(Received for publication, January 23, 1995; and in revised form, April 11, 1995)
From the
The amyloid
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
Mature
Anterograde axonal transport of neuronal
In order to
examine the generation of potentially amyloidogenic fragments of
We have utilized the retinal ganglion cell/ON preparation
combined with 10-20% Tris-Tricine gradient gels to detect A
Figure 1:
Generation of amyloidogenic and
non-amyloidogenic fragments during axonal transport of
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,
We have subfractionated the
Figure 2:
Subcellular location of
We also carried out immune precipitation of
Figure 3:
Generation of non-amyloidogenic fragments
by
Figure 4:
The use of a monoclonal antibody specific
to A
The results presented have demonstrated that there is a rapid
(<3 h) metabolism of the retinal ganglion cell produced
The 12-kDa C-terminal fragment recognized only by C8 may be
produced by an
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
Using an equilibrium sucrose gradient, we were able to
fractionate
It is
likely that
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
In
cultured cells, amyloidogenic fragments as well as A
In 6-h post-labeled ON,
C-terminal fragments are detected in both peaks 2 and 3 (Fig. 2), thereby indicating intracellular
In conclusion with regard to
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
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
The above results may help to
elucidate the normal processing of
We thank Dr. Peter Morin, Dr. Robin Johnson, and Hae
Yung Pyun for their invaluable advice and assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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.
-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)).
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).
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).
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.
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).
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.
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).
APP
was shown to be anterogradely axonally transported in small
vesicles of the plasma membrane type (Morin et al., 1993).
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).
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.
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).
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.
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.
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).
-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.
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.
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).
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)
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.
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).
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.
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.
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.
than cultured neurons
(Busciglio et al., 1993).
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.
, 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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.