an exposure time between 2 days bp): 5'-GATCTCAGAAGTGAAGATGGATGC-3'(nucleotides 1995recorded by 2019); ar664 (24 bp): 5'-GGCTACITCTACGACl"GTCITC-3' (nucleo- and 3 weeks. Imagesof ethidium bromide-stained gels were tides 664-687); ax1810 (22bp):5'-CAn'CACGGGAAGGAGCTCCAC-3' means of a n electronic video imaging system (Cybertech CS1) andan video copy processor (Mitsubishi). Purificationof unlabeled PCR prod(nucleotides 1810-1831); ar1970 (24 bp): 5'-C'ITCTGTCn'GATGTTI" ucts by low melting point-agarose gel electrophoresis(1%) and phenolGTCAACC-3' (nucleotides 1970-1993); ax2360 (22 bp): 5"CGATGGGchloroform extraction was performed as described (49). TAGTGAAGCAATGGT-3' (nucleotides 2360-2381). For labeling and quantitation of PCR products only the primer pair RESULTS sr841 and ar1970 was used. 5'-End-labelingof ar1970 was performed with polynucleotide kinase(BoehringerMannheim)and [Y-~~P]ATP RT-PCR Can Be Used to Amplify APP cDNA Fragments Com(Amersham or DuPont-New England Nuclear) to a specific activity of about 0.6 pCi/pmol primer, as determined after purification of the la- prising the Alternative Splice Sites at Exon 718 and Exon 15 "Several APP isoforms are generatedby alternative splicing of beled oligonucleotide (Bio-Spin 6 columns, Bio-Rad) (49). Human APP cDNA encoding plasmids used as APP standards in PCR the 19 exonsencoded by the APP gene (24).Fig. 1illustrates the experiments were previously described (41). exon-structure of the full-length APP transcript,among which Reverse Dunscription of RNA a n d Polymerase Chain Reaction-In exon 7,8, and 15 are known to be alternatively spliced (13,28, general 2 pgof total RNA, in some cases (meninges, plexus choroideus,29, 41). In order todevelop a RT-PCR assay for distinguishing parathyreoidea) 1pg, and in the case of brain arteries the whole RNA preparation were reverse-transcribed with200 units of Super-ScriptTM between the different APP splice products, we performed a RNA extracted (GIBCOBRL) usingoligo(dT)17as primer following the manufacturer's series of PCRs using different primer pairs with from perfused rat brain and thymus.For analysis, 2 pg of RNA protocol. One-hundredth of this cDNA preparation was amplified by PCR (48). PCR was carried out in a Perkin ElmerDNA thermal cycler were reverse transcribed and an aliquot amplified in 35 cycles.

-

Ubiquitous Expression of L-APP mRNAs

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A B C D E F G M ---- Cx Thy Cx Thy Cx Thy Cx Thy Cx Thy Cx Thy Cx Thy [bPl 16361

517E06 1°18] 396 298

"_""'

"1

1

FIG.2. PCR products obtained with cortical and thymuscDNA preparations by use of different primer pairs. TotalRNA was extracted from perfused rat brain and thymus, and 2 pg were reverse transcribed and aliquots amplified for 35 cycles using the primer pairs described in Fig. 1. PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide. Cx, cortex; Thy, thymus; M,DNA molecular weight marker.

-

I 20

no

22

24

26

28

30

Number of PCR cycles

FIG.3. Quantitation of APP mRNA isoforms using RT-PCR 2 pg of hippocampal RNA (Sprague-Dawley rat, 1 year of age) were reverse transcribed, the resulting cDNAs amplified for the stated number of cycles using unlabeled sr841 and 33P-end-labeledar1970 as PCR primers, and the products separated by denaturing polyacrylamide gel electrophoresis. Individual were quantitated using a phosphorimager. This logarithmic plot of incorporated radioactivity as a function of the PCR cycle number demonstrates that the amplification efficiencies remain constant through 24 cycles and were about equal for all bands analyzed.

In Fig. 2 the results are shown. PCR analysis A and B were performed to evaluate whether there is significant alternative splicing 5' to exon 6. Only the expected PCR products were illustrated, theslopes of the curves remain constantthrough 24 observed. The same holds true for PCR analysis G, which cov- cycles, corresponding to anamplification efficiency of 0.59-0.62 ers the region 3' to the exon 15 splice site. PCR analysis C, for all assessed APP mRNA isoforms (16). This conformity is using sx1200 and ax1810 as primers, produced the predicted presumably due to the similarity in length of the different PCR 632-bp fragment, indicating the absence of alternative splicing products (Fig. 1).The initial relative amounts of the different in between exon 9 and 14. PCR analysis D was performed using APP transcripts in the sample should therefore be identical to the sr841 primer on exon 6 and ax1810 on exon 14. In both the numerical values as determined for the corresponding PCR cDNA preparations, i.e. from brain and from thymus, several products, which remained constant even through 26cycles PCR products were detected, corresponding to the transcript (data not shown). We then evaluated thereproducibility of the datagained by including exon 7 and 8 (991 bp), and the transcripts lacking either exon 8 (934 bp)or exon 7 (823bp) or both exons (766 bp). our PCR assay (24 cycles). For this, we analyzed RNA prepaThese results are consistent with the presence of alternatively rations extracted from hippocampi of six different animals spliced mRNA isoforms previously described as APP 770, 751, (Sprague-Dawley, about 1 year of age). The following results 714, and 695 (13,16,27-29). The weak band at 840 bp canbe were obtainedfor the described four bands:APP770,l.g 0.3%; explained as heteroduplex form resulting from the high num- APP751 + L-APP752, 6.8 2 0.5%;APP733, 2.4 2 0.4%; and ber of PCR cycles (50). PCR analysis E demonstrates the re- APP695, 88.4 2 1.2%. This indicates a high accuracy of the sults obtained when a sense primer on exon 9 (sx1200) and an relative amounts as obtained by our assay. antisense primeron exon 16 (ar1970)were applied. Two differPeripheral Tissues Contain Significant Amounts of L-APP ent products of 771 and 717 bp (and an intermediate hetero- mRNAs-For a detailed analysis of APP mRNA isoforms in duplex form) were detected using thymuscDNA, corresponding peripheraltissues,thequantitative PCR assay, described to the transcripts with and without exon 15 (41). This result above, was used. In Fig. 4, top, corresponding autoradiograms clearly indicates thepresence of significant L-APP mRNAs in are shown for 11of the examined tissues. In general, five difthis tissue. The717-bp fragment (L-APP mRNA isoforms) was ferent bands arevisible, corresponding to different APPmRNA detectable as a very faint band even in the brain sample. For isoforms. These are identified by comparison with PCR proddetailed analysis of individual APP splice products, we em- ucts of APP plasmid standards. In some of the samples, espeployed a sense primer on exon 6(sr841) and an antisense cially skeletal muscle, a n additional signal is detectable, correprimer on exon 16 (ar1970)as shown in PCR F (Figs. 1and 2): sponding to APP714 mRNA (16, 27). Direct inspection of the a complex splice pattern arises reflecting the eight different amplified APP cDNA fragments showed that in all peripheral possible mRNA isoforms illustrated in Fig. 1. In order to fur- tissues LAPP733mRNA was expressed. LAPP677 mRNA was ther analyze and to quantitate the relative proportion of the alsodetectablein allperipheraltissues examined. Since different APP transcripts in perfused rat tissues, this PCR APP751 and L-APP752 comigrate as a single band, we further assay was applied in a radioactive version as described below. analyzed the amplified APP cDNA fragments by restriction RT-PCR Assay for Quantitation ofAPPmRNA Isoforms-For digestion with BgZI (Fig. 1).The resulting 5"digestion fragquantitation of APP mRNAs by RT-PCR using the primer pair ments, i.e. the exon 15 containing fragment of 538 bp and the sr841 (exon 6) and ar1970(exon 16), ar1970 wasradioactively fragment without exon 15 of 484 bp, were then quantitated labeled on its 5'-end with polynucleotide kinase and (Fig. 4). The relative amount of LAPP mRNAs in the periph[Y-~~PIATP. Initially, we tested the PCR amplification rate of eral tissues examined varies between 25% (skeletal muscle, 72% (pancreas, followed by aorta the different productsfor different cycling times, using a cDNA then placenta and testis) and pool from perfused rat hippocampus. Four major bands were and lung)of total APP transcripts (Table I). This is even higher detected on the autoradiogram which were identified by use of than the result obtained for blood (56%), indicating that the plasmid standards (see also Figs. 4 and 6a).As expected, the observed L-APP transcripts areindeed due toendogeneous exmost intense band corresponds to APP695 mRNA. Another pression by the perfused tissues themselves. Thus, alternative band corresponds to APP751 and LAPP752 together, and two splicing of exon 15 seems to occur in all peripheral tissues. weaker bands are made up by APP770 and GAPP733. Hence, Comparison with results obtained for unperfused tissues reat least one L-APP isoform is readily detectable in perfused rat veals that the relative amount ofL-APPmRNA isoforms is brain hippocampus. The amount of radioactivity incorporated virtually independent of perfusion. As assessed for liver, kidin theindividual bands was then measured and the logarithms ney, and heart, the differences are usually smaller than 3%. plotted as a function of the PCR cycle number (Fig. 3). As This is in excellent agreement with data from Northern blot

*

Ubiquitous Expression of L-APP mRNAs

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predominant APP695 mRNA, while the 393-bp and the538-bp fragments are derived from the weaker expressed L-APP696 mRNA. The additional band intermediate to the fragments of 538 and 481bp most probably arises from heteroduplex formation due to the high numbers of PCR cycles (50).Thus, all four APP mRNA isoform: possible L-APP transcripts areexpressed in skeletal muscle to a detectable level, and therefore exist in vivo. 751l752 Assessment of All Eight Possible Isoforms Arising from Alternative Splicingof Exon 7,8,and 1.5-Our assay can be used 695/696 to quantitate all eightAPP mRNA isoforms generated by alternative splicing of exon 7,8, or 15. For this, cDNA fragments are amplified as described with radioactive labeling of the sense primer (exon 6). Aliquots of the PCR products are then anaNon-L-APP lyzed both directly without digestion, and after restriction digestion in between exon 9-14, revealing the amounts of the L-APP corresponding 5' digestion products. From this, the relative amount of all eight APPtranscript can be calculated.2 FIG.4. RT-PCR analysis of APP mRNAs in peripheral tissues. Using a labeled antisense primer, a good estimation of the Autoradiograms for 11 of the examined tissues are shown. For each tissue, 2 pg of total RNA were reverse transcribed, and aliquotsof the contribution of APP751 mRNA and L-APP752 mRNA can also resulting cDNAs wereamplifiedusing sr841 andsnP-end-labeled be made. The relative amounts of the undigested PCR products ar1970 a s PCR primers. PCR products were either directly separated by and, afterdigestion, of total L-APP and total exon 15 encoding denaturing polyacrylamide gel electrophoresis (top) or digested with BglI before analysis (bottom).The total number of cycles was 24; only APP transcripts are determined as described. From this, the among the unresolved cDNAs from blood and pancreas wereamplified in 26 cycles in order to portion of APP751 andLAPP752 obtain comparable signals. A water control was performed which in- APP751+ L-APP752 band of the undigested PCR products can cluded all steps of RNA extraction, cDNA synthesis, and PCR, and no approximately be ~ a l c u l a t e d Corresponding .~ values for APP detectable signals wereobserved (data not shown).GI., gland;parathyc, 751 and L-APP752 are given in Table I. For some samples, the parathyroid gland. first method mentioned above was also employed. Because the results obtained did not differ significantly, and since our inanalysis of total APPexpression in peripheral tissues including terest mainly focuses on L-APP mRNAexpression in thiswork, blood. Hereby, the level of total APP transcripts in blood was we further applied the second method as described. Tissue-specific APP mRNA Splice Patterns-Application of assessed to be much lower than inevery other organ analyzed the methods described revealed different patterns of APP (data not shown). All Four Possible L-APP mRNAs Exist in Vivo-Four possible mRNA expression in the examined tissues (Table I). Skeletal L-APP mRNA isoforms can theoretically arise from independ- muscle is striking in expressing more than half of its APP ent splicing of exon 7, 8, and 15 (Fig. 1).The presence of L transcripts as APP770. Among the peripheral tissues the lowAPP733 and L-APP677 was readily confirmed as described est level of exclusion of exon 15 was alsoobserved in this tissue above. In order to verify the identity of the individual bands (25% L-APP transcripts). Interestingly, in skeletal muscle the and to check them for existence of L-APP752 and L-APP 696, highest amount of APP714 mRNA was detected which is in unlabeled PCR products from skeletal muscle and heart were agreement with the observation of well detectable APP714 separated by agarose gel electrophoresis. These two tissues transcripts in related organs such as tongue and heart. In testis a very high amount of APP770 mRNA was also were chosen because of their relativehigh proportion of APP770 and APP714 (Fig. 4, Table I). From the gel, the ampli- detected (47%), while in epididymis APP752 was the transcript fied cDNA fragments were individually purified and reampli- with the highest relative amount. In the latter tissue, more tied by PCR using the same primer pair sr814/ar1970. Aliquots than 80%ofAPP mRNA splicing isoforms contain exon 7 and 8, were compared by gel electrophoresis with PCR products of to about equal parts as transcriptswith and without exon 15. APP transcripts with exon 7, but lacking exon 8, were maxiAPP cDNA containing plasmids confirming the correct size of the excised and reamplified material (Fig. 5a).Further aliquots mally expressed in kidney, contributing to -80%. Most of these were then digested with FspI, producing fragments of charac- are exon 15containingAPP mRNAs: APP751 mRNArepresents of APP teristic length for each of the different APP mRNA isoforms. about 50%of total APP mRNA in kidney. A high content These digestion products are shown in Fig. 5b in comparison to splice isoforms coding for exon 7 but not exon 8 was also obidentically digested amplified fragments of APP plasmids. As served for classical endocrine organs like thyroid gland(69%), expected, digestion of a plasmid coding for a L-APP cDNA pro- adrenal gland (61%), as well as for pancreas (66%), and intesduced a 3' fragment of 393 bp. A sequence containingexon 15 tinal tissues (about60%). Pancreatic tissueexhibited the highwas identified by appearance of a band of 447 bp. In each of the plasmid standard lanes, the5' fragment is visible as a second APP770, LAPP733, APP714, and LAPP677 are directly obtained band of either 706 bp (with exon 7 and 81,649 bp (without exon from quantitation of undigested products. The valuesof the otherAPP 81,538 bp (without exon 7; data notshown), or 481 bp (without mRNA isoforms are calculated as follows ( U means value obtainedfrom exon 7 and 8). By comparison, the L-APP677, APP714, quantitation of undigestedproducts, D from digested products): LAPP733, andAPP770 cDNA fragments from skeletal muscle LAPP752 = [770 + 7521(0) - 770(U), APP751 = L751 + 7331(D) could unequivocally be verified. The excised and digested 733(U), LAPP696 = [714 + 696](D) - 714(U),APP695 = [695 + 677KD) - 677(U). APP751 + L-APP752 band from heart as well as from skeletal APP770, LAPP733, APP714, and LAPP677 are obtained from muscle mainly produced the 393 and the706 bp bands indicat- quantitation of undigested products. The relative amounts of ApP751 ing a predominance of the L-APP752 mRNA isoform in this and LAPP752 are then calculated by (Umeans value obtained from band. In a similar way, the presence of L-APP696 mRNA was quantitation of undigested products,D from digested products): a = 751 714 =total APPwithexon 15(0)- 770(U)- 695 total APP with exon proven, at least for the skeletal muscle. Among the digestion +15(0) -770( U )4695 + 6961(U);6 = 752 + 696 = total LAPP@) products of the excised and reamplified APP695+L-APP696 677(U) - 733(U); then: APP751 a/(a + b ) * [751 + 752l(U), and: band, the two major bands (481 and 447 bp) represent the LAPP752 = b/(a + b ) * [751 + 752l(U).

I

1

f

1514

Ubiquitous Expression of L-APP mRNAs

TABLEI Differential expression of APP mRNAs in peripheral tissues, brain tissues, and primary cultured brain cells Radiolabeled and electrophoretically separated RT-PCR products (Figs. 4 and 6) were analyzed on a phosphorimager and relative amounts calculated in percent. For each band, a corresponding background value was subtracted. 770,751&752,733,714,695&696,and 677 represent the corresponding APP cDNA bands from analysis of undigested PCR products, while L-APP denotes the value obtained after BglI digestion. In 6958~696,the portion of L-APP696 is generally very low except from skeletal muscle and the non-neuronal primary cell cultures, as can be assumed from analysis of APP714 and total L-APP mRNA expression. KF’I represents the Kunitz-type protease inhibitor domain encoding APP mRNA isoforms calculated as the sumof APP770, AF’P751&L-APP752, and L-APP733. 752and 751 indicate estimated valuesfor the relative amountsof L-APP752 and APP751.. remectivelv (see text). APP mRNAisoform

770

751 714 &752

733

18 31 22 22 18 40 19 11 15 58 21 15 24 10 21 9 22 13 33 19 35 31 47 38 25 25 28 19 31

48 44 49 45 52 30 52 46 48 26 47 45 43 33 49 58 51 51 46 51 48 47 40 50 51 45 50 26 45

27 15 25 24 16 17 22 19 25 9 25 26 23 49 26 27 19 25 17 23 12 14 7 5 18 23 16 33 14