Characterization and Use of the NSC-34Cell Line for Study of NeurotrophinReceptor Trafficking

Dusan Matusica,* Matthew P. Fenech, Mary-Louise Rogers, and Robert A. Rush

Centre for Neuroscience, Department of Human Physiology, Flinders University, Bedford Park,South Australia, Australia

This study addressed the suitability of the NSC-34 cellline as a motor neuron-like model for investigating neu-rotrophin receptor trafficking and associated subcellularprocesses. Initially, culture conditions were optimizedfor the use of NSC-34 cells in confocal microscopy.Cell surface markers, as well as markers associatedwith the regulated endosomal pathway thought tobe associated with neurotrophin receptor transport,were identified. The study revealed the presence of anumber of molecules previously not described in theliterature, including the tropomyosin-like receptor ki-nase C (TrkC), sortilin, the vesicular acetylcholinetransporter (VAChT), and the lipid raft-associated gan-glioside GT1b. The presence of both sortilin and Gt1bwas of special interest, insofar as these markers havebeen implicated in direct relationships with the p75NTRreceptor. Evidence is provided for neurotrophin-de-pendent internalization of p75NTR and TrkB. Bothnerve growth factor (NGF) and brain-derived neurotro-phic factor (BDNF) increased the rate of internalizationof p75NTR, with internalization dynamics comparableto those described for other cell lines. Thus, thesestudies not only describe components of the regulatoryprocess governing the trafficking of this important re-ceptor but also clearly demonstrate the value of NSC-34 cells as a suitable motor neuron model for the studyof internalization and trafficking of cell surface mole-cules. VVC 2007 Wiley-Liss, Inc.

Key words: motor neuron; neurotrophin receptors;receptor endocytosis; trafficking

Neurotrophins are involved in several functions inthe developing and mature nervous system, includingsurvival, proliferation, differentiation, myelination, apo-ptosis, axonal growth, and synaptic plasticity (for reviewssee Thoenen, 1995; Kaplan and Miller, 2000; Lee et al.,2001; Huang and Reichardt, 2001). The effects of NGFwere originally discovered in an in vivo model, but invitro cell studies have provided a valuable and reliable,parallel experimental paradigm. Cells used to studyneurotrophin function include the well-characterized ratPC12 pheochromocytoma cell line (Greene and Tischler,1976), various neuroblastoma cell lines (Biedler et al.,

1978; Encinas et al., 2000), and primary neuronal cultures(Delcroix et al., 2003; Ye et al., 2003). Established celllines have the advantage of being relatively easy and inex-pensive to grow and can be readily manipulated, trans-fected, infected, or microinjected for the expression ofintroduced products (Kaplan, 1998). In particular, PC12cells have provided a unique and powerful system forstudying nerve growth factor (NGF) signalling and traf-ficking for the past 3 decades (Greene and Kaplan, 1995)and have been instrumental in the recent development ofthe ‘‘signalling endosome hypothesis’’ (Grimes et al.,1996; Beattie et al., 1996; Howe and Mobley, 2004).

Despite its importance in neuronal physiology anddisease, a very limited number of studies have focusedon neurotrophin trafficking and regulated pathway sort-ing in motor neurons. Major reasons for this trend arethe difficulty of obtaining and growing primary motorneuron cultures (Deinhardt and Schiavo, 2005) and thelack of existing, well-characterized motor neuron-likecell lines equivalent to the PC12 cell. Available methodsfor purifying motor neurons to homogeneity fromrodent spinal cords involve retrograde labeling coupledwith fluorescence-activated cell sorting (Eagleson andBennett, 1983; Calof and Reichardt, 1984) and isolationand purification by panning against p75NTR antibodies(Camu and Henderson, 1992). Both methods are costlyand time consuming and provide only limited numbersof neurons, which rarely survive for longer than 7 daysin culture.

Immortalized and clonally uniform murine neuro-blastoma 3 spinal cord (NSC) hybrid cell lines weredeveloped as a model relevant to the study of motorneuron biology (Cashman et al., 1992). All NSC hybridswere produced through somatic fusion between theN18TG2 aminopterin-sensitive neuroblastoma and motor

*Correspondence to: Dusan Matusica, Centre for Neuroscience, Depart-

ment of Human Physiology, Flinders University, Flinders Drive, Bedford

Park 5042, South Australia, Australia.

E-mail: dusan.matusica@flinders.edu.au

Received 16 April 2007; Revised 8 July 2007; Accepted 12 July 2007

Published online 26 September 2007 in Wiley InterScience (www.

interscience.wiley.com). DOI: 10.1002/jnr.21507

Journal of Neuroscience Research 86:553–565 (2008)

' 2007 Wiley-Liss, Inc.

neuron-enriched embryonic day 12–14 spinal cord cells(Cashman et al., 1992). One of these clones, NSC-34,expresses many of the morphological and physiologicalproperties of motor neurons, including extension ofprocesses, formation of contacts with cultured myotubes,synthesis and storage of acetylcholine (ACh), support ofaction potentials, induction of myotube twitching, andexpression of neurofilament proteins (Cashman et al.,1992). In addition, NSC-34 cells are also able to induceacetylcholine receptor (AChR) clustering upon coculturewith myotubes, suggesting that these cells might be ableto model aspects of early neuromuscular synapse forma-tion (Martinou et al., 1991). It has also been shown thatNSC-34 cells adhere specifically to the leucine-arginine-glutamate (LRE) motif of S-laminin, a specific basallamina glycoprotein concentrated at the neuromuscularsynapse (Hunter et al., 1991). With the availability ofthe NSC-34 cell line, several studies have reported thepresence of numerous motor neuron-specific markers(summarized in Table I).

Developing and mature motor neurons respond tomultiple growth factors, including neurotrophins (forreviews see Thoenen et al., 1993; Oppenheim, 1996;Henderson et al., 1998; Sendtner et al., 2000). Our in-terest is in the study of receptor trafficking within motorneurons, with a view to a better understanding of themechanisms by which the neurotrophins elicit their vari-ous roles. In particular, it is important to determine theinteractions of neurotrophins with the p75NTR receptorand its numerous associated endosomal partners and sig-nalling molecules (see Barker, 2004).

This paper describes the neurotrophin-associatedtrafficking of p75NTR from the cell surface into endo-somal compartments and identifies a number of othercell surface molecules that are both associated with andindependent of p75NTR trafficking. Furthermore, thecharacterization of these markers on NSC-34 cells high-lights the value of this cell line for studies examining therole of neurotrophins in motor neuron function.

MATERIALS AND METHODS

Materials

Dulbecco’s modified Eagle’s medium (DMEM), Ham’sF-12 medium, RPMI 1640 medium, fetal bovine serum(FBS), nonessential amino acids (NEAA), L-glutamine,penicillin-streptomycin-glutamine (PSG), HEPES buffer,DAPI, Alexa Fluor 488 and 568 dyes, Lysosensor Dextran,and Lysotracker Red DN-99 were obtained from InvitrogenLife Technologies (Mulgrave, Australia). Fluorescein isothio-cyanate (FITC) isomer I, (4)-nitrophenyl phosphate disodiumsalt hexahydrate, dimethyl sulfoxide (DMSO), Triton X-100,and retinoic acid (RA) were purchased from Sigma (CastleHill, Australia). Protein-G fast-flow media, recombinanthuman BDNF, NT-3, and NT-4 were purchased fromChemicon (Boronia, Australia). Secondary antibody conjugateswere supplied by Jackson Immunoresearch Laboratories (WestGrove, PA). Vesicular ACh transporter (VAChT) antibody(V-007) was purchased from Phoenix Pharmaceuticals

(Belmont, CA). Sephadex G-25 Fastflow columns and chro-matography reagents were acquired from GE Healthcare Bio-Sciences (Uppsala, Sweden). Tissue cultureware was purchasedfrom Interpath Services (West Heidelberg, Australia). MLR-1,MLR-2, and MLR-3 antibodies against mouse p75NTR wereproduced in our laboratory (Rogers et al., 2006). Polyclonalantibody 9560 against p75NTR was kindly supplied by Dr.Moses Chao (NYU, New York, NY). Gt1b hybridomaclones were a kind gift from Dr. Ronald Schnaar (John Hop-kins University, Baltimore, MD). Purified mouse NGF was akind gift from Dr. Ian Hendry (ANU, Australia). The sheepantibody against HB9 protein was provided by Flinders Tech-nology (Bedford Park, Australia). All other antibodies were pro-duced in our laboratory. Data analysis and graphs were com-piled in GraphPad Prism software (GraphPad, San Diego, CA).

NSC-34 Cell Culture

Neuroblastoma 3 spinal cord cells (NSC-34; kindlyprovided by Dr. Neil Cashman, University of Toronto) weremaintained in Dulbecco’s modified Eagle’s medium (DMEM)supplemented with 10% fetal bovine serum (FBS) and 1%penicillin/streptomycin/glutamine solution (PSG) as previ-ously described (Cashman et al., 1992). Cells were subculturedevery 3–4 days. To slow the proliferation and enhance thedifferentiation, cells were grown to 60% confluence, and themaintenance medium (DMEM, 10% FBS, 1% PSG) wasexchanged for differentiating medium comprising 1:1 DMEMplus Ham’s F12, 1% FBS, 1% PSG, and 1% modifiedEagle’s medium nonessential amino acids (NEAA; previouslydescribed by Eggett et al., 2000). After 24–48 hr of culture inthe differentiating medium, there was a large amount of celldeath. At this stage, the medium was replaced with fresh dif-ferentiating medium, and the remaining populations of cellswere allowed to proliferate for up to �7 days, with freshmedia change every 2 days. Differentiated cells (NSC-34D)survived in the reduced-serum medium and were subsequentlypassaged for up to two times, without loss of viability (Eggettet al., 2000).

Cell Viability Assays Measuring Effects ofLow-Serum Media Conditions

NSC-34 and NSC-34D cells were seeded in 96-wellmicrotiter plates at 1 3 104 per well in 100 ll of DMEMcontaining one of the following: 1% FBS, 2% FBS, or 3%FBS supplemented with 2 mM L-glutamine, 20 mM HEPES,1% NEAA, 1% PSG and incubated in time intervals of 24,48, 72, and 96 hr at 378C at 5.1% atmospheric CO2. Cell via-bility was assessed as described below.

Cell Viability Assays Measuring the Effectsof Neurotrophins

NSC-34 and NSC-34D cells were seeded in 96-wellmicrotiter plates at 1 3 104 per well in 100 ll of DMEMcontaining one of the following: 1% FBS, 2% FBS, or 3%FBS supplemented with 2 mM L-glutamine, 20 mM HEPES,1% NEAA, 1% PSG and incubated for 18 hr at 378C at 5.1%atmospheric CO2. Cells were then incubated in serum-deficient DMEM for 4 hr prior to application of growth fac-

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tors. Cells were treated with NGF, BDNF, NT-3, and NT-4at 200, 100, 50, 20, and 10 ng/ml concentrations in 100 llfresh differentiating media for up to 48 hr.

Acid Phosphatase Hydrolysis Assay

Cell viability for all of the assays described above wasassessed by (4)-nitrophenyl phosphate hydrolysis. The assay is

based on hydrolysis of the p-nitrophenyl phosphate by intra-cellular acid phosphatases in viable cells to produce p-nitro-phenol. Absorbance of p-nitrophenol at 405 nm is directlyproportional to the cell number in the range of 103–105 cells.The assay can quantify as few as 1,000 cells per well in 96-well microtiter plates, as previously described by Yang et al.(1996). Treated plates were gently washed with 100 ll of PBS

TABLE I. Neuronal Properties and Markers of NSC-34 Cell Line*

N18TG2 NSC-34 NSC-34D Method Reference

Growth Factor Receptors

Trk A ND 2 2 Western Blot (Turner et al., 2004)

Trk B 2 1 1 Western Blot (Turner et al., 2004)

p-75NTR 2 1 1 Western Blot (Turner et al., 2004)

NRH2 2 1 1 Western Blot (Turner et al., 2004)

CNTF-r 2 1 1 IF (Usuki et al., 2001)

Gp-130 2 1 ND IF (Usuki et al., 2001)

TNF-aR ND 1 1 Viability Assay (He et al., 2002)

Cytoskeletal Proteins

NF-68 2 1 1 IHC (Cashman et al., 1992)

NF-150 2 1 1 IHC (Cashman et al., 1992)

NF-200 2 1 1 IHC (Cashman et al., 1992)

MAP-1a 2 1 1 IHC (Cashman et al., 1992)

MAP-2 2 2 2 IHC (Cashman et al., 1992)

GFAP 2 2 2 IHC (Cashman et al., 1992)

Vimentin 2 1 2 IHC (Cashman et al., 1992)

b-tubulin III ND 1 1 Western Blot (Rembach et al., 2004)

Cholinergic Markers

Ach 2 1 1 [14C] Choline (Cashman et al., 1992)

ChAT 2 1 1 Fonnum Assay (Cashman et al., 1992)

nAChR 2 1 1 IHC (Rembach et al., 2004)

Gangliosides

GM-1 1 1 1 CT IHC (Matsumoto et al., 1995)

GM-2 1 1 1 IHC (Matsumoto et al., 1995)

GM-3 1 ND ND IHC (Matsumoto et al., 1995)

GD1a 1 1 1 IHC (Matsumoto et al., 1995)

GD1b ND ND ND Western Blot (Matsumoto et al., 1995)

Glutamate Receptor Proteins

NMDAR1 2 2 1 IHC (Eggett et al., 2000)

NMDAR2a/b 2 2 1 IHC (Eggett et al., 2000)

GluR1 2 2 1 IHC (Eggett et al., 2000)

GluR2/3 2 2 1 IHC (Eggett et al., 2000)

GluR2 2 2 1 IHC (Eggett et al., 2000)

GluR4 2 2 1 IHC (Eggett et al., 2000)

KA2 2 2 1 IHC (Eggett et al., 2000)

Cell Adhesion Molecules (CAMs)

L1 1 1 ND mRNA/PCR (Moscoso and Sanes, 1995)

Nr-CAM/Bravo 2 1 ND mRNA/PCR (Moscoso and Sanes, 1995)

Neurofascin/ABGP 1 1 ND mRNA/PCR (Moscoso and Sanes, 1995)

N-CAM 2 1 ND mRNA/PCR (Moscoso and Sanes, 1995)

Neuronal Nuclear Markers

NeuN-nuclear marker ND 1 1 Western Blot (Rembach et al., 2004)

Other Neuronal Properties

Process extension 2 1 1 PCM (Cashman et al., 1992)

Myotube twitch induction 2 1 1 dbcAMP add. (Cashman et al., 1992)

Action potential generation 2 1 1 ES (Cashman et al., 1992)

AchR clusters on myotubes 2 1 ND Co-culture (Cashman et al., 1992)

Ach release 2 ND ND [14C] Choline (Cashman et al., 1992)

*ND, not determined; N18TG2, parent neuroblastoma, NSC-34D, differentiated NSC-34 cells; IF, immuno-

fluorescence; IHC, immunohistochemistry; CT, cholera toxin; ES, electrical stimulation; PCM, phase contrast

microscopy.

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three times and then incubated with 100 ll of 5 mM p-(4)-nitrophenyl phosphate, 0.1 M sodium acetate, 0.1% (vol/vol)Triton X-100, pH 5.0, for �30 min to 1 hr at 378C and5.1% atmospheric CO2. Cell viability was expressed as a per-centage of untreated cell cultures (100%).

Conjugation of Monoclonal AntibodiesWith Fluorophores

Antibodies were concentrated using a Nanosep 100Kcentrifugal device (Pall) to a final concentration >2.0 mg/ml,and conjugations with FITC (Sigma Aldrich, Australia) andAlexa Fluor 488 and 568 (Invitrogen, Australia) were per-formed according to the manufacturer’s instructions. Conju-gates that stained intensely on positive cells and were negativeon negative cells were used for the experiments.

Immunofluorescence Staining and SortingFlow Cytometry

All procedures were carried out at 48C (or on ice) andin the dark. Single cell suspensions were prepared in 0.15 MPBS, 2% FBS, 10 mM HEPES, at a concentration of 1 3 107

cells/ml. One hundred microliters of cell suspension were ali-quoted into 5-ml polystyrene (FACS) tubes. Primary antibod-ies including fluorescent conjugates were added at �10–20lg/ml to the cells and mixed gently, followed by incubationfor 30–60 min. Cells were washed twice by adding 2 ml 0.15 MPBS, 0.2% BSA, 10 mM HEPES, 0.02% NaN3, and centri-fuged for 5 min at 1,600 rpm (300g). Cells were resuspended,and appropriate secondary antibodies (when required) wereadded at 1:400–1:1,000, followed by a further incubationfor 30–60 min. Stained cell pellets were resuspended in 0.15M PBS, 0.2% BSA, 10 mM HEPES, 0.02% NaN3 for flowcytometry. Twenty microliters of propidium iodide (PI) so-lution was added 5 min prior to analysis to detect deadcells.

Effects of Neurotrophins on Antibody Binding

The experimental procedure was carried out asdescribed above with the addition of a 1-hr preincubationstep with NGF, BDNF, NT-3, or NT-4 at 100 ng/ml addedto antibody treatment groups. Counting events of viable cellswere gated at 20,000, and mean fluorescence measurementswere plotted against antibody-treated samples not treated withneurotrophins.

Western Blotting

Cells were lysed using chilled lysis buffer containing10 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1% (vol/vol)Triton X-100 with 1% (vol/vol) protease inhibitor cocktailfor 10 min on ice. Lysates were solubilized in an equal vol-ume of 23 SDS sample buffer containing 4% (wt/vol) SDS,2% (wt/vol) glycine, 0.015% (wt/vol) bromophenol blue,20% (vol/vol) glycerol, and 10% (vol/vol) b-mercaptoethanolin 100 mM Tris-HCl buffer, pH 6.8. Cell lysates were elec-trophoresed through 12.5% Tris-glycine-buffered SDS gels.Proteins from the gels were electroblotted onto nitrocellulosemembrane at 30 V for 1 hr. The membranes were blocked in4% (wt/vol) skim milk powder, 0.1% (vol/vol) Tween-20,

and 0.02% (wt/vol) NaN3 in Tris-buffered saline (TBS), pH8.0, for 1 hr at room temperature and then incubated over-night with primary antibodies at 48C. Neuron-specific anti-bodies in this study included the tyrosine kinase receptor(TrkB, 5 lg/ml); TrkC (Ab07-226, 5 ll/ws); p75NTR ratspecific antibody (Ab9560 2 lg/ml); p75NTR specific anti-bodies MLR-1, MLR-2, and MLR-3 (2 lg/ml); sortilin(Ab16640, 2 lg/ml); choline acetyltransferase (ChAT, 2 ll/ws); vesicular acetylcholine transporter (VAChT, V-007,2 lg/ml); gangliosides GT1b (AbGt1b, 2 lg/ml) and GQ1b(MAb321, 2 lg/ml); HB9 (5 ll/ws); and BDNF (5 lg/ml).Blots were then washed three times in Tris-buffered saline-Tween 20 (TBST), pH 8.0, for 10 min and incubated for1 hr with appropriate horseradish peroxidase (HRP)-conju-gated secondary antibodies (1:2,500) in TBS at room tempera-ture and then washed three times in TBS for 10 min. Blotswere developed with enhanced chemiluminescence (ECL).

NSC-34 Immunoendocytosis Assay

Cells were cultured on poly-D-lysine/collagen/laminin-coated coverslips in 24-well plates as described above but inDMEM/F-12 medium containing no phenol red. After prein-cubation for 1 hr at 378C with incubation buffer (DMEM/F-12 HEPES, 1 mg/ml BSA) to deplete endogenous transferrinand growth factors, cells were treated with primary antibodiesfor 1 hr at 48C in the presence of (100 ng/ml NGF orBDNF). Cells were then washed with ice-cold incubationmedia three times and incubated with secondary antibodiesand Lysosensor Blue/Green or Lysotracker Red DN-99 for afurther 1 hr at 48C. Cells were subsequently washed with ice-cold incubation media, followed by prewarmed incubationbuffer for 15–180 min at 378C in the presence of 100 ng/mlNGF or BDNF, followed by three washes in ice-cold PBSand fixation in 3% paraformaldehyde for 20 min. The fixationreaction was quenched in 0.1 mM glycine for 10 min. Thepresence of vesicles positive for p75 (MLR-3, 3 lg/ml) andTrkB (Trk-B, 5 ll/ws), or both, was quantified by measuringthe fluorescence intensity levels for each dye (FITC and Cy-3) along a line through the respective vesicles. Results werepresented as a percentage of total intracellular vesicles contain-ing the receptors per cell; 8–12 different cells were analyzedper time point in each experiment. Confocal imaging with anOlympus AX70 microscope fitted with a krypton/argon laser.Analysis was performed by using Bio-Rad 1024 LaserSharpsoftware.

RESULTS

Effects of Low-Serum Media on NSC-34 Cells

NSC-34 cells can be identified as two morphologi-cally distinct populations: parent-like neuroblastic (N-type) cells with minimal projections and substrate adher-ent (S-type) cells that undergo differentiation and havelong processes. NSC-34 cells differentiate in low (1%)FBS-supplemented media (Cashman et al., 1992). How-ever, this differentiation is preceded by a massive loss ofcells (Eggett et al., 2000), with the surviving cells sprout-ing extensive neuron-like projections over 5–8 days(NSC-34D).

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Seeding NSC-34 cells on substrate-coated cover-slips (poly-D-lysine and/or rat tail collagen) under thelow-serum conditions had negative effects on cell adhe-sion to the substrate, in many cases not even permittinggentle wash cycles between various treatments. Thisproblem was further complicated by the need to washout the dead cells after the first 48 hr of low-serumtreatment. Because low-serum conditions are necessaryfor the culture of motor neuron-like cell morphology,we tested the viability of NSC-34 cells in 1–3% FBS-containing media, paying particular attention to cell ad-hesion and sprouting of neuron-like processes at timeintervals up to 96 hr. After culture in normal growthmedia (10% FBS), cells were seeded at 1,000 cells perwell in 96-well microtiter plates in either normal growthmedia or media containing 1%, 2%, or 3% FBS(Fig. 1A). Culture with 1% or 2% FBS-supplementedmedia resulted in a significant loss of viable cells in thefirst 48 hr of treatment (*P < 0.005, paired t-test), withsigns of cell viability recovery by 96 hr posttreatment.

Treating cells with 3% FBS supplemented media, how-ever, almost completely reduced serum-withdrawal-asso-ciated cell death within the first 48 hr, including theenhancement of the sprouting of neuron-like processes(Fig. 1C). Increasing the serum content of culture mediafrom 1% to 3% also significantly increased the adhesionof these cells to poly-D-lysine and/or rat tail collagen,making it possible to treat cells with various experimen-tal procedures.

Analysis of NSC-34 Cell Surface andCytosolic Proteins

NSC-34 cells express a number of standard markersfor motor neurons. Neurotrophin receptors p75NTR,TrkB, TrkC and ciliary neurotrophic factor receptor-a(CNTFR-a) were identified. The most recently in-cluded member of the neurotrophin receptor family, thesortilin receptor, was also identified. The motor neuroncholinergic marker VAChT and cell membrane lipid raft

Fig. 1. Effects of low-serum growth mediaconditions on NSC-34 viability. A: Cell via-bility in NSC-34 populations treated withnormal (10% fetal calf serum) growth mediavs. low-serum media conditions was com-pared in 24-hr intervals for up to 96 hr (n 56, mean 6 SEM); 1% and 2% FCS-supple-mented media resulted in a significant loss ofviable cells in the first 48 hr of treatment(*P < 0.005, paired t-test difference fromnormal growth conditions), with signs of cellviability recovery by 96 hr posttreatment. B:Growth rate of NSC-34 cells treated withnormal and low-serum media. Cell numberwas calculated by detaching cells at indicatedtime points, and counts were performed witha hemocytometer (n 5 3, mean 6 SEM). C:Cells grown in 10% FBS are mainly neuro-blastic (N-type, white arrowheads), withoccasional substrate-adherent cells (S-type,white arrows). Treating cells with 1% FBSmedia resulted in serum-withdrawal-associatedcell death, but the surviving cells have ahigher population of S-type cells, with neu-rites starting to sprout; 3% FBS-supplementedmedia reduced serum-withdrawal-associatedcell death, resulted in a high ratio of S-typecells, and enhanced the sprouting of neuron-like processes. Maintenance of cells in 3%FBS media for 7 days results in extension oflong processes (black arrows) with visiblegrowth cones (black arrowheads). Scale bar 550 lm.

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ganglioside subtypes Gt1b, which appeared to beexpressed at higher levels (median cell counts), andGQ1b were also detected (Fig. 2). Flow cytometrymeasurements represent only viable gated populations.X-63FITC control IgG (Fig. 2, solid line) was includedas the negative control for all experiments.

Western blot analysis confirmed the presence ofneurotrophin receptors detected by flow cytometry in-cluding p75NTR and sortilin; however, the TrkB andTrkC receptors expressed are predominantly the trun-

cated isoforms at �100 kDa (Fig. 3A), but with somefull-length (�140 kDa) protein being detected. Analysisof both normal and differentiated NSC-34 cells revealedthe presence of endogenous BDNF (Fig. 3B). Two

Fig. 3. Western blot analysis of neurotrophin receptors and intracel-lular transport proteins. A: NSC-34 (10% FBS media) and NSC-34D(3% and 1% FBS media) cells express a neurotrophin receptor profileconsistent with developing motor neurons, including the presence ofp75NTR, TrkB, TrkC, and sortilin, but no TrkA (data not shown).TrkB and TrkC receptors are present in both full-length (140-kDa)and truncated (100-kDa) isoforms. The presence of BDNF (B), butno NGF, NT-3, or NT-4 (data not shown), was detected. BDNFwas detected at 26 kDa, indicating the homodimer, and also at35 kDa, consistent with prepro-BDNF. Both isoforms of BDNFwere detected in normal (NSC-34) and differentiated (NSC-34D)cells, but only the homodimer was detected in these cells after 7 daysof culture. NSC-34 cells express motor neuron-specific markers HB9and ChAT in cells lysates from normal (10%) and low-serum (1–3%)cultures. Other important motor neuron markers such as the cholin-ergic marker VAChT and ganglioside Gt1b are expressed. C: Criticalmarkers of the endocytic pathway including EEA-1, clathrin heavychain (clathrin-HC), adaptor protein-1 (AP-1), and the lysosomalmarkers Lamp-1 and Limp-II were also shown to be present.

Fig. 2. Flow cytometry fluorescent analysis of NSC-34 cell surfacemarkers. NSC-34 cells express a number of markers for motor neu-rons. Neurotrophin receptors p75NTR (MLR-2, 20 lg/ml), tyrosinekinase B (Trk-B, 10 ml/ws), tyrosine kinase C (Trk-C; Ab07–226,20 lg/ml), and ciliary neurotrophic factor receptor-a (CNTFR-a)were identified. The most recent member of the neurotrophin fam-ily, the sortilin receptor, was also identified (Ab16640, 20 lg/ml).The motor neuron cholinergic marker vesicular acetylcholine trans-porter (VAChT; V-007, 20 lg/ml) and cell membrane lipid raftminor ganglioside subtypes Gt1b (AbGt1b, 20 lg/ml) and GQ1b(MAb321, 20 lg/ml) were also detected. Flow cytometry measure-ments represent only viable gated populations. X-63 FITC (solidline)-conjugated control IgG represents negative control for allexperiments. X-axis represents FITC log fluorescence intensity, andY-axis represents number of FITC-positive cells (dashed line).

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bands were detected that correlate with the known mo-lecular weights for the BDNF homodimer and prepro-BDNF at 26 kDa and 35 kDa, respectively. Interestingly,the prepro-BDNF was detected only in the early stagesfollowing subculture and was absent in cells cultured for7 days (Fig. 3B). NGF, NT-3, and NT-4 were notdetected in any NSC-34 or NSC-34D lysates or cellmembrane preparations (data not shown).

In addition to the many neuronal antigens identi-fied in both the Western blot and FACS analyses, thepresence of motor neuron-specific markers HB9 andChAT confirmed the motor-neuronal phenotype ofNSC-34 cells grown under standard and serum-reducedconditions (Fig. 3C). Also detected were intracellularmarkers of the regulated traffic pathways, including thesynaptic vesicle marker VAChT and the ganglioside lipidraft component Gt1b not previously reported in theNSC-34 cells. Components of the clathrin-mediatedendocytosis pathways associated with neurotrophin sig-nalling events such as clathrin (heavy chain), adaptorprotein AP-1, early endosomal autoantigen-1 (EEA-1),and lysosomal markers Lamp-1 and Limp-II were alsodetectable in cytosolic lysates (Fig. 3C).

Effect of Neurotrophins on Normal andDifferentiated NSC-34 Cells

To establish that neurotrophin receptors expressedon both NSC-34 and NSC-34D cells were physiologi-cally active and sensitive to their respective neurotro-phins, cells were exposed to these ligands in concentra-tions ranging from 10 to 200 ng/ml for 48 hr. 4-N-nitrophenol reduction in acid phosphatase, as an indexof cell viability, was then assessed (Fig. 4). Treatment ofNSC-34 cells with neurotrophins failed to produce anysignificant effects within 48 hr, with the exception of aslight increase in viability at 10 ng/ml of BDNF treat-ment (Fig. 4A,B). Significant dose-dependent cell deathin 48 hr of 55–60% after addition of 200 ng/ml of NGFwas observed in NSC-34D but not NSC-34 cells(P < 0.01 difference compared with untreated controls).However, the rate of cell death did not reach 40% inany individual experiment (Fig. 4C). There was a smallbut significant increase in proliferation with BDNF (Fig.4C) and NT-4 (Fig. 4D) treatments at 10 ng/ml (P <0.05 difference compared with untreated controls) butnot at higher concentrations. NT-3 treatment (Fig. 4D)elicited a pattern similar to that of NGF but not as sig-nificant a response at 200 ng/ml (P < 0.05 differencecompared with untreated controls), with no cell deathobserved over 40%.

Effects of NGF and BDNF on Binding ofp75NTR, TrkB, and Gt1b Antibodies

To investigate the effect of NGF and BDNF onthe binding ability of p75NTR, TrkB, and Gt1b anti-bodies, NSC-34D cells were grown in 3% FBS-contain-ing media for 5 days and incubated in serum-free mediafor 1 hr to remove any growth factors before experi-

Fig. 4. Effects of neurotrophins on NSC-34 and NSC-34D cell sur-vival. NSC-34 and NSC-34D cells were treated with 10–200 ng/mlconcentrations NGF or BDNF (A,C), NT-3, or NT-4 (B,D) in for48 hr. Treatment of NSC-34 cells with neurotrophins under normal-serum conditions (10% FBS) failed to produce any significant effects inthese cells within 48 hr, with the exception of slight increase in viabilityat 10 ng/ml of BDNF treatment (A). In NSC-34D cells, high doses ofNGF and NT-3 induced significant cell death, whereas BDNF andNT-4 stimulated marginal proliferation at small doses (C,D). Cell via-bility under these conditions was assessed by acid phosphatase reduction(n 5 5, mean6 SEM, *P < 0.05, **P < 0.01 vs. untreated cells).

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mental treatment. Cells were then treated in 1% BSA-containing media with either the control antibody X-63or antibodies to p75NTR, MLR-2 (Fig. 5A), or MLR-3 (Fig. 5B), TrkB (Fig. 5C), and Gt1b (Fig. 5D) aloneor in coincubation with 100 ng/ml of NGF or BDNFfor 1 hr on ice. Antibody binding was determined byusing median fluorescence of individual cell countsdetermined by flow cytometry (n 5 4, mean 6 SEM, P< 0.05, NGF, BDNF, NT-3, or NT-4 vs. antibody-only treated cells). Both NGF and BDNF failed to pro-duce significant effects on MLR-2 (Fig. 5A), TrkB (Fig.5C), and Gt1b (Fig. 5D) antibodies, indicating that NGFand BDNF do not actively compete for receptor bindingsites with MLR-2, TrkB, or Gt1b antibodies. However,coincubation of NGF (100 ng/ml) in the presenceof p75NTR antibody MLR-3, produced a significantincrease in median fluorescence counts (B; P < 0.05, vs.NGF untreated cells), indicating that the presence ofNGF could be responsible for the increase in binding ofMLR-3 antibody.

Time-Lapse Ligand-Dependent and-Independent Internalization of p75NTR

To test whether mouse monoclonal antibodyMLR-3 was suitable as an intracellular probe for NGF-and BDNF-dependent internalization, MLR-3 FITCwas incubated on NSC-34D cells in the presence or ab-sence of NGF and BDNF for up to 90 min at 378C.Treatment with MLR-3 was able to induce internaliza-tion of p75NTR in the absence of neurotrophins(Fig. 6A,D). However, the time for internalization wasfaster in the presence of 100 ng/ml of NGF (Fig. 6B,D) orBDNF (Fig. 6C,D). The treatment with NGF appearedalso to increase the total surface and intracellular stainingwith MLR-3 FITC, compared with BDNF treatment(Fig. 6D), but this was not quantified. Furthermore, therate of p75NTR internalization in the presence of NGFwas slightly faster compared with BDNF. The faster rateof internalization of p75NTR in the presence of NGFand MLR-3 compared with BDNF and MLR-3 couldindicate a possible synergistic effect of the antibodyinteracting specifically with NGF but not BDNF tointernalize the receptor-ligand complex. Internalizationwas defined as any subcellular surface staining in a subsetof randomly selected cells (n 5 12).

Time-Lapse Ligand-Dependent and-Independent Internalization of TrkB

To test whether rabbit polyclonal antibody to TrkBwas suitable as an intracellular probe for NGF- andBDNF-dependent internalization, anti-TrkB was incu-bated on NSC-34D cells in the presence or absence of100 ng/ml NGF or BDNF for up to 90 min at 378C.As with the p75NTR antibody, anti-TrkB was able toinduce internalization of TrkB in the absence of neuro-trophins (Fig. 7A,D); however, the time of internaliza-tion was increased with the addition of 100 ng/ml of

Fig. 5. Binding of p75NTR (MLR-2 and -3), TrkB, and Gt1b anti-bodies in the presence of neurotrophins. The binding of antibodiesalone or in the presence of 100 ng/ml NGF, BDNF, NT-3, or NT-4 in NSC-34D cells was analyzed by flow cytometry. A: MLR-2FITC (10 lg/ml). B: MLR-3 FITC (10 lg/ml). C: Anti-TrkB(20 ll/whole serum 1 anti-rabbit FITC secondary 1:500). D: Anti-Gt1b FITC (10 lg/ml; median fluorescence measured by flowcytometry; n 5 4). X-63 is a control IgG. No significant statisticaldifference was observed with MLR-2 (A), TrkB (C), or Gt1b (D);however, MLR-3 binding was significantly increased with the addi-tion of 100 ng/ml NGF (n 5 4, mean 6 SEM, *P < 0.05, vs. NGFand BDNF untreated cells).

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NGF (Fig. 7B,D), but decreased with the addition of100 ng/ml BDNF (Fig. 7C,D). The addition of BDNFdid not appear to increase the binding and rate of inter-nalization of anti-TrkB, compared with untreated cells

(Fig. 7D). One notable observation was the behavior ofanti-TrkB under NGF treatment. Anti-TrkB internaliza-tion decreased when treated with NGF compared withuntreated cells. Internalization was defined as any subcel-lular surface staining in a subset of randomly selectedcells (n 5 12).

Fig. 7. A–D: Time-lapse ligand-dependent and -independent inter-nalization of neurotrophin receptor TrkB. Anti-TrkB sera were ableto induce ligand-independent internalization of TrkB in NSC-34Dcells (A,D). The presence of 100 ng/ml NGF (B,D), and BDNF(C,D) resulted in altered internalization rates of the TrkB receptor.The presence of BDNF increased the speed of TrkB internalizationbut did not appear to increase the cell surface binding, comparedwith untreated cells (D). One notable observation was the behaviorof TrkB in the presence NGF. The speed TrkB internalizationdecreased when treated with NGF compared with untreated cells.Internalization was defined as any subcellular surface staining in asubset of randomly selected cells (n 5 12). Scale bar 5 50 lm.

Fig. 6. A–D: Time-lapse ligand-dependent and -independent inter-nalization of p75NTR. Monoclonal antibody MLR-3 FITC was ableto induce ligand-independent internalization of p75NTR (A,D), butthe time of internalization was faster with the addition of 100 ng/mlNGF (B,D) or BDNF (C,D). The addition of NGF appeared toincreased total surface binding and rate of internalization of MLR-3FITC, compared with BDNF treated cells (D), even thoughp75NTR has similar affinities for both ligands. The difference ofp75NTR internalization rates in NGF- compared with BDNF-treated cells could possibly indicate a synergistic effect of the MLR-3antibody with NGF on the p75NTR internalization. Internalizationwas defined as any subcellular surface staining in a subset of randomlyselected cells (n 5 12). Scale bars 5 50 lm.

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DISCUSSION

In the present study, we used an existing, readilyavailable and partially characterized cell line NSC-34(Cashman et al., 1992), to determine its suitability foruse in the study of neurotrophin receptor trafficking inmotor neurons. The use of the NSC-34 cell line wasbased on many of its inherent morphological and physi-ological properties resembling motor neurons in neuro-nal development (summarized in Table I).

Although the growth and maturation under low-serum (1% FBS) conditions for the NSC-34 cells havebeen described previously (Hunter et al., 1991; Cashmanet al., 1992; Durham et al., 1993), we experienced thesame difficulties in maintaining cell adhesion and cellpopulation numbers while duplicating these protocols.The significant cell death and retarded cell division ofNSC-34 cells after their removal from 10% serum couldbe tolerated, but the severe reduction in cell adhesionwas not suitable for immunofluorescence and live-cellimaging studies with multiple experimental steps.

These difficulties were overcome by increasing theamount of FBS in the maturation media from 1% to 3%.The treatment of NSC-34 cells with 3% FBS-supple-mented media almost completely reduced the amount ofserum-withdrawal-associated cell death and increased theoverall cell adhesion on an identical growth matrix, butdid not effect the sprouting of neuritic processes, a mor-phological marker of neuronal cell maturation and differ-entiation.

As would be predicted, Western blot and flowcytometry results confirmed a neurotrophin receptorprofile typical of a developing motor neuron with thecommon neurotrophin receptor p75NTR and tyrosinekinase receptors TrkB and TrkC present, but the Trk Areceptor was not expressed (Yan et al., 1993). NSC-34 cells also express the sortilin receptor, whichwas detected in a cell lysate preparation but showed arelatively weak signal when detected by fluorescence-activated cell counting.

This observation appears consistent with reportsthat sortilin is expressed primarily in the cytoplasm(Petersen et al., 1997), with significantly fewer receptorsbeing expressed on the extracellular membrane (Nielsenet al., 1999). Although sortilin has not previously beenreported in NSC-34 cells, its presence has been detectedby immunohistochemistry in rat spinal cord lysates(Petersen et al., 1997) and cultured embryonic motorneurons, localized in the cell body and the neurites(Domeniconi et al., 2006). Furthermore, sortilin is notdetected in PC12 cells (unpublished observation), whichhave served as a reliable model in the study of p75NTRtrafficking (Saxena et al., 2004). Sortilin has recentlyemerged as a coreceptor for p75NTR for pro-NGF(Nykjaer et al., 2004)- and pro-BDNF (Teng andHempstead, 2004)-mediated apoptotic signalling. How-ever, completely opposing reports have already emerged(Fahnestock et al., 2004) regarding the functional mech-anisms and biological outcomes of this receptor-ligandcomplex. To date, no neuron-like cell lines that endoge-

nously express both p75NTR and the sortilin receptorhave been used to study this key interaction. Becausethese two receptors appear to be involved in mediatingcell death via proneurotrophin activation, and this is anemerging field in the study of neurotrophins, this cellline could be useful.

The motor neuron-like neurotrophin receptor pro-file of the NSC-34 cells is further validated by the pres-ence of the motor neuron-specific markers HB9 andChAT. Homeodomain factor HB9 is critical in establish-ing motor neuron identity and differentiation (Arberet al., 1999). Developing motor neurons lacking HB9have a significantly altered genetic profile, more typicalof interneurons (Thaler et al., 1999). Motor neurons arethe first population of spinal neurons to express ChAT,and this enzyme is readily detected in motor neuronsfrom embryonic day 13 onward (E13; Phelps et al.,1990).

Other important extracellular and intracellular markersimplicated in neurotrophin receptor trafficking were alsoidentified. These included two members from the gan-glioside b-series family, Gt1b and GQ1b, which aredirect analogues of GD1b. Gangliosides, are broadly dis-tributed in the nervous system and are sometimes associ-ated with clinical symptoms that imply selective nervedamage (Allende and Proia, 2002; Gong et al., 2002).They have been reported to act as functional nerve cellligands involved in inhibition of nerve regeneration(Vyas et al., 2002) but have also been implicated in thebinding and transport of tetanus (Sinha et al., 2000) andcholera toxins (Torgersen et al., 2001). Members of theb-series family of gangliosides have been implicated asessential components of the tetanus toxin receptor com-plex (Kitamura et al., 1999), and the tetanus toxin frag-ment TeNT has been reported to associate withp75NTR in lipid raft-mediated endocytic transport (Lalliand Schiavo, 2002). A partial characterization of NSC-34 gangliosides has been previously described (Matsu-moto et al., 1995), but the presence of Gt1b and GQ1bwas not examined.

The essence of the neurotrophin hypothesis rests inneurotrophins supplied in limited quantities by the targettissue being able to bind to their receptors at the nerveterminal and subsequently elicit a molecular response atthe cell body following retrograde transport of the neu-rotrophin-receptor complex (Sofroniew et al., 2001).Physiological markers of interest in intracellular traffick-ing, in particular markers of the regulated endocyticpathway, such as clathrin, AP-1, EEA-1, and acidic or-ganelle/lysosomal markers Lamp-1 and Limp-II, wereconfirmed in cellular lysate preparations.

Of particular interest is the presence of both pro-and mature forms of BDNF in NSC-34 cell lysates.BDNF is well recognized as a neurotrophic factor formotor neurons. However, there is also evidence forBDNF synthesis within developing (Buck et al., 2000)or injured (Kobayashi et al., 1996; Qin et al., 2006) ratmotor neurons. Thus, the detection of BDNF proteinwithin the NSC-34 cells is not surprising and is consist-

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ent with a possible autocrine role for motor neurons(Lindsay, 1996).

To determine whether NSC-34 cells were suitablecandidates for the study of neurotrophin receptor traf-ficking and to confirm that neurotrophin receptorsexpressed on both immature and mature NSC-34 cellswere physiologically active and sensitive to the neurotro-phins NGF, BDNF, NT-3 and NT-4, cells wereexposed to these ligands for 48 hr. A significant dose-dependent cell death in 48 hr of 55–60% at 200ng/ml ofNGF in NSC-34D occurred, but not in NSC-34 cells,supporting a previous report that NSC-34D cells lack theTrkA receptor (Turner et al., 2004) typical of motorneurons but expresses biologically active and functionalp75NTR receptor, which may be involved in mediatingcell death through NGF activation.

This study has detected both full-length and trun-cated forms of the TrkB receptor in Western blots.Truncated isoforms of TrkB receptors that lack a tyro-sine kinase domain (Middlemas et al., 1991) predominatein the adult mammalian CNS, yet their roles remaincontroversial. Coexpression of the full-length TrkB to-gether with the truncated TrkB.T1 isoform results indecreased levels of full-length TrkB on the cell surface(Haapasalo et al., 2002). This effect is specific to the T1isoform, because coexpression of a kinase-inactive TrkBmutant or another kinase domain-lacking TrkB form,truncated T-Shc, leads to increased surface levels ofTrkB (Haapasalo et al., 2002). The presence of a cur-rently unidentified isoform of truncated TrkB and subse-quent down-regulation of the full-length TrkB receptorresponsible for mediation of survival and differentiationcould explain the insignificant responses of NSC-34 cellsto BDNF and NT-4. The increase in proliferationresulting from BDNF and NT-4 treatments at 10 ng/ml,but not higher concentrations, possibly indicates a smallamount of intrinsic ligand activity consistent with therole of these neurotrophins in motor neurons (Hugheset al., 1993). NT-3 treatment elicited a smaller responseat 200 ng/ml than NGF at equal doses. However, it isnot clear from the current experiments whether it isp75NTR activation or other mechanisms downstream ofTrkC receptor signalling that are responsible for thissmall effect.

Despite the lack of a highly significant responseregarding proliferation and morphological differentiationin immature and mature cells at high neurotrophin con-centrations, some response was evident at low doses ofcells treated with BDNF and NT-4. This was initiallysurprising, considering that BDNF supports the survivalof motor neurons in vitro (Hughes et al., 1993), duringthe period of naturally occurring cell death (Oppenheimet al., 1993), after axotomy (Yan et al., 1992) or rootavulsion (Novikov et al., 1997). Both NT-3 and NT-4support motor neurons in vitro (Hughes et al., 1993),during the period of naturally occurring cell death(Oppenheim et al., 1993) and after axotomy (Vejsadaet al., 1995). However, the ratio of full-length and trun-cated isoforms of the TrkB and TrkC receptors has not

been quantified, and the presence of endogenous BDNFwas detected, so it is plausible that these factors contrib-ute to the lack of cellular responses typically associatedwith these neurotrophins in motor neurons.

One important requirement for the study of recep-tor internalization in the presence of the native ligand isthe ability of the antibody and the ligand to bind to thereceptor simultaneously and the ability of that receptorto carry out its function. To investigate the interactionsof our antibodies to p75NTR and TrkB with neurotro-phins, we tested the ability of the antibodies to bind inthe presence of neurotrophins. NSC-34D cells preincu-bated with NGF, BDNF, NT-3, or NT-4 for 1 hr didnot significantly affect the cell surface binding ofp75NTR and TrkB antibodies. This result indicates thatthere is very little, or no, hindrance to the binding ofthese specific antibodies to their receptors when neuro-trophins occupy their binding sites.

To test whether our mouse monoclonal MLR-3and polyclonal TrkB antibodies were suitable as intracel-lular probes for the study of neurotrophin receptor-mediated NGF- and BDNF-dependent internalization,we tested the ability of each antibody to internalize inNSC-34D cells in the presence as well as absence ofNGF and BDNF. MLR-3 was able to induce ligand-in-dependent internalization of p75NTR, but thetime of internalization was decreased with the additionof 100 ng/ml of NGF or BDNF. The addition of NGFappeared to increase binding and rate of internalizationof MLR-3 FITC, compared with BDNF, even thoughp75NTR has similar affinities for both ligands. Theslower rate of p75NTR internalization under BDNFcould indicate a possible synergistic effect of MLR-3antibody with NGF.

As with the p75NTR antibodies, polyclonal TrkBantibodies were able to induce ligand-independent inter-nalization of TrkB, but the time of internalization wasincreased with the addition of 100 ng/ml NGF anddecreased with the addition of 100 ng/ml BDNF. Theaddition of BDNF did not seem to increase the bindingand rate of internalization of TrkB compared withuntreated cells. One notable observation was the behav-ior of TrkB under NGF treatment. TrkB internalizationdecreased when treated with NGF compared withuntreated cells. The internalization rates observed in ourexperiments, for both p75NTR and TrkB, were consist-ent with the internalization rates for these receptorsreported in other cell lines (Grimes et al., 1996; Iwasakiet al., 1997; Saxena et al., 2004).

The experiments reported here highlight thepotential of this cell line in dissecting the complex re-ceptor mechanisms that occur as a result of neurotrophinbinding, leading to multiple biological responses, includ-ing cell death or survival. The spontaneous differentia-tion of the NSC-34 cells, including the presence of longprocesses, make this cell line attractive for the study ofreceptor trafficking in neurites or axons and subsequentlong-range signalling. The current results are supportiveof the role of the NSC-34 cell line in experiments

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studying receptor function and for several neurotrophicfactors in a context of a model for motor neuron devel-opment. In particular, the presence of functional neuro-trophin receptors resembling developing motor neuronswill allow the exploitation of this cell line for studiesof receptor trafficking, signalling, and downstreamresponses, including those initiated by the proneurotro-phins through the sortilin and p75 receptor.

ACKNOWLEDGMENTS

The generosity and help of the following people isgratefully acknowledged. The NSC-34 cell line was sup-plied by Prof. Neil Cashman, University of Toronto,Centre for Research in Neurodegenerative Diseases, To-ronto. Purified NGF was obtained from Prof. Ian Hen-dry, Developmental Neurobiology Group, John CurtinSchool of Medical Research, Australian National Uni-versity. Prof. Ronald Schnaar, Department of Pharma-cology, Johns Hopkins School of Medicine, Baltimore,developed hybridoma clones producing the Gt1b anti-bodies. Antibodies against clathrin HC and AP-1 wereprovided by Antibody Technology Australia Pty. Ltd.,Adelaide, Australia. Monoclonal antibodies against EEA-1, Limp-II, and Lamp-1 were produced by Dr. D.A.Brooks, Department of Chemical Pathology, Women’sand Children’s Hospital, Adelaide, Australia. Polyclonalantibody 9560 against p75NTR used for Western blotswas provided by Prof. Moses Chao, Departments of CellBiology (Skirball) and Physiology and Neuroscience,Skirball Institute Program of Molecular Neurobiology,NYU Medical Center, New York). The authors alsothank Bradley Turner at the Motor Neuron DiseaseResearch Laboratory, Howard Florey Institute, Univer-sity of Melbourne, for help with the NSC-34 cell line.

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Journal of Neuroscience Research DOI 10.1002/jnr