Retinal Muller glia secrete apolipoproteins E and J which are efficiently assembled into lipoprotein particles

Molecular Brain Research 50 Ž1997. 113–120

Research report

Retinal Muller glia secrete apolipoproteins E and J which are efficiently assembled into lipoprotein particles Janani Shanmugaratnam a , Eric Berg a , Lauren Kimerer c , Robin J. Johnson a , Anil Amaratunga a , Barbara M. Schreiber a , Richard E. Fine a,b,c,) a

Department of Biochemistry, Boston UniÕersity School of Medicine, 80 E. Concord Street K-124C, Boston, MA 02118, USA b Department of Neurology, Boston UniÕersity School of Medicine, Boston, MA 02118, USA c Edith Nourse Rodgers Memorial Veterans Hospital, Bedford, MA 01730, USA Accepted 30 April 1997

Abstract We have shown that apolipoprotein E ŽApoE. is synthesized by Muller cells, the major glial cell of the rabbit retina, and secreted into the vitreous after which it is taken up by retinal ganglion cells and rapidly transported into the optic nerve wAmaratunga et al., J. Biol. Chem. 271 Ž1996. 5628–5632x. In this report we demonstrate that the ApoE secreted by Muller cells in vivo and in culture is efficiently assembled into lipoprotein particles. Apolipoprotein J ŽApoJ. is also synthesized by these cells and assembled into lipoprotein particles. The lipoproteins are triglyceride-rich and contain cholesterol esters and free cholesterol. They are heterogeneous, with densities between 1.006 and 1.18 and diameters between 14 and 45 nm. We discuss the possible role of these lipoproteins in supplying the needs of neurons for lipids, especially long axonal projection neurons such as retinal ganglion cells, which are vulnerable to age-related neurodegenerative diseases including Alzheimer’s disease. q 1997 Elsevier Science B.V. Keywords: Rabbit; Retinal ganglion cell; Vitreous; Alzheimer’s disease; Cholesterol; Triglyceride

1. Introduction: Apolipoprotein E ŽApoE., 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 w19x. Its primary function appears to be that of a recognition ligand for the receptor-specific removal of cholesteryl ester-rich lipoproteins from the circulation w35,39x. It also plays a role in local transport of cholesterol, as seen in nerves of both peripheral w4,12x and central nervous systems ŽCNS. Žreviewed in w28x.; and has been implicated in mediating immune responses and cell proliferation, processes that may not be related to its association with lipid Žfor review, see w19x.. Abbreviations: ApoE, Apolipoprotein E; ApoJ, Apolipoprotein J; CNS, central nervous system; LDL, low density lipoprotein; LRP, low density lipoprotein related protein; TLC, thin layer chromatography. ) Corresponding author, at address ‘a’. Fax: q1 Ž617. 638-5339; E-mail: [email protected]

In plasma, ApoE transports lipoproteins containing cholesterol, triglycerides and other lipids to various cells via binding to the low density lipoprotein ŽLDL. receptor w11x or the LDL receptor-related proteinra 2-macroglobulin receptor ŽLRP. w16x. In the CNS of rodents and humans, ApoE is primarily synthesized by the major glial cell, the astrocyte w26,31x, and is found in significant quantities in the cerebrospinal fluid w26x. Since apolipoprotein B, the other molecule which can mediate the internalization of lipoproteins via association with the LDL receptor, is not synthesized in the CNS Žreviewed in w28x., it is highly likely that ApoE plays a major role in cholesterol and lipid transport in this compartment. ApoE is the most abundant apolipoprotein in the CNS w28x. It plays a major role in cholesterol and other lipid transport processes in both the periphery and in the brain w19x. Recently, Poirier has proposed a central role for ApoE in lipid transfer between glial cells, in which synthesis occurs, and neurons w28x. Consistent with this proposal, ApoE synthesis increases in situations of excitotoxic or ischemic injury to the brain when a great increase in lipid

114

J. Shanmugaratnam et al.r Molecular Brain Research 50 (1997) 113–120

synthesis occurs associated with gliosis and collateral formation w30x. Also ApoE synthesis is increased tremendously following the axotomy of peripheral nerves, suggesting a key role for ApoE in lipid transport involved in nerve repair Žreviewed in w19x.. Evidence of a major role for ApoE in the growth and branching of cultured peripheral neurons has been published w25x. Another major CNS apolipoprotein which has been implicated in the physiology and potentially, the pathophysiology of the retina is apoJ, a 70–80 kDa protein which is cleaved posttranslationally to form a disulfide linked complex of two approximately 40 kDa polypeptides. This protein, which is known by a variety of names including clusterin and sulfated glycoprotein 2, has a number of activities including the ability to form a lipoprotein by binding lipid moieties, and the ability to inhibit complement activation Žreviewed in w13x.. It is synthesized by epithelial cells in various parts of the body. Recently it has been found that mRNA for ApoJ is present in the retinal ganglion cell ŽRGC. and inner nuclear layer of mouse and human retina, and ApoJ has been identified in the vitreous by Western blotting w14,33x. Immunoreactivity for ApoJ is present in both the RGC and inner nuclear layers. ApoJ synthesis, like that of ApoE, is induced in CNS cells, mainly astrocytes, by injury to the brain Žreviewed in w20,21x.. We have employed the in vivo rabbit retina to probe the synthesis, intracellular transport and metabolism of CNS proteins including kinesin, the motor for anterograde rapid transport w3,23x and the b-amyloid precursor protein w2,22x. Since it has been shown recently that individuals having the e-4 allele of ApoE are at high risk for Alzheimer’s disease ŽAD. w37x, we decided to use this widely accepted model of normal adult retinal protein synthesis and metabolism Že.g. w36,38x. to examine the synthesis and transport of ApoE. Using this model and cultured adult rabbit Muller cells, we have recently generated evidence that there is a transfer of newly synthesized ApoE from Muller cells to RGCs and subsequently the anterograde rapid transport of intact ApoE into the optic nerve in the in vivo rabbit retina w1x. In this report we provide evidence, using both the in vivo retinarvitreous and cultured Muller cells, that newly synthesized ApoE, ApoJ and lipid moieties are incorporated into lipoprotein particles with characteristics different than plasma lipoproteins.

2. Materials and methods 2.1. Injection of [ 35S]methionine, cysteine (Met,Cys) into the Õitreous Adult male albino rabbits Ž6 lb. were anesthetized with 1 ml of 5% sodium pentobarbital Žintravenous., and two drops of the local anesthetic, 0.5% proparacaine-HCl, were

put in each eye. U-100 insulin syringes with 28-gauge needles were used to introduce 0.5 mCi Ž50 ml. w 35 SxMet,Cys into the vitreous of one or both eyes. The animals awakened within 1–2 h and showed no signs of discomfort or redness of the eye. Within 3 min of sacrifice by intravenous injection of 100 mgrkg sodium pentobarbital, the vitreous was removed from each injected eye. 2.2. Biochemical and immunochemical methods Protein determinations were carried out by the method of Bradford w5x. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis ŽSDS–PAGE. was performed on 10% gels by the method of Laemmli w17x. Gels were prepared for autoradiography by immersing them in an enhancing solution followed by drying for 2 h under vacuum as described w24x. Films were exposed for appropriate times and developed in an automatic processor. Densitometry was carried out using an LKB laser densitometer. Immunoprecipitations were performed as described previously w24x. Briefly, samples were dissolved in 0.1% SDS, 1.0% Triton X-100 in PBS and undissolved material removed by centrifugation at 15 000 = g for 1 min in an Eppendorf centrifuge. Samples were incubated overnight at 48C with agitation with the appropriate primary antibody previously bound to Sepharose bead linked anti-IgG. The beads were then centrifuged in an Eppendorf centrifuge and washed six times in PBS containing 1% Triton X-100 and 1 M NaCl. The final samples were then subjected to SDS– PAGE. Quantitation of gels was performed using a Packard Instant Imager or of films using a Molecular Dynamics laser densitometer. 2.3. Muller glial cell culture Muller glial cells were cultured from adult albino rabbits, as previously described by Edwards et al. w6x, which was modified from the method of Lewis et al. w18x. Animals were killed by intravenous sodium pentobarbital Ž100 mgrkg.. Eyes were enucleated and the anterior half and vitreous discarded. The retinas were removed, rinsed, cut into 4 = 4 mm fragments, and incubated for 30 min in 0.5 mgrml protease Nagarse ŽSigma. in Ca2qrMgq2-free balanced salt solution at 378C. The tissue was rinsed, resuspended in Hank’s balanced salt solution that contained 0.1 mgrml DNase and 0.5 mgrml bovine serum albumin, and dissociated into a single-cell suspension by passing the retinal fragments through a 10 ml plastic sterile pipet 10 times. The cells were gently centrifuged, resuspended in culture medium ŽDulbecco’s Modified Eagle medium with 10% fetal bovine serum., seeded in one 25-cm2 laminin-coated culture flask per two retinas, and maintained at 378C in 5% CO 2r95% air. The medium was changed every 3–4 days. Retinal neurons that initially adhered to the surface of Muller cells were rinsed off with medium changes by 10 days in vitro, leaving a monolayer

J. Shanmugaratnam et al.r Molecular Brain Research 50 (1997) 113–120

of glial cells as demonstrated by uniform immunostaining with an antibody against glial filament associated protein w1x. 2.4. [ 35S]Met,Cys labeling of cultured Muller cells Cells on 25-cm2 culture flasks were washed with Hank’s buffered saline and incubated with methionine free culture medium ŽSigma. for 30 min. Then 0.2 mCirml w 35 SxMet,Cys was added for 4 h. The medium was collected and the cells rinsed once with Hank’s and dissolved in immunoprecipitation buffer. 2.5. Preparation of lipoproteins To separate total lipid-bound proteins from soluble proteins as described w10x, the density was adjusted to 1.215 grml with solid KBr and the samples subjected to ultracentrifugation at 40 000 rpm in a Beckman SW41 rotor at 158C for 48–72 h. Alternatively, conditioned media were subjected to density gradient ultracentrifugation in a discontinuous KBr gradient as described w32x. Ultracentrifugation was performed for 24 h at 40 000 rpm in an SW41 rotor. Fractions were collected sequentially from the top of the tubes. Densities of the fraction were determined by use of a refractometer ŽAmerican Optical Corp... 2.6. Lipid analysis The medium was collected from Muller cells in culture which had been exposed to w 14 Cxacetate Ž15 mCirml. for 24 h. The medium was dialyzed against PBS overnight at 48C and was then brought to a density of 1.215 grml by adding KBr and was fractionated by layering 1.75 ml of dense medium with KBr solutions of varying density Ž1.25 ml, d s 1.115; 3 ml, d s 1.019; and 2 ml, d s 1.006.. The gradient was centrifuged at 150 000 = g for 22 h at 48C using a Beckman SW40 rotor. One-ml fractions were collected and along with unfractionated medium, ApoE was immunoadsorbed by adding goat anti-rabbit ApoE antibody w34x in nondenaturing conditions at a 1:500 dilution. Immunoadsorbed ApoE was removed from solution via centrifugation by adding 2 mg of mouse anti-goat antibody coupled to Sepharose beads. Sepharose pellets were washed with PBS and lipids were extracted using the Folch Method w8x. Samples were then allowed to evaporate to dryness and resuspended in 30 ml of toluene. Samples were spotted on Baker Silica gel IB2 thin layer chromatography ŽTLC. plates. Neutral lipids were separated using a running solvent of hexanerethyl etherracetic acid Ž69:30:1. and polar lipids were separated by using a running solvent of chloroformrmethanolrwaterracetic acid Ž25:15:3:1.. Plates were imaged and analyzed on a Packard InstantImager or exposed on film.

115

2.7. Electron microscopy Lipoproteins were isolated from rabbit vitreous by KBr density gradient centrifugation as described w10x. The lipoproteins were dialyzed against PBS overnight at 48C. An aliquot was applied to a carbon-coated electron microscopy grid, and negative-stained with 1% uranyl acetate. To determine the mean particle size and the range, five 1-inch2 templates were placed on random areas of the micrograph, and the diameters of each particle measured.

3. Results It is known that ApoE is recognized by its receptors only when it is bound to a lipoprotein particle w19,27,28x. Since our evidence indicates that ApoE is taken up by RGCs after synthesis and secretion by Muller cells w1x, we hypothesized that newly secreted ApoE is found in the form of lipoprotein particles. We therefore labeled cultured Muller cells grown in serum free medium with w 35 SxMet,Cys. The medium was fractionated on a KBr gradient followed by immunoprecipitation with a polyclonal goat antibody against rabbit ApoE w34x. As shown in Fig. 1A, a major fraction of the total 35 S-labeled ApoE secreted by these cells Ž63%. is present in the lipoproteincontaining fractions of the gradient, indicating it is packaged into a lipoprotein. We have also characterized the vitreous from a w 35 SxMet,Cys-labeled rabbit ŽFig. 1B.. We see a significant fraction of the total w 35 SxMet,Cys-labeled ApoE in the vitreal lipoprotein 4 h after w 35 SxMet,Cys injection. We do, however, see several higher molecular mass bands in the protein-containing region of the gradient. These may represent tight complexes of ApoE with the proteoglycans, which comprise a major fraction of the proteins found in the vitreous. These results demonstrate that Muller cells can produce ApoE-containing lipoprotein particles in culture and that the Muller cells in vivo are likely to produce lipoproteins as well. In order to more precisely characterize the class of lipoprotein particle with which ApoE is associated in the medium from cultured Muller cells, we carried out equilibrium centrifugation employing a discontinuous 1.006–1.21 grml KBr gradient w32x. As shown in Fig. 2, we see ApoE associated with material in different density regions of the gradient: fraction 10 corresponds to intermediate density lipoprotein ŽIDL. Ž1.006 - d - 1.02., fractions 6 to 9 correspond to LDL Ž1.02 - d - 1.063. and fractions 1 to 5 correspond to high density lipoprotein ŽHDL. Ž1.063 - d - 1.18. regions. This is in striking contrast to the situation in the plasma in which ApoE is only associated with the VLDL and HDL particles. As expected, there is more ApoE, as well as total protein, associated with the denser LDL and HDL particles than with the IDL particles ŽFig. 2..

116

J. Shanmugaratnam et al.r Molecular Brain Research 50 (1997) 113–120

As can be seen in the lanes marked with a T, there are several other peptide bands associated with the lipoproteins. One of these, approximately as abundant as ApoE, migrates as a tight doublet with molecular masses of 40

Fig. 2. Muller cell derived ApoE is found in lipoprotein particles from 35 S-labeled cell medium with densities between 1.06 ŽVLDL. and 1.18 ŽHDL.. We labeled Muller cells with w 35 SxMet,Cys for 16 h, collected the medium and subjected it to a discontinuous KBr gradient equilibrium centrifugation in 12-ml tubes as described w34x. Ten fractions were collected. A 250-ml aliquot of each fraction was subjected to immunoprecipitation with anti-ApoE serum and the immunoprecipitates subjected to 10% SDS–PAGE followed by autoradiography. Lanes designated by the fraction number followed by a ‘T’ show the total labeled protein found in a 50-ml aliquot of the fraction. Fraction 1 is the densest fraction, which contains lipoproteins, 10 is the least dense. Fractions 1 to 5 correspond to the density of HDL; fractions 6 to 9 correspond to the density of LDL; and fraction 10 to the density of IDL. The arrow shows the mobility of ApoE; the arrowhead shows the mobility of ApoJ.

Fig. 1. Lipoproteins isolated from the medium of cultured Muller cells and from the vitreous of the eye contain newly synthesized ApoE. A: Muller glial cells were placed in serum free DMEM without Met and Cys. After 30 min, 100 mCir ml w 35 SxMet,Cys was added. Four hours later the medium was removed, adjusted to a density of 1.21 with solid KBr and centrifuged for 48 h in an SW50 rotor at 47 000 rpm. The lipoprotein Žtop 25%. and protein Žbottom 25%. layers were collected and equal aliquots were subjected to immunoprecipitation with anti ApoE or control antibodies, followed by SDS–PAGE and autoradiography. Lanes: 1, the lipoprotein fraction precipitated with normal rabbit serum; 2, the lipoprotein fraction precipitated with anti-ApoE; 3, the protein fraction immunoprecipitated with normal rabbit serum; 4, the protein fraction precipitated with anti ApoE. The figure shown is made from the phosphoimager. Only the results from the top and bottom quarters are shown since the other two layers contain negligible 35 S-labeled material. B: 0.5 mCi w 35 SxMet,Cys were injected into both vitreae of an anesthetized rabbit. Four hours later the vitreae were removed, fractionated and immunoprecipitated as described in A. Lanes are as in A. Again the two middle fractions contain negligible 35 S-labeled material and are not shown.

and 41 kDa. These are precisely the Mr of ApoJ, another abundant CNS apolipoprotein which was recently shown to be present in vitreous w14,33x. In order to convincingly demonstrate that this bandŽs. is, indeed, ApoJ we obtained a monospecific anti-ApoJ antibody from Dr. Michael Griswold. As shown in Fig. 3, a doublet of 40–41 kDa is specifically immunoprecipitated by anti-ApoJ from labelled Muller cells. We also see an approximately 70 kDa band specifically precipitated as well. This could represent ApoJ molecules that were not cleaved w13x. Several other less abundant polypeptides are also associated with 35 S-labeled lipoprotein particles. We are obtaining monospecific or monoclonal antibodies against potential candidates including ApoA1, ApoD, ApoH and SAA to hopefully identify these polypeptides. We have partially characterized the lipid moieties associated with the lipoprotein particles. We have immunoprecipitated KBr separated lipoprotein particles from w 14 Cxacetate-labeled Muller cell cultures with anti ApoE serum. The resultant lipids have been solubilized in chloro-

J. Shanmugaratnam et al.r Molecular Brain Research 50 (1997) 113–120

Fig. 3. Newly synthesized ApoJ is immunoprecipitated from the medium of cultured Muller cells. Muller cells were labeled with w 35 SxMet,Cys for 12 h. Equal aliquots of the labeled medium and cell lysate were subjected to immunoprecipitation with anti-rat ApoJ Žlanes 2 and 4. or with normal rabbit serum Žlanes 1 and 3., respectively. The precipitates were subjected to 10% SDS–PAGE followed by autoradiography. The marks on the right designate the molecular mass of the marker proteins, 140, 83, 35, 29 kDa, respectively. Arrows designate the mobility of reduced and unreduced rat ApoJ Žkind gift of Dr. M. Griswold..

formrmethanol and subjected to TLC as described in Section 2. The results are shown in Fig. 4. Comparison with standards identified two major spots corresponding to cholesterol ester and triglycerides. A third spot corresponding to free cholesterol is also seen, as well as a spot corresponding to diacyl glycerol. As expected, the least dense lipoproteins contain the highest quantity of labeled lipids. We do not detect 14 C-labeled lipids in the HDL region of the gradient. We assume that because of the low

Fig. 4. ApoE containing lipoproteins contain triglycerides, cholesterol and cholesterol esters. w 14 CxAcetate Ž15 mCirml. was added to the medium of confluent Muller cell cultures in serum free medium. After 12 h the medium was collected and subjected to discontinuous KBr gradient centrifugation as described in Section 2. Ten fractions were collected and each fraction was subjected to immunoprecipitation with anti-ApoE followed by extraction with chloroformrmethanol and TLC. TG designates the mobility of standard triglycerides; CE, the mobility of cholesterol esters; C, the mobility of free cholesterol; and DAG, the mobility of diacyl glycerol. Fractions are numbered from most dense, 1, through least dense, 10.

117

Fig. 5. Newly synthesized ApoJ is found in the lipoprotein fraction isolated from the vitreous. The lipoprotein fraction from 4 h w 35 SxMet,Cys-labeled vitreae was isolated and immunoprecipitations, gel electrophoresis and autoradiography carried out as described in Fig. 1. Lines on the left of the gel correspond to the mobilities of 50 and 35 kDa marker proteins, respectively. Lane 1santi ApoJ; lane 2 s normal rabbit serum.

specific activity of the w 14 Cxacetate compared to w 35 SxMet,Cys. We are unable to detect 14 C-labeled lipids associated with the denser regions of the gradient even though their density is that of HDL particles. When the extracted lipids from the medium are chromatographed with a more polar solvent, we see labeled spots corresponding in mobility to phosphatidylcholine and sphingomyelin Ždata not shown.. We have also begun to characterize the newly synthesized lipoproteins found in the vitreous 3 h after w 35 SxMet,Cys injection. Several major polypeptides are present in the lipoprotein fraction isolated from the vitreous after equilibrium centrifugation. One corresponds in mobility to ApoE which is immunoprecipitated from the lipoprotein-containing fraction from vitreous ŽFig. 1A.. Another major protein with the mobility of ApoJ is also present. As shown in Fig. 5, we have confirmed that this peptide is, in fact, ApoJ by immunoprecipitation. We have characterized the lipid moieties found in the vitreal lipoprotein fraction by TLC. The lipids found in the lipoprotein-containing fraction are triglyceride and cholesterol-containing and correspond closely in intensity and mobility to lipids extracted from the Muller cell medium Ždata not shown.. We have also examined the lipoprotein particles we obtained from the vitreous by negative-stain electron microscopy ŽFig. 6A.. Fairly uniform-sized particles with an average diameter of 28 nm are seen. The size range is between 11 and 54 nm. We see similar-sized particles in

118

J. Shanmugaratnam et al.r Molecular Brain Research 50 (1997) 113–120

Fig. 6. Negative-stained electron micrographs of the lipoproteins isolated from the rabbit vitreous and Muller cell medium. A: lipoproteins were isolated as described w35x from the rabbit vitreous. An aliquot was placed on a carbon coated EM grid and negatively stained with 1% uranyl acetate. =71 280. Bar s100 nm. B: lipoproteins were isolated and negatively stained as in A, from the serum-free medium of cultured rabbit Muller cells. =71 280.

negatively stained preparations of lipoproteins isolated from Muller cell medium ŽFig. 6B.. The average diameter of these particles is 26 nm with a range between 15 and 43 nm.

4. Discussion The data presented here indicate that the adult Muller glial cell in cell culture and, as we discuss below, in vivo,

has the capacity to synthesize and secrete lipoprotein particles. Alternately it is possible that the apolipoproteins and lipids are secreted independently, and assemble in the extracellular medium, or vitreous. We are currently using electron microscopy to look for lipoprotein formation in secretory compartments in Muller cells, to distinguish between these possibilities. The lipoprotein particles secreted by the Muller cells appear to differ significantly from those found in plasma, in that they contain two major apolipoproteins, ApoE and ApoJ, which are found in very small amounts in plasma lipoproteins, while containing no apolipoprotein B and very little if any A and C type apolipoproteins, the major constituents of plasma lipoproteins. The two major apolipoproteins are also homogeneously distributed within the various density lipoproteins isolated here; as opposed to the situation in plasma, with ApoE found only on VLDL and subpopulations of HDL and ApoJ found only on subfractions of HDL. On the other hand, the lipid composition of the newly synthesized lipoproteins is similar to that of plasma lipoproteins, composed mainly of triglycerides and cholesterol esters, with smaller quantities of free cholesterol, diacylglycerol and phospholipids. Since there is evidence based on a study of cultured peripheral neurons that the axon cannot synthesize significant cholesterol and sphingolipids w41x, our evidence that ApoE is synthesized by Muller cells, taken up by RGCs and rapidly transported down the axon leads to the hypothesis that ApoE-containing lipoproteins secreted by Muller cells are taken up by receptors on the RGCs and axonally transported to help supply the needs of the axon for lipids w1,7x. The evidence presented here that Muller cells synthesize and secrete ApoE and J-containing lipoproteins into the vitreous where they are in contact with the axons of the RGCs which run along the surface of the vitreous is consistent with this hypothesis. We also have recently obtained evidence that LRPs are synthesized by retinal ganglion cells in vivo and rapidly transported into the optic nerve ŽShanmugaratnam and Fine, unpublished data.. These data may also provide insight into why individuals who possess the e 4 allele of ApoE are at high risk for developing late-onset AD. If ApoE4-containing lipoproteins are not as efficient donors of lipids to myelinated projection neurons as are ApoE3 or ApoE2-containing lipoproteins, then with aging, the neurons from individuals with the e 4-4 or to a lesser extent e 4-3 phenotype may become more susceptible to injury and death. In this regard there are recent data indicating that ApoE4-containing lipoproteins do not bind to ApoE receptors on cultured neurons as well as do apoE3-containing lipoproteins w9,15x. The recent finding that cholinergic neuronal death is much greater in AD patients with e 4-4 and e 4-3 genotypes than in those without, is also significant with respect to this possibility w29x. Acetylcholine is synthesized in the nerve terminals from the large pool of phosphatidylcholine w40x. It is therefore likely that this class of neuron would be very

J. Shanmugaratnam et al.r Molecular Brain Research 50 (1997) 113–120

susceptible to depletion of lipids at the nerve terminal due to inefficient transport.

Acknowledgements We acknowledge the expert secretarial assistance of Simona Zacarian. The research described here was supported by PHS Grants RO1EY-08535 and R37 AG-05894 Žto R.E.F.., PO1HL-13262 Žto B.M.S.., by an Alzheimer’s Association Research Grant Žto R.E.F.. and a Merit Award grant from the Dept. of Veterans Affairs.

References w1x A. Amaratunga, C.R. Abraham, R.B. Edwards, J.H. Sandell, B.M. Schreiber, R.E. Fine, Apolipoprotein E is synthesized in the retina by Muller glial cells, secreted into the vitreous, and rapidly transported into the optic nerve by retinal ganglion cells, J. Biol. Chem. 271 Ž1996. 5628–5632. w2x A. Amaratunga, R.E. Fine, Generation of amyloidogenic C-terminal fragments during rapid axonal transport in vivo of b-amyloid precursor protein in the optic nerve, J. Biol. Chem. 270 Ž1995. 17268– 17272. w3x A. Amaratunga, P.J. Morin, K.S. Kosik, R.E. Fine, Inhibition of kinesin synthesis and rapid anterograde axonal transport in vivo by an antisense oligonucleotide, J. Biol. Chem. 268 Ž1993. 17427– 17430. w4x J. Boyles, C. Zoellner, L. Anderson, L. Kosik, R. Pitas, K. Weisgraber, D. Hui, R. Mahley, P. Gebicke-Harter, M. Ignatius, E. Shooter, A role for apolipoprotein E, apolipoprotein A-1, and low density lipoprotein receptors in cholesterol transport during regeneration and remyelination of the rat sciatic nerve, J. Clin. Invest. 83 Ž1989. 1015–1031. w5x M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 Ž1976. 248–254. w6x R.B. Edwards, A.J. Adler, S. Dev, R.C. Claycomb, Synthesis of retinoic acid from retinol by cultured rabbit Muller cells, Exp. Eye Res. 54 Ž1992. 481–490. w7x A.M. Fagan, G. Bu, Y. Sun, A. Daugherty, D.M. Holtzman, Apolipoprotein E-containing high density lipoprotein promotes neurite outgrowth and is a ligand for the low density lipoprotein receptor-related protein, J. Biol. Chem. 271 Ž1996. 30121–30125. w8x J. Folch, M. Lees, G.H. Sloane-Stanley, A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem. 226 Ž1957. 497–509. w9x D. Guillame, P. Betrand, D. Dea, J. Davignon, J. Poirier, Apolipoprotein E and low-density lipoprotein binding and internalization in primary cultures of rat astrocytes: isoform specific alterations, J. Neurochem. 66 Ž1996. 2410–2418. w10x H. Herscovitz, D. Gantz, A. Tereyok, V. Zannis, D. Small, Expression of human apolipoprotein E but not that of apolipoprotein A-1 by mouse C-127 cells is associated with increased secretion of lipids in the form of vesicles and discs, J. Lipid Res. 33 Ž1992. 791–803. w11x D. Hui, T. Innerarity, R. Mahley, Detective hepatic lipoprotein receptor binding of b-very low density lipoproteins from type III hypolipoproteinemic patients. Importance of apolipoprotein E, J. Biol. Chem. 256 Ž1981. 5646–5655. w12x M. Ignatius, P. Gebicki-Harter, J. Pate Skeen, J. Schilling, K. Weisgraber, R. Mahley, E. Shooter, Expression of apolipoprotein E during nerve degeneration and regeneration, Proc. Natl. Acad. Sci. USA 83 Ž1986. 1125–1129.

119

w13x D.E. Jenne, J. Tschopp, Clusterin: the intriguing guises of a widely expressed glycoprotein, Trends Biochem. Sci. 17 Ž1992. 154–159. w14x C. Jomary, B. Murphy, M. Neal, S.E. Jones, Abnormal distribution of retinal clusterin in retinitis pigmentosa, Brain Res. Mol. Biol. 20 Ž1993. 274–278. w15x M.Z. Kounnas, E.B. Loukinova, S. Stefansson, J.A.K. Harmony, B.H. Brewer, D.K. Strickland, W.S. Argraves, Identification of glycoprotein 330 as an endocytic receptor for apolipoprotein Jrclusterin, J. Biol. Chem. 270 Ž1995. 13070–13075. w16x R. Kowal, J. Herz, J. Goldstein, V. Esser, M. Brown, Low density lipoprotein receptor-related protein mediates uptake of cholesteryl esters derived from apoprotein E-enriched lipoprotein, Proc. Natl. Acad. Sci. USA 86 Ž1989. 5810–5814. w17x U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 Ž1970. 680–685. w18x G.P. Lewis, P. Erickson, D. Kaska, S.K. Fisher, An immunocytochemical comparison of Muller cells and astrocytes in the cat retina, Exp. Eye Res. 47 Ž1988. 855–868. w19x R. Mahley, Apolipoprotein E: cholesterol transport protein with expanding roles in cell biology, Science 240 Ž1988. 622–630. w20x P.C. May, C.E. Finch, Sulfated glycoprotein 2: new relationships of this multifunctional protein to neurodegeneration, Trends Neurosci. 15 Ž1992. 391–396. w21x P. May, M. Lambert-Etchells, S. Johnson, J. Poirier, J. Masters, C. Finch, Dynamics of gene expression for a hippocampal glycoprotein elevated in Alzheimer’s disease and in response to experimental lesions in rats, Neuron 5 Ž1990. 831–839. w22x P.J. Morin, C.R. Abraham, A. Amaratunga, R.J. Johnson, G. Huber, J.H. Sandell, R.E. Fine, Amyloid precursor protein is synthesized by retinal ganglion cells, rapidly transported to the optic nerve plasma membrane and nerve terminals and metabolized, J. Neurochem. 61 Ž1993. 464–473. w23x P.J. Morin, R.J. Johnson, R.E. Fine, Kinesin is rapidly transported in the optic nerve as a membrane associated protein, Biochim. Biophys. Acta 1146 Ž1993. 275–281. w24x P.J. Morin, N. Liu, R.J. Johnson, S.E. Leeman, R.E. Fine, Isolation and characterization of rapid transport vesicle subtypes from rabbit optic nerve, J. Neurochem. 56 Ž1991. 415–427. w25x B. Nathans, S. Bellosta, A. Sanan, K. Weisgraber, R. Mahley, R. Pitas, Differential effects of apolipoproteins E3 and E4 on neuronal growth in vitro, Science 264 Ž1994. 850–852. w26x R. Pitas, J. Boyles, S. Lee, D. Foss, R. Mahley, Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins, Biochim. Biophys. Acta 917 Ž1987. 148–161. w27x W. Pitas, J. Boyles, D. Lee, S. Hui, K. Weisgraber, Lipoproteins and their receptors in the central nervous system, J. Biol. Chem. 262 Ž1987. 14352–14360. w28x J. Poirier, ApoE in animal models of CNS injury and in AD, Trends Neurosci. 17 Ž1994. 525–530. w29x J. Poirier, M.-C. Delisle, R. Quirion, I. Aubert, M. Farlow, D. Lahiri, S. Hui, P. Bertrand, J. Nalbantoglu, B.M. Gilfix, S. Gauthier, Apolipoprotein E4 allele as a predictor of cholinergic deficiency and treatment outcome in Alzheimer’s disease, Proc. Natl. Acad. Sci. USA 92 Ž1995. 12260–12264. w30x J. Poirier, M. Haass, P. May, C. Finch, Astrocytic apolipoprotein E mRNA and GFAP mRNA in hippocampus after entorhinal cortex lesioning, Mol. Brain Res. 11 Ž1991. 97–106. w31x G. Rebeck, J. Reiter, D. Strickland, B. Hyman, ApoE in sporadic AD: allelic variation and receptor interactions, Neuron 11 Ž1993. 575–580. w32x T.G. Redgrave, D.C.K. Roberts, C.F. West, Separation of plasma lipoproteins by density-gradient ultracentrifugation, Anal. Biochem. 65 Ž1975. 42–49. w33x D.J. Reeder, W.D. Stuart, D.P. Witte, T.K. Brown, J.A. Harmony, Local synthesis of apolipoprotein J in the eye, Exp. Eye Res. 60 Ž1995. 495–505. w34x B. Schreiber, H. Jones, C. Franzblau, Apolipoprotein E expression in

120

w35x w36x w37x

w38x

J. Shanmugaratnam et al.r Molecular Brain Research 50 (1997) 113–120 aortic smooth muscle cells: the effect of b VLDL, J. Lipid Res. 35 Ž1994. 1177–1186. B. Sherrill, T. Innerarity, R. Mahley, Rapid hepatic clearance of the canine lipoproteins containing only the E apoprotein, J. Biol. Chem. 255 Ž1980. 1807–8304. J. Sjostrand, J.-O. Karlsson, Axoplasmic transport in the optic nerve and tract of the rabbit: a biochemical and radiographic study, J. Neurochem. 16 Ž1969. 833–844. W.J. Strittmatter, A.M. Saunders, D. Schmechel, L. Dong, G. Salvesen, M. Pericak-Vance, D. Goldgaber, A. Roses, Apolipoprotein E: high avidity binding to B-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease, Proc. Natl. Acad. Sci. USA 90 Ž1993. 1977–1981. M. Willard, W.M. Cowan, P.R. Vagelos, The polypeptide composi-

tion of intra-axonally transported proteins: evidence of 4 transport velocities, Proc. Natl. Acad. Sci. USA 71 Ž1974. 2183–2187. w39x E. Windler, Y. Chao, R. Havel, Regulation of the hepatic uptake of triglyceride-rich lipoproteins in the rat. Opposing effects of homologous apolipoprotein E and individual apoproteins, J. Biol. Chem. 255 Ž1980. 8303–8307. w40x R.J. Wurtman, Choline metabolism as a basis for the selective vulnerability of cholinergic receptors, Trends Neurosci. 15 Ž1992. 117–122. w41x J. Vance, D. Pan, R. Campenot, M. Bussiere, D. Vance, Evidence that the major membrane lipids, except cholesterol, are made in axons of cultured rat sympathetic neurons, J. Neurochem. 62 Ž1994. 329–337.