©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Generation of Amyloidogenic C-terminal Fragments during Rapid Axonal Transport in Vivo of -Amyloid Precursor Protein in the Optic Nerve (*)

(Received for publication, January 23, 1995; and in revised form, April 11, 1995)

Anil Amaratunga , Richard E. Fine (§)

From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118 and the Eleanor Norse Rodgers Veterans Administration Hospital, Bedford, Massachusetts 01730

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The amyloid -protein (A) is a major component of extracellular deposits that are characteristic features of Alzheimer's disease. A is derived from the large transmembrane -amyloid precursor protein (APP). In the rabbit optic nerve/optic tract (ON), APP is synthesized in vivo in retinal ganglion cell perikarya, rapidly transported into the ON axons in small transport vesicles and is subsequently transferred to the axonal plasma membrane as well as to the presynaptic nerve terminals (Morin, P. J., Abraham, C. R., Amaratunga, A., Johnson, R. J., Huber, G., Sandell, J. H., and Fine, R. E.(1993) J. Neurochem. 61, 464-473). Present results indicate that there is rapid processing of APP in the ON to generate a 14-kDa C-terminal membrane-associated fragment that contains the A sequence. By using equilibrium sucrose density gradient fractionation, this fragment, as well as non-amyloidogenic C-terminal fragments and intact APP, are detected in at least two classes of transport vesicles destined for the plasma membrane and the presynaptic nerve terminal. The two classes of transported vesicles are distinguished by labeling kinetics as well as by density. In contrast to the ON, only non-amyloidogenic C-terminal fragments are generated in the retina, which contains the perikarya of retinal ganglion cells and glial (Muller) cells which also produce APP.


INTRODUCTION

Alzheimer's disease is characteristic of neuronal loss, neurofibrillary tangles, and senile plaques in selective brain regions. Mature senile plaques contain a central core of extracellular amyloid, the major component being the 4-kDa amyloid -protein (A)()(reviewed in Price(1986), Muller-Hill and Beyreuther(1989), and Selkoe(1989)). A is derived from the large transmembrane -amyloid precursor protein (APP) which is found in many cell types. There are three major alternatively spliced transcripts of the APP gene that are 695, 751, and 770 amino acids long; the two larger forms contain a Kunitz-type protease inhibitor domain. A variety of post-translational modifications of APP have been characterized in cultured cells (reviewed in Selkoe (1994)).

Mature APP can undergo proteolytic cleavage within the A domain and this results in the secretion of soluble APP and a 9-12-kDa non-amyloidogenic fragment which remains membrane-bound (Selkoe et al., 1988; Schubert et al., 1989; Weidemann et al., 1989; Esch et al., 1990; Sisodia et al., 1990). APP is also processed and degraded within an endosome/lysosome pathway, following reinternalization of full-length APP from the cell surface via clathrin-coated vesicles (Golde et al., 1992; Haass et al., 1992a). C-terminal fragments containing the complete A sequence of APP (of 11 kDa and larger) have been detected in endosomes/lysosomes in cultured cells and brain tissue (Nordstedt et al., 1991; Estus et al., 1992; Tamaoka et al., 1992) and could thus serve as potential intermediates for A formation. Both neurons and astrocytes in cell culture also generate and secrete soluble A (Haass et al., 1992b; Seubert et al., 1992; Shoji et al., 1992), which is also found in the cerebral spinal fluid of normal individuals (Seubert et al., 1992).

Anterograde axonal transport of neuronal APP in the central nervous system is likely to be a pathway by which A is released to extracellular regions of brain, following its generation from APP. APP was shown to be the predominant form that is expressed in rat sensory ganglia and transported along the sciatic nerve (Koo et al., 1991; Sisodia et al., 1993). Employing a central nervous system neuron of rabbit, we demonstrated that APP as well as APP are synthesized in the retina; APP is rapidly transported in vivo into the optic nerve/optic tract (ON) in small vesicles and is subsequently transferred to a membrane fraction containing glucose transporters and other axonal plasma membrane markers, as well as to the presynaptic nerve terminals in the lateral geniculate (Morin et al., 1993). Axonal transport of APP was also reported in the developing hamster ON where APP was the prominent form(s) detected in the lateral geniculate (Moya et al., 1994). Anterograde axonal transport of APP may occur via a kinesin based motor, as shown in cultured neurons (Ferreira et al., 1993) and in the ON in vivo (Amaratunga et al., 1995). Following transport to the plasma membrane, APP is degraded with a t of less than 5 h. Protease activity that can potentially generate amyloidogenic APP-derived peptides is also present in the same ON membrane fractions as APP (Morin et al., 1993).

In order to examine the generation of potentially amyloidogenic fragments of APP in the ON in vivo, we carried out a series of studies employing gel electrophoresis conditions capable of detecting small molecular weight fragments. In this report, we describe results indicating that there is rapid processing of APP in the ON to generate a C-terminal membrane-associated fragment that contains the A sequence. This fragment, as well as non-amyloidogenic fragments and intact APP, are seen in regions of an equilibrium sucrose density gradient that contain axonal plasma membrane and at least two classes of transport vesicles, destined for the plasma membrane and axon terminal, respectively. This finding suggests that there is significant processing of APP to an amyloidogenic C-terminal-containing species before it reaches the axonal plasma membrane and the nerve terminal. We also report that in the retina, where APP and APP are also present, APP is processed differently from that which occurs in the ON, generating only non-amyloidogenic C-terminal fragments.


EXPERIMENTAL PROCEDURES

Labeling of ON and Retina

Adult male albino rabbits (6 lb) were anesthetized with 1 ml of 5% sodium pentobarbital (intravenous), and two drops of 0.5% proparacaine HCL (topical). U-100 insulin syringes with 28-gauge needles are used to introduce 0.5 mCi (50 µl) of [S]methionine/cysteine (DuPont NEN) into the vitreous chamber of each eye. All animal use procedures were in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the local Animal Care Committee.

Preparation of Membranes from Rabbit Optic Nerve and Retina

Animals were sacrificed 3, 6, 9, or 12 h following radiolabel injection and retinas and ONs were dissected out. Tissues were homogenized in ice-cold phosphate-buffered saline (PBS) (containing 30 µg/ml phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, and 0.5 µg/ml aprotinin) in a motor-driven Teflon glass homogenizer and centrifuged at 45,000 rpm for 1 h in a Ti 70 rotor. Pellets were resuspended in PBS and diluted 1:1 by adding 2 immunomix (1.0% (v/v) Triton X-100, 0.5% (v/v) deoxycholate, and 0.1% (w/v) sodium dodecyl sulfate (SDS) in PBS) for solubilization, followed by centrifuging at 25,000 rpm in a Ti 70 rotor for 10 min for clarification. The resulting solubilized membrane protein preparations were used for immune precipitations.

Density Gradient Separation of ON Membranes

Three- or 6-h post-S-labeled ONs were dissected out (described above). Density gradient separations were carried out as described earlier (Morin et al., 1991a, 1993). Briefly, ONs were homogenized in 7 ml of ice-cold homogenization (H) buffer (1 mM triethanolamine, 320 mM sucrose), containing 30 µg/ml phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, and 0.5 µg/ml aprotinin). Homogenates were diluted to 40 ml with H buffer and centrifuged at 1,200 g for 7 min. The resulting supernatant was then centrifuged at 100,000 g for 60 min. The resulting pellet was resuspended in 2 ml of H buffer and loaded onto a discontinuous gradient consisting of 2 ml each of 20/26/31/37/45% sucrose (w/w) in H buffer. These gradients were centrifuged at 150,000 g in a SW 40 rotor (Beckman) for 16 h. Twenty-four 0.5-ml fractions were collected from each gradient, and each fraction was assayed for protein and radioactivity to determine specific activity. Fractions containing the highest specific activities were pooled, diluted to 5 ml with PBS, and pelleted at 150,000 g in an SW 50.1 rotor for 60 min. The pellets were resuspended in PBS, solubilized with immunomix and used for immune precipitations.

Immune Precipitation

Antibodies were added at appropriate dilutions to aliquots of equal volume and incubated overnight at 4 °C (Amaratunga et al., 1993). Sepharose-linked anti-rabbit or anti-mouse immunoglobulin G beads (Organon Teknika) were then added to samples (15 µl of beads/1 µl of primary antibody) and incubated for 3 h at 4 °C with agitation. Beads were separated in an Eppendorf centrifuge and washed five times using immunomix. Washed beads were suspended in SDS sample buffer and analyzed by 10-20% Tris-Tricine polyacrylamide gel electrophoresis. Gels were incubated in an enhancing solution for 1 h, dried under vacuum for 2 h, and exposed to films.

Antibodies

Rabbit sera made against a synthetic peptide corresponding to the C-terminal 20 amino acids of all isoforms of APP, C8 (Selkoe et al., 1988), and corresponding to 1-40 amino acids of A, R1280 (Tamaoka et al., 1992) (gifts of Dr. D. Selkoe), were used at dilutions of 1:1000 and 1:200, respectively. R1280 recognizes A (4 kDa) peptide as well as a 3-kDa peptide starting at residue 16 of the A near the secretory cleavage site of APP that ends near residue 40 (Haass et al., 1992b). A monoclonal antibody specific to the N-terminal 17 amino acids of A, 6E10 (Kim et al., 1988), was used at a dilution of 1:300. Control experiments to determine nonspecific immune precipitations were carried out by using normal rabbit serum (Rockland Co.) and a monoclonal antibody against insulin-regulatable glucose transporter, 1F8, which is not found in brain (James et al., 1987).

Quantitative Analysis of Radioactive Proteins in Gels

Scanning of autoradiograms was performed by volume integration of protein bands using a densitometer (Molecular Dynamics). Direct determination of radioactive counts in dried gels was performed by using an instant imager (Packard).


RESULTS

We have utilized the retinal ganglion cell/ON preparation combined with 10-20% Tris-Tricine gradient gels to detect A containing APP-derived fragments in vivo of the [S]methionine/cysteine-labeled ON. ON membranes were taken at various times after S injection, and aliquots were subjected to immune precipitation with either a C-terminal-recognizing antibody, C8, or with a polyclonal antibody against A, R1280. As early as 3 h after S injection, a fragment with an apparent molecular mass of 14 kDa is specifically precipitated with both antibodies, whereas several other fragments are only detected with C8 (Fig. 1). The 14-kDa fragment precipitated by both antibodies is maximally labeled at 3 and 6 h following S injection and then disappears gradually at 9 and 12 h after injection. Of the C8-precipitated proteins, fragments of 12-16 kDa are detected at 3 h after S injection, and smaller fragments of 10-14 kDa are detected at 6 h, which also gradually disappear at later time points (Fig. 1). We also observe the full-length APP (molecular mass of 110 kDa) at all these times but a considerably lesser amount at 12 h.


Figure 1: Generation of amyloidogenic and non-amyloidogenic fragments during axonal transport of APP. Rabbits were given vitreal injections of [S]methionine/cysteine and sacrificed thereafter. Three, 6-, 9-, and 12-h post-[S]methionine/cysteine-labeled ON membranes were immune-precipitated with an antibody against the APP C-terminal (C8), an antibody against A (R1280), and with normal rabbit serum (nrs) and subjected to SDS-polyacrylamide gel electrophoresis (10-20% Tris-Tricine gradient gels) and autoradiography. Autoradiograms were exposed for long periods to clearly demonstrate the presence of smaller bands. APP full-length form (110 kDa) and the 14-kDa amyloidogenic fragment are indicated by large and small arrowheads, respectively. Marks(-) on the left refer to molecular mass markers: 205, 116, 80, 45, 32, 15, 6, and 3 kDa, respectively. Post 3-h autoradiogram shown in the figure was scanned to quantitatively compare the intensities of 14-kDa bands. The intensities of C8- and R1280-precipitated 14-kDa bands are: 2345 and 1317 OD units, respectively. Post 3-h gel exposed on a film for a shorter period (therefore, not overexposed for 110-kDa bands) was scanned by a densitometer to quantitatively compare the intensities of 110-kDa bands. The intensities of C8- and R1280-precipitated 110-kDa bands are: 5438 and 782 OD units, respectively.



It is expected that the cleaved N-terminal fragments are present in the soluble fraction of our ON preparation and therefore should only be recognized by antibodies against the APP N terminus. Consistent with this expectation, we have not observed any C-terminal fragments released into the soluble fraction of ON by using C8 and R1280 (data not shown).

We have previously demonstrated that in the rabbit ON in vivo, there are at least three types of anterograde rapidly transported vesicles, two types of synaptic vesicle precursors, one for classic transmitters (small, light vesicles) and one for peptides (large, dense vesicles), and a small, light vesicle that carries plasma membrane proteins to the axonal and presynaptic nerve terminal plasma membrane (Morin et al., 1991a). In the rabbit ON in vivo, APP was shown to be anterogradely axonally transported in small vesicles of the plasma membrane type (Morin et al., 1993).

We have subfractionated the S-labeled membranes into the three major membrane containing peaks as described earlier (Morin et al., 1991a, 1993). At 3-h post-injected ON, we detect 10-14-kDa fragments in peaks 1 (light fraction) and 2 (intermediate fraction) in the gradient, which correspond to the plasma membrane, and small transport vesicles destined for the plasma membrane and the nerve terminal, respectively (Fig. 2).


Figure 2: Subcellular location of APP processing in the ON. Three-h and 6-h post [S]methionine/cysteine-labeled ON membranes were subjected to sucrose density gradient separation. Fractions from peak 1 (light membranes), peak 2 (intermediate membranes), and peak 3 (heavy membranes) were separately pooled and subjected to immune precipitation using antibodies C8, and R1280, and with normal rabbit serum (nrs). Electrophoresis and autoradiography were performed as described in the legend to Fig. 1. The location of full-length APP (110 kDa) and 12-14-kDa fragments are indicated by large and small arrowheads, respectively. Marks(-) on the left refer to molecular mass markers: 205, 116, 80, 45, 32, and 15 kDa, respectively.



According to previous results, peak 3 (dense fraction) shows delayed transport kinetics and contains substance P/K containing dense transport vesicles destined for the nerve terminal (Morin et al., 1991b, 1993). In 6-h post-injected ON, in addition to peaks 1 and 2, peak 3 also contains C-terminal fragments (Fig. 2). The full-length APP is present in all three peaks at 3 h as well as at 6 h.

We also carried out immune precipitation of APP and its C-terminal fragments from the S-labeled retina. S-Labeled amino acids injected into the vitreous of the eye can be utilized by retinal ganglion cells as well as by Muller glial cells, which send their processes to the ganglion cell layer (Morin et al., 1993). Previous results indicate that the synthesis of APP (molecular mass, 110 kDa) as well as APP isoforms containing the Kunitz-type protease inhibitor domain occurs in the retina (Morin et al., 1993). A sizable amount of a 12-kDa C-terminal fragment is produced in the membrane fraction of retina at 3 h (Fig. 3) and also at 6 h (data not shown) following S injection. This fragment does not appear to be amyloidogenic as indicated by the inability of R1280 to precipitate this component (Fig. 3).


Figure 3: Generation of non-amyloidogenic fragments by APP processing in the retina. Rabbits were injected with [S]methionine/cysteine and sacrificed 3 h later. Retinal membranes were immune-precipitated with antibodies C8 and R1280 and with normal rabbit serum (nrs). Electrophoresis and autoradiography were performed as described in the legend to Fig. 1. APP full-length forms (110 kDa and higher) and the 12-kDa non-amyloidogenic fragment are indicated by large and small arrowheads, respectively. Marks(-) on the left refer to molecular mass markers: 205, 116, 80, 45, 32, 15, 6, and 3 kDa, respectively.



We examined the possibility that the recognition of C-terminal fragments by the antibody C8 may be due to the metabolism of APPLP2, a recently cloned APP-like protein. APPLP2 is highly homologous to APP at the C terminus, but does not contain the extracellular A sequence (Slunt et al., 1994). When a monoclonal antibody specific for the N-terminal 17 amino acids of A, 6E10, was used in immune precipitations, a 14-kDa fragment was detected only in the ON and not in the retina (Fig. 4). This confirms that the 14-kDa fragment is derived from APP as well as containing the entire A sequence.


Figure 4: The use of a monoclonal antibody specific to A demonstrates that an amyloidogenic C-terminal fragment of APP is generated in the ON but not in the retina. Three h after the vitreal injection of [S]methionine/cysteine, membranes were isolated from retina and ON. Equal aliquots were immune-precipitated with C8 and anti-A, 6E10. Control immune precipitations were carried out with normal rabbit serum (nrs) (control for C8) and with 1F8 (control for 6E10) (see ``Experimental Procedures''). Electrophoresis and autoradiography were performed as described in the legend to Fig. 1. Autoradiograms were exposed for long periods to clearly show the smaller bands. APP and the 14-kDa fragment are indicated by large and small arrows, respectively. Marks(-) on the left refer to molecular weight markers: 205, 116, 80, 45, 32, 15, 6, and 3 kDa, respectively. Autoradiogram of the ON was scanned by a densitometer to quantitatively compare the intensities of 14-kDa bands. The intensities of C8- and 6E10-precipitated 14-kDa bands are: 302 and 233 OD units, respectively. To compare the intensities of 110-kDa bands, the radioactive counts of the bands in the gel were measured by an instant imager. The ratio of counts of C8- and 6E10-precipitated 110-kDa bands is: 1:0.86.



We compared the C8 and 6E10 immune-precipitated protein bands shown in Fig. 4by quantitating radioactive counts in the gel of the 110-kDa band and by densitometric analysis of the 14-kDa band. With respect to the 110-kDa species, 6E10 immune-precipitated 86% of that precipitated by C8 (Fig. 4). Although at present no data are available on the expression of APPLP2 in the rabbit retinal ganglion cell, this result may suggest that APP is the predominant APP form that is axonally transported in the ON. With respect to the 14-kDa fragment, 6E10 precipitated 77% of that precipitated by C8 (Fig. 4), thereby indicating that this fragment is produced predominantly by the cleavage of APP to generate a fragment containing the entire A sequence.


DISCUSSION

The results presented have demonstrated that there is a rapid (<3 h) metabolism of the retinal ganglion cell produced APP in the ON to generate a C-terminal 14-kDa fragment containing the A sequence. This fragment is a likely precursor to A (Dyrks et al., 1993), although we have no evidence as yet that A is produced in this system in vivo. It is possible that in this system the incorporation of S into A, which has only one methionine residue and no cysteine, might be insufficient for detecting A. The cell type which produces A in the central nervous system is not known, and it is also possible that the retinal ganglion cells do not produce A. However, the amyloidogenic fragment(s) is metabolized fairly rapidly, since it disappears by 12 h after labeling (Fig. 1). This is to be expected, since there is evidence that overproduction of a C-terminal A-containing fragment in transfected cells can produce neurotoxicity when these cells are differentiated into neuron-like cells (Kammesheidt et al., 1992).

The 12-kDa C-terminal fragment recognized only by C8 may be produced by an -secretase that cleaves within the A sequence to generate a C-terminal fragment containing the transmembrane portion and cytoplasmic domain (Selkoe et al., 1988). It is possible that in this system R1280 precipitates only the 14-kDa amyloidogenic C-terminal fragment. The nondetection of a 12-kDa fragment by R1280 may be due to the fact that R1280 appears to be a weak immune-precipitating antibody consistent with peptide antibodies against the A region of APP (Tamaoka et al., 1992), compared with C8 and 6E10. When simultaneously used in equal aliquots of an ON membrane preparation, R1280 precipitated only 14% of the 110-kDa species precipitated by C8 (Fig. 1). Since over 80% of the C8-precipitated 110-kDa species is APP (Fig. 4), this shows that R1280 precipitates only a minor fraction of intact APP. Therefore, it is possible that the detection of the 12-kDa fragment by R1280 was beyond the limit of sensitivity.

We also simultaneously detect other C-terminal fragments that are not recognized by R1280 (Fig. 1) and 6E10 (Fig. 4). The cleavage of APPLP2 could generate these fragments, which are recognized by C8 (Slunt et al., 1994). If it is assumed that both APP and APPLP2 are expressed in the rabbit retinal ganglion cell, then APPLP2 represents less than one-fifth of the total that is axonally transported (Fig. 4). Therefore, it is unlikely that the C-terminal fragments recognized exclusively by C8 are generated entirely from APPLP2.

Using an equilibrium sucrose gradient, we were able to fractionate APP-derived C-terminal fragments in the ON into three separate membrane compartments (Fig. 2) (Morin et al., 1993). At 3 h following S injection, some APP has already reached the axonal plasma membrane (peak 1) and C-terminal fragments are also detected in this fraction (Fig. 2). The observation of C-terminal fragments (and APP) in peak 2 indicates that APP processing to generate C-terminal fragments occurs within transport vesicles bound for the axonal plasma membrane. The fact that protease activity specific for the generation of amyloidogenic fragments from APP has been co-localized in peak 2 vesicles (Morin et al., 1993) further supports this possibility. Also in PC12 cells, APP cleavage was shown to occur intracellularly following glycosylation and sulfation within the vesicles that transport proteins to the plasma membrane (Sambamurti et al., 1992).

It is likely that APP-containing vesicles bound for the plasma membrane (peak 2) consist of a subpopulation of small, light vesicles, as was shown to be the case for glucose transporter-containing vesicles (Morin et al., 1991a). Also, immune staining experiments did not co-localize APP and synaptic vesicle markers in the same vesicles (Schubert et al., 1991; Ferreira et al., 1993; Caporaso et al., 1994)

It is possible that fragments observed in peaks 1 and 2 of 3-h post-labeled ON (Fig. 2) could be derived from internalization of APP from the plasma membrane into an endosome-lysosome-like compartment (Haass et al., 1992a; Golde et al., 1992). This is supported by the fact that intact APP is detected in the plasma membrane fraction (peak 1) (Fig. 2). Immune staining in cultured neurons indicated APP to be associated with clathrin-coated vesicles and endosome-like vesicles, some of which may be involved in retrograde transport (Ferreira et al., 1993; Caporaso et al., 1994). Also, APP has been localized within clathrin-coated vesicles in mammalian brain (Sapirstein et al., 1994). However, clathrin-coated vesicles are much too dense to be present in this region of the 20-45% gradient used in these experiments.

In cultured cells, amyloidogenic fragments as well as A appear to be generated within an acidic compartment in the secretory pathway (Shoji et al., 1992; Haass et al., 1993). Axonal endosomes appear not to be acidified until reaching the cell body, making it unlikely that proteolysis in endosomes occurs to any great extent in the axon (Augenbraun et al., 1993). It is, however, likely that the axonal secretory vesicles are acidified as are other secretory vesicles (Anderson and Pathek, 1985).

In 6-h post-labeled ON, C-terminal fragments are detected in both peaks 2 and 3 (Fig. 2), thereby indicating intracellular APP processing in two classes of vesicles of different densities bound for the nerve terminal. The peak 2 fraction at 6 h contains transport vesicles carrying plasma membrane proteins as well as small synaptic vesicle precursors (Morin et al., 1993). The peak 3 fraction at 6 h contains dense neuropeptide (substance P/K) containing vesicles (Morin et al., 1991a, 1991b). The possibility that APP and substance P/K are co-localized in these dense vesicles is suggested by immune localization results (Schubert et al., 1991), in which case they would be bound for the nerve terminal. It is also possible that APP is localized in a separate class of dense vesicle which is bound for the plasma membrane.

In conclusion with regard to APP processing in the transport vesicles, the data strongly support the possibility that axonally transported APP is processed intracellularly to generate C-terminal fragments in at least two classes of vesicle, which are distinguished by labeling kinetics as well as by density. These vesicles are bound for both axonal plasma membrane and nerve terminal.

In contrast with the ON, in which both amyloidogenic and non- amyloidogenic C-terminal fragments are produced, we only detect the production of a non-amyloidogenic C-terminal fragment in the retina ( Fig. 3and Fig. 4). The two cell types in the retina which produce APP are the retinal ganglion cells that produce APP (and rapidly axonally transport it) and the Muller glial cells that produce the Kunitz-type protease inhibitor-containing APP isoforms, APP and APP, as well (Morin et al., 1993). Also, cultured rabbit Muller cells synthesize 120- and 140-kDa species of APP, corresponding to APP and APP, respectively.()Because of the rapid clearance of neuronally produced APP into the ON (Morin et al., 1993), it is likely that the retinal non-amyloidogenic C-terminal metabolite is produced in the Muller cell. Therefore, this result may indicate differential processing of APP with respect to the central nervous system cell type and/or to the APP isoform.

This result also suggests that in the normal central nervous system, only neurons are capable of generating amyloidogenic fragments. This is in contrast to the reported results that cultured astrocytes, which are similar in certain ways to Muller cells, produce more A than cultured neurons (Busciglio et al., 1993).

The above results may help to elucidate the normal processing of APP in vivo in a central nervous system neuron. In contrast to rat and mouse, the A sequence of the rabbit is identical to that of human (Johnstone et al., 1991), making it a more appropriate system to study APP processing.


FOOTNOTES

*
This work was supported by an Alzheimer's Association/Polygram Pilot Research Grant (to A. A.) and a National Institutes of Health Grant RO1 EY 08535 (to R. E. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Boston University School of Medicine, 80 E. Concord St., K-124C, Boston, MA 02118. Tel.: 617-638-4190; Fax: 617-638-5339.

The abbreviations used are: A, amyloid -protein; APP, -amyloid precursor protein; ON, optic nerve/optic tract; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

A. Amaratunga, C. R. Abraham, R. B. Edwards, J. H. Sandell, B. M. Shreiber and R. E. Fine, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. Peter Morin, Dr. Robin Johnson, and Hae Yung Pyun for their invaluable advice and assistance.


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