A Potential Role for APLP2 in Axogenesis

The Journal of Neuroscience, October 1995, 15(10): 63144326

Distribution of an APP Homolog, APLP2, in the Mouse Olfactory System: A Potential Role for APLP2 in Axogenesis

Gopal Thinakaran,1,5 Cheryl A. Kitt, 1,5 A. Jane I. Roskams,” Hilda H. Slunt,1,5 Eliezer Masliah,’ Cornelia von Koch,2,5 Stephen D. Ginsberg,i,5 Gabriele V. Ronnett,‘s4 Randall Ft. Reed,233I6 Donald L. Price,‘,2.4,5 and Sangram S. Sisodiaiz5 ‘Departments of Pathology, 2Neuroscience, 3Molecular Biology and Genetics, and 4Neurology, 5Neuropathology Laboratory, and the 6Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland, 21205 and ‘Departments of Neurosciences and Pathology and Neuroscience, University of California at San Diego, La Jolla, California 92093

Deposition of l3-amyloid (AS) in senile plaques is a major APLP2, alternative splicing, chondroitin sulfate glycosami- pathological characteristic of Alzheimer’s disease. Ap is noglycan, glomeruli, olfactory epithelium, o/factory sen- generated by proteolytic processing of amyloici precursor sory neurons] proteins (APP). APP is a member of a family of related polypeptides that includes amyloid precursor-like proteins A principal pathological feature of Alzheimer’s disease is the APLPl and APLPS. To examine the distribution of APLP2 deposition of Al3 in brain parenchyma (Glenner and Wong, in the nervous system, we generated antibodies specific 1984; Masters et al., 1985). Al3, an -4 kDa polypeptide, is for APLPS and used these reagents in immunocytochemi- derived from larger type I integral membrane glycoproteins, cal and biochemical studies of the rodent nervous system. termed the amyloid precursor proteins (APP) (Kang et al., 1987; In this report, we document that in cortex and hippocam- Kitaguchi et al., 1988; Ponte et al., 1988; Tanzi et al., 1988; pus, APLPS is enriched in postsynaptic compartments. In Golde et al., 1990; K&rig et al., 1992). APP is a member of a the olfactory system, however, APLPS is abundant in ol- family of homologous amyloid precursor-like proteins (APLPs), factory sensory axons, and axon terminals in glomeruli. including APLPl (Wasco et al., 1992) and APLP2 (Sprecher et Confocal microscopy revealed that APLPP is present in al., 1993; Wasco et al., 1993; Slunt et al., 1994). Although the both pre- and postsynaptic compartments in the olfactory members of this gene family share considerable homology in the bulb. Notably, mRNA encoding chondroitin sulfate glycos- amino- and carboxyl-terminal domains, the APLPs differ from aminoglycan (CS GAG)-modified forms of APLPS are en- APP by the absence of the Al3 region. riched in the olfactory epithelium, relative to alternatively- At present, APLP2 is the best characterized member of spliced mRNA, encoding CS GAG-free forms of APLPP. In APLPs. Like APP APLP2 is encoded by several mRNAs de- addition, we demonstrate that CS-modified APLPS forms rived by alternative splicing (Wasco et al., 1993; Sandbrink et accumulate in the olfactory bulb. CS proteoglycans are al., 1994; Slunt et al., 1994). Both APP and APLP2 are ex- known to play an important role in regulating cell migration pressed in brain and peripheral tissues (Wasco et al., 1993; Slunt and neuronal outgrowth. Since sensory neurons in the ol- et al., 1994), and in situ hybridization studies disclose similari- factory epithelium are in a state of continual turnover, ax- ties in the distributions of APP and APLP2 mRNAs in mouse ons of newly generated cells must establish synaptic con- (Slunt et al., 1994) and human (Wasco et al., 1993) CNS. Fi- nections with neurons in the olfactory bulb in adult life. The nally, APLP2 matures through the same unusual secreto- presence of APLPS in olfactory sensory axons and glo- ry/cleavage pathway as APP (Slunt et al., 1994). However, in meruli is consistent with the view that this protein may play contrast to APP-695 and APP-7511770, APLP2-751 is a sub- an important role in axonal pathfinding and/or synaptoge- strate for modification by a single CS GAG chain (Thinakaran nesis. and Sisodia, 1994). [Key words: amyloid precursor-like protein 2, APP, In this study, we report on the distribution of APLP2 in the rodent nervous system using newly generated antibodies specific for APLP2. Consistent with previous in situ hybridization stud- Received Apr. 3, 1995; revised May 22, 1995; accepted May 31, 1995. The CT12 peptide was obtained from the Protein Sequencing and Peptide ies (Slunt et al., 1994), our investigations with the APLPZspe- Synthesis Core maintained by the Center for AIDS Research at Johns Hopkins cific antibodies revealed that APLP2 is expressed in a variety of University School of Medicine under Contract Number 1P30 Al28748 from regions, including cerebral cortex, hippocampus, and thalamus. the National Institute for Allergy and Infectious Diseases of the NIH. We thank Drs. Samuel Gandy, Mike Lee, and Philip Wong for providing antibodies 369, Confocal microscopy revealed prominent colocalization of TuJl, and Myc-I, respectively; Karen Schrader for providing mouse olfactory APLP2 with MAP-2, a postsynaptic marker, in cortex and hip- epithelium RNA; Dr. David Borchelt for providing APP-695 cDNA; and Dr. pocampus. Moreover, APLP2 mRNA is highly expressed in sen- Wilma Wasco for providing the APLPI cDNA. This work was supported by grants from the NIA, NINDS, American Health Assistance Foundation, Al- sory neurons in the olfactory epithelium, and the encoded poly- zheimer’s Association, and the Adler Foundation. peptide is abundant in olfactory sensory axons, and axon ter- Corresoondence should be addressed to Dr. Sanaram S. Sisodia. The Johns Hopkins University School of Medicine, Neuropathilogy Laboratory, 558 Ross minals in glomeruli. In contrast to its predominant postsynaptic Research Building, 720 Rutland Avenue, Baltimore, MD 21205-2196. localization in neurons in cortex and hippocampus, APLP2 is Copyright 0 1995 Society for Neuroscience 0270-6474/95/156314-13$05.00/O present in both pre- and postsynaptic compartments in the ol- The Journal of Neuroscience, October 1995, 75(10) 6315 factory bulb. Notably, mRNA encoding CS GAG-modified cells,an African greenmonkey kidney line, weretransiently transfected forms of APLP2 are highly represented in the olfactory epithe- in 60 mm plates with 10 pg of CsCl-purified plasmid DNA using a high-efficiency calcium phosphate method (Chen and Okayama, 1987). hum, and CS GAG modified forms of APLP2 accumulate in the Thirty-six hours after transfection, cells were labeled with 200 l&i/ml olfactory bulb. Since olfactory sensory neurons are continually 35S-methionine (Du Pont NEN, Wilmington, DE) for 3 hr in Dulbecco’s turned over, axons of newly generated neurons must establish modified Eagle medium lacking methionine and supplemented with 1% synaptic connections with neurons in the olfactory bulb in adult dialyzed fetal calf serum. Detergent lysates and conditioned medium prepared from labeled cells were subject to immunoprecipitation anal- life. Taken together with the view that CS proteoglycans play isis as described (Sisodia et al., 1996; Slunt et al., 1994):CT12, D2-I, an important role in regulating cell migration and neuronal out- and D2-II antibodies were used at 1:2500 dilution for Western blot growth (for review, see Wight et al., 1992), the unique localiza- analysis of lysates prepared from cells transiently expressing APP, tion of APLP2 in olfactory sensory axons and glomemli is con- APLPl, or APLP2. sistent with the notion that this protein may play an important In situ hybridization. Sequences that encode residues 568-681 of mouse APLP2 were cloned into Bluescript KS+ (Slunt et al., 1994) and role in axonal pathfinding and/or synaptogenesis. used as templates to generate sense or antisense 33P-labeled riboprobes using a RNA transcription kit (Stratagene, La Jolla, CA). Sections (10 Materials and Methods pm) cut from fixed frozen mouse nasal epithelium were dried onto slides coated with Fol’s mounting medium, fixed in 4% paraformalde- Antibodies. Ab369 is a polyclonalantibody raised against the entire hyde (PFA) for 20 min and rinsed in phosphate-buffered saline (PBS) cytoplasmic domain (residues 645-694) of APP-695 (Buxbaum et al., and deionized water. After acetylation in 0.25% acetic anhydride pre- 1990), and was kindly provided by Dr. Samuel Gandy (Cornell Uni- pared in 0.1 M triethanolamine, sections were rinsed in PBS and de- versity Medical College, New York). c-Myc-modified APLP2 polypep- hydrated through a graded series of alcohol, and then dried. Sections tides (Thinakaran and Sisodia, 1994) were immunoprecipitated using were hybridized overnight at 55°C in a buffer containing 50% formam- polyclonal antiserum Myc-I (provided by Dr. Philip Wong, Department ide, 10% dextran sulfate, 0.3 M NaCl, 20 mu Tris (pH 7.5), 10 mu of Pathology, The Johns Hopkins University School of Medicine, Bal- Dm, 5 mu EDTA, 1 X Denhardt’s solution and 0.4 mg/ml yeast tRNA, timore), raised against the synthetic peptide MEQKLISEEDLN (Lee et X al., 1993). and 1 rig/slideof labeledprobe (-2 lo6 cpm/ng).Slides were washed asfollows: 5 X SSC (1 X SSC = 0.15 M NaCl, 0.015 M sodium citrate) To generate polyclonal APLP2-specific antibody CT12, a synthetic X peptide corresponding to the C-terminal 12 amino acids (residues 740- and 1 mu D’IT at 55°Cfor 20 min; 50% formamide,2 SSC and 1 mu D’lT at 60°C for 30 min; 10 mu Tiis (pH 7.5), 0.5 M NaCl, and 5 75 1) of mouse APLP2-75 1 with an additional cysteine at the N-terminus mu EDTA (TNE buffer) at 37°C three times 10 min each; TNE buffer (CNPTYKYLEQMQI) was coupled to maleimide-activated keyhole containing 20 kg/ml RNase A at 37°C for 45 min; TNE buffer at 37°C limpet hemocyanin (Imject, Pierce Chemical Co., Rockford, IL). The for 15 min: 50% formamide. 2 X SSC and 1 mM DTT at 60°C for 30 conjugate was purified by gel filtration and subsequently injected into min; 2 X SSC at 37°C for 20 min; and 0.1 X SSC at 37°C for 20 min. New Zealand White rabbits. Polyclonal antibodies D2-I and D2-II were After dehydration, slides were dipped into Kodak emulsion NTB-2 for raised in two separate rabbits using full. length mouse APLP2-75 1 synthesized in Sf9 cells as the antigen, as described previously (Thinakaran 3 d, and developed in Kodak D-19. and Sisodia, 1994). PCR analysis of APLP2 mlUVA. Total RNA was isolated by homoge- Purijcation of CT12, 02-J and 02-U antibodies. For immunocyto- nization of mouse olfactory epithelium and whole brain in 4 M guanidine chemical studies, we prepared an IgG fraction of CT12 antibody using thiocyanate and centrifugation of the lysate over a 5.7 M cesium chloride a Protein A agarose affinity column (Pierce Chemical Co., Rockford, cushion (Chirgwin et al., 1979). Reverse-transcribed RNA was used in IL). D2-I and D2-II antibodies were affinity purified on a column con- PCR to amplify alternatively spliced APLPZ mRNA. The primer pair taining a glutathione S-transferase (GST)-APLP2 fusion protein. Briefly, SD2, S’-CCGCTCGAGGAACAGCGAGCG-3’ (encoding amino acids sequences that encode residues 562-681 of mouse APLP2-751 (Slunt 567-570 of APLP2, and containing an X/z01 site at its 5’ end) and anti- et al., 1994) were generated by PCR and subcloned into plasmid pGEX- sense primer AsD2, S’-CCGGAAlTCATTGCTGCTCAAACTGAAAT- 2T (Smith and Johnson, 1988). The resulting plasmid, pGEXAAPLP2, CC-3’ (complementary to sequences encoding amino acids 675481 of encodes a fusion of GST with a 120 amino acid region of APLP2-751 APLP2-751 and containing an EcoRI site at its 5’ end) was used to which occurs immediately N-terminal to the transmembrane domain. analyze the presence of alternatively spliced transcripts which encode a The GST fusion protein (GSTAAPLP2) was expressed in bacteria and 12 amino acid peptide, DTQPELYHPMKK, primer pair SKPI, 5’- immobilized onto glutathione agarose beads as described (Smith and GTGCTGCCCTCAGACAAAG-3’ (encoding amino acids 202-207 of Johnson, 1988). For affinity purification, an IgG fraction of the D2-I or APLP2) and antisense primer AsD2 (above) was used to analyze alter- DZII antiserum was incubated at 4°C for 16 hr with glutathione agarose natively spliced transcripts which encode the KPI domain and/or the 12 beads containing bound GSTAAPLP2. The beads were washed twice amino acid peptide (see above). In order to identify PCR products gen- in a buffer containing 50 mu Tris-HCl (pH 7.5) and 0.3 M NaCl. Bound erated from the four different mRNA isofoxms of APLP2, cloned cDNA antibodies were eluted with 2 ml of 100 mu glycine-HCl (pH 2.5) and corresponding to APLP2-694, APLP2-751, or APLP2-763 were used as neutralized. templates in parallel PCR reactions. The PCR products were separated Plasmid construction. To generate pEF695, an expression construct on 1.3% agarose or 3% NuSieve GTG agarose (FMC Bioproducts, Rock- containing the mouse APP695 cDNA, plasmid pFLAPP (provided by land, ME) and visualized by ethidium bromide staining. Dr. Bruce T. Lamb, Department of Gynecology and Obstetrics, The Zmmunocytochemistry. C57/B 16 mice (Jackson Laboratories, Bar Johns Hopkins University School of Medicine, Baltimore) was incu- Harbor, Maine) or adult Sprague-Dawley rats were administered lethal bated in a polymerase chain reaction with sense primer 5’-CCGCTC- doses of the anestheticXylaket (25%ketamine, 2.5% xylazine in 15% GAGACGATGCTGCCCAGCTTGG-3’, antisense primer 5 ‘-CCGCTC- ethanol, 0.9% saline) and perfused intracardially with PBS, followed by GAGGCTTAGTTCTGCATTTGCTC-3’ and a third primer 5’-CGCC- 4% freshly depolymerized paraformaldehyde in 0.1 M PBS. The olfac- TGGACCTGGAGAATTACATCATCGC-3’ in order to modify the in- tory epithelium was dissected following removal of craniofacial skeletal ternal X/z01 site. The PCR product was digested with XhoI and the APP tissue and fascia and postfixed in 4% paraformaldehyde in PBS for 2 cDNA fragment was ligated to an unique Sal1 site in a modified hr. Olfactory bulbs were dissected and postfixed as above. After fixa- pEFBOS vector (Mizushima and Nagata, 1990). Plasmids pSVAPLP1 tion, dissected tissue was equilibrated and cryoprotected in 10% sucrose and pSVAPLP2 which contain the mouse APLPl and APLP2-75.1 in PBS at 4”C, then transferred to plastic molds and embedded in OCT cDNA, respectively, placed downstream of the SV40 promoter/enhancer medium (Tissue Tek, Miles, Inc., Elkhart, IN) on dry ice. Tissue was were generated as follows. Sequences encoding mouse APLPl were stored at -20°C until cryostat sections (10 pm) were cut onto gelatin- excised from a Bluescript vector (Wasco et al., 1992; kindly provided subbed glass slides and stored at -20°C. by Dr. Wilma Wasco, Harvard Medical School) and inserted in a pUC For immunocytochemical analysis, sections were first blocked for 1 19-based vector from p77OSP (Sisodia et al., 1990). The APLP2 cDNA hr in PBS containing 10% normal goat serum, 1% bovine serum albu- insert was isolated from plasmid pD2 (Slunt et al., 1994), and ligated min and oermeabilized in 0.1% Triton X-100 in PBS. Primarv antibod- to a vector molecule derived from plasmid p77OSP (Sisodia et al., ies were-incubated with sections overnight at 4°C in 4% no&al goat 1990). serum.CT12 IgG was usedat 6 kg/ml, and control reactionswere Transfection, immunoprecipitation, and Western blot analysis. COS-1 performed by preincubating an equivalent aliquot of antibody overnight 6316 Thinakaran et al. - APLPP in Olfactory System at 4°C with 60 pg/ml of CT12 peptide. D2-I and D2-II antibodies were that each of the transgene-derivedpolypeptides were expressed affinity purified on a GST-fusion protein matrix (see above) and used in transiently transfectedcells, we immunoprecipitatedeach spe- at 0.5 p,g/ml. For control reactions, the appropriate dilution of antibody was preincubated overnight with 25 pg of GST-fusion protein bound to cies from 35S-methionine-labeleddetergent lysates using an an-, matrix and used as indicated above. Primary antibodies were recognized tibody Ab369, raisedagainst the entire cytoplasmic tail (residues and subsequently visualized using an avidin-biotin-peroxidase kit (Vec- 645-694) of APP-695 (Buxbaum et al., 1990). We anticipated tor, Burlingame, CA) according to the manufacturer’s instructions and that Ab369 would recognize APLPl basedon our earlier studies visualized with diaminobenzidine. Sections were viewed and photo- graphed using a Zeiss Axiophot microscope and camera and images documentingthat Ab369 recognizesAPLP2 (Slunt et al., 1994). were processed and compiled using an AGFA color scanner and image Ab369 immunoprecipitated low levels of endogenously ex- analysis system. pressed- 110, -115, and -130 kDa APPIAPLP polypeptides Laserscanning confocal microscopy. Forty micrometer thick para- from extracts of COS-1 cells transfected with the expression formaldehyde-fixed brain sections were double immunolabeled using a plasmid which lacked a cDNA (Fig. lA, lane 1). As expected, combination of oolvclonal anti-APLP2 suecific antibody, D2-I (at 1:500), with moiocfonal anti-synaptophys;n (at 1:20) or monoclonal COS-1 cells transfected with plasmid pEF695 overexpressed anti-MAP-2 antibodies (at 1:20) (anti-synaptophysin and anti-MAP-2 - 110 kDa APP-695 (Fig. lA, lane 2). Furthermore,cells trans- antibodies were from Boehringer-Mannheim Labs, Indianapolis, IN). fected with plasmidspSVAPLP1 or pSVAPLP2 expressed- 100 Bound antibodies were subsequently reacted with a FITC-conjugated kDa APLPl or - 120 kDa APLP2 polypeptides, respectively horse anti-mouse IgG (1:75) and biotinylated goat anti-rabbit IgG sec- ondarv antibodies (1:lOO). followed bv avidin-Texas red (1:lOO) (Vec- (Fig. lA, lanes3 and 4, respectively); the heterogeneoussmear tar, Burlingame, Ck). tie double stained sections were transferred to of polypeptides extending from -125 kDa to -200 kDa in lane SuperFrost plus slides (Fisher, Tustin, CA) and mounted under glass 4 representsCS GAG-modified forms of APLP2-75 1 (seebelow, coverslips with anti-fading media containing 4% n-propyl gallate (Sig- Thinakaran and Sisodia, 1994). ma, St. Louis, MO). Sections were examined using the Bio-Rad MRC- Antibody D2-I, raised againstfull-length mouseAPLP2-75 1 600 laser scanning microscope mounted on an Zeiss Axiovert 35 microscope and optical Z-sections 0.5 p,rn in thickness were collected and detected low levels of endogenouslysynthesized - 110 and 115 stored as described (Masliah et al., 1993). kDa polypeptides in COS-1 cells (Fig. lC, lane 1) and high Preparationofbruin exrracrs.Adult Sprague-Dawley rats were deep- levels of - 115-200 kDa forms of APLP2 from extracts of cells ly anesthetized and perfused transcardially with PBS. The brain was transfected with APLP2-75 1 cDNA (Fig. 1C, lane 4). Similarly, then removed and different regions were dissected quickly and frozen at -70°C. Soluble and membrane protein extracts were prepared as antibody D2-II recognized high levels of - 115-200 kDa forms described (Blackstone et al., 1992). Briefly, tissues were homogenized of APLP2 in extracts of cells expressingAPLP2-751 (Fig. lC, with a Brinkmann Polytron in 10 volumes of ice-cold 10 mu Tris-HCl lane 8). Neither D2-I nor DZII antibodies detected transiently (pH 7.4) containing 10% (wt/vol) sucrose and protease inhibitors (20 expressedmouse APP-695 or APLPl (Fig. lC, lanes 2,3 and pg/ml each of leupeptin, pepstatin, antipain, and chymostatin, 0.2 U/ml 6,7, respectively). Antibody CT12, raised againstthe C-terminal aprotinin, 10 mu benzamidine, 0.1 mu PMSF, 1 mu EGTA, and 1 mu EDTA). The homogenate was centrifuged at 1000 X g for 10 min to 12 amino acids of APLP2, also detected low levels of endoge- remove nuclei and-cell debris, and the resulting supernatant fraction nously synthesized - 110 and 115 kDa polypeptides in COS-1 was centrifuged at 114.000 X ,e for 20 min at 4°C. The supernatant cells (Fig. lC, lane 9) and an -115 kDa form of APLP2 in fraction (S2)Yfrom the high speed centrifugation containing soluble pro- extracts of cells overexpressing APLPZ (Fig. lC, lane 12). teins was saved. The pellet fraction (P2) containing membrane proteins was washed twice by resuspension in homogenization buffer (lacking Moreover, CT12 failed to detect either APP-695 or APLPl (Fig. sucrose), centrifuged at 114,000 X g for 20 min and finally resuspended lC, lanes 10 and 11, respectively). The heterogeneity of the in homogenization buffer. Glycerol was added to both S2 and P2 frac- mature forms of APLP2 (Fig. lC, lanes 4, 8) is the result of tions to a final concentration of 20% and the extracts were stored at addition of variable-length CS GAG chainsto APLP2 core pro- -70°C. teins (Thinakaran and Sisodia, 1994). The apparent failure of Chondroirinasedigestion. Ten microgram aliquots of the S2 fraction prepared from olfactory bulb and hippocampus were digested with 5 CT12 antibody to detect the heterogeneousAPLP2 speciesis mU of protease-free chondroitinase ABC (Seikagaku America, Inc., due to the insensitivity of this reagent relative to the D2-I and Rockville, MD) in 100 mu Tris-HCl (pH 8.0) and 30 mu sodium ac- D2-II antibodies. Thus, theseresults unambiguouslydocument etate for 1 hr at 37°C. As a positive control for chondroitinase diges- that antibodies D2-I, DZII, and CT12 specifically recognize tions, we used serum-free conditioned medium prepared from a CHO cell line, B2, that constitutively expresses mouse APLP2-751 (Thinak- APLP2, but not its homologousproteins, APP and APLPl. aran and Sisodia, 1994) and which secretes soluble CS GAG-modified APLP2 derivatives. Localization of APLP2 in cortical and hippocampalneurons Consistent with our earlier in situ hybridization analyses of Results APLP2 mRNA expression (Slunt et al., 1994), immunocyto- Biochemical characterization of anti-APLP2 antibodies chemical mappingwith antibodiesD2-I, D2-II, and CT12 veri- In an earlier report (Slunt et al., 1994), we noted that several fied that APLP2 is expressedin neuronal populationsin many widely utilized antibodies raised against APP are highly cross- regions of mousebrain, including cerebral cortex, hippocampus, reactive with APLP2, a result due in large part to the significant and thalamus.To examine the subcellulardistribution of APLP2 sequencehomology between the two proteins. In the present in neuronalpopulations in cortex and hippocampus,we double- study, we report on the characterization of APLPZspecific an- stainedsections with D2-I and synaptophysin,a marker for pre- tibodies and the use of thesereagents in immunocytochemical synaptic boutons, or for D2-I and MAP-2, a dendritic cytoskel- and biochemical studiesof the distribution of APLP2 in the ro- eton-associatedmarker. Sections were analyzed by laser scan- dent nervous system.To assessthe specificity of antibodiesD2-I ning confocal microscopy. In the hippocampus,predominant (Thinakaran and Sisodia, 1994) and D2-II, raised against full- APLP2 immunoreactivity (IR) was observed in apical dendrites length mouse APLP2-751 or CT12 antisera, generated against (Fig. 2b), structureswhich were strongly stainedwith antibodies the C-terminal 12 amino acids of APLP2, we performed im- specific to the dendritic marker, MAP-2 (Fig. 2a,c). Parallel co- munoblot analysesof lysates prepared from COS-1 cells tran- localization studiesof APLP2 IR (Fig. 2e) and synaptophysin siently transfected with expression plasmids encoding mouse IR (Fig. 2d) againrevealed prominent localization of APLP2 in APP-695, mouse APLPl, or mouse APLP2-751. To document apical dendrites,but someoverlap in IR was evident in the neu- The Journal of Neuroscience, October 1995, 15(10) 6317

116- 116- @mm 91- 97- -

68 -

1 2 3 4 1 2 3 4 5678 9 10 11 12 Ab 369 Coomassie D2-I CT12 staining Figure 1. Biochemical characterization of APLP2 antibodies. COS-1 cells were transfected with a plasmid vector or plasmids encoding mouse APP-695, mouse APLPl or mouse APLP2-751. Detergent lysates of cells were used for immunoprecipitation and Western blot analysis. A, Ex- pression of APP and the homologues in COS-1 cells. Lanes 1-4 represent Ab369 immunoprecipitates prepared from COS-1 cells transfected with plasmid vector or cells transiently expressing mouse APP, APLPl, or APLP2, respectively. Exogenous APP APLPl and APLP2 polypeptides are indicated by arrowheads. B, Coomassie blue staining of lysates used for Western blotting experiments shown in C. C, Western blot analysis of APLP2 antibody specificity. Lysates of COS-1 cells transfected with plasmid vector (lanes IS, and 9) or plasmids encoding APP-695 (lanes 26, and IO), APLPl (lanes 3,7, and II), and APLP2-751 (lanes 48, and 12) were probed with APLP2 antibodies D2-I (lanes l-4), DZII (lanes .5-8), and CT12 (lanes 9-12). Exogenous APLP2-751 is marked by arrowheads.

Figure 2. Laser scanning confocal images of APLP2 IR in mouse hippocampus. a, Anti-MAP-2 antibody staining of mouse hippocampus. b, Costaining of the section shown in A with antibody D2-I. c, Overlap of the images presented in a and b. Overlap of the two markers confirms the predominant dendritic localization of APLP2. d, Anti-synaptophysin antibody staining of mouse hippocampus. e, Costaining of the section shown in d with antibody D2-I. f; Overlap of the images shown in d and e. 6316

gion of the olfactory epithelium that contains sensoryneurons and sustentacularcells. On the basis of earlier studies which documentedthat the primary APLP2 transcript undergoesalternate splicing (Sprecher et al., 1993; Wasco et al., 1993; Sandbrink et al., 1994; Slunt et al., 1994), we carried out RT-PCR analysisof mRNA prepared from olfactory epithelium. The alternatively splicedAPLP2 transcripts encode APLP2 polypeptides of 694, 706, 751, or 763 amino acids (Fig. 4). The 751 and 763 isoforms contain a domain homologousto the Kunitz proteaseinhibitors, while the 694 and 706 forms lack this region. Moreover, the 706 and 763 forms contain an additional 12 amino acid sequenceencoded by an alternatively spliced exon. We recently documentedthat APLP2-75 1 undergoesmodification by the addition of CS GAG at a single site, serine-614(Thinakaran and Sisodia, 1994). The amino acid sequencenear the CS GAG attachment site is ENEGSGMAEQ (residues61 O-6 19 of the APLP2-75 1 isoform). Notably, the additional 12 amino acid sequencein the APLP2- 706 and APLP2-763 isoforms is insertedimmediately upstream of the CS attachment site. The sequenceof the insert and residues flanking the CS GAG attachment site is ENEDTQPE- LYHPMKKGSGMAEQ. Indeed, we have recently demonstrated Figure 3. In situ hybridizationof APLP2 mRNA in mouseolfactory that the APLP2-763 isoform fails to be modified by CS GAG epithelium.Shown are dark-fieldphotomicrographs of olfactoryepithe- hum (externalsurface facing towardsbottom) hybridized with APLP2 (Thinakaran et al., 1995) suggestingthat the insert disrupts the antisenseand senseriboprobes. optimal recognition sequencefor CS GAG addition. For exam- ple, in transiently transfected COS-1 cells, APLP2-751 is synthesized as an -120 kDa polypeptide, and maturesinto hetero- ropil, as well (Fig. 2f). Similarly, in cortex, we observed an geneous CS GAG-modified polypeptides of - 130-200 kDa essentially identical colocalization pattern with APLP2 and (Fig. 5A, lane 1). In contrast, APLP2-763 is synthesized as a MAP-2 antibodiesin dendrites,with modest overlap in staining - 120 kDa molecule, and maturesinto a homogeneouspolypep- usingAPLP2 and synaptophysinantibodies in the neuropil (data tide of -130 kDa (Fig. 5A, lane 2). Furthermore, immunopre- not shown). As part of our effort to map the distributions of cipitation of solubleAPLPZderivatives from conditionedmedia APLP2 in brain, we noted an interesting distribution of this mol- also revealed that APLP2-75 1 is secretedas heterogeneouspop- ecule in the rodent olfactory system. Hence, we have focused ulation of polypeptides of -100-150 kDa, while APLP2-763 is our attention on the distribution of APLP2 mRNA and proteins secretedas a -100 kDa polypeptide (Fig. 5A, lanes 3 and 4). in cells within this circuit. These results demonstratethat unlike APLP2-75 1, the APLP2- 763 isoform fails to undergo modification by CS GAG. Thus, Expression of APLPZ mRNA in olfactory epithelium we conclude that only two 7f the alternatively spliced isoforms To examine the cellular expressionof APLP2 mRNA in the ol- of APLP2, APLP2-694, and APLP2-75 1 are substratesfor mod- factory epithelium, we used in situ hybridization analysis.We ification by the addition of CS GAG chains. generated33P-labeled riboprobes complementary to the sequenc- In order to determine whether APLP2 mRNA expressedin es that encode a llO-amino acid stretch that immediately pre- olfactory epithelium contains alternatively spliced sequenceen- cedes the transmembranedomain of APLP2; this sequenceof coding the 12 amino acid peptide, we performed RT-PCR anal- APLP2 is highly divergent from an analogousregion of APP ysis with primers that flank the sequenceand we expected two (Slunt et al., 1994). In Figure 3, we demonstratethat mRNAs products. We performed a parallel analysisof RNA isolatedfrom encoding APLP2 are abundantly expressedthroughout the re- mouse brain. Equal amounts of RNA from each tissue were

12aa KPI ISOFORM KPI TM peptide I Y///J i Ra I + + 763 NH2 COOH A I Y//A m I + - 751

I I m I - + 706

r IAl ml I - - 694

Figure 4. Structureof APLP2isoforms encoded by alternativelyspliced transcripts. KPZ, Kunitz proteaseinhibitor domain; stippled box, 12amino acidpeptide; TM, transmembranedomain. The Journal of Neuroscience, October 1995, 75(10) 6319

Figure 5. Posttranslationalprocessing of APLP2isofomrs and APLP2 mRNA expressionin mousebrain and olfactory epithelium.A, Maturation of APLP2 B isoformsin transfectedCOS-1 cells. Lanes I and 2 representexogenous Myc-taggedAPLP2 immunopretcipitat- ed usingantibody Myc-I, from COS-1 bp cells transfectedwith cDNA encoding APLP2-751 or APLP2-763,respective- 506\ ly. Lanes 3 and 4 representsoluble de- rivativesof APLP2-751or APLP2-763, respectively,immunoprecipitated from conditionedmedium using antibody D2- I. B, Ethidiumbromide stained gel of PCR productsgenerated using primers that flank the sequencesencoding the 12 aminoacid peptide.Lanes 1 and2 represent PCR productsderived from cDNA which encodeAPLP2-751 and AF’LP2-763,respectively. Lanes 3 and4 representPCR products generated from C reverse-transcribedRNA isolatedfrom olfactory epithelium(OE) and brain (H?), respectively.C, Ethidiumbromide 97 - Kb stainedgel of F’CRproducts generated usinga senseprimer upstream of the KPI domain(XH) andantisense prim- 2.0 - er (AsD2) downstreamof the sequences encodingthe 12 amino acid peptide. 1.6 - LanesI and 2 representRT-PCR products generatedfrom olfactory epitheli- 68 - urn (OE) and brain (HZ), respectively. Lanes 3-5 representPCR products derived from cDNA which encode APLP2-694, APLP2-751, or AF’LP2- 763, respectively. reverse transcribed, and the resulting cDNA subject to PCR for chemical analysis of the mouse olfactory system, we affinity an identical number of cycles. We demonstrate that In brain, the purified D2-I and DZII antibodies on a bacterially expressed vast majority of PCR product is the longer form and thus con- fusion protein (GSTAAPLP2) that contained a 120 amino acid tains sequences which encode the 12 amino acid peptide (Fig. region of APLP2-751 which occurs immediately N-terminal to 5B, lane 4). These results are essentially indistinguishable from the transmembranedomain. The affinity purified antibodiesde- quantitative RT-PCR studies of mRNA from rat cortex, hippo- tect both full-length and soluble (C-terminal truncated) APLP2 campus, and cerebellum in which transcripts encoding the 12 derivatives (G. Thinakaran and S. Sisodia, unpublishedobser- amino acid peptide represented 87%, 92%, and 95%, respec- vations), similar to those detected with unpurified APLP2 anti- tively, of total APLP2 mRNA in these tissues (Sandbrink et al., sera(Thinakaran and Sisodia,1994). Immunocytochemicalanal- 1994). On the other hand, the shorter PCR product is the pre- ysis of the mouseolfactory epithelium with affinity purified D2-I dominant species in olfactory epithelium and hence lacks se- antibody revealed remarkable APLP2 IR in the sensory axons quences that encode the peptide (Fig. 5B, lane 3). To distinguish of olfactory receptor cells that are arrangedin parallel fiber bun- between APLP2 mRNA that contained both, or either the 12 dles and destinedfor the olfactory bulb (Fig. 6A); an essentially amino acid peptide and KPI sequences, we performed RT-PCR indistinguishablepattern of IR was obtainedwith antibody D2- analysis with a sense primer upstream of the KPI domain and II (Fig. 6C). We demonstratedthat the D2-I labeled structures an antisense primer downstream of the insertion site. PCR anal- are axon bundles by demonstratingcclocalization with the neu- ysis revealed that the vast majority of APLP2 mRNA expressed ron-specific ClassIII P-tubulin monoclonal antibody, TuJl (Lee in olfactory epithelium does not contain the sequences encoding et al., 1990) (data not shown). Moreover, immunostainingpat- the KPI domain (Fig. 5C, lane l), whereas, the majority of the terns obtained with D2-I and D2-II antibodieswere fully com- APLP2 mRNA expressed in brain contains this sequence (Fig. petable by prior incubation of the antibodies with the GSTA- 5C, lane 2). Thus, we conclude that the CS GAG modified iso- APLP2 fusion protein (Fig. 6B,D). Finally, we stainedparallel forms, APLP2-694 and APLP2-751, are the preponderant sectionswith CT12 antisera, raised against the C-terminal 12 APLP2 isoforms expressed in olfactory epithelium. amino acids of APLP2 and which only recognize full-length APLP2 and its membrane-boundmetabolites. As was observed Immunocytochemical localization of APLPZ in the rodent with the D2-I and DZII antibodies, CT12 antiserum strongly olfactory system stainedthe sensoryaxon bundles (Fig. 6E) in the olfactory ep- We performed immunocytochemical analysis of the distribution ithelium. Immunostaining was competable by prior incubation of APLP2 in the olfactory system of mouse and rat using the of the CT12 antiserumwith CT12 peptide (Fig. 6F). Given the APLPZspecific antibodies described above. For immunocyto- strong staining in the sensoryaxon bundlesin the olfactory ep- 6320 Thinakaran et al. l APLP2 in Olfactory System

Figure 6. APLP2 IR in mouse olfactory epithelium; 10 pm sections of olfactory neuroepithelium were examined immunohistochemically for APLP2 IR using affinity purified antibodies D2-I (A), DZII (C), and CT12 antisera (E). In all cases, only light APLP2 IR was detected in the olfactory receptor neurons (ORN). However, APLP2 IR was highly enriched in afferent axons (Ax) projecting to the olfactory bulb. APLP2 IR was abolished by preincubation of D2-I and D2-II antibodies with GSTAAPLPZ fusion protein (B and D) and CT12 antisera with immunizing peptide (F). ithelium, we anticipated that APLP2 would also be localized to and granule cells, along with scattered APLP2 IR throughout the the terminal fields of the sensory afferents in the olfactory bulb. neuropil of the external plexiform layer. Indeed, the glomeruli Using antibody D2-I, we show remarkable APLP2 IR in the were also strongly stained with DZII antibody and CT12 anti- glomeruli of the olfactory bulb (Fig. 7A). In addition, fine serum (Fig. 7,C and E, respectively). Moreover, staining in the APLP2 IR is seen in dendrites and cell bodies of mitral, tufted olfactory bulb by all three antibodies was competable with re- The Journal of Neuroscience, October 1995, 75(10) 6321

Figure 7. APLP2 IR in mouse olfactory bulb; 10 pm sections of olfactory bulb were examined immunohistochemically for APLP2 IR using affinity purified antibodies D2-I (A), D2-II (C), and CT12 antisera (E). APLPZ IR was detected primarily in glomeruli (Gr) and mitral cells of the olfactory bulb, where axons from the neuroepithelium terminate and synapse. APLP2 IR was abolished by preincubation of D2 antibodies with GSTAAPLP2 fusion protein (B and D) and CT12 antisera with immunizing peptide (F). spective antigens (Fig. 7B, 0, F, respectively). Taken together idence that APLP2 and/or its metabolitesare being specifically with our observation that antibodiesagainst nonoverlapping epi- recognizedby each of the antibodiesin situ. topesof APLP2 recognize an essentiallyindistinguishable set of To demonstratethat the pattern of immunostainingin mice structuresin the olfactory system (i.e., in sensoryaxon bundles detected with the APLP2 antibodies was conserved in another and glomeruli), the competition studiesprovide compelling ev- rodent species,we immunostainedsections of rat olfactory ep- 6322 Thinakaran et al. l APLP2 in Olfactoi

Figure 8. APLP2 IR in rat olfactory epithelium and bulb. A, A cross-section of rat olfactory epithelium (UE) and olfactory bulb (OBu) was examined for APLP2 expression using affinity pun- fied D2-I antibodies. Axons bundles (&) are clearly seen underlying the olfactory receptor neuron layer (ORN) and bundles of axons (arrow) are seen projecting through the cribiform plate (CP) into the glomemlar layer (CT) of the olfactory bulb. B, Higher power view of APLP2 IR in axons projecting through the cribiform plate into the glomerular layer. C, Higher power view of APLP2 IR in the glomerular layer. ithelium and olfactory bulb with affinity purified antibody D2- ithelium through the cribiform plate towards the olfactory bulb I. A low power view of a cross-sectionthrough the olfactory (Fig. 8B). Finally, a higher power view of the glomeruli shows epithelium and bulb showsstrong staining of the sensoryaxons strong D2-1 IR in this structure (Fig. 8C). and glomeruli (Fig. 8A). A higher power view illuminatescours- In order to define the subcellularlocalization of APLP2 in the ing of D2-I immunoreactivesensory axon bundlesfrom the ep- glomeruli, we double-stainedsections of the olfactory bulb with The Journal of Neuroscience, October 1995, 15(10) 6323

e APLP2

-2 antibody staining of mouse olfactory bulb (green). MAP-2 staining is only light in glomeruli but is abundant in the outer plexiform layer b. Costaining of the section shown in A with antibody D2-I (r-ed). D2-1 stains both the outer plexiform layers and glomentli. c, Overlap of the images presented in a and b. Yellon. signifies overlap of the two markers. d, Anti-synaptophysin antibody staining of mouse olfactory bulb (green). e, Costaining of the section shown in D with D2-I antibodies (red). f; Overlap of the images shown in d and e. Yellow signifies overlap of the two markers: considerable staining of both the glcmeruli and outer plexiform layer is apparent confirming that APLPZ is expressed in pre- and postsynaptic structures in the glomeruli.

D2-I and synaptophysin,or for D2-I and MAP-2, and analyzed olfactory bulb samplethat migrate between the -120-l 60 kDa optical sectionsby laser scanningconfocal microscopy. As ex- (Fig. lOA, lane 2) representaccumulated CS GAG-modified spe- pected, synaptophysinIR was presentboth in the terminal fields cies synthesized by olfactory receptor neuronsand transported of the olfactory sensory afferents within the glomeruli and the to the olfactory bulb. Moreover, our resultsare fully consistent incoming fiber tracts (Fig. 9d). Costainingwith the APLP2-spe- with the earlier RT-PCR analysisin which we demonstratethat cific antibody, D2-I, revealed conspicuous colocalization in the vast majority of APLP2 mRNA in the olfactory epithelium structuresstained with synaptophysinantibodies (Fig. 9ef). On encodesisoforms postulated to undergo CS GAG modification the other hand, MAP-2 antibody stained structures contained while brain primarily expressesAPLP2 mRNA encodingthe CS primarily in the external plexiform layer with negligible staining GAG inhibitory 12 amino acid peptide(see Fig. 5&C). To con- in the glomeruli (Fig. 90). Similar to our observationsin cortex firm that accumulatedAPLP2 in the olfactory bulb was indeed and hippocampus.APLP2 IR strongly colocalized with MAP-2 modified by CS GAG. we treated the solublefraction (S2) with immunoreactivestructures in the external plexiform layer (Fig. chondroitinaseABC and subject the resulting preparationsto SC). Nevertheless,it is clear that APLP2 IR extended into the immunoblot analysiswith antibody D2-II. Soluble APLP2 de- glomeruli, a region which was essentiallydevoid of MAP-2 IR rivatives in the S2 fraction from olfactory bulb migratesas a (Fig. 9b,c). doublet of -85 kDa and - 105 kDa and a heterogeneousspecies Characterization of APLPZ proteoglycan in olfactoc bulb between -90-l 15 kDa (Fig. lOB, lane 1). ChondroitinaseABC treatment of the S2 fraction converted the heterogeneouspopu- Analysis of APLP2 mRNA in the olfactory epithelium (Fig. lation of moleculesto fairly discreteband that overlapsthe -85 .5&C) strongly suggestedthat the encodedisoforms would be kDa specieswith little changein the levels of the -105 kDa substratesfor CS GAG addition. To assessthe level of CS GAG species(Fig. lOB, lane 2). Thus. we argue that the heterogeneity modification of APLP2 in the olfactory system, we usedD2-II of the -90-l 15 kDa APLP2-related moleculesin the S2 fraction antiserumin Westernblot analysisof membrane-boundand soluble extracts preparedfrom olfactory bulb. a region which is is the result of CS GAG modification of a C-terminally truncated enriched in APLP2 IR (seeabove). The D2-II antibody is spe- APLP2 core protein of -85 kDa. In contrast.the APLP2 forms cific for APLP2 (seeFig. 1). Immunoblotanalysis of membrane in the S2 fraction from hippocampusappear as -85 kDa and fractions from whole brain, cortex, hippocampus,cerebellum, -105 kDa species(Fig. lOB, lane 3) which, as expected. are and spinal cord (Fig. lOA, lanes 1. 3-6, respectively) revealed insensitiveto digestion with chondroitinaseABC (Fig. lOB, lane the presenceof discrete APLP2-related speciesof -95 kDa, 4). Finally, as a control for chondroitinasedigestions. we pre- - 100 kDa, and - 115 kDa. On the other hand. D2-II recognized pared conditioned medium from CHO cells that constitutively the -95 kDa, - 100 kDa. and - 115 kDa speciesand a hetero- expressAPLP2-75 1. As we previously documented(Thinakaran geneouspattern of polypeptides with apparentmolecular weight and Sisodia, 1994): solubleAPLP2-751 derivatives secretedby of -120-160 kDa range in the olfactory bulb (Fig. lOA, lane CHO cells migrate as a smear between - 120-150 kDa (Fig. 2). We suggestthat the membrane-boundAPLP2 forms in the lOB, lane 5) and theseforms are fully sensitiveto chondroitinase 6324 Thinakaran et al. l APLP2 in Olfactoty System A

Figure 10. Expression of CS GAG- modified APLP2 in the olfactory system. A, Tbe P2 fraction (10 kg per lane) from whole brain, olfactory bulb, cortex, hippocampus, cerebellum, and spinal cord were fractionated by SDS- PAGE and subject to immunoblotting with APLP2-specific antibody, D2-II. -+-+ - + Ch. ABC Arrowhead indicates the APLPZ proteoglycan expressed in the olfactory bulb. B, The S2 fraction (10 kg) prepared from olfactory bulb and hippo- 200 - campus, or an aliquot of conditioned medium prepared from CHO cells sta- 116- r . ..* bly expressing mouse APLP2-75 1 were 97- incubated at 37°C in the presence (+) or absence (-) of chondroitinase ABC. After digestion, the samples were frac- 68- tionated by SDS-PAGE and immuno- blotted using APLP2 antibody, D2-II. Tbe position of the chondroitinase-sen- 43- sitive -90-120 kDa APLP2 species 43- are marked by a bracket. Note that following chondroitinase digestion of soluble APLP2 forms from olfactorv bulb, a marked increase in the amobnt of -85 kDa APLP2 polypeptide (marked 29- 29- by an arrowhead) is observed with little change in the - 105 kDa APLP2 polypeptide (marked by an asterisks). 1 2 3 4 5 6 123456 digestion, resulting in the production of a discrete species of pared from COS-1 cells transiently transfected with cDNA en- -105 kDa (Fig. lo& lane 6). coding each of the known membersof the APP family of proteins revealed that the APLP2 antibodies D2-I and DZ-II and Discussion CT12 specifically recognize APLP2. APP, the precursor of the A@ peptide, is a member of a family Previous in situ hybridization studieshave shown that APLP2 of closely related transmembraue glycoproteins that includes is ubiquitously expressedin CNS. Indeed, our present immu- APLPl and APLPZ (Wasco et al., 1992, 1993; Sprecher et al., nocytochemical investigationsconfirm the in situ results, and in 1993; Slunt et al., 1994). At present, APLP2 is the best char- addition revealed that the preponderantsubcellular localization acterized member of the APLPs. APLP2 mRNAs are expressed of APLP2 in neuronsof the cortex and hippocampusis in post- in most, if not all, neuronal populations in the CNS and at high synaptic elements.Most provocative is our finding that APLP2, levels in peripheral tissues (Wasco et al., 1993; Slunt et al., synthesized in sensory neurons of the olfactory epithelium, is 1994). However, it is likely that the distribution of APLP2 in enriched in sensory axons and sensory axon terminals in the the CNS may show significant differences as compared to the glomeruli. In the olfactory bulb, colocalization studiesof APLP2 mRNA expression patterns observed using in situ hybridization, with markersof synaptic (synaptophysin) or dendritic (MAP-2) particularly since APLP2, like APP (Koo et al., 1990; Sisodia structures reveal that APLP2 is present in presynaptic sites in et al., 1993), is transported in axons by the rapid anterograde the glomeruli as well as in postsynaptic elementsin the outer component (G. Thinakaran and S. Sisodia, unpublishedobser- plexiform layer. vations). Furthermore, it is conceivable that differences in pro- Although speculative, we suggestthat the unique distributions teolytic processing and/or secretion of APLP2 between cell of APLP2 to sensoryafferents and terminals may be linked to types may lead to differential accumulationof the precursor iso- its generalizedrole in processformation or pathfinding, and con- forms and its derivatives. Immunocytochemical methodshave nectivity. Olfactory sensoryneurons are the only neuronalpop- been usedto study the distribution of APP in CNS (Card et al., ulation in the adult mammalian CNS which are continuously 1988; Shivers et al., 1988; Kawarabayashiet al., 1991; Martin renewed from a population of mitotic precursors,with replace- et al., 1991; Imaizumi et al., 1993; Okuda et al., 1994; Ouimet ment of neuronsin the olfactory epithelium occurring every 4- et al., 1994; Trapp and Hauer, 1994) but becauseantibodies 6 weeks (Graziadei and Monti Graziadei, 1979). Hence, axonal raised against independent epitopes of APP cross-react with outgrowth from newly born sensoryneurons towards the olfac- APLP2 (Slunt et al., 1994), it is quite likely that the distributions tory bulb is a processthat requiresmolecular clues involved in of APP IR describedin a variety of reports, including our own guidance, adhesion, and synaptogenesis.We recently demon- (Martin et al., 1991) reflect IR to APP, APLP2, and perhaps stratedthat APLP2 is a substratefor modification by the addition APLPl, as well. To define the distribution of APLP2 in the CNS, of CS GAG chains (Thinakaran and Sisodia,1994). Altemative- we generateda seriesof antibodiesagainst nonoverlapping epi- ly-spliced mRNA encodingisoforms of APLP2 (APLP2-706 and topes of mouse APLP2. Western blot analysesof lysates pre- APLP2-763) that do not undergomodification by CS GAG (Thi- The Journal of Neuroscience, October 1995, 75(10) 6325 nakaranet al., 1995) are the predominantly expressedin cortex, precursor of Alzheimer’s disease amvloid protein shows protease inhibitory activity. Nature 331:530-532. - hippocampus,and cerebellum (Sandbrink et al., 1994), an ob- Koniz G. Miinninz U. Czech C. Prior R. Banati R. Schreiter-Gasser U. servation we have now confirmed by biochemicalanalysis with BaGer J, Masteg CL, Beyreuther K (1992) Identification and differ- our newly generatedantibodies. However, the APLP2 isoforms ential expression of a novel alternative splice isoform of the PA4 which undergoCS GAG modification (APLP2-694 and APLPZ amyloid precursor protein (APP) mRNA in leukocytes and brain mi- 75 1) are selectively expressedin olfactory epithelium. Although croglial cells. J Biol Chem 267:10804-10809. Koo EH, Sisodia SS, Archer DR, Martin LJ, Weidemann A, Beyreuther CS proteoglycans expressedby non-neuronal cells along path- K, Fischer R Masters CL, Price DL (1990) Precursor of amyloid ways of axon outgrowth in developing retina (Brittis et al., 1992) proteinin Alzheimerdisease undergoes fast anterogradeaxonal trans- and cerebellum (Friedlander et al., 1994) have been shown to port. Proc Nat1 Acad Sci USA 87:1561-1565. repel axons, little is known regarding the role(s) of CS proteo- Lee MK, Tuttle JB, Rebhun LI, Cleveland DW, Frankfurter A (1990) glycans expressedintrinsically by neurons.Some studies,how- The expression and posttranslational modification of a neuron-spe-

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