(Received for publication, June 14, 1995; and in revised form, December 27, 1995)
From the
We have investigated the synthesis and transport of apoE, the
major apolipoprotein of the central nervous system, in the retina of
the living rabbit. Four hours after the injection of
[S]methionine/cysteine into the vitreous, 44% of
[
S]Met/Cys-labeled apoE is in soluble and
membrane-enclosed retinal fractions, while 50% is in the vitreous. A
significant amount of intact [
S]Met/Cys-labeled
apoE is rapidly transported into the optic nerve and its terminals in
the lateral geniculate and superior colliculus within 3-6 h in
two distinguishable vesicular compartments. Müller
glia in cell culture also synthesize and secrete apoE. Taken together,
these results suggest that apoE is synthesized by
Müller glia and secreted into the vitreous. ApoE is
also internalized by retinal ganglion cells and/or synthesized by these
cells and rapidly transported into the optic nerve and brain as an
intact molecule. We discuss the possible roles of retinal apoE in
neuronal dynamics.
ApoE, a 36-kDa glycoprotein, is a component of a number of circulating plasma lipoproteins, including very low density lipoproteins, high density lipoproteins, and chylomicron remnants(1) . Its primary function appears to be that of a recognition ligand for the receptor-specific removal of cholesteryl ester-rich lipoproteins from the circulation(2, 3) . It also plays a role in local transport of cholesterol, as seen in nerves of both peripheral (4, 5) and central nervous systems (reviewed in (6) ) and has been implicated in mediating immune responses and cell proliferation, processes that may not be related to its association with lipid (for review, see (1) ).
In plasma, apoE transports lipoproteins containing cholesterol,
triglycerides and other lipids to various cells via binding to the low
density lipoprotein (LDL) ()receptor (7) or the LDL
receptor-related protein (LRP)/
-macroglobulin
receptor(8, 9) . In the central nervous system, apoE
is primarily synthesized by the major glial cell, the astrocyte, in
rodents and humans (10, 11) and is found in
significant quantities in the cerebrospinal fluid(10) . Since
apolipoprotein B, the other molecule that can mediate the
internalization of lipoproteins via association with the LDL receptor,
is not synthesized in the central nervous system (reviewed in (6) ), it is highly likely that apoE plays a major role in
cholesterol and lipid transport in this compartment.
We have
employed the in vivo rabbit retina to probe the synthesis,
intracellular transport, and metabolism of central nervous system
proteins including kinesin, the motor for anterograde rapid
transport(12, 13) , and the -amyloid precursor
protein (APP)(14, 15) . Since it has been shown
recently that individuals having the
4 allele of apoE are at high
risk for Alzheimer's disease(16) , we decided to use this
widely accepted model of normal adult retinal protein synthesis and
metabolism (e.g.(17) and (18) ) to examine
the synthesis and transport of apoE. In this report, we present
evidence, obtained by the injection of
[
S]methionine/cysteine into the vitreous chamber
that overlies the retina, that apoE is synthesized in significant
quantities in vivo by rabbit Müller cells,
the predominant glial cell of the retina. ApoE is then transferred,
presumably after secretion, into the vitreous. A substantial quantity
of newly synthesized apoE is also subsequently transported as an intact
molecule into the optic nerve by retinal ganglion cells (RGCs) through
vesicular anterograde rapid transport. We discuss the possible role of
this pathway in axonal lipid metabolism and the possible interactions
of apoE with APP or its metabolites.
At the various times
following injection of label, subcellular fractionation was done using
a modification of the method of Lorenz and Willard(20) . Within
10 min of sacrifice by intravenous injection of 100 mg/kg sodium
pentobarbital, optic nerves and optic tracts (ON/OT) were rapidly
dissected and placed in 7 ml of ice-cold homogenization buffer (1
mM triethanolamine, 320 mM sucrose) containing 30
units/ml phenylmethysulfonyl fluoride, 0.5 µg/ml leupeptin, and 0.5
µg/ml aprotinin. Homogenization consisted of three strokes in a
motor-driven Teflon/glass homogenizer (0.005-inch clearance) in a
volume of 7 ml, followed by three strokes in a glass Dounce homogenizer
in a volume of 10 ml. Homogenates were diluted to 40 ml with
homogenization buffer and spun at 1200 g for 7 min.
The resulting supernatant was then spun at 100,000
g for 60 min. The resulting pellet was resuspended in 2 ml of
homogenization buffer and either used for immunoprecipitations directly
or loaded onto a discontinuous gradient consisting of 2 ml each of
20/26/31/37/45% sucrose (w/w) in homogenization buffer. These gradients
were spun at 150,000
g in an SW 40 rotor (Beckman
Instruments) for 16 h. Twenty-four 0.5-ml fractions were collected from
each gradient. A very similar protocol was employed for the
retina(21) .
The primary antibody used was rabbit anti-bovine GFAP (1:100 dilution; Dako Corp., Carpinteria, CA). Purified nonimmune rabbit IgG (ICN) was used as a control in place of a GFAP. The secondary antibody used was horseradish peroxidase-conjugated goat anti-rabbit IgG (1:100 dilution; Cappel, Rockville, MD). Cultures were photographed using phase-contrast optics or conventional bright-field optics when only the opaque precipitate of the peroxidase stain needed to be photographed.
Figure 1:
ApoE synthesis in the retina and rapid
axonal transport into the optic nerve at 3 h. Rabbits were given
vitreal injections of [S]Met/Cys and sacrificed
3 h later. A retinal membrane fraction (R-M) and a retinal
soluble fraction (R-S) were immunoprecipitated with a goat
anti-rabbit apoE antibody (I) and with nonimmune normal goat
serum (P). Similarly, vitreous humor (V) was
immunoprecipitated with the apoE antibody and with normal goat serum.
[
S]Met/Cys-labeled optic nerve membranes (ON-M), a soluble fraction (ON-S), and LGN were
immunoprecipitated with the apoE antibody and with normal goat serum.
Immunoprecipitates were separated on 10% acrylamide gels, followed by
autoradiography. The location of apoE (
36 kDa) is indicated by arrows. Bars indicate the positions of molecular mass
protein markers: 80, 49, 32, and 18 kDa.
We repeated the experiment
using a 6-h interval between [S]Met/Cys
injection and sacrifice. A similarly significant quantity of
specifically immunoprecipitated 36-kDa polypeptide was still found in
the ON/OT membrane fraction and in the LGN, in addition to a
significant amount of the immunoprecipitated 36-kDa polypeptide in the
retinal fractions (Fig. 2). These results indicate that there is
very active synthesis of apoE in the retina, and some of this apoE is
rapidly transported down the ON/OT in transport vesicles. We still did
not, however, find a detectable quantity of soluble apoE in the ON/OT.
This suggests that apoE does not gain access to the axonal cytoplasm or
the extracellular milieu of the ON/OT, at least for 6 h following
synthesis and transport.
Figure 2:
ApoE synthesis in the retina and rapid
axonal transport into the optic nerve at 6 h. Rabbits were given
vitreal injections of [S]Met/Cys and sacrificed
6 h later. A retinal membrane fraction (R-M), optic nerve
membranes (ON-M), a soluble fraction (ON-S), and LGN
were immunoprecipitated with apoE antibody (I) and with normal
goat serum (P), followed by electrophoresis and
autoradiography as described for Fig. 1. The location of apoE
(
36 kDa) is indicated by arrows. Bars denote the
positions of molecular mass protein standards: 106, 80, 49, and 32
kDa.
These data provide strong evidence for the
rapid transport of apoE into the optic nerve since detectable apoE
reached the termination of the 3-cm long optic nerve within 3 h after
synthesis, conforming to the known speed of rapid transport, 1 cm/h.
Because we found little apparent quantitative difference in the amounts
of apoE transported into the ON/OT at 3 and 6 h after
[S]Met/Cys injection, we chose an intermediate
time point (4 h) to carry out a quantitative distribution of apoE in
the various retinal and ON/OT fractions. Table 1shows the
results. As can be seen, over 50% of the total newly synthesized apoE
was in the vitreous, while a slightly smaller quantity was in the
retinal membranes, presumably from either the
Müller cells or the RGCs and their proximal axons.
A small but significant amount was also found in the ON/OT and LGN,
indicating RGC uptake and/or synthesis and rapid transport. No counts
were found in fractions taken from the uninjected vitreous, retina, and
ON/OT, indicating that no transfer of apoE from blood or vitreal
leakage occurred.
To characterize the type of transport vesicle that
carries apoE into the optic nerve, we fractionated the ON/OT
preparations isolated 3 and 6 h after [S]Met/Cys
injection by sucrose density gradient centrifugation as described
previously(14, 19) . We obtained three discrete peaks
of radioactivity. We have demonstrated previously that the peak with
the lowest density (peak 1) contains plasma membrane; the peak of
intermediate density (peak 2) contains transport vesicles bound for the
plasma membrane as well as synaptic vesicle precursors that go to the
nerve terminals; and the peak with the greatest density (peak 3)
contains peptide granules that are also transported to the nerve
terminals, but with significantly different kinetics(19) .
When we immunoprecipitated apoE from the three peaks described above, we obtained the results shown in Fig. 3. At 3 h after labeling, all the apoE was found in peak 2, while after 6 h, radioactive apoE was found in both peaks 2 and 3. The most reasonable interpretation of these results is that apoE is carried into the axon in two distinct transport vesicles with different kinetic and biochemical properties.
Figure 3:
Subcellular location of apoE processing in
the optic nerve. Three- and 6-h post
[S]Met/Cys-labeled optic nerve membranes were
subjected to sucrose density gradient separation(18) .
Fractions from peak 1 (light membranes), peak 2 (intermediate
membranes), and peak 3 (heavy membranes) were separately pooled and
subjected to immunoprecipitation using a goat anti-rabbit apoE
antibody. Immunoprecipitates were separated on 10% acrylamide gels,
followed by autoradiography. Lane 1, peak 1; lane 2,
peak 2; lane 3, peak 3. The location of apoE is indicated by arrows. Bars denote the positions of molecular mass
protein markers: 80, 49, 32, and 18 kDa.
We therefore cultured Müller cells from adult rabbits by the technique described by one of us(24) . These cells at early stages of growth have the bipolar appearance of Müller cells in vivo(24) . They also stain positively for GFAP, a widely accepted marker for these cells. Routinely, our cultures are over 95% positive for this marker (Fig. 4). They are also positive for retinal-binding protein(24) .
Figure 4:
Immunostaining of cultured adult rabbit
Müller glia with anti-GFAP. Cultures of
Müller glia were immunostained with anti-GFAP serum
followed by a horseradish peroxidase-labeled second antibody as
described under ``Materials and Methods.'' a, cells
incubated with anti-GFAP and viewed with a phase-contrast micrograph; b, cells incubated with anti-GFAP and viewed with bright-field
optics to accentuate the peroxidase-labeled filamentous network; c, cells stained with nonimmune IgG and viewed with
phase-contrast optics, showing no reaction product. Magnification
200.
We labeled
confluent Müller cells for 4 h with
[S]Met/Cys and carried out immunoprecipitation
with anti-apoE with both cells and medium. The cells synthesized a
36-kDa polypeptide and secreted it into the medium (Fig. 5). The
cells also produced a significant quantity of two high molecular mass
isoforms of APP, which is also characteristic of
Müller cells in vivo(14) .
Figure 5:
Immunoprecipitation of apoE and APP from
rabbit Müller cells and medium. Cultured
Müller cells were metabolically labeled for 4 h by
adding 0.2 mCi of [S]Met/Cys per ml. Labeled
cells (lanes 1, 2, 5, and 6) and
medium (lanes 3, 4, 7, and 8) were
immunoprecipitated for apoE (lanes 1-4) and APP (lanes 5-8). Precipitates were run on a 10-20%
gradient acrylamide gel, followed by autoradiography to detect labeled
proteins. A goat anti-rabbit apoE antibody (lanes 2 and 4) and a rabbit anti-APP C-terminal antibody (C8) (lanes 6 and 8) were used for detecting apoE and APP,
respectively. Control immunoprecipitates were carried out using normal
goat serum (lanes 1 and 3) and normal rabbit serum (lanes 5 and 7). Bars denote the positions
of molecular mass protein markers: 205, 116, 80, 49, 32, 27, and 18
kDa. The locations of the apoE bands (lanes 2 and 4)
and APP bands (lane 6) are indicated by arrows.
The in vivo data presented here suggest that retinal apoE is synthesized at least in part by Müller glial cells and secreted into the vitreous and extracellular compartment of the retina. The apoE synthesized in the retina is also internalized by RGCs from the vitreous and extracellular space and/or synthesized by the RGCs. It is then transported into the axon by a vesicular anterograde rapid mechanism.
The bipolar shape of the Müller cell, with a long process extending in each direction, allows it to secrete apoE at essentially any point in the retina, and Müller cells directly contact all nerve and photoreceptor cells in the retina. It is therefore ideally situated to supply apoE and any lipid moieties it associates with to all the cells of the retina including the RGCs. Since there appears to be a large pool of newly synthesized, soluble apoE in the retina itself and an even larger pool in the vitreous, it is likely that apoE is released directly into the vitreous as well as into the extracellular space of the retina. The axons of the RGCs, which are non-myelinated until they pass through the retina, lie in the perfect position to internalize the vitreal apoE, via surface receptors. Müller cells synthesize and secrete apoE in cell culture. Also in other regions of the central nervous system as well as in the periphery, apoE is synthesized mainly by glial cells and to some degree by macrophages, but not by neurons(6) .
However, there is still the possibility that the apoE we detected in the optic nerve in the brain has been synthesized by the RGCs themselves, although our data provide no evidence for this possibility. We hope to resolve this question by use of in situ RNA hybridization. We should also note that some of the apoE could be synthesized by retinal astrocytes that are associated with the RGC axons located near the center of the retina(27) . These cells are, however, much less abundant than Müller cells.
There are three possible sites of uptake for apoE on the retinal ganglion cell: the dendrites, the cell body, and the proximal axons of the RGCs, which are non-myelinated. It is very likely that apoE is taken up via a high affinity receptor, either the LDL receptor or LRP. While the distribution of these two receptors in the retina is not known, it is known that in the brain, the LRP is present on neuronal cell bodies, dendrites, and proximal axons(11) . LDL receptors are found on astrocytes, but not on neurons(11) .
We are now determining the distribution of the LRP and LDL receptor in the retina. We have also attempted to obtain direct evidence for receptor-mediated uptake of apoE into retinal ganglion cells by the injection of radiolabeled apoE into the vitreous with and without excess unlabeled apoE. These experiments have been unsuccessful, probably because of the large quantity of endogenous apoE in the vitreous.
Whichever site(s) of uptake are utilized, the vesicular transport pathway utilized by apoE (and potentially by its receptor) appears likely to be one of two types that are not mutually exclusive. One is a transcytotic pathway involving internalization into endosomal vesicles in either dendrites and/or the cell soma and transfer to axonally bound transport vesicles. This pathway is analogous to the basolateral-to-apical transcytosis pathway utilized by epithelial cells (28) . There is recent evidence, utilizing the transfected IgA receptor, for the existence of a dendritic/cell body-to-axonal transcytotic pathway in cultured neurons(29) . Also very recently, evidence for APP transcytosis has been obtained in cultured hippocampal neurons (30) .
The delayed appearance of radioactive apoE in peak 3 of the sucrose density gradient-separated transport vesicles may be explained by the existence of such a transcytotic pathway. However, it also could be explained by the delayed export of peptide-containing secretory vesicle precursors that has been previously described in this system(19, 21) . It is possible that all apoE molecules are taken up into the proximal axons and some are immediately transported in a forward direction, while the other portion is taken back to the Golgi apparatus and sorted into a peptide granule.
Another pathway would involve direct uptake of apoE into the proximal axon and anterograde transport toward the axon terminal. This is likely to be the pathway utilized by the plasma membrane-targeted transport vesicle found in peak 2 of these gradients. This pathway is very rapid as opposed to the peak 3 pathway, with detectable apoE in the LGN by 3 h after labeling. To our knowledge, there is no evidence at present for the existence of such a pathway in neurons. However, there are apoE receptors localized on the proximal axons in the brain(11) .
Finally, apoE in either peak 2- or 3-containing transport vesicles could result from the synthesis of apoE in the RGCs, followed by packaging into secretory vesicles with different densities and kinetic behavior. We have recently found that newly synthesized APP is rapidly transported in two discrete classes of secretory vesicle as well (15) .
Irrespective of the pathway
involved, what would be the function of such a pathway? One possibility
would be the use of apoE as a lipid carrier to supply the axon's
needs for particular lipids. Consistent with this possibility, we have
recently found that a high percentage of newly synthesized apoE is
associated with lipids in the medium of cultured
Müller cells as well as in the vitreous. ()There is recent evidence that while axons and nerve
terminals in particular can resynthesize phospholipids, they cannot
synthesize cholesterol and glycolipids and therefore depend on
transport from the cell body to supply these essential
molecules(31) , analogous to the situation with regard to
proteins(32) . There is evidence that apoE produced by
astrocytes donates lipids and cholesterol to central nervous system
neurons engaged in extensive axon growth and synaptic remodeling
following lesioning (reviewed in (6) ). Also, it was very
recently reported that homozygous apoE-deficient mice display a
significant loss of synapses and disruption of the dendritic
cytoskeleton with age as well as decreased hippocampal compensatory
synaptogenesis after lesioning of the projections from the entorhinal
cortex(33) . These changes would be consistent with long-term
damage to these cells caused by a deficit in apoE-supplied lipid
moieties, although other explanations cannot be ruled out.
Our finding that apoE is rapidly transported in the axon of a long projection neuron, the neuronal class that is selectively vulnerable to Alzheimer's disease, may offer some insight into why the E4 isoform is a significant risk factor for Alzheimer's disease(16, 34) . If the E4 form of apoE is unable to supply lipid to neurons as well as the other two isoforms of apoE, as a recent experiment with cell-cultured dorsal root ganglia indicates(35) , people with the E4 isoform may be more susceptible to the development of Alzheimer's disease. Other evidence supporting this conjecture has recently been reviewed(6) .
Our apoE data combined with our results
demonstrating APP transport (14, 15) also suggest the
possibility that apoE and APP could directly interact and be
transported together in both Müller glia and RGCs.
Since apoE4 and apoE3 appear to interact differently in vitro with the -amyloid portion of APP(15) , it is possible
that they together perform some as yet unknown function in the central
nervous system. In this regard, it has recently been demonstrated that
the LRP can internalize and mediate degradation of APP as well as apoE
in the context of a lipoprotein particle, suggesting internalization of
both molecules into the same compartment(36) . We are presently
carrying out experiments investigating possible interactions of these
molecules in the in vivo retina.