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Uptake of Lipoproteins for Axonal Growth of Sympathetic Neurons

Uptake of Lipoproteins for Axonal Growth of Sympathetic Neurons THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 26, Issue of June 30, pp. 19883–19890, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Received for publication, January 12, 2000, and in revised form, February 18, 2000) Elena I. Posse de Chaves‡§, Dennis E. Vance¶i, Robert B. Campenoti**, Robert S. Kiss¶, and Jean E. Vance‡ ‡‡ From the Departments of ¶Biochemistry, **Cell Biology, and ‡Medicine, University of Alberta, Edmonton, Alberta T6G 2S2, Canada Lipoproteins originating from axon and myelin break- Instead, cholesterol from degenerating axons and myelin is proposed to be retained within the nerve and re-utilized via a down in injured peripheral nerves are believed to sup- ply cholesterol to regenerating axons. We have used lipoprotein-mediated process (8), although cholesterol uptake compartmented cultures of rat sympathetic neurons to by neurons was not directly demonstrated. The presence of investigate the utilization of lipids from lipoproteins for endoneural lipoproteins in regenerating, but not non-injured, axon elongation. Lipids and proteins from human low nerves is well documented. These lipoproteins contain apoli- density lipoproteins (LDL) and high density lipopro- poprotein (apo) E and apoAI but not apoB (9 –11). After pe- teins (HDL) were taken up by distal axons and trans- ripheral nerve injury, apoE synthesis by resident macrophages ported to cell bodies, whereas cell bodies/proximal ax- increases greatly, and apoE accumulates within the nerve (12– ons internalized these components from only LDL, not 14) supporting the concept that apoE is involved in re-utiliza- HDL. Consistent with these observations, the impair- tion of lipids from degenerating nerves for axonal regeneration ment of axonal growth, induced by inhibition of choles- and myelin production. terol synthesis, was reversed when LDL or HDL were The receptors involved in lipoprotein uptake by axons and added to distal axons or when LDL, but not HDL, were Schwann cells have not been fully characterized, nor has the added to cell bodies. LDL receptors (LDLRs) and LR7/8B uptake and utilization of lipids from lipoproteins for axonal (apoER2) were present in cell bodies/proximal axons regeneration been directly demonstrated. The low density li- and distal axons, with LDLRs being more abundant in poprotein receptor (LDLR) has been reported to be used by the former. Inhibition of cholesterol biosynthesis in- PC12 cells, Schwann cells, and sensory neurons in vitro for creased LDLR expression in cell bodies/proximal axons uptake of plasma lipoproteins and endoneural lipoproteins iso- but not distal axons. LR11 (SorLA) was restricted to cell lated from crushed sciatic nerves (15, 16). In vivo, the tips of bodies/proximal axons and was undetectable in distal regenerating axons contain a high concentration of LDLRs (9), axons. Neither the LDL receptor-related protein nor the and LDLRs are distributed throughout all regions of PC12 cells HDL receptor, SR-B1, was detected in sympathetic neu- rons. These studies demonstrate for the first time that (15). Thus, the model originally proposed for re-utilization or lipids are taken up from lipoproteins by sympathetic “salvage” of cholesterol during nerve regeneration involved neurons for use in axonal regeneration. apoE-containing lipoproteins and LDLRs. More recently, other lipoprotein receptors have been de- tected in neurons. In adult rats, the LDLR-related protein Axonal elongation requires the expansion of axonal mem- (LRP), a multifunctional apoE receptor, was found to be highly branes by addition of new membrane materials (proteins and expressed in neurons of brain and spinal cord (17, 18) and was lipids) to the growing axon. In sheep (1) and rats (2, 3) the brain shown to modulate hippocampal neurite development (19). and peripheral nerves synthesize all the cholesterol needed for LRP was also detected in neuronal cell bodies and proximal development without requiring cholesterol from circulating li- processes in adult human brain (20, 21). In cultured hippocam- poproteins. Moreover, after birth, cholesterol used for myelin pal neurons, LRP is restricted to the somatodendritic domain production is made locally (4). In contrast, fetal liver supplies (22). Two other receptors of the LDLR family that are predom- about 50% of the cholesterol needed for development of heart, inantly expressed in the brain, LR11 (SorLA) (23–25) and lung, and kidney (3). Surprisingly, during peripheral nerve LR7/8B (apoER2) (26 –28), have been identified. Since LR11 regeneration, cholesterol synthesis in the nerve is down-regu- and LR7/8B are highly expressed in neurons, and LR11 expres- lated (5), yet serum-derived cholesterol does not contribute sion is regulated during central nervous system (CNS) devel- significantly to myelin synthesis or axonal regeneration (6, 7). opment (24), these receptors have been suggested to be in- volved in neural organization, synaptic formation, and neurodegenerative diseases such as Alzheimer’s disease (29). * This work was supported in part by grants from the Medical Re- search Council of Canada and the Heart and Stroke Foundation of The lipoprotein receptors present in neurons of the peripheral Alberta/Northwest Territories. The costs of publication of this article nervous system (PNS) have not yet been rigorously examined were defrayed in part by the payment of page charges. This article must although both the LDLR and LRP have been detected in rabbit therefore be hereby marked “advertisement” in accordance with 18 dorsal root ganglia neurons (30). A reasonable prediction is U.S.C. Section 1734 solely to indicate this fact. that a different spectrum of lipoprotein receptors might be § Supported by postdoctoral fellowships from the Alberta Heritage Foundation for Medical Research and the Alberta Paraplegic Founda- present in CNS and PNS neurons since the classes of lipopro- tion. Present address: Dept. of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2S2, Canada. Medical Scientists of the Alberta Heritage Foundation for Medical The abbreviations used are: apo, apolipoprotein; CNS, central nerv- Research. ous system; DiI, 1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocyanine ‡‡ To whom correspondence should be addressed: 328 Heritage Med- perchlorate; HDL, high density lipoproteins; LDL, low density lipopro- ical Research Centre, Faculty of Medicine, University of Alberta, Ed- teins; LDLR, low density lipoprotein receptor; LRP, low density lipopro- monton, Alberta T6G 2S2, Canada. Tel.: 780-492-7250; Fax: 780-492- tein receptor-related protein; PNS, peripheral nervous system; SR-B, 3383; E-mail: [email protected]. scavenger receptor class B; h, human; c, chicken. This paper is available on line at http://www.jbc.org 19883 This is an Open Access article under the CC BY license. 19884 Lipoproteins and Axonal Regeneration man HDL (hHDL ) were isolated from plasma by sequential ultracen- teins that these two types of neurons would encounter are 3 3 trifugation and affinity chromatography (36). Lipoprotein preparations different. For example, in the PNS, distal axons would be were monitored by SDS-polyacrylamide gel electrophoresis to confirm exposed to the same types of lipoproteins as in the circulation the expected apoprotein compositions and to ensure that hHDL did not (i.e. very low density lipoproteins, LDL, and HDL). In contrast, contain apoE. Chicken HDL (cHDL) was isolated by sequential ultra- apoB-containing lipoproteins (LDL and very low density li- centrifugation as the fraction of density between 1.12 and 1.21 g/ml poproteins) appear to be absent from cerebrospinal fluid. In- (41). Lipoproteins were labeled with DiI as described previously (42). stead, lipoproteins in the vicinity of CNS neurons are HDL- Briefly, 4 mg of hLDL, hHDL , or cHDL were added to 8 ml of lipopro- sized and contain apoE, apoAI, apoD, and/or apoJ (31–34). ml, 3 mg/ml) tein-deficient calf serum. DiI in dimethyl sulfoxide (200 We have previously shown that cholesterol synthesis is re- was added, and the mixture was incubated at 37 °C for 18 h. Subse- stricted to cell bodies/proximal axons of sympathetic neurons quently, the density was adjusted to 1.063 g/ml for hLDL and 1.21 g/ml and is not detectable in distal axons (35). When cholesterol for hHDL and cHDL, by addition of KBr. Lipoproteins were re-isolated synthesis was inhibited by pravastatin, axonal growth was at 4 °C by centrifugation at 150,000 3 g for 24 h in a Beckman Ti50.2 rotor. The top 2 ml, containing DiI-labeled lipoproteins, were collected severely impaired. However, normal growth was restored when by aspiration and dialyzed against 0.9% NaCl and 0.01% EDTA. Sub- cholesterol was added to either cell bodies or distal axons (36). mm filters, stored sequently, DiI-lipoproteins were filtered through 0.45- Serum lipoproteins also restored normal axonal growth to under sterile conditions at 4 °C, and used within a month. DiI-lipopro- these neurons. When LDL were given to either cell bodies or teins were subjected to SDS-polyacrylamide electrophoresis on 3–15% axons of pravastatin-treated neurons, axonal elongation pro- gradient gels. Gels were analyzed visually for DiI fluorescence and ceeded normally, presumably because the LDL supplied cho- Coomassie Blue staining for identification of major apoproteins. In hHDL, bands corresponding to apoAI and apoAII were not labeled with lesterol. In contrast, HDL restored axonal growth when added DiI; the same was true for apoAI in cHDL. For hLDL, most fluorescence only to distal axons but not to cell bodies. These observations was at the gel front as free DiI. However, some DiI fluorescence was suggest that sympathetic neurons express multiple lipoprotein associated with apoB100 since some lipids remain associated with apoB receptors and that these receptors have a polarized 2 under these conditions. distribution. Alexa 488-labeled lipoproteins were prepared according to manufac- We now show that lipids can be supplied to sympathetic turer’s instructions. Alexa Fluor 488 dye reacts with primary amines of proteins forming stable dye-protein conjugates. In HDL, Alexa 488 neurons from lipoproteins for use in axonal growth. Our data binds covalently to apoAI and apoAII (43). SDS-polyacrylamide gel are consistent with the model that the uptake of lipoprotein electrophoresis confirmed that apoB, apoAI, apoAII, and chicken apoAI components by these neurons occurs via receptor-mediated en- were labeled. All lipoprotein preparations were essentially free of un- docytosis. We show that the LDLR is present in both cell bodies bound Alexa. Recovery of Alexa fluorescence in the aqueous phase after and axons of rat sympathetic neurons, and we have identified extraction of Alexa lipoproteins with organic solvents was greater than two additional members of the LDL receptor superfamily, 95% providing evidence that the label was primarily associated with proteins. LR7/8B (apoER2) and LR11 (SorLA), in these neurons. How- Lipoproteins were labeled with [ H]cholesterol as described else- ever, neither LRP (which is present in CNS neurons) nor the mCi of [1a,2a- where (44). Briefly, the solvent from 200 H]cholesterol HDL receptor, SR-BI, was detected. In addition, the experi- was evaporated to form a thin film. The lipoprotein preparation (3 mg ments show that lipoprotein receptors are differentially distrib- of protein) was added in 0.9% NaCl, and the mixture was shaken uted and regulated in cell bodies and distal axons of sympa- overnight at 4 °C, after which lipoproteins were re-isolated by flotation thetic neurons. mg total choles- as above. The specific radioactivity of cholesterol (dpm/ terol) was determined. Cholesterol mass was determined by an enzy- EXPERIMENTAL PROCEDURES matic kit (Sigma). Typically, the specific radioactivity of cholesterol was 29,700 dpm/mg for hLDL, 150,200 dpm/mg for hHDL Materials—[1a,2a- H]Cholesterol (specific activity 45 mCi/mmol) , and 64,100 mg for cHDL. was purchased from Amersham Pharmacia Biotech. Pravastatin was a dpm/ Analysis of Lipoprotein Uptake by Fluorescence Microscopy—Neu- gift from Dr. S. Yokoyama, Nagoya City University Medical School, rons were plated in the center compartment of compartmented dishes Japan. 1,19-Dioctadecyl-3,3,39,39- tetramethylindocarbocyanine per- and cultured for 14 days. DiI or Alexa lipoproteins were added to the chlorate (DiI) and the Alexa 488 protein labeling kit were from Molec- cell body- or distal axon-containing compartments as indicated. Li- ular Probes, Inc. (Eugene, OR). L15 medium without antibiotics was poprotein concentration in media was normalized to 100 mg of total purchased from Life Technologies, Inc. Mouse 2.5 S nerve growth factor cholesterol/ml. In addition, fluorescence units were normalized to was from Alomone Laboratories Ltd. (Jerusalem, Israel). Rat serum achieve the same fluorescence in all media by addition of unlabeled was provided by the University of Alberta Laboratory Animal Services. lipoproteins. After 18 h, the neurons were washed extensively with cold The anti-rat LDLR polyclonal antibody was a gift from Dr. G. Ness, phosphate-buffered saline containing methylcellulose (3 g/500 ml) and University of South Florida (37). A polyclonal antibody directed against fixed for 20 min in 4% paraformaldehyde. Coverslips were secured the carboxyl-terminal 14 amino acids of the chicken LR7/8B (apoER2) using para-phenylenediamine in glycerol as mounting medium. Fluo- receptor (28) was generously provided by Dr. J. Nimpf, University of rescence microscopy was performed with an Olympus BX-SOF fluores- Vienna, Austria. The anti-murine SorLA (LR11) polyclonal antibody, cence microscope equipped with a 100-watt gas mercury lamp and raised against a fusion protein containing the fibronectin domain of rhodamine filter (for DiI). Alternatively, neurons were analyzed by SorLA, was a gift from Dr. H. Schaller, University of Hamburg, Ger- confocal laser scanning microscopy using a Leica microscope illumi- many (23). The anti-SR-BI antibody, directed against a peptide corre- nated by a 100-watt mercury burner for direct observation, a Ar/Kr sponding to amino acids 495–509 from murine SR-BI was kindly pro- laser with major emissions at 488, 568 and 647 nm for scanning, and vided by Dr. M. Krieger, Massachusetts Institute of Technology (38). 63 3 1.40 NA oil immersion objectives. Figs. 2, 4, and 5 were prepared The rabbit anti-LRP polyclonal antibody was generated against the using Adobe Photoshop software. 85-kDa fragment of rat LRP and was a gift from Dr. J. Herz, University Association of DiI and [1a,2a- of Texas Southwestern Medical Center (39). Electrophoresis reagents H]Cholesterol with Neurons—Neurons were supplied by Bio-Rad. Polyvinylidene difluoride membranes were were incubated with DiI lipoproteins in the cell body-containing com- from Millipore. All other reagents were from Sigma or Fisher. partment for 18 h and then washed three times with ice-cold phosphate- Preparation of Neuronal Cultures—Procedures for growing rat sym- buffered saline. Cellular material was harvested from the center com- pathetic neurons in compartmented cultures have been previously re- partment in phosphate-buffered saline. The detached neurons were ported (40). Briefly, superior cervical ganglia from newborn Harlan sonicated for 10 s with a tip probe. An aliquot was added to 2 ml of Sprague-Dawley rats were dissected and enzymatically and mechani- methanol, and fluorescence was analyzed in a Hitachi F-2000 spec- cally dissociated. The cells were plated in either the center or the left trofluorometer with excitation light of 550 nm and emission at 565 nm. compartment of three-compartmented culture dishes (36). Neurons Protein content was determined using the BCA protein kit (Pierce). DiI were cultured for 10 –14 days prior to the start of experiments. For did not interfere with the protein determinations. In experiments with measurements of axonal extension, left-plated cultures were used; all other experiments were performed using center-plated cultures. Lipoprotein Isolation and Labeling—Human LDL (hLDL) and hu- J. Vance, unpublished results. Lipoproteins and Axonal Regeneration 19885 H]cholesterol-labeled lipoproteins, neurons were given labeled li- poproteins in the cell body-containing compartment. The concentration of lipoproteins was adjusted so that the cholesterol content was 100 mg/ml, and the same specific radioactivity of cholesterol was achieved for all lipoproteins by addition of unlabeled lipoproteins. After 18 h, the cells were washed, and material from the center compartment was harvested, and radioactivity was measured. The amount of cell-associ- ated lipoprotein-derived cholesterol was calculated from the specific radioactivity of the lipoprotein [ H]cholesterol. Measurement of Axonal Extension—Neurons were plated in the left compartment of compartmented dishes. Distal axons were mechani- cally removed from the right-hand compartment with a jet of sterile distilled water delivered with a syringe through a 22-gauge needle. The water was aspirated and the wash repeated twice, after which fresh FIG.1. Utilization of lipoproteins for axonal elongation. Neu- medium was added. This procedure, termed axotomy, effectively re- rons were cultured in the left-hand compartment of compartmented moves all visible traces of axons from the right-side compartment. dishes. Distal axons were removed from the right-hand compartment, Axonal growth was measured as described previously (45). and the cell body-containing compartment was supplied with 50 mM Analysis of Lipoprotein Receptors by Immunoblotting—Material from pravastatin, as well as hLDL, hHDL , or cHDL (100 mg of cholesterol/ ml). After 4 days, axonal extension was measured in three cultures (a distal axon- and cell body-containing compartments was harvested total of 45– 48 tracks) for each treatment. Data are means 6 S.E. separately in 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 1 mM Statistical significance of differences between untreated and treated EDTA, 0.05 mM phenylmethylsulfonyl fluoride. Cellular material was neurons is indicated by * and was evaluated by the Student’s t test (p # sonicated for 10 s with a probe sonicator and centrifuged at 350,000 3 0.05). The experiment was repeated three times using different lipopro- g for 20 min. Membrane pellets were dissolved in 50 mM Tris-HCl (pH tein preparations with similar results. 7.5) containing 150 mM NaCl, 1 mM EDTA, 0.05 mM phenylmethylsul- fonyl fluoride. The protein content was determined using the BCA protein kit (Pierce). Proteins were separated by electrophoresis on an cholesterol synthesis was inhibited by addition of 50 mM 8% SDS-polyacrylamide gel for LDLR and SRBI, a 5% gel for LR11 and pravastatin to the cell body-containing compartment. This con- LR7/8B, and a 3–15% gradient gel for LRP. For LR7/8B, electrophoresis centration of pravastatin inhibits the incorporation of [ C]ac- was performed under non-reducing conditions (28). Proteins were transferred to polyvinylidene difluoride membranes for 18 h at 25 V etate into cholesterol by ;80% (50). The concentration of each (36). Membranes were incubated in 20 mM Tris-HCl (pH 7.5), 500 mM lipoprotein preparation added to cell bodies was equalized in NaCl, 0.1% (v/v) Tween 20 (TTBS) with 5–10% (w/v) non-fat dried milk terms of cholesterol content (100 mg/ml). Axonal extension was for at least 2 h and then incubated for1hat room temperature with measured after 4 days (Fig. 1). In agreement with our previous primary antibody in TTBS containing 1% non-fat dried milk using the results (36), axonal extension was reduced by ;50% in pravas- following dilutions of primary antibodies: LDLR, 1:1000; LRP, 1:5000; tatin-treated cells compared with untreated cells, whereas the SR-BI, 1:1000; LR11, 1:1500; and LR7/8B, 1:1500. Membranes were addition of hLDL, but not hHDL then incubated for 1 h with horseradish peroxidase linked to anti-rabbit , to cell bodies/proximal axons IgG secondary antibody (Pierce, diluted 1:10,000), and immunoreactive restored axonal elongation. In addition, Fig. 1 shows that axons proteins were detected by enhanced chemiluminescence. extended normally when cHDL were added to cell bodies/prox- imal axons of pravastatin-treated neurons. These data suggest RESULTS that hHDL cannot provide cholesterol to cell bodies for axonal Lipoproteins and Axonal Growth—We have previously growth, possibly because cell bodies lack a receptor for hHDL . shown that inhibition of cholesterol synthesis by pravastatin In accordance with our previous observations (36), when any of impairs axonal elongation of compartmented cultures of rat the lipoproteins was given to distal axons alone, the axons sympathetic neurons. Addition of cholesterol to axons or cell extended normally (data not shown). bodies restored normal axonal growth to pravastatin-treated Uptake of Lipids and Proteins from Lipoproteins—We inves- neurons. Similarly, when human lipoproteins (hLDL, hHDL , tigated whether or not lipids and proteins from lipoproteins or hHDL ) were supplied to distal axons of pravastatin-treated were taken up by cell bodies of sympathetic neurons. hLDL, neurons, normal axonal growth occurred. In contrast, normal cHDL, and hHDL were labeled with DiI, a hydrophobic fluo- axonal elongation occurred only when hLDL, but not hHDL or rescent dye that intercalates into the neutral lipid core of hHDL , were added to the cell body-containing compartment lipoproteins without affecting receptor binding (51). Rat sym- (36). These studies suggested that cholesterol can be taken up pathetic neurons were maintained for 14 days and then incu- from lipoproteins by these neurons and used for axonal growth. bated with DiI lipoproteins in the center compartment contain- The observations also raised the possibility that LDL and HDL, ing the cell bodies. The concentration of lipoproteins in the or the cholesterol therein, are taken up by different mecha- media was adjusted so that the total cholesterol concentration nisms, potentially involving distinct lipoprotein receptors. (100 mg/ml medium) and specific fluorescence (fluorescence We have now extended these studies and analyzed the ca- intensity units/ml of medium) were the same for all lipopro- pacity of hLDL, hHDL , and chicken HDL (cHDL) to deliver teins. After 18 h, the neurons were extensively washed and lipids, including cholesterol, for axonal growth of rat sympa- examined by fluorescence microscopy. Fig. 2, a, c, and e, shows thetic neurons. hHDL lacks apoE and therefore would not be the bright field images of cell bodies/proximal axons of neurons expected to interact with the apoB/apoE receptor (i.e. the given hLDL, cHDL, and hHDL , respectively. Fig. 2, b, d, and LDLR). cHDL was selected because chicken apoAI has been f, shows the corresponding fluorescence images. Cell bodies of proposed to play a role in nerve regeneration in chickens sim- neurons given DiI-hLDL or DiI-cHDL exhibited intense fluo- ilar to that of apoE in mammals (46 – 48). Consequently, we rescence (Figs. 2, b and d), whereas almost no fluorescence was expected cHDL and hLDL to be similarly taken up by rat visible in cell bodies of neurons given hHDL (Fig. 2f). For sympathetic neurons. For these experiments, rat sympathetic quantification of the fluorescence associated with cell bodies, neurons were plated in the left side compartment of three- cellular material was harvested from the center compartment, compartmented culture dishes. In these cultures (49), all com- and DiI fluorescence intensity was measured. Neurons treated partments contain independent fluid environments, and the with hLDL and cHDL contained 4- and 3-fold, respectively, cell bodies/proximal axons reside in a compartment completely more fluorescence than did neurons given hHDL (Fig. 3a). separate from that containing distal axons. After 14 days, Assuming that DiI is a valid marker for uptake of neutral lipids axons in the right side compartment were axotomized, and from lipoproteins (52), these data suggest that cell bodies/ 19886 Lipoproteins and Axonal Regeneration ies, since the only way in which components of lipoproteins that have been added to distal axons can enter the cell bodies is from retrograde, intracellular transport. Thus, the appearance of fluorescence in cell bodies, after addition of lipoproteins con- taining fluorescent lipids and proteins to distal axons, would unambiguously demonstrate that lipids and proteins, respec- tively, had been taken up from lipoproteins by distal axons and transported to cell bodies. For these experiments, lipoproteins were labeled with either DiI or Alexa 488 (a fluorescent label of proteins) and added to either distal axons or cell bodies/proxi- mal axons. Lipoprotein concentrations were adjusted in terms of total cholesterol content (100 mg/ml medium) and specific fluorescence. The protein components of the lipoproteins did not bind significant amounts of DiI, and conversely, Alexa 488 associated almost exclusively with proteins of the lipoproteins (see “Experimental Procedures”). After 18 h, cell bodies were examined for fluorescence. All images were analyzed on a plane where nuclei were visible to ensure that the fluorescent li- FIG.2. Labeling of sympathetic neurons with DiI lipoproteins. poprotein did not only adhere to the cell surface but was truly Neurons were plated in the center compartment of compartmented intracellular. dishes. After 14 days, DiI lipoproteins (hLDL, cHDL, or hHDL ) were When cell bodies/proximal axons were incubated with fluo- added to the cell body-containing compartment. Lipoprotein concentra- rescent hLDL or cHDL, intense DiI (Fig. 4, B and D) and Alexa tion was adjusted to 100 mg of cholesterol/ml and 136,000 fluorescence intensity units/ml. After 18 h, neurons were extensively washed, fixed, (Fig. 4, H and J) fluorescence, likely representing aggregates of and examined by fluorescence microscopy. Left panels (a, c, and e) are lipoproteins, was observed inside the cell bodies. In contrast, bright field images, and right panels (b, d, and f) are fluorescence when either DiI- or Alexa-hHDL was given to the cell body- images of cell bodies of neurons given hLDL (a and b), cHDL (c and d), containing compartment, little fluorescence was visible in cell and hHDL (e and f). The horizontal “rope-like” lines shown in the bright field images are scratches in the collagen surface of the culture dish and bodies (Fig. 4, F and L). The ring of fluorescence in Fig. 4L create tracks along which the axons extend (*). Proximal neurites and probably represents Alexa lipoproteins remaining on the cell clusters of cell bodies are indicated with arrowheads and arrows, re- surface after the extensive washing procedure. Even when the spectively. Images are representative of 50 fields analyzed per treat- experiments were repeated using a 10-fold higher concentra- ment. The experiment was repeated three times with equivalent results. tion of DiI- or Alexa-hHDL , the fluorescence inside the cell bodies remained at basal levels. These experiments show that lipids and proteins from hLDL and cHDL, but not hHDL , are proximal axons internalize lipids from hLDL and cHDL but not internalized by cell bodies/proximal axons. hHDL . In parallel experiments, neurons that were given DiI-hLDL We next measured the uptake of [ H]cholesterol from li- (Fig. 4A), DiI-cHDL (Fig. 4C), or DiI-hHDL (Fig. 4E) to distal poproteins by cell bodies. Neurons were plated in the center axons exhibited profuse punctate fluorescence in the cell bod- compartment and maintained for 14 days, after which cell 3 ies, suggesting that lipids from all the lipoproteins were taken bodies/proximal axons were incubated with [ H]cholesterol-la- up by distal axons and transported to cell bodies. These results beled lipoproteins for 18 h. The cells were then washed and are in accordance with our observations that all these lipopro- harvested, and radioactivity was determined. Neurons incu- teins, given to distal axons, can overcome the inhibition of bated with hLDL and cHDL contained approximately 70% axonal growth induced by pravastatin (36). The uptake of more radioactivity than did neurons incubated with hHDL Alexa-labeled hLDL, cHDL, and hHDL by distal axons was (Fig. 3b). Although Fig. 3, a and b, indicates that the associa- similarly examined. Fig. 4, G, I, and K, shows that each of the tion of lipids from hLDL and cHDL by cell bodies is greater 3 Alexa lipoproteins was taken up by distal axons and trans- than from hHDL , relatively more [ H]cholesterol-labeled ported to cell bodies. These data demonstrate that lipids and hHDL than DiI-labeled hHDL associated with the neurons. 3 3 proteins from hLDL, cHDL, and hHDL can be internalized by One likely explanation for this difference is that in addition to distal axons and transported to cell bodies. In addition, lipids hHDL specifically binding to the cell surface, [ H]cholesterol and proteins from hLDL and cHDL, but not hHDL , were on the HDL surface might exchange with cell membranes via internalized by cell bodies/proximal axons. Our findings also aqueous diffusion. In contrast, DiI is present in the neutral confirm that the various lipoprotein classes are differentially lipid core of HDL and would, therefore, not exchange via taken up by cell bodies and distal axons of sympathetic neu- aqueous diffusion on to the cell surface but would associate rons. Previous studies have reported that in several cell types with cells only via a specific receptor-mediated interaction. a selective uptake of lipids, particularly cholesteryl esters, oc- The experiments described above suggest that lipids from curs from HDL (43), whereas the uptake of HDL holoparticles hLDL and cHDL, but not hHDL , are taken up by the neuronal has been less well documented (53, 54). Therefore, our obser- cell bodies. Since in our experience these neurons are particu- vation that distal axons of sympathetic neurons were able to larly sensitive to cold temperatures, we were unable to perform internalize both lipids and proteins from hHDL was somewhat classical lipoprotein binding experiments at 4 °C to differenti- ate between uptake and nonspecific binding of lipoproteins. To unexpected. We next investigated whether or not uptake of hLDL and exclude the possibility that the observations described above could be explained solely by the lipoproteins binding to the cell hHDL occurred via receptor-mediated processes. Neurons were given DiI- or Alexa-labeled lipoproteins to either distal surface, without their internalization, we used confocal laser scanning microscopy to examine more directly fluorescent li- axons or cell bodies/proximal axons. Some cultures were given poprotein uptake. By using this technique we were also able to a 50-fold excess of unlabeled lipoproteins for competition of determine whether or not distal axons can take up lipids and uptake. Unlabeled hLDL competed effectively for uptake of proteins from lipoproteins and transport them to the cell bod- fluorescent hLDL by cell bodies (compare Fig. 5, A with D, and Lipoproteins and Axonal Regeneration 19887 FIG.3. Quantitation of fluorescent and radiolabeled lipids associated with cell bodies. a, neurons were incubated with DiI-lipoproteins as in Fig. 2 legend. After extensive washing, material from the center compartment was harvested and DiI fluorescence measured. b, neurons cultured as in Fig. 2 were incubated with [ H]cholesterol-lipoproteins for 18 h, then washed, harvested and the amount of cell-associated, lipoprotein-derived cholesterol was calculated from the specific radioactivity of [ H]cholesterol in the lipoprotein. Values are means 6 S.E. of six determinations for each treatment. Statistically significant differences compared with treatment with hLDL are indicated by * and were evaluated by the Student’s t test (p # 0.05). The experiment was repeated twice with similar results. FIG.4. Uptake of fluorescent lipids and proteins from lipoproteins. Cen- ter-plated neurons were incubated with DiI-or Alexa-lipoproteins in either the cell body-containing compartment (B, D, F, H, J, and L) or distal axon-containing com- partment (A, C, E, G, I, and K). Lipopro- tein concentration was adjusted to 100 mg of cholesterol/ml and 136,000 fluorescence intensity units/ml for DiI lipoproteins or 180,000 fluorescence intensity units/ml for Alexa lipoproteins. After 18 h, neurons were processed for confocal microscopy. The images shown for each group (DiI and Alexa) were captured under identical con- ditions, and 50 –100 fields were analyzed for each treatment. The experiment was repeated twice with comparable results. FIG.5. Competition of fluorescent lipoprotein uptake by unlabeled li- poproteins. Neurons were incubated in either the cell body-containing (A, D, G, and J) or distal axon-containing (B, C, E, F, H, I, K, and L) compartment with flu- orescent lipoproteins, as in Fig. 4 legend. Some cultures were given a 50-fold excess of unlabeled lipoproteins. Cell bodies were visualized by confocal microscopy, and the images shown for each group (DiI and Alexa) were captured under identical conditions. The experiment was repeated twice with similar results. Fig. 5, G with J) and distal axons (compare Fig. 5, B with E, “mass cultures”) and compartmented dishes. Cellular material and H with K). Similarly, excess unlabeled hHDL blocked the was harvested, a membrane-enriched fraction was prepared, uptake of fluorescent hHDL by distal axons (compare Fig. 5, C and membrane proteins were separated by SDS-polyacryl- with F, and I with L), supporting the idea that both hLDL and amide gel electrophoresis, after which immunoblotting was hHDL are internalized by receptor-mediated mechanisms. performed. Proteins from homogenates of rat liver and rat Lipoprotein Receptors in Sympathetic Neurons—The distri- brain were used as controls. Fig. 6a shows that the LDLR (M bution of lipoprotein receptors in cell bodies and distal axons of ;130) is widely distributed throughout cell bodies and axons of sympathetic neurons was investigated by immunoblot analy- sympathetic neurons but is more abundant in cell bodies/prox- sis. Neurons were cultured both in 24-well dishes (designated imal axons than in distal axons. As a positive control, the 19888 Lipoproteins and Axonal Regeneration FIG.7. Expression of LR11 and LR7/8B in sympathetic neu- rons. Neurons were cultured in three-compartmented dishes. Mem- brane proteins (30 mg) from neurons, rat brain, and rat liver were separated by SDS-polyacrylamide electrophoresis and analyzed by im- munoblotting using antibodies directed against LR11 or LR7/8B. imal axons and distal axons, as well as in rat brain but not in FIG.6. Distribution of LDLR, LRP, and SR-BI in sympathetic rat liver. neurons. a, neurons were cultured in 24-well dishes and in compart- mented dishes. Cellular material from 24-well dishes (Mass), the cell DISCUSSION body-containing compartment (CB), and distal axon-containing com- Our previous experiments indicated that lipoproteins given partments (AX), was harvested. A membrane-enriched fraction was prepared, and membrane proteins (20 mg) were separated by SDS- to distal axons of rat sympathetic neurons can provide choles- polyacrylamide gel electrophoresis and immunoblotted using antibodies terol, but not phospholipids, for axonal regeneration (36). We directed against one of the LDLR, the 85-kDa subunit of LRP, or SR-BI. now show for the first time that distal axons internalize pro- Proteins (20 mg) from homogenates of rat liver and rat brain were used teins and lipids from lipoproteins and transport these compo- as controls. * indicates that membranes from rat brain and neuronal mass cultures were mixed (see text). b, compartmented cultures of nents retrogradely to cell bodies. In contrast, cell bodies/prox- neurons were incubated with (1) or without (2)50 mM pravastatin imal axons take up lipids and proteins from LDL but not HDL . (Prav) for 48 h. Cellular material was harvested, and the presence of the The results are consistent with the idea that both lipid and LDLR (LDLr) analyzed by immunoblotting. This experiment was per- protein components of lipoproteins are taken up by axons via formed four times with independent preparations of neurons. In addi- tion, two different amounts of axonal proteins (20 and 60 mg) were receptor-mediated endocytosis and transported to cell bodies, applied to the gel. The same result was obtained for each experiment. the site of lysosomal processing. However, the possibility exists that some components (e.g. cholesteryl esters and/or choles- LDLR was also detected in rat liver and rat brain. In contrast, terol) remain in axons and are directly used for growth. the 85-kDa fragment of LRP was not detected in any prepara- The LDL Receptor in Sympathetic Neurons—Lipoprotein re- tion of sympathetic neurons. However, this receptor was pres- ceptors are known to play an important role in cellular choles- ent in rat liver and rat brain, which were used as positive terol homeostasis (55), and our experiments indicate that li- controls. We were surprised that we were unable to detect LRP poprotein uptake by sympathetic neurons is receptor-mediated. in sympathetic neurons since LRP has been reported to be Many of these receptors belong to the LDLR superfamily of abundant in CNS neurons (20) and brain (18, 21). We consid- which the LDLR, which has been detected in PNS and CNS ered the possibility that our inability to detect LRP was due to neurons (9, 16, 30, 57), is the prototype. Our data show that the a factor in the neuron samples that interfered with immuno- LDLR is present in cell bodies/proximal axons and distal axons detection of LRP. This possibility was eliminated when a rat of rat sympathetic neurons, with an apparent enrichment in brain homogenate was mixed with membranes from neuronal cell bodies. It is likely, therefore, that in our studies the inter- mass cultures (shown as Rat Brain* in Fig. 6a), and the inten- nalization of hLDL was mediated primarily by the LDLR. How- sity of the band representing brain LRP was not diminished. ever, since the LDLR only binds lipoproteins that contain apoB Fig. 6 also shows that the HDL receptor, SR-BI, is present in and/or apoE (55), this receptor probably does not participate in rat liver but not brain, in agreement with the distribution of the uptake of hHDL , which contain neither apoB nor apoE. SR-BI mRNA (38). No immunoreactive SR-BI was detected in A spatially distinct expression of the LDLR has been ob- sympathetic neurons. served in other polarized cells. For example, in Madin-Darby In many cell types, expression of the LDLR is regulated by canine kidney cells expression of recombinant LDLRs resulted cellular cholesterol content (55). We therefore investigated in their preferential targeting to basolateral membranes (58). whether or not inhibition of cholesterol biosynthesis increased In addition, using adenovirus expression the LDLR was highly expression of the LDLR in cell bodies/proximal axons and distal expressed in the somatodendritic region of hippocampal neu- axons of sympathetic neurons. Compartmented neuron cul- rons but was undetectable in distal axons. In contrast, mutant tures were treated with 50 mM pravastatin in the cell body- LDLRs lacking a putative basolateral targeting sequence were containing compartment for 48 h after which cellular material most highly concentrated in growth cones and distal axons (59). was isolated. Immunoblotting of proteins with anti-LDLR an- The distribution of endogenous LDLRs was not examined in tibody revealed that pravastatin treatment markedly up-regu- this report. However, Li et al. (60) reported the presence of a lated LDLR expression in cell bodies but not in distal axons cell surface receptor that bound LDL with the characteristics of (Fig. 6b). the LDL receptor in both apical and basolateral domains of The presence of two other receptors of the LDL receptor Madin-Darby canine kidney cells. Studies on the sorting of superfamily that are abundant in brain, LR7/8B (also known as viral proteins (61) and endogenously expressed proteins (62, apoER2) (27, 28) and LR11 (also known as SorLA) (23, 25, 26, 63) in hippocampal neurons and Madin-Darby canine kidney 56), was also examined by immunoblotting. Fig. 7 shows the cells have suggested that neurons and polarized epithelial cells presence of LR11 (M ;240) in rat brain and cell bodies/prox- share common mechanisms of protein targeting and sorting, imal axons but the absence of LR11 from distal axons and rat with the axonal membrane being equivalent to the apical do- liver. LR7/8B (M ;130) was detected in both cell bodies/prox- main of epithelial cells and the somatodendritic region being r Lipoproteins and Axonal Regeneration 19889 equivalent to the basolateral domain. Our data are, in general, creases dramatically after peripheral nerve injury (46). Our consistent with this hypothesis. data show that cHDL are internalized by sympathetic neurons Expression of the LDLR in many cell types is regulated by in a manner analogous to that of hLDL but unlike that of cellular cholesterol levels (55); when cholesterol synthesis is hHDL . Uptake of HDL by Distal Axons—The finding that HDL are inhibited, expression of LDLRs increases. In the present study, 3 3 we inhibited cholesterol synthesis in sympathetic neurons and internalized by distal axons is intriguing although the distri- bution of lipoprotein receptors that we report here does not found that the amount of the LDLR in cell bodies/proximal explain how this uptake occurs. Presumably, other receptors axons, but not in distal axons, increased. Spatially independent are present that mediate the uptake of hHDL by distal axons. regulation of expression of the LDLR has been previously ob- Since cell bodies did not internalize either lipids or proteins of served in other polarized cells. For example, the number of hHDL , our data imply that cell bodies lack a functional hHDL LDLRs is regulated by cellular cholesterol content on only 3 3 receptor. The best characterized HDL receptor is SR-BI (72). basolateral, but not apical, membranes of Madin-Darby canine SR-BI binds HDL via apoAI (73) and mediates a selective kidney cells (60). One possible explanation for the lack of up- uptake of lipids, particularly cholesteryl esters, from HDL (52, regulation of expression of the LDLR in distal axons in re- 74 –76) and LDL (77, 78), without a concomitant uptake of sponse to pravastatin is that the reduced cholesterol content apoproteins. In rat adrenal cells, the selective uptake of cho- might impair anterograde vesicular transport that would pre- lesteryl esters occurs by a mechanism distinct from the classi- vent LDLRs, which are synthesized in the cell bodies, from cal endosomal/lysosomal pathway (79). Similarly, in rat hepa- being exported to distal axons. toma cells HDL cholesteryl esters are hydrolyzed extra- Other Lipoprotein Receptors in Sympathetic Neurons—Sev- lysosomally (80). SR-BI is abundantly expressed in liver and eral members of the LDLR superfamily have been proposed to steroidogenic tissues but has not been found in brain (81), and be involved in neuronal metabolism and pathogenesis of neu- we were unable to detect SR-BI in sympathetic neurons. In rodegenerative diseases (29). For example, LRP, a multifunc- addition to selective lipid uptake from HDL, the endocytosis tional receptor that mediates the endocytosis of several ligands and lysosomal degradation of HDL holoparticles have previ- (64), is expressed in brain and spinal cord (18), as well as in the ously been observed (53, 54). In our experiments, not only lipids somatodendritic domain of cultured hippocampal neurons (22). but also proteins of hHDL were internalized by axons and The LRP and one of its ligands, apoE, have been reported to 3 transported to cell bodies, indicating holoparticle uptake. How- play an important role in early hippocampal development (19). ever, our data are also consistent with the possibility that Moreover, apoE synthesis dramatically increases in the CNS distal axons participate in a selective uptake of some lipids and PNS after nerve injury (10, 12, 46), and apoE-containing from HDL, in addition to holoparticle uptake. lipoproteins have been proposed to play a role in peripheral Since hHDL lack apoE they are unlikely to be internalized nerve regeneration (8) and dendritic remodeling in the CNS by the LDLR although receptors participating in the uptake of (65). Consequently, we were surprised that we could detect no intact HDL particles have not yet been clearly defined. Re- LRP in sympathetic neurons. Our result is, however, consistent cently, cubilin has been identified as a receptor that mediates with the idea that CNS and PNS neurons might express dis- the endocytosis/lysosomal degradation of HDL (82, 83). Since tinct populations of lipoprotein receptors because the types of cubilin lacks a membrane-spanning domain, it is considered to lipoproteins to which they would be exposed in their native be a peripheral membrane protein (84) that requires a co- environments are different. receptor, probably megalin, to mediate endocytosis (82). Mega- LR11 is a recently discovered member of the LDLR super- lin (also known as gp330 or LRP2) is a member of the LDLR family (23–25). In mammals, LR11 is abundant in the brain family and binds multiple ligands including apoE and apoJ and binds apoE-containing lipoproteins (26). This receptor has (reviewed in Ref. 64). This finding is of particular interest in been suggested to play a role in lipoprotein metabolism in the relation to cholesterol homeostasis in the brain and during CNS (25). We found that LR11 is present in cell bodies/proxi- PNS nerve regeneration since lipoproteins involved in those mal axons of sympathetic neurons but, interestingly, not in processes are thought to contain apoE and/or apoJ (32). Cubilin distal axons. is localized to apical membranes of polarized endothelial cells Our experiments also show that LR7/8B, for which apoE is a of the kidney (85), and megalin is also expressed on the apical ligand, is expressed throughout cell bodies and axons of sym- surfaces of many epithelial cells (86, 87). Therefore, since neu- pathetic neurons. cDNAs encoding this receptor have been rons are polarized cells in which the distal axons are, in some cloned from brain of human, mouse, and chicken (27, 28). ways, equivalent to the apical domain of other polarized cells Interestingly, in chickens, which lack apoE, LR11 and LR7/8B (62, 63), one might predict that if megalin and cubilin were are abundant in the brain. The importance of LR7/8B in CNS present they would be concentrated in distal axons. If this were development is underscored by the finding that mice lacking the case, megalin and cubilin would be viable candidates for both LR7/8B and the very low density lipoprotein receptor mediating HDL uptake by distal axons. Whether or not cubilin (another LDLR family member) (66) display an inversion of and megalin are present in distal axons, and absent from cell cortical layers during development (67, 68). Some receptors of bodies, of sympathetic neurons warrants further investigation. the LDLR superfamily have recently been implicated in intra- In summary, we have shown that cultured rat sympathetic cellular signaling events because their cytosolic domains inter- neurons internalize lipids and proteins of lipoproteins by re- act with signaling molecules such as mDab1 (68). Whether or ceptor-mediated mechanisms. Although LRP was absent from not these receptors function in vivo for endocytosis of lipopro- these neurons, related receptors such as the LDLR, LR7/8B, teins and/or as signaling receptors is not yet known. and LR11 were detected. These receptors were differentially Uptake of Chicken HDL—cHDL were used as model lipopro- distributed, and expression of the LDLR was differentially teins because they exhibit some similarities to mammalian regulated in cell bodies and distal axons of sympathetic neu- LDL. Although chickens lack apoE, their HDL contain apoAI rons. Our studies also show that distal axons, but not cell that has been proposed to be a “surrogate” for mammalian bodies, of sympathetic neurons have the capacity to internalize apoE (47, 69, 70). 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Uptake of Lipoproteins for Axonal Growth of Sympathetic Neurons

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 26, Issue of June 30, pp. 19883–19890, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Received for publication, January 12, 2000, and in revised form, February 18, 2000) Elena I. Posse de Chaves‡§, Dennis E. Vance¶i, Robert B. Campenoti**, Robert S. Kiss¶, and Jean E. Vance‡ ‡‡ From the Departments of ¶Biochemistry, **Cell Biology, and ‡Medicine, University of Alberta, Edmonton, Alberta T6G 2S2, Canada Lipoproteins originating from axon and myelin break- Instead, cholesterol from degenerating axons and myelin is proposed to be retained within the nerve and re-utilized via a down in injured peripheral nerves are believed to sup- ply cholesterol to regenerating axons. We have used lipoprotein-mediated process (8), although cholesterol uptake compartmented cultures of rat sympathetic neurons to by neurons was not directly demonstrated. The presence of investigate the utilization of lipids from lipoproteins for endoneural lipoproteins in regenerating, but not non-injured, axon elongation. Lipids and proteins from human low nerves is well documented. These lipoproteins contain apoli- density lipoproteins (LDL) and high density lipopro- poprotein (apo) E and apoAI but not apoB (9 –11). After pe- teins (HDL) were taken up by distal axons and trans- ripheral nerve injury, apoE synthesis by resident macrophages ported to cell bodies, whereas cell bodies/proximal ax- increases greatly, and apoE accumulates within the nerve (12– ons internalized these components from only LDL, not 14) supporting the concept that apoE is involved in re-utiliza- HDL. Consistent with these observations, the impair- tion of lipids from degenerating nerves for axonal regeneration ment of axonal growth, induced by inhibition of choles- and myelin production. terol synthesis, was reversed when LDL or HDL were The receptors involved in lipoprotein uptake by axons and added to distal axons or when LDL, but not HDL, were Schwann cells have not been fully characterized, nor has the added to cell bodies. LDL receptors (LDLRs) and LR7/8B uptake and utilization of lipids from lipoproteins for axonal (apoER2) were present in cell bodies/proximal axons regeneration been directly demonstrated. The low density li- and distal axons, with LDLRs being more abundant in poprotein receptor (LDLR) has been reported to be used by the former. Inhibition of cholesterol biosynthesis in- PC12 cells, Schwann cells, and sensory neurons in vitro for creased LDLR expression in cell bodies/proximal axons uptake of plasma lipoproteins and endoneural lipoproteins iso- but not distal axons. LR11 (SorLA) was restricted to cell lated from crushed sciatic nerves (15, 16). In vivo, the tips of bodies/proximal axons and was undetectable in distal regenerating axons contain a high concentration of LDLRs (9), axons. Neither the LDL receptor-related protein nor the and LDLRs are distributed throughout all regions of PC12 cells HDL receptor, SR-B1, was detected in sympathetic neu- rons. These studies demonstrate for the first time that (15). Thus, the model originally proposed for re-utilization or lipids are taken up from lipoproteins by sympathetic “salvage” of cholesterol during nerve regeneration involved neurons for use in axonal regeneration. apoE-containing lipoproteins and LDLRs. More recently, other lipoprotein receptors have been de- tected in neurons. In adult rats, the LDLR-related protein Axonal elongation requires the expansion of axonal mem- (LRP), a multifunctional apoE receptor, was found to be highly branes by addition of new membrane materials (proteins and expressed in neurons of brain and spinal cord (17, 18) and was lipids) to the growing axon. In sheep (1) and rats (2, 3) the brain shown to modulate hippocampal neurite development (19). and peripheral nerves synthesize all the cholesterol needed for LRP was also detected in neuronal cell bodies and proximal development without requiring cholesterol from circulating li- processes in adult human brain (20, 21). In cultured hippocam- poproteins. Moreover, after birth, cholesterol used for myelin pal neurons, LRP is restricted to the somatodendritic domain production is made locally (4). In contrast, fetal liver supplies (22). Two other receptors of the LDLR family that are predom- about 50% of the cholesterol needed for development of heart, inantly expressed in the brain, LR11 (SorLA) (23–25) and lung, and kidney (3). Surprisingly, during peripheral nerve LR7/8B (apoER2) (26 –28), have been identified. Since LR11 regeneration, cholesterol synthesis in the nerve is down-regu- and LR7/8B are highly expressed in neurons, and LR11 expres- lated (5), yet serum-derived cholesterol does not contribute sion is regulated during central nervous system (CNS) devel- significantly to myelin synthesis or axonal regeneration (6, 7). opment (24), these receptors have been suggested to be in- volved in neural organization, synaptic formation, and neurodegenerative diseases such as Alzheimer’s disease (29). * This work was supported in part by grants from the Medical Re- search Council of Canada and the Heart and Stroke Foundation of The lipoprotein receptors present in neurons of the peripheral Alberta/Northwest Territories. The costs of publication of this article nervous system (PNS) have not yet been rigorously examined were defrayed in part by the payment of page charges. This article must although both the LDLR and LRP have been detected in rabbit therefore be hereby marked “advertisement” in accordance with 18 dorsal root ganglia neurons (30). A reasonable prediction is U.S.C. Section 1734 solely to indicate this fact. that a different spectrum of lipoprotein receptors might be § Supported by postdoctoral fellowships from the Alberta Heritage Foundation for Medical Research and the Alberta Paraplegic Founda- present in CNS and PNS neurons since the classes of lipopro- tion. Present address: Dept. of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB T6G 2S2, Canada. Medical Scientists of the Alberta Heritage Foundation for Medical The abbreviations used are: apo, apolipoprotein; CNS, central nerv- Research. ous system; DiI, 1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocyanine ‡‡ To whom correspondence should be addressed: 328 Heritage Med- perchlorate; HDL, high density lipoproteins; LDL, low density lipopro- ical Research Centre, Faculty of Medicine, University of Alberta, Ed- teins; LDLR, low density lipoprotein receptor; LRP, low density lipopro- monton, Alberta T6G 2S2, Canada. Tel.: 780-492-7250; Fax: 780-492- tein receptor-related protein; PNS, peripheral nervous system; SR-B, 3383; E-mail: [email protected]. scavenger receptor class B; h, human; c, chicken. This paper is available on line at http://www.jbc.org 19883 This is an Open Access article under the CC BY license. 19884 Lipoproteins and Axonal Regeneration man HDL (hHDL ) were isolated from plasma by sequential ultracen- teins that these two types of neurons would encounter are 3 3 trifugation and affinity chromatography (36). Lipoprotein preparations different. For example, in the PNS, distal axons would be were monitored by SDS-polyacrylamide gel electrophoresis to confirm exposed to the same types of lipoproteins as in the circulation the expected apoprotein compositions and to ensure that hHDL did not (i.e. very low density lipoproteins, LDL, and HDL). In contrast, contain apoE. Chicken HDL (cHDL) was isolated by sequential ultra- apoB-containing lipoproteins (LDL and very low density li- centrifugation as the fraction of density between 1.12 and 1.21 g/ml poproteins) appear to be absent from cerebrospinal fluid. In- (41). Lipoproteins were labeled with DiI as described previously (42). stead, lipoproteins in the vicinity of CNS neurons are HDL- Briefly, 4 mg of hLDL, hHDL , or cHDL were added to 8 ml of lipopro- sized and contain apoE, apoAI, apoD, and/or apoJ (31–34). ml, 3 mg/ml) tein-deficient calf serum. DiI in dimethyl sulfoxide (200 We have previously shown that cholesterol synthesis is re- was added, and the mixture was incubated at 37 °C for 18 h. Subse- stricted to cell bodies/proximal axons of sympathetic neurons quently, the density was adjusted to 1.063 g/ml for hLDL and 1.21 g/ml and is not detectable in distal axons (35). When cholesterol for hHDL and cHDL, by addition of KBr. Lipoproteins were re-isolated synthesis was inhibited by pravastatin, axonal growth was at 4 °C by centrifugation at 150,000 3 g for 24 h in a Beckman Ti50.2 rotor. The top 2 ml, containing DiI-labeled lipoproteins, were collected severely impaired. However, normal growth was restored when by aspiration and dialyzed against 0.9% NaCl and 0.01% EDTA. Sub- cholesterol was added to either cell bodies or distal axons (36). mm filters, stored sequently, DiI-lipoproteins were filtered through 0.45- Serum lipoproteins also restored normal axonal growth to under sterile conditions at 4 °C, and used within a month. DiI-lipopro- these neurons. When LDL were given to either cell bodies or teins were subjected to SDS-polyacrylamide electrophoresis on 3–15% axons of pravastatin-treated neurons, axonal elongation pro- gradient gels. Gels were analyzed visually for DiI fluorescence and ceeded normally, presumably because the LDL supplied cho- Coomassie Blue staining for identification of major apoproteins. In hHDL, bands corresponding to apoAI and apoAII were not labeled with lesterol. In contrast, HDL restored axonal growth when added DiI; the same was true for apoAI in cHDL. For hLDL, most fluorescence only to distal axons but not to cell bodies. These observations was at the gel front as free DiI. However, some DiI fluorescence was suggest that sympathetic neurons express multiple lipoprotein associated with apoB100 since some lipids remain associated with apoB receptors and that these receptors have a polarized 2 under these conditions. distribution. Alexa 488-labeled lipoproteins were prepared according to manufac- We now show that lipids can be supplied to sympathetic turer’s instructions. Alexa Fluor 488 dye reacts with primary amines of proteins forming stable dye-protein conjugates. In HDL, Alexa 488 neurons from lipoproteins for use in axonal growth. Our data binds covalently to apoAI and apoAII (43). SDS-polyacrylamide gel are consistent with the model that the uptake of lipoprotein electrophoresis confirmed that apoB, apoAI, apoAII, and chicken apoAI components by these neurons occurs via receptor-mediated en- were labeled. All lipoprotein preparations were essentially free of un- docytosis. We show that the LDLR is present in both cell bodies bound Alexa. Recovery of Alexa fluorescence in the aqueous phase after and axons of rat sympathetic neurons, and we have identified extraction of Alexa lipoproteins with organic solvents was greater than two additional members of the LDL receptor superfamily, 95% providing evidence that the label was primarily associated with proteins. LR7/8B (apoER2) and LR11 (SorLA), in these neurons. How- Lipoproteins were labeled with [ H]cholesterol as described else- ever, neither LRP (which is present in CNS neurons) nor the mCi of [1a,2a- where (44). Briefly, the solvent from 200 H]cholesterol HDL receptor, SR-BI, was detected. In addition, the experi- was evaporated to form a thin film. The lipoprotein preparation (3 mg ments show that lipoprotein receptors are differentially distrib- of protein) was added in 0.9% NaCl, and the mixture was shaken uted and regulated in cell bodies and distal axons of sympa- overnight at 4 °C, after which lipoproteins were re-isolated by flotation thetic neurons. mg total choles- as above. The specific radioactivity of cholesterol (dpm/ terol) was determined. Cholesterol mass was determined by an enzy- EXPERIMENTAL PROCEDURES matic kit (Sigma). Typically, the specific radioactivity of cholesterol was 29,700 dpm/mg for hLDL, 150,200 dpm/mg for hHDL Materials—[1a,2a- H]Cholesterol (specific activity 45 mCi/mmol) , and 64,100 mg for cHDL. was purchased from Amersham Pharmacia Biotech. Pravastatin was a dpm/ Analysis of Lipoprotein Uptake by Fluorescence Microscopy—Neu- gift from Dr. S. Yokoyama, Nagoya City University Medical School, rons were plated in the center compartment of compartmented dishes Japan. 1,19-Dioctadecyl-3,3,39,39- tetramethylindocarbocyanine per- and cultured for 14 days. DiI or Alexa lipoproteins were added to the chlorate (DiI) and the Alexa 488 protein labeling kit were from Molec- cell body- or distal axon-containing compartments as indicated. Li- ular Probes, Inc. (Eugene, OR). L15 medium without antibiotics was poprotein concentration in media was normalized to 100 mg of total purchased from Life Technologies, Inc. Mouse 2.5 S nerve growth factor cholesterol/ml. In addition, fluorescence units were normalized to was from Alomone Laboratories Ltd. (Jerusalem, Israel). Rat serum achieve the same fluorescence in all media by addition of unlabeled was provided by the University of Alberta Laboratory Animal Services. lipoproteins. After 18 h, the neurons were washed extensively with cold The anti-rat LDLR polyclonal antibody was a gift from Dr. G. Ness, phosphate-buffered saline containing methylcellulose (3 g/500 ml) and University of South Florida (37). A polyclonal antibody directed against fixed for 20 min in 4% paraformaldehyde. Coverslips were secured the carboxyl-terminal 14 amino acids of the chicken LR7/8B (apoER2) using para-phenylenediamine in glycerol as mounting medium. Fluo- receptor (28) was generously provided by Dr. J. Nimpf, University of rescence microscopy was performed with an Olympus BX-SOF fluores- Vienna, Austria. The anti-murine SorLA (LR11) polyclonal antibody, cence microscope equipped with a 100-watt gas mercury lamp and raised against a fusion protein containing the fibronectin domain of rhodamine filter (for DiI). Alternatively, neurons were analyzed by SorLA, was a gift from Dr. H. Schaller, University of Hamburg, Ger- confocal laser scanning microscopy using a Leica microscope illumi- many (23). The anti-SR-BI antibody, directed against a peptide corre- nated by a 100-watt mercury burner for direct observation, a Ar/Kr sponding to amino acids 495–509 from murine SR-BI was kindly pro- laser with major emissions at 488, 568 and 647 nm for scanning, and vided by Dr. M. Krieger, Massachusetts Institute of Technology (38). 63 3 1.40 NA oil immersion objectives. Figs. 2, 4, and 5 were prepared The rabbit anti-LRP polyclonal antibody was generated against the using Adobe Photoshop software. 85-kDa fragment of rat LRP and was a gift from Dr. J. Herz, University Association of DiI and [1a,2a- of Texas Southwestern Medical Center (39). Electrophoresis reagents H]Cholesterol with Neurons—Neurons were supplied by Bio-Rad. Polyvinylidene difluoride membranes were were incubated with DiI lipoproteins in the cell body-containing com- from Millipore. All other reagents were from Sigma or Fisher. partment for 18 h and then washed three times with ice-cold phosphate- Preparation of Neuronal Cultures—Procedures for growing rat sym- buffered saline. Cellular material was harvested from the center com- pathetic neurons in compartmented cultures have been previously re- partment in phosphate-buffered saline. The detached neurons were ported (40). Briefly, superior cervical ganglia from newborn Harlan sonicated for 10 s with a tip probe. An aliquot was added to 2 ml of Sprague-Dawley rats were dissected and enzymatically and mechani- methanol, and fluorescence was analyzed in a Hitachi F-2000 spec- cally dissociated. The cells were plated in either the center or the left trofluorometer with excitation light of 550 nm and emission at 565 nm. compartment of three-compartmented culture dishes (36). Neurons Protein content was determined using the BCA protein kit (Pierce). DiI were cultured for 10 –14 days prior to the start of experiments. For did not interfere with the protein determinations. In experiments with measurements of axonal extension, left-plated cultures were used; all other experiments were performed using center-plated cultures. Lipoprotein Isolation and Labeling—Human LDL (hLDL) and hu- J. Vance, unpublished results. Lipoproteins and Axonal Regeneration 19885 H]cholesterol-labeled lipoproteins, neurons were given labeled li- poproteins in the cell body-containing compartment. The concentration of lipoproteins was adjusted so that the cholesterol content was 100 mg/ml, and the same specific radioactivity of cholesterol was achieved for all lipoproteins by addition of unlabeled lipoproteins. After 18 h, the cells were washed, and material from the center compartment was harvested, and radioactivity was measured. The amount of cell-associ- ated lipoprotein-derived cholesterol was calculated from the specific radioactivity of the lipoprotein [ H]cholesterol. Measurement of Axonal Extension—Neurons were plated in the left compartment of compartmented dishes. Distal axons were mechani- cally removed from the right-hand compartment with a jet of sterile distilled water delivered with a syringe through a 22-gauge needle. The water was aspirated and the wash repeated twice, after which fresh FIG.1. Utilization of lipoproteins for axonal elongation. Neu- medium was added. This procedure, termed axotomy, effectively re- rons were cultured in the left-hand compartment of compartmented moves all visible traces of axons from the right-side compartment. dishes. Distal axons were removed from the right-hand compartment, Axonal growth was measured as described previously (45). and the cell body-containing compartment was supplied with 50 mM Analysis of Lipoprotein Receptors by Immunoblotting—Material from pravastatin, as well as hLDL, hHDL , or cHDL (100 mg of cholesterol/ ml). After 4 days, axonal extension was measured in three cultures (a distal axon- and cell body-containing compartments was harvested total of 45– 48 tracks) for each treatment. Data are means 6 S.E. separately in 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 1 mM Statistical significance of differences between untreated and treated EDTA, 0.05 mM phenylmethylsulfonyl fluoride. Cellular material was neurons is indicated by * and was evaluated by the Student’s t test (p # sonicated for 10 s with a probe sonicator and centrifuged at 350,000 3 0.05). The experiment was repeated three times using different lipopro- g for 20 min. Membrane pellets were dissolved in 50 mM Tris-HCl (pH tein preparations with similar results. 7.5) containing 150 mM NaCl, 1 mM EDTA, 0.05 mM phenylmethylsul- fonyl fluoride. The protein content was determined using the BCA protein kit (Pierce). Proteins were separated by electrophoresis on an cholesterol synthesis was inhibited by addition of 50 mM 8% SDS-polyacrylamide gel for LDLR and SRBI, a 5% gel for LR11 and pravastatin to the cell body-containing compartment. This con- LR7/8B, and a 3–15% gradient gel for LRP. For LR7/8B, electrophoresis centration of pravastatin inhibits the incorporation of [ C]ac- was performed under non-reducing conditions (28). Proteins were transferred to polyvinylidene difluoride membranes for 18 h at 25 V etate into cholesterol by ;80% (50). The concentration of each (36). Membranes were incubated in 20 mM Tris-HCl (pH 7.5), 500 mM lipoprotein preparation added to cell bodies was equalized in NaCl, 0.1% (v/v) Tween 20 (TTBS) with 5–10% (w/v) non-fat dried milk terms of cholesterol content (100 mg/ml). Axonal extension was for at least 2 h and then incubated for1hat room temperature with measured after 4 days (Fig. 1). In agreement with our previous primary antibody in TTBS containing 1% non-fat dried milk using the results (36), axonal extension was reduced by ;50% in pravas- following dilutions of primary antibodies: LDLR, 1:1000; LRP, 1:5000; tatin-treated cells compared with untreated cells, whereas the SR-BI, 1:1000; LR11, 1:1500; and LR7/8B, 1:1500. Membranes were addition of hLDL, but not hHDL then incubated for 1 h with horseradish peroxidase linked to anti-rabbit , to cell bodies/proximal axons IgG secondary antibody (Pierce, diluted 1:10,000), and immunoreactive restored axonal elongation. In addition, Fig. 1 shows that axons proteins were detected by enhanced chemiluminescence. extended normally when cHDL were added to cell bodies/prox- imal axons of pravastatin-treated neurons. These data suggest RESULTS that hHDL cannot provide cholesterol to cell bodies for axonal Lipoproteins and Axonal Growth—We have previously growth, possibly because cell bodies lack a receptor for hHDL . shown that inhibition of cholesterol synthesis by pravastatin In accordance with our previous observations (36), when any of impairs axonal elongation of compartmented cultures of rat the lipoproteins was given to distal axons alone, the axons sympathetic neurons. Addition of cholesterol to axons or cell extended normally (data not shown). bodies restored normal axonal growth to pravastatin-treated Uptake of Lipids and Proteins from Lipoproteins—We inves- neurons. Similarly, when human lipoproteins (hLDL, hHDL , tigated whether or not lipids and proteins from lipoproteins or hHDL ) were supplied to distal axons of pravastatin-treated were taken up by cell bodies of sympathetic neurons. hLDL, neurons, normal axonal growth occurred. In contrast, normal cHDL, and hHDL were labeled with DiI, a hydrophobic fluo- axonal elongation occurred only when hLDL, but not hHDL or rescent dye that intercalates into the neutral lipid core of hHDL , were added to the cell body-containing compartment lipoproteins without affecting receptor binding (51). Rat sym- (36). These studies suggested that cholesterol can be taken up pathetic neurons were maintained for 14 days and then incu- from lipoproteins by these neurons and used for axonal growth. bated with DiI lipoproteins in the center compartment contain- The observations also raised the possibility that LDL and HDL, ing the cell bodies. The concentration of lipoproteins in the or the cholesterol therein, are taken up by different mecha- media was adjusted so that the total cholesterol concentration nisms, potentially involving distinct lipoprotein receptors. (100 mg/ml medium) and specific fluorescence (fluorescence We have now extended these studies and analyzed the ca- intensity units/ml of medium) were the same for all lipopro- pacity of hLDL, hHDL , and chicken HDL (cHDL) to deliver teins. After 18 h, the neurons were extensively washed and lipids, including cholesterol, for axonal growth of rat sympa- examined by fluorescence microscopy. Fig. 2, a, c, and e, shows thetic neurons. hHDL lacks apoE and therefore would not be the bright field images of cell bodies/proximal axons of neurons expected to interact with the apoB/apoE receptor (i.e. the given hLDL, cHDL, and hHDL , respectively. Fig. 2, b, d, and LDLR). cHDL was selected because chicken apoAI has been f, shows the corresponding fluorescence images. Cell bodies of proposed to play a role in nerve regeneration in chickens sim- neurons given DiI-hLDL or DiI-cHDL exhibited intense fluo- ilar to that of apoE in mammals (46 – 48). Consequently, we rescence (Figs. 2, b and d), whereas almost no fluorescence was expected cHDL and hLDL to be similarly taken up by rat visible in cell bodies of neurons given hHDL (Fig. 2f). For sympathetic neurons. For these experiments, rat sympathetic quantification of the fluorescence associated with cell bodies, neurons were plated in the left side compartment of three- cellular material was harvested from the center compartment, compartmented culture dishes. In these cultures (49), all com- and DiI fluorescence intensity was measured. Neurons treated partments contain independent fluid environments, and the with hLDL and cHDL contained 4- and 3-fold, respectively, cell bodies/proximal axons reside in a compartment completely more fluorescence than did neurons given hHDL (Fig. 3a). separate from that containing distal axons. After 14 days, Assuming that DiI is a valid marker for uptake of neutral lipids axons in the right side compartment were axotomized, and from lipoproteins (52), these data suggest that cell bodies/ 19886 Lipoproteins and Axonal Regeneration ies, since the only way in which components of lipoproteins that have been added to distal axons can enter the cell bodies is from retrograde, intracellular transport. Thus, the appearance of fluorescence in cell bodies, after addition of lipoproteins con- taining fluorescent lipids and proteins to distal axons, would unambiguously demonstrate that lipids and proteins, respec- tively, had been taken up from lipoproteins by distal axons and transported to cell bodies. For these experiments, lipoproteins were labeled with either DiI or Alexa 488 (a fluorescent label of proteins) and added to either distal axons or cell bodies/proxi- mal axons. Lipoprotein concentrations were adjusted in terms of total cholesterol content (100 mg/ml medium) and specific fluorescence. The protein components of the lipoproteins did not bind significant amounts of DiI, and conversely, Alexa 488 associated almost exclusively with proteins of the lipoproteins (see “Experimental Procedures”). After 18 h, cell bodies were examined for fluorescence. All images were analyzed on a plane where nuclei were visible to ensure that the fluorescent li- FIG.2. Labeling of sympathetic neurons with DiI lipoproteins. poprotein did not only adhere to the cell surface but was truly Neurons were plated in the center compartment of compartmented intracellular. dishes. After 14 days, DiI lipoproteins (hLDL, cHDL, or hHDL ) were When cell bodies/proximal axons were incubated with fluo- added to the cell body-containing compartment. Lipoprotein concentra- rescent hLDL or cHDL, intense DiI (Fig. 4, B and D) and Alexa tion was adjusted to 100 mg of cholesterol/ml and 136,000 fluorescence intensity units/ml. After 18 h, neurons were extensively washed, fixed, (Fig. 4, H and J) fluorescence, likely representing aggregates of and examined by fluorescence microscopy. Left panels (a, c, and e) are lipoproteins, was observed inside the cell bodies. In contrast, bright field images, and right panels (b, d, and f) are fluorescence when either DiI- or Alexa-hHDL was given to the cell body- images of cell bodies of neurons given hLDL (a and b), cHDL (c and d), containing compartment, little fluorescence was visible in cell and hHDL (e and f). The horizontal “rope-like” lines shown in the bright field images are scratches in the collagen surface of the culture dish and bodies (Fig. 4, F and L). The ring of fluorescence in Fig. 4L create tracks along which the axons extend (*). Proximal neurites and probably represents Alexa lipoproteins remaining on the cell clusters of cell bodies are indicated with arrowheads and arrows, re- surface after the extensive washing procedure. Even when the spectively. Images are representative of 50 fields analyzed per treat- experiments were repeated using a 10-fold higher concentra- ment. The experiment was repeated three times with equivalent results. tion of DiI- or Alexa-hHDL , the fluorescence inside the cell bodies remained at basal levels. These experiments show that lipids and proteins from hLDL and cHDL, but not hHDL , are proximal axons internalize lipids from hLDL and cHDL but not internalized by cell bodies/proximal axons. hHDL . In parallel experiments, neurons that were given DiI-hLDL We next measured the uptake of [ H]cholesterol from li- (Fig. 4A), DiI-cHDL (Fig. 4C), or DiI-hHDL (Fig. 4E) to distal poproteins by cell bodies. Neurons were plated in the center axons exhibited profuse punctate fluorescence in the cell bod- compartment and maintained for 14 days, after which cell 3 ies, suggesting that lipids from all the lipoproteins were taken bodies/proximal axons were incubated with [ H]cholesterol-la- up by distal axons and transported to cell bodies. These results beled lipoproteins for 18 h. The cells were then washed and are in accordance with our observations that all these lipopro- harvested, and radioactivity was determined. Neurons incu- teins, given to distal axons, can overcome the inhibition of bated with hLDL and cHDL contained approximately 70% axonal growth induced by pravastatin (36). The uptake of more radioactivity than did neurons incubated with hHDL Alexa-labeled hLDL, cHDL, and hHDL by distal axons was (Fig. 3b). Although Fig. 3, a and b, indicates that the associa- similarly examined. Fig. 4, G, I, and K, shows that each of the tion of lipids from hLDL and cHDL by cell bodies is greater 3 Alexa lipoproteins was taken up by distal axons and trans- than from hHDL , relatively more [ H]cholesterol-labeled ported to cell bodies. These data demonstrate that lipids and hHDL than DiI-labeled hHDL associated with the neurons. 3 3 proteins from hLDL, cHDL, and hHDL can be internalized by One likely explanation for this difference is that in addition to distal axons and transported to cell bodies. In addition, lipids hHDL specifically binding to the cell surface, [ H]cholesterol and proteins from hLDL and cHDL, but not hHDL , were on the HDL surface might exchange with cell membranes via internalized by cell bodies/proximal axons. Our findings also aqueous diffusion. In contrast, DiI is present in the neutral confirm that the various lipoprotein classes are differentially lipid core of HDL and would, therefore, not exchange via taken up by cell bodies and distal axons of sympathetic neu- aqueous diffusion on to the cell surface but would associate rons. Previous studies have reported that in several cell types with cells only via a specific receptor-mediated interaction. a selective uptake of lipids, particularly cholesteryl esters, oc- The experiments described above suggest that lipids from curs from HDL (43), whereas the uptake of HDL holoparticles hLDL and cHDL, but not hHDL , are taken up by the neuronal has been less well documented (53, 54). Therefore, our obser- cell bodies. Since in our experience these neurons are particu- vation that distal axons of sympathetic neurons were able to larly sensitive to cold temperatures, we were unable to perform internalize both lipids and proteins from hHDL was somewhat classical lipoprotein binding experiments at 4 °C to differenti- ate between uptake and nonspecific binding of lipoproteins. To unexpected. We next investigated whether or not uptake of hLDL and exclude the possibility that the observations described above could be explained solely by the lipoproteins binding to the cell hHDL occurred via receptor-mediated processes. Neurons were given DiI- or Alexa-labeled lipoproteins to either distal surface, without their internalization, we used confocal laser scanning microscopy to examine more directly fluorescent li- axons or cell bodies/proximal axons. Some cultures were given poprotein uptake. By using this technique we were also able to a 50-fold excess of unlabeled lipoproteins for competition of determine whether or not distal axons can take up lipids and uptake. Unlabeled hLDL competed effectively for uptake of proteins from lipoproteins and transport them to the cell bod- fluorescent hLDL by cell bodies (compare Fig. 5, A with D, and Lipoproteins and Axonal Regeneration 19887 FIG.3. Quantitation of fluorescent and radiolabeled lipids associated with cell bodies. a, neurons were incubated with DiI-lipoproteins as in Fig. 2 legend. After extensive washing, material from the center compartment was harvested and DiI fluorescence measured. b, neurons cultured as in Fig. 2 were incubated with [ H]cholesterol-lipoproteins for 18 h, then washed, harvested and the amount of cell-associated, lipoprotein-derived cholesterol was calculated from the specific radioactivity of [ H]cholesterol in the lipoprotein. Values are means 6 S.E. of six determinations for each treatment. Statistically significant differences compared with treatment with hLDL are indicated by * and were evaluated by the Student’s t test (p # 0.05). The experiment was repeated twice with similar results. FIG.4. Uptake of fluorescent lipids and proteins from lipoproteins. Cen- ter-plated neurons were incubated with DiI-or Alexa-lipoproteins in either the cell body-containing compartment (B, D, F, H, J, and L) or distal axon-containing com- partment (A, C, E, G, I, and K). Lipopro- tein concentration was adjusted to 100 mg of cholesterol/ml and 136,000 fluorescence intensity units/ml for DiI lipoproteins or 180,000 fluorescence intensity units/ml for Alexa lipoproteins. After 18 h, neurons were processed for confocal microscopy. The images shown for each group (DiI and Alexa) were captured under identical con- ditions, and 50 –100 fields were analyzed for each treatment. The experiment was repeated twice with comparable results. FIG.5. Competition of fluorescent lipoprotein uptake by unlabeled li- poproteins. Neurons were incubated in either the cell body-containing (A, D, G, and J) or distal axon-containing (B, C, E, F, H, I, K, and L) compartment with flu- orescent lipoproteins, as in Fig. 4 legend. Some cultures were given a 50-fold excess of unlabeled lipoproteins. Cell bodies were visualized by confocal microscopy, and the images shown for each group (DiI and Alexa) were captured under identical conditions. The experiment was repeated twice with similar results. Fig. 5, G with J) and distal axons (compare Fig. 5, B with E, “mass cultures”) and compartmented dishes. Cellular material and H with K). Similarly, excess unlabeled hHDL blocked the was harvested, a membrane-enriched fraction was prepared, uptake of fluorescent hHDL by distal axons (compare Fig. 5, C and membrane proteins were separated by SDS-polyacryl- with F, and I with L), supporting the idea that both hLDL and amide gel electrophoresis, after which immunoblotting was hHDL are internalized by receptor-mediated mechanisms. performed. Proteins from homogenates of rat liver and rat Lipoprotein Receptors in Sympathetic Neurons—The distri- brain were used as controls. Fig. 6a shows that the LDLR (M bution of lipoprotein receptors in cell bodies and distal axons of ;130) is widely distributed throughout cell bodies and axons of sympathetic neurons was investigated by immunoblot analy- sympathetic neurons but is more abundant in cell bodies/prox- sis. Neurons were cultured both in 24-well dishes (designated imal axons than in distal axons. As a positive control, the 19888 Lipoproteins and Axonal Regeneration FIG.7. Expression of LR11 and LR7/8B in sympathetic neu- rons. Neurons were cultured in three-compartmented dishes. Mem- brane proteins (30 mg) from neurons, rat brain, and rat liver were separated by SDS-polyacrylamide electrophoresis and analyzed by im- munoblotting using antibodies directed against LR11 or LR7/8B. imal axons and distal axons, as well as in rat brain but not in FIG.6. Distribution of LDLR, LRP, and SR-BI in sympathetic rat liver. neurons. a, neurons were cultured in 24-well dishes and in compart- mented dishes. Cellular material from 24-well dishes (Mass), the cell DISCUSSION body-containing compartment (CB), and distal axon-containing com- Our previous experiments indicated that lipoproteins given partments (AX), was harvested. A membrane-enriched fraction was prepared, and membrane proteins (20 mg) were separated by SDS- to distal axons of rat sympathetic neurons can provide choles- polyacrylamide gel electrophoresis and immunoblotted using antibodies terol, but not phospholipids, for axonal regeneration (36). We directed against one of the LDLR, the 85-kDa subunit of LRP, or SR-BI. now show for the first time that distal axons internalize pro- Proteins (20 mg) from homogenates of rat liver and rat brain were used teins and lipids from lipoproteins and transport these compo- as controls. * indicates that membranes from rat brain and neuronal mass cultures were mixed (see text). b, compartmented cultures of nents retrogradely to cell bodies. In contrast, cell bodies/prox- neurons were incubated with (1) or without (2)50 mM pravastatin imal axons take up lipids and proteins from LDL but not HDL . (Prav) for 48 h. Cellular material was harvested, and the presence of the The results are consistent with the idea that both lipid and LDLR (LDLr) analyzed by immunoblotting. This experiment was per- protein components of lipoproteins are taken up by axons via formed four times with independent preparations of neurons. In addi- tion, two different amounts of axonal proteins (20 and 60 mg) were receptor-mediated endocytosis and transported to cell bodies, applied to the gel. The same result was obtained for each experiment. the site of lysosomal processing. However, the possibility exists that some components (e.g. cholesteryl esters and/or choles- LDLR was also detected in rat liver and rat brain. In contrast, terol) remain in axons and are directly used for growth. the 85-kDa fragment of LRP was not detected in any prepara- The LDL Receptor in Sympathetic Neurons—Lipoprotein re- tion of sympathetic neurons. However, this receptor was pres- ceptors are known to play an important role in cellular choles- ent in rat liver and rat brain, which were used as positive terol homeostasis (55), and our experiments indicate that li- controls. We were surprised that we were unable to detect LRP poprotein uptake by sympathetic neurons is receptor-mediated. in sympathetic neurons since LRP has been reported to be Many of these receptors belong to the LDLR superfamily of abundant in CNS neurons (20) and brain (18, 21). We consid- which the LDLR, which has been detected in PNS and CNS ered the possibility that our inability to detect LRP was due to neurons (9, 16, 30, 57), is the prototype. Our data show that the a factor in the neuron samples that interfered with immuno- LDLR is present in cell bodies/proximal axons and distal axons detection of LRP. This possibility was eliminated when a rat of rat sympathetic neurons, with an apparent enrichment in brain homogenate was mixed with membranes from neuronal cell bodies. It is likely, therefore, that in our studies the inter- mass cultures (shown as Rat Brain* in Fig. 6a), and the inten- nalization of hLDL was mediated primarily by the LDLR. How- sity of the band representing brain LRP was not diminished. ever, since the LDLR only binds lipoproteins that contain apoB Fig. 6 also shows that the HDL receptor, SR-BI, is present in and/or apoE (55), this receptor probably does not participate in rat liver but not brain, in agreement with the distribution of the uptake of hHDL , which contain neither apoB nor apoE. SR-BI mRNA (38). No immunoreactive SR-BI was detected in A spatially distinct expression of the LDLR has been ob- sympathetic neurons. served in other polarized cells. For example, in Madin-Darby In many cell types, expression of the LDLR is regulated by canine kidney cells expression of recombinant LDLRs resulted cellular cholesterol content (55). We therefore investigated in their preferential targeting to basolateral membranes (58). whether or not inhibition of cholesterol biosynthesis increased In addition, using adenovirus expression the LDLR was highly expression of the LDLR in cell bodies/proximal axons and distal expressed in the somatodendritic region of hippocampal neu- axons of sympathetic neurons. Compartmented neuron cul- rons but was undetectable in distal axons. In contrast, mutant tures were treated with 50 mM pravastatin in the cell body- LDLRs lacking a putative basolateral targeting sequence were containing compartment for 48 h after which cellular material most highly concentrated in growth cones and distal axons (59). was isolated. Immunoblotting of proteins with anti-LDLR an- The distribution of endogenous LDLRs was not examined in tibody revealed that pravastatin treatment markedly up-regu- this report. However, Li et al. (60) reported the presence of a lated LDLR expression in cell bodies but not in distal axons cell surface receptor that bound LDL with the characteristics of (Fig. 6b). the LDL receptor in both apical and basolateral domains of The presence of two other receptors of the LDL receptor Madin-Darby canine kidney cells. Studies on the sorting of superfamily that are abundant in brain, LR7/8B (also known as viral proteins (61) and endogenously expressed proteins (62, apoER2) (27, 28) and LR11 (also known as SorLA) (23, 25, 26, 63) in hippocampal neurons and Madin-Darby canine kidney 56), was also examined by immunoblotting. Fig. 7 shows the cells have suggested that neurons and polarized epithelial cells presence of LR11 (M ;240) in rat brain and cell bodies/prox- share common mechanisms of protein targeting and sorting, imal axons but the absence of LR11 from distal axons and rat with the axonal membrane being equivalent to the apical do- liver. LR7/8B (M ;130) was detected in both cell bodies/prox- main of epithelial cells and the somatodendritic region being r Lipoproteins and Axonal Regeneration 19889 equivalent to the basolateral domain. Our data are, in general, creases dramatically after peripheral nerve injury (46). Our consistent with this hypothesis. data show that cHDL are internalized by sympathetic neurons Expression of the LDLR in many cell types is regulated by in a manner analogous to that of hLDL but unlike that of cellular cholesterol levels (55); when cholesterol synthesis is hHDL . Uptake of HDL by Distal Axons—The finding that HDL are inhibited, expression of LDLRs increases. In the present study, 3 3 we inhibited cholesterol synthesis in sympathetic neurons and internalized by distal axons is intriguing although the distri- bution of lipoprotein receptors that we report here does not found that the amount of the LDLR in cell bodies/proximal explain how this uptake occurs. Presumably, other receptors axons, but not in distal axons, increased. Spatially independent are present that mediate the uptake of hHDL by distal axons. regulation of expression of the LDLR has been previously ob- Since cell bodies did not internalize either lipids or proteins of served in other polarized cells. For example, the number of hHDL , our data imply that cell bodies lack a functional hHDL LDLRs is regulated by cellular cholesterol content on only 3 3 receptor. The best characterized HDL receptor is SR-BI (72). basolateral, but not apical, membranes of Madin-Darby canine SR-BI binds HDL via apoAI (73) and mediates a selective kidney cells (60). One possible explanation for the lack of up- uptake of lipids, particularly cholesteryl esters, from HDL (52, regulation of expression of the LDLR in distal axons in re- 74 –76) and LDL (77, 78), without a concomitant uptake of sponse to pravastatin is that the reduced cholesterol content apoproteins. In rat adrenal cells, the selective uptake of cho- might impair anterograde vesicular transport that would pre- lesteryl esters occurs by a mechanism distinct from the classi- vent LDLRs, which are synthesized in the cell bodies, from cal endosomal/lysosomal pathway (79). Similarly, in rat hepa- being exported to distal axons. toma cells HDL cholesteryl esters are hydrolyzed extra- Other Lipoprotein Receptors in Sympathetic Neurons—Sev- lysosomally (80). SR-BI is abundantly expressed in liver and eral members of the LDLR superfamily have been proposed to steroidogenic tissues but has not been found in brain (81), and be involved in neuronal metabolism and pathogenesis of neu- we were unable to detect SR-BI in sympathetic neurons. In rodegenerative diseases (29). For example, LRP, a multifunc- addition to selective lipid uptake from HDL, the endocytosis tional receptor that mediates the endocytosis of several ligands and lysosomal degradation of HDL holoparticles have previ- (64), is expressed in brain and spinal cord (18), as well as in the ously been observed (53, 54). In our experiments, not only lipids somatodendritic domain of cultured hippocampal neurons (22). but also proteins of hHDL were internalized by axons and The LRP and one of its ligands, apoE, have been reported to 3 transported to cell bodies, indicating holoparticle uptake. How- play an important role in early hippocampal development (19). ever, our data are also consistent with the possibility that Moreover, apoE synthesis dramatically increases in the CNS distal axons participate in a selective uptake of some lipids and PNS after nerve injury (10, 12, 46), and apoE-containing from HDL, in addition to holoparticle uptake. lipoproteins have been proposed to play a role in peripheral Since hHDL lack apoE they are unlikely to be internalized nerve regeneration (8) and dendritic remodeling in the CNS by the LDLR although receptors participating in the uptake of (65). Consequently, we were surprised that we could detect no intact HDL particles have not yet been clearly defined. Re- LRP in sympathetic neurons. Our result is, however, consistent cently, cubilin has been identified as a receptor that mediates with the idea that CNS and PNS neurons might express dis- the endocytosis/lysosomal degradation of HDL (82, 83). Since tinct populations of lipoprotein receptors because the types of cubilin lacks a membrane-spanning domain, it is considered to lipoproteins to which they would be exposed in their native be a peripheral membrane protein (84) that requires a co- environments are different. receptor, probably megalin, to mediate endocytosis (82). Mega- LR11 is a recently discovered member of the LDLR super- lin (also known as gp330 or LRP2) is a member of the LDLR family (23–25). In mammals, LR11 is abundant in the brain family and binds multiple ligands including apoE and apoJ and binds apoE-containing lipoproteins (26). This receptor has (reviewed in Ref. 64). This finding is of particular interest in been suggested to play a role in lipoprotein metabolism in the relation to cholesterol homeostasis in the brain and during CNS (25). We found that LR11 is present in cell bodies/proxi- PNS nerve regeneration since lipoproteins involved in those mal axons of sympathetic neurons but, interestingly, not in processes are thought to contain apoE and/or apoJ (32). Cubilin distal axons. is localized to apical membranes of polarized endothelial cells Our experiments also show that LR7/8B, for which apoE is a of the kidney (85), and megalin is also expressed on the apical ligand, is expressed throughout cell bodies and axons of sym- surfaces of many epithelial cells (86, 87). Therefore, since neu- pathetic neurons. cDNAs encoding this receptor have been rons are polarized cells in which the distal axons are, in some cloned from brain of human, mouse, and chicken (27, 28). ways, equivalent to the apical domain of other polarized cells Interestingly, in chickens, which lack apoE, LR11 and LR7/8B (62, 63), one might predict that if megalin and cubilin were are abundant in the brain. The importance of LR7/8B in CNS present they would be concentrated in distal axons. If this were development is underscored by the finding that mice lacking the case, megalin and cubilin would be viable candidates for both LR7/8B and the very low density lipoprotein receptor mediating HDL uptake by distal axons. Whether or not cubilin (another LDLR family member) (66) display an inversion of and megalin are present in distal axons, and absent from cell cortical layers during development (67, 68). Some receptors of bodies, of sympathetic neurons warrants further investigation. the LDLR superfamily have recently been implicated in intra- In summary, we have shown that cultured rat sympathetic cellular signaling events because their cytosolic domains inter- neurons internalize lipids and proteins of lipoproteins by re- act with signaling molecules such as mDab1 (68). Whether or ceptor-mediated mechanisms. Although LRP was absent from not these receptors function in vivo for endocytosis of lipopro- these neurons, related receptors such as the LDLR, LR7/8B, teins and/or as signaling receptors is not yet known. and LR11 were detected. These receptors were differentially Uptake of Chicken HDL—cHDL were used as model lipopro- distributed, and expression of the LDLR was differentially teins because they exhibit some similarities to mammalian regulated in cell bodies and distal axons of sympathetic neu- LDL. Although chickens lack apoE, their HDL contain apoAI rons. Our studies also show that distal axons, but not cell that has been proposed to be a “surrogate” for mammalian bodies, of sympathetic neurons have the capacity to internalize apoE (47, 69, 70). 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