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• W2132983892 abstract "Lipoproteins originating from axon and myelin breakdown in injured peripheral nerves are believed to supply cholesterol to regenerating axons. We have used compartmented cultures of rat sympathetic neurons to investigate the utilization of lipids from lipoproteins for axon elongation. Lipids and proteins from human low density lipoproteins (LDL) and high density lipoproteins (HDL) were taken up by distal axons and transported to cell bodies, whereas cell bodies/proximal axons internalized these components from only LDL, not HDL. Consistent with these observations, the impairment of axonal growth, induced by inhibition of cholesterol synthesis, was reversed when LDL or HDL were added to distal axons or when LDL, but not HDL, were added to cell bodies. LDL receptors (LDLRs) and LR7/8B (apoER2) were present in cell bodies/proximal axons and distal axons, with LDLRs being more abundant in the former. Inhibition of cholesterol biosynthesis increased LDLR expression in cell bodies/proximal axons but not distal axons. LR11 (SorLA) was restricted to cell bodies/proximal axons and was undetectable in distal axons. Neither the LDL receptor-related protein nor the HDL receptor, SR-B1, was detected in sympathetic neurons. These studies demonstrate for the first time that lipids are taken up from lipoproteins by sympathetic neurons for use in axonal regeneration. Lipoproteins originating from axon and myelin breakdown in injured peripheral nerves are believed to supply cholesterol to regenerating axons. We have used compartmented cultures of rat sympathetic neurons to investigate the utilization of lipids from lipoproteins for axon elongation. Lipids and proteins from human low density lipoproteins (LDL) and high density lipoproteins (HDL) were taken up by distal axons and transported to cell bodies, whereas cell bodies/proximal axons internalized these components from only LDL, not HDL. Consistent with these observations, the impairment of axonal growth, induced by inhibition of cholesterol synthesis, was reversed when LDL or HDL were added to distal axons or when LDL, but not HDL, were added to cell bodies. LDL receptors (LDLRs) and LR7/8B (apoER2) were present in cell bodies/proximal axons and distal axons, with LDLRs being more abundant in the former. Inhibition of cholesterol biosynthesis increased LDLR expression in cell bodies/proximal axons but not distal axons. LR11 (SorLA) was restricted to cell bodies/proximal axons and was undetectable in distal axons. Neither the LDL receptor-related protein nor the HDL receptor, SR-B1, was detected in sympathetic neurons. These studies demonstrate for the first time that lipids are taken up from lipoproteins by sympathetic neurons for use in axonal regeneration. apolipoprotein central nervous system 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate high density lipoproteins low density lipoproteins low density lipoprotein receptor low density lipoprotein receptor-related protein peripheral nervous system scavenger receptor class B human chicken Axonal elongation requires the expansion of axonal membranes by addition of new membrane materials (proteins and lipids) to the growing axon. In sheep (1.Turley S.D. Burns D.K. Rosenfeld C.R. Dietschy J.M. J. Lipid. Res. 1996; 37: 1953-1961Abstract Full Text PDF PubMed Google Scholar) and rats (2.Jurevics H.A. Morell P. J. Lipid. Res. 1994; 35: 112-120Abstract Full Text PDF PubMed Google Scholar, 3.Jurevics H.A. Kidwai F.Z. Morell P. J. Lipid. Res. 1997; 38: 723-733Abstract Full Text PDF PubMed Google Scholar) the brain and peripheral nerves synthesize all the cholesterol needed for development without requiring cholesterol from circulating lipoproteins. Moreover, after birth, cholesterol used for myelin production is made locally (4.Jurevics H. Morell P. J. Neurochem. 1995; 64: 895-901Crossref PubMed Scopus (248) Google Scholar). In contrast, fetal liver supplies about 50% of the cholesterol needed for development of heart, lung, and kidney (3.Jurevics H.A. Kidwai F.Z. Morell P. J. Lipid. Res. 1997; 38: 723-733Abstract Full Text PDF PubMed Google Scholar). Surprisingly, during peripheral nerve regeneration, cholesterol synthesis in the nerve is down-regulated (5.Goodrum J.F. J. Neurochem. 1990; 54: 1709-1715Crossref PubMed Scopus (45) Google Scholar), yet serum-derived cholesterol does not contribute significantly to myelin synthesis or axonal regeneration (6.Goodrum J.F. J. Neurochem. 1993; 60: 1564-1566Crossref PubMed Scopus (16) Google Scholar, 7.Jurevics H. Bouldin T.W. Toews A.D. Morell P. Neurochem. Res. 1998; 23: 401-406Crossref PubMed Scopus (18) Google Scholar). Instead, cholesterol from degenerating axons and myelin is proposed to be retained within the nerve and re-utilized via a lipoprotein-mediated process (8.Mahley R.W. Science. 1988; 240: 622-630Crossref PubMed Scopus (3376) Google Scholar), although cholesterol uptake by neurons was not directly demonstrated. The presence of endoneural lipoproteins in regenerating, but not non-injured, nerves is well documented. These lipoproteins contain apolipoprotein (apo)1E and apoAI but not apoB (9.Boyles J.K. Zoellner C.D. Anderson L.J. Kosik L.M. Pitas R.E. Weisgraber K.H. Hui D.Y. Mahley R.W. Gebicke-Haerter P.J. Ignatius M.J. et al.J. Clin. Invest. 1989; 83: 1015-1031Crossref PubMed Scopus (444) Google Scholar, 10.Boyles J.K. Notterpek L.M. Anderson L.J. J. Biol. Chem. 1990; 265: 17805-17815Abstract Full Text PDF PubMed Google Scholar, 11.Goodrum J.F. J. Neurochem. 1991; 56: 2082-2086Crossref PubMed Scopus (57) Google Scholar). After peripheral nerve injury, apoE synthesis by resident macrophages increases greatly, and apoE accumulates within the nerve (12.Ignatius M.J. Gebicke-Harter P.J. Skene J.H. Schilling J.W. Weisgraber K.H. Mahley R.W. Shooter E.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1125-1229Crossref PubMed Scopus (496) Google Scholar, 13.Snipes G.J. McGuire C.B. Norden J.J. Freeman J.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1130-1134Crossref PubMed Scopus (224) Google Scholar, 14.LeBlanc A.C. Poduslo J.F. J. Neurosci. Res. 1990; 25: 162-171Crossref PubMed Scopus (65) Google Scholar) supporting the concept that apoE is involved in re-utilization of lipids from degenerating nerves for axonal regeneration and myelin production. The receptors involved in lipoprotein uptake by axons and Schwann cells have not been fully characterized, nor has the uptake and utilization of lipids from lipoproteins for axonal regeneration been directly demonstrated. The low density lipoprotein receptor (LDLR) has been reported to be used by PC12 cells, Schwann cells, and sensory neuronsin vitro for uptake of plasma lipoproteins and endoneural lipoproteins isolated from crushed sciatic nerves (15.Ignatius M.J. Shooter E.M. Pitas R.E. Mahley R.W. Science. 1987; 236: 959-962Crossref PubMed Scopus (170) Google Scholar, 16.Rothe T. Muller H.W. J. Neurochem. 1991; 57: 2016-2025Crossref PubMed Scopus (28) Google Scholar). In vivo, the tips of regenerating axons contain a high concentration of LDLRs (9.Boyles J.K. Zoellner C.D. Anderson L.J. Kosik L.M. Pitas R.E. Weisgraber K.H. Hui D.Y. Mahley R.W. Gebicke-Haerter P.J. Ignatius M.J. et al.J. Clin. Invest. 1989; 83: 1015-1031Crossref PubMed Scopus (444) Google Scholar), and LDLRs are distributed throughout all regions of PC12 cells (15.Ignatius M.J. Shooter E.M. Pitas R.E. Mahley R.W. Science. 1987; 236: 959-962Crossref PubMed Scopus (170) Google Scholar). Thus, the model originally proposed for re-utilization or “salvage” of cholesterol during nerve regeneration involved apoE-containing lipoproteins and LDLRs. More recently, other lipoprotein receptors have been detected in neurons. In adult rats, the LDLR-related protein (LRP), a multifunctional apoE receptor, was found to be highly expressed in neurons of brain and spinal cord (17.Bu G. Maksymovitch E.A. Nerbonne J.M. Schwartz A.L. J. Biol. Chem. 1994; 269: 18521-18528Abstract Full Text PDF PubMed Google Scholar, 18.Ishiguro M. Imai Y. Kohsaka S. Mol. Brain Res. 1995; 33: 37-46Crossref PubMed Scopus (41) Google Scholar) and was shown to modulate hippocampal neurite development (19.Narita M. Bu G. Holtzman D.M. Schwartz A.L. J. Neurochem. 1997; 68: 587-595Crossref PubMed Scopus (94) Google Scholar). LRP was also detected in neuronal cell bodies and proximal processes in adult human brain (20.Moestrup S.K. Gliemann J. Pallesen G. Cell Tissue Res. 1992; 269: 375-382Crossref PubMed Scopus (356) Google Scholar, 21.Wolf B.B. Lopes M.B. VandenBerg S.R. Gonias S.L. Am. J. Pathol. 1992; 141: 37-42PubMed Google Scholar). In cultured hippocampal neurons, LRP is restricted to the somatodendritic domain (22.Brown M.D. Banker G.A. Hussaini I.M. Gonias S.L. VandenBerg S.R. Brain Res. 1997; 747: 313-317Crossref PubMed Scopus (33) Google Scholar). Two other receptors of the LDLR family that are predominantly expressed in the brain, LR11 (SorLA) (23.Hermans-Borgmeyer I. Hampe W. Schinke B. Methner A. Nykjaer A. Susens U. Fenger U. Herbarth B. Schaller H.C. Mech. Dev. 1998; 70: 65-76Crossref PubMed Scopus (60) Google Scholar, 24.Kanaki T. Bujo H. Hirayama S. Tanaka K. Yamazaki H. Seimiya K. Morisaki N. Schneider W.J. Saito Y. DNA Cell Biol. 1998; 17: 647-657Crossref PubMed Scopus (24) Google Scholar, 25.Motoi Y. Aizawa T. Haga S. Nakamura S. Namba Y. Ikeda K. Brain Res. 1999; 833: 209-215Crossref PubMed Scopus (63) Google Scholar) and LR7/8B (apoER2) (26.Yamazaki H. Bujo H. Kusunoki J. Seimiya K. Kanaki T. Morisaki N. Schneider W.J. Saito Y. J. Biol. Chem. 1996; 271: 24761-24768Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 27.Kim D.H. Iijima H. Goto K. Sakai J. Ishii H. Kim H.J. Suzuki H. Kondo H. Saeki S. Yamamoto T. J. Biol. Chem. 1996; 271: 8373-8380Abstract Full Text Full Text PDF PubMed Scopus (349) Google Scholar, 28.Novak S. Hiesberger T. Schneider W.J. Nimpf J. J. Biol. Chem. 1996; 271: 11732-11736Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), have been identified. Since LR11 and LR7/8B are highly expressed in neurons, and LR11 expression is regulated during central nervous system (CNS) development (24.Kanaki T. Bujo H. Hirayama S. Tanaka K. Yamazaki H. Seimiya K. Morisaki N. Schneider W.J. Saito Y. DNA Cell Biol. 1998; 17: 647-657Crossref PubMed Scopus (24) Google Scholar), these receptors have been suggested to be involved in neural organization, synaptic formation, and neurodegenerative diseases such as Alzheimer's disease (29.Schneider W.J. Nimpf J. Bujo H. Curr. Opin. Lipidol. 1997; 8: 315-319Crossref PubMed Scopus (62) Google Scholar). The lipoprotein receptors present in neurons of the peripheral nervous system (PNS) have not yet been rigorously examined although both the LDLR and LRP have been detected in rabbit dorsal root ganglia neurons (30.Handelmann G.E. Boyles J.K. Weisgraber K.H. Mahley R.W. Pitas R.E. J. Lipid. Res. 1992; 33: 1677-1688Abstract Full Text PDF PubMed Google Scholar). A reasonable prediction is that a different spectrum of lipoprotein receptors might be present in CNS and PNS neurons since the classes of lipoproteins that these two types of neurons would encounter are different. For example, in the PNS, distal axons would be exposed to the same types of lipoproteins as in the circulation (i.e. very low density lipoproteins, LDL, and HDL). In contrast, apoB-containing lipoproteins (LDL and very low density lipoproteins) appear to be absent from cerebrospinal fluid. Instead, lipoproteins in the vicinity of CNS neurons are HDL-sized and contain apoE, apoAI, apoD, and/or apoJ (31.Chiba H. Mitamura T. Fujisawa S. Ogata A. Aimoto Y. Tashiro K. Kobayashi K. Neurosci. Lett. 1991; 133: 207-210Crossref PubMed Scopus (23) Google Scholar, 32.LaDu M.J. Gilligan S.M. Lukens J.R. Cabana V.G. Reardon C.A. Van Eldik L.J. Holtzman D.M. J. Neurochem. 1998; 70: 2070-2081Crossref PubMed Scopus (242) Google Scholar, 33.Rebeck G.W. Alonzo N.C. Berezovska O. Harr S.D. Knowles R.B. Growdon J.H. Hyman B.T. Mendez A.J. Exp. Neurol. 1998; 149: 175-182Crossref PubMed Scopus (57) Google Scholar, 34.Yamauchi K. Tozuka M. Hidaka H. Hidaka E. Kondo Y. Katsuyama T. Clin. Chem. 1999; 45: 1431-1438Crossref PubMed Scopus (38) Google Scholar). We have previously shown that cholesterol synthesis is restricted to cell bodies/proximal axons of sympathetic neurons and is not detectable in distal axons (35.Vance J.E. Pan D. Campenot R.B. Bussiere M. Vance D.E. J. Neurochem. 1994; 62: 329-337Crossref PubMed Scopus (94) Google Scholar). When cholesterol synthesis was inhibited by pravastatin, axonal growth was severely impaired. However, normal growth was restored when cholesterol was added to either cell bodies or distal axons (36.Posse de Chaves E.I. Rusinol A.E. Vance D.E. Campenot R.B. Vance J.E. J. Biol. Chem. 1997; 272: 30766-30773Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Serum lipoproteins also restored normal axonal growth to these neurons. When LDL were given to either cell bodies or axons of pravastatin-treated neurons, axonal elongation proceeded normally, presumably because the LDL supplied cholesterol. In contrast, HDL restored axonal growth when added only to distal axons but not to cell bodies. These observations suggest that sympathetic neurons express multiple lipoprotein receptors and that these receptors have a polarized distribution. We now show that lipids can be supplied to sympathetic neurons from lipoproteins for use in axonal growth. Our data are consistent with the model that the uptake of lipoprotein components by these neurons occurs via receptor-mediated endocytosis. We show that the LDLR is present in both cell bodies and axons of rat sympathetic neurons, and we have identified two additional members of the LDL receptor superfamily, LR7/8B (apoER2) and LR11 (SorLA), in these neurons. However, neither LRP (which is present in CNS neurons) nor the HDL receptor, SR-BI, was detected. In addition, the experiments show that lipoprotein receptors are differentially distributed and regulated in cell bodies and distal axons of sympathetic neurons. [1α,2α-3H]Cholesterol (specific activity 45 mCi/mmol) was purchased from Amersham Pharmacia Biotech. Pravastatin was a gift from Dr. S. Yokoyama, Nagoya City University Medical School, Japan. 1,1′-Dioctadecyl-3,3,3′,3′- tetramethylindocarbocyanine perchlorate (DiI) and the Alexa 488 protein labeling kit were from Molecular Probes, Inc. (Eugene, OR). L15 medium without antibiotics was purchased from Life Technologies, Inc. Mouse 2.5 S nerve growth factor was from Alomone Laboratories Ltd. (Jerusalem, Israel). Rat serum was provided by the University of Alberta Laboratory Animal Services. The anti-rat LDLR polyclonal antibody was a gift from Dr. G. Ness, University of South Florida (37.Ness G.C. Lopez D. Borrego O. Gilbert-Barness E. Am. J. Med. Genet. 1997; 68: 294-299Crossref PubMed Google Scholar). A polyclonal antibody directed against the carboxyl-terminal 14 amino acids of the chicken LR7/8B (apoER2) receptor (28.Novak S. Hiesberger T. Schneider W.J. Nimpf J. J. Biol. Chem. 1996; 271: 11732-11736Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) was generously provided by Dr. J. Nimpf, University of Vienna, Austria. The anti-murine SorLA (LR11) polyclonal antibody, raised against a fusion protein containing the fibronectin domain of SorLA, was a gift from Dr. H. Schaller, University of Hamburg, Germany (23.Hermans-Borgmeyer I. Hampe W. Schinke B. Methner A. Nykjaer A. Susens U. Fenger U. Herbarth B. Schaller H.C. Mech. Dev. 1998; 70: 65-76Crossref PubMed Scopus (60) Google Scholar). The anti-SR-BI antibody, directed against a peptide corresponding to amino acids 495–509 from murine SR-BI was kindly provided by Dr. M. Krieger, Massachusetts Institute of Technology (38.Acton S.L. Scherer P.E. Lodish H.F. Krieger M. J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar). The rabbit anti-LRP polyclonal antibody was generated against the 85-kDa fragment of rat LRP and was a gift from Dr. J. Herz, University of Texas Southwestern Medical Center (39.Rohlmann A. Gotthardt M. Hammer R.E. Herz J. J. Clin. Invest. 1998; 101: 689-695Crossref PubMed Scopus (399) Google Scholar). Electrophoresis reagents were supplied by Bio-Rad. Polyvinylidene difluoride membranes were from Millipore. All other reagents were from Sigma or Fisher. Procedures for growing rat sympathetic neurons in compartmented cultures have been previously reported (40.Campenot R.B. Walji A.H. Draker D.D. J. Neurosci. 1991; 11: 1126-1139Crossref PubMed Google Scholar). Briefly, superior cervical ganglia from newborn Harlan Sprague-Dawley rats were dissected and enzymatically and mechanically dissociated. The cells were plated in either the center or the left compartment of three-compartmented culture dishes (36.Posse de Chaves E.I. Rusinol A.E. Vance D.E. Campenot R.B. Vance J.E. J. Biol. Chem. 1997; 272: 30766-30773Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Neurons were cultured for 10–14 days prior to the start of experiments. For measurements of axonal extension, left-plated cultures were used; all other experiments were performed using center-plated cultures. Human LDL (hLDL) and human HDL3 (hHDL3) were isolated from plasma by sequential ultracentrifugation and affinity chromatography (36.Posse de Chaves E.I. Rusinol A.E. Vance D.E. Campenot R.B. Vance J.E. J. Biol. Chem. 1997; 272: 30766-30773Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Lipoprotein preparations were monitored by SDS-polyacrylamide gel electrophoresis to confirm the expected apoprotein compositions and to ensure that hHDL3 did not contain apoE. Chicken HDL (cHDL) was isolated by sequential ultracentrifugation as the fraction of density between 1.12 and 1.21 g/ml (41.Kiss R.S. Kay C.M. Ryan R.O. Biochemistry. 1999; 38: 4327-4334Crossref PubMed Scopus (44) Google Scholar). Lipoproteins were labeled with DiI as described previously (42.Pitas R.E. Innerarity T.L. Weinstein J.N. Mahley R.W. Arteriosclerosis. 1981; 1: 177-185Crossref PubMed Google Scholar). Briefly, 4 mg of hLDL, hHDL3, or cHDL were added to 8 ml of lipoprotein-deficient calf serum. DiI in dimethyl sulfoxide (200 μl, 3 mg/ml) was added, and the mixture was incubated at 37 °C for 18 h. Subsequently, the density was adjusted to 1.063 g/ml for hLDL and 1.21 g/ml for hHDL3 and cHDL, by addition of KBr. Lipoproteins were re-isolated at 4 °C by centrifugation at 150,000 × g for 24 h in a Beckman Ti50.2 rotor. The top 2 ml, containing DiI-labeled lipoproteins, were collected by aspiration and dialyzed against 0.9% NaCl and 0.01% EDTA. Subsequently, DiI-lipoproteins were filtered through 0.45-μm filters, stored under sterile conditions at 4 °C, and used within a month. DiI-lipoproteins were subjected to SDS-polyacrylamide electrophoresis on 3–15% gradient gels. Gels were analyzed visually for DiI fluorescence and Coomassie Blue staining for identification of major apoproteins. In hHDL, bands corresponding to apoAI and apoAII were not labeled with DiI; the same was true for apoAI in cHDL. For hLDL, most fluorescence was at the gel front as free DiI. However, some DiI fluorescence was associated with apoB100 since some lipids remain associated with apoB under these conditions. 2J. Vance, unpublished results. Alexa 488-labeled lipoproteins were prepared according to manufacturer's instructions. Alexa Fluor 488 dye reacts with primary amines of proteins forming stable dye-protein conjugates. In HDL, Alexa 488 binds covalently to apoAI and apoAII (43.Gu X. Trigatti B. Xu S. Acton S. Babitt J. Krieger M. J. Biol. Chem. 1998; 273: 26338-26348Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). SDS-polyacrylamide gel electrophoresis confirmed that apoB, apoAI, apoAII, and chicken apoAI were labeled. All lipoprotein preparations were essentially free of unbound Alexa. Recovery of Alexa fluorescence in the aqueous phase after extraction of Alexa lipoproteins with organic solvents was greater than 95% providing evidence that the label was primarily associated with proteins. Lipoproteins were labeled with [3H]cholesterol as described elsewhere (44.Hara H. Yokoyama S. J. Biol. Chem. 1991; 266: 3080-3086Abstract Full Text PDF PubMed Google Scholar). Briefly, the solvent from 200 μCi of [1α,2α-3H]cholesterol was evaporated to form a thin film. The lipoprotein preparation (3 mg of protein) was added in 0.9% NaCl, and the mixture was shaken overnight at 4 °C, after which lipoproteins were re-isolated by flotation as above. The specific radioactivity of cholesterol (dpm/μg total cholesterol) was determined. Cholesterol mass was determined by an enzymatic kit (Sigma). Typically, the specific radioactivity of cholesterol was 29,700 dpm/μg for hLDL, 150,200 dpm/μg for hHDL3, and 64,100 dpm/μg for cHDL. Neurons were plated in the center compartment of compartmented dishes and cultured for 14 days. DiI or Alexa lipoproteins were added to the cell body- or distal axon-containing compartments as indicated. Lipoprotein concentration in media was normalized to 100 μg of total cholesterol/ml. In addition, fluorescence units were normalized to achieve the same fluorescence in all media by addition of unlabeled lipoproteins. After 18 h, the neurons were washed extensively with cold phosphate-buffered saline containing methylcellulose (3 g/500 ml) and fixed for 20 min in 4% paraformaldehyde. Coverslips were secured usingpara-phenylenediamine in glycerol as mounting medium. Fluorescence microscopy was performed with an Olympus BX-SOF fluorescence microscope equipped with a 100-watt gas mercury lamp and rhodamine filter (for DiI). Alternatively, neurons were analyzed by confocal laser scanning microscopy using a Leica microscope illuminated by a 100-watt mercury burner for direct observation, a Ar/Kr laser with major emissions at 488, 568 and 647 nm for scanning, and 63 × 1.40 NA oil immersion objectives. Figs. 2, 4, and 5 were prepared using Adobe Photoshop software.Figure 4Uptake of fluorescent lipids and proteins from lipoproteins. Center-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 compartment (A, C, E, G, I, andK). Lipoprotein concentration was adjusted to 100 μg 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 conditions, and 50–100 fields were analyzed for each treatment. The experiment was repeated twice with comparable results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Competition of fluorescent lipoprotein uptake by unlabeled lipoproteins. 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 fluorescent 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Neurons were incubated with DiI lipoproteins in the cell body-containing compartment for 18 h and then washed three times with ice-cold phosphate-buffered saline. Cellular material was harvested from the center compartment in phosphate-buffered saline. The detached neurons were sonicated for 10 s with a tip probe. An aliquot was added to 2 ml of methanol, and fluorescence was analyzed in a Hitachi F-2000 spectrofluorometer with excitation light of 550 nm and emission at 565 nm. Protein content was determined using the BCA protein kit (Pierce). DiI did not interfere with the protein determinations. In experiments with [3H]cholesterol-labeled lipoproteins, neurons were given labeled lipoproteins in the cell body-containing compartment. The concentration of lipoproteins was adjusted so that the cholesterol content was 100 μg/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-associated lipoprotein-derived cholesterol was calculated from the specific radioactivity of the lipoprotein [3H]cholesterol. Neurons were plated in the left compartment of compartmented dishes. Distal axons were mechanically 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 medium was added. This procedure, termed axotomy, effectively removes all visible traces of axons from the right-side compartment. Axonal growth was measured as described previously (45.Campenot R.B. Stevenson R.B. Gallin W. Paul D. Cell-Cell Interactions: A Practical Approach. IRL Press at Oxford University Press, Oxford1992: 275-298Google Scholar). Material from distal axon- and cell body-containing compartments was harvested separately in 50 mm Tris-HCl (pH 7.5) containing 150 mm NaCl, 1 mm EDTA, 0.05 mm phenylmethylsulfonyl fluoride. Cellular material was sonicated for 10 s with a probe sonicator and centrifuged at 350,000 × g for 20 min. Membrane pellets were dissolved in 50 mm Tris-HCl (pH 7.5) containing 150 mm NaCl, 1 mm EDTA, 0.05 mm phenylmethylsulfonyl fluoride. The protein content was determined using the BCA protein kit (Pierce). Proteins were separated by electrophoresis on an 8% SDS-polyacrylamide gel for LDLR and SRBI, a 5% gel for LR11 and LR7/8B, and a 3–15% gradient gel for LRP. For LR7/8B, electrophoresis was performed under non-reducing conditions (28.Novak S. Hiesberger T. Schneider W.J. Nimpf J. J. Biol. Chem. 1996; 271: 11732-11736Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Proteins were transferred to polyvinylidene difluoride membranes for 18 h at 25 V (36.Posse de Chaves E.I. Rusinol A.E. Vance D.E. Campenot R.B. Vance J.E. J. Biol. Chem. 1997; 272: 30766-30773Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Membranes were incubated in 20 mm Tris-HCl (pH 7.5), 500 mm NaCl, 0.1% (v/v) Tween 20 (TTBS) with 5–10% (w/v) non-fat dried milk for at least 2 h and then incubated for 1 h at room temperature with primary antibody in TTBS containing 1% non-fat dried milk using the following dilutions of primary antibodies: LDLR, 1:1000; LRP, 1:5000; SR-BI, 1:1000; LR11, 1:1500; and LR7/8B, 1:1500. Membranes were then incubated for 1 h with horseradish peroxidase linked to anti-rabbit IgG secondary antibody (Pierce, diluted 1:10,000), and immunoreactive proteins were detected by enhanced chemiluminescence. We have previously shown that inhibition of cholesterol synthesis by pravastatin impairs axonal elongation of compartmented cultures of rat sympathetic neurons. Addition of cholesterol to axons or cell bodies restored normal axonal growth to pravastatin-treated neurons. Similarly, when human lipoproteins (hLDL, hHDL2, or hHDL3) were supplied to distal axons of pravastatin-treated neurons, normal axonal growth occurred. In contrast, normal axonal elongation occurred only when hLDL, but not hHDL3 or hHDL2, were added to the cell body-containing compartment (36.Posse de Chaves E.I. Rusinol A.E. Vance D.E. Campenot R.B. Vance J.E. J. Biol. Chem. 1997; 272: 30766-30773Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). These studies suggested that cholesterol can be taken up from lipoproteins by these neurons and used for axonal growth. The observations also raised the possibility that LDL and HDL, or the cholesterol therein, are taken up by different mechanisms, potentially involving distinct lipoprotein receptors. We have now extended these studies and analyzed the capacity of hLDL, hHDL3, and chicken HDL (cHDL) to deliver lipids, including cholesterol, for axonal growth of rat sympathetic neurons. hHDL3 lacks apoE and therefore would not be expected to interact with the apoB/apoE receptor (i.e. the LDLR). cHDL was selected because chicken apoAI has been proposed to play a role in nerve regeneration in chickens similar to that of apoE in mammals (46.Dawson P.A. Schechter N. Williams D.L. J. Biol. Chem. 1986; 261: 5681-5684Abstract Full Text PDF PubMed Google Scholar, 47.Rajavashisth T.B. Dawson P.A. Williams D.L. Shackleford J.E. Lebherz H. Lusis A.J. J. Biol. Chem. 1987; 262: 7058-7065Abstract Full Text PDF PubMed Google Scholar, 48.LeBlanc A.C. Foldvari M. Spencer D.F. Breckenridge W.C. Fenwick R.G. Williams D.L. Mezei C. J. Cell Biol. 1989; 109: 1245-1256Crossref PubMed Scopus (23) Google Scholar). Consequently, we expected cHDL and hLDL to be similarly taken up by rat sympathetic neurons. For these experiments, rat sympathetic neurons were plated in the left side compartment of three-compartmented culture dishes. In these cultures (49.Campenot R.B. Methods Enzymol. 1979; 58: 302-307Crossref PubMed Scopus (56) Google Scholar), all compartments contain independent fluid environments, and the cell bodies/proximal axons reside in a compartment completely separate from that containing distal axons. After 14 days, axons in the right side compartment were" @default.
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• W2132983892 date "2000-06-01" @default.
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• W2132983892 title "Uptake of Lipoproteins for Axonal Growth of Sympathetic Neurons" @default.
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• W2132983892 doi "https://doi.org/10.1074/jbc.275.26.19883" @default.
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