Apolipoproteins D and E3 exert neurotrophic and synaptogenic effects in dorsal root ganglion cell cultures

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Neuroscience 162 (2009) 282–291

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POLIPOPROTEINS D AND E3 EXERT NEUROTROPHIC ANDYNAPTOGENIC EFFECTS IN DORSAL ROOT GANGLION CELL
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. KOSACKA,a,d* M. GERICKE,a M. NOWICKI,a

. KACZA,b J. BORLAKc AND K. SPANEL-BOROWSKIa

Institute of Anatomy, University of Leipzig, Liebigstra�e 13, D-04103eipzig, Germany

Institute of Veterinary Anatomy, University of Leipzig, An den Tierkliniken3, D-04103, Leipzig, Germany

Fraunhofer Institute of Toxicology and Experimental Medicine, Nico-ai-Fuchs-Stra�e 1, D-30625 Hannover, Germany

Translational Centre for Regenerative Medicine-Leipzig, University ofeipzig, Phillipp-Rosenthal-Stra�e 55, D-04103 Leipzig, Germany

bstract—Co-cultures of 3T3-L1 adipocytes with neuronsrom the rat dorsal root ganglia (DRG) showed enhancedeuritogenesis and synaptogenesis. Microarray analysis forpregulated genes in adipocyte/DRG co-cultures currentlyoints to apolipoproteins D and E (ApoD, ApoE) as influentialroteins. We therefore tested adipocyte-secreted cholesterolnd the carrier proteins ApoD and ApoE3. Cholesterol, ApoD,nd ApoE3 each increased neurite outgrowth and upregu-

ated the expression of presynaptic synaptophysin and syn-ptotagmin, as well as the postsynaptic density protein 95.he neurotrophic effects of ApoD and ApoE3 were associ-ted with an increased expression of the low-density lipopro-ein receptor and apolipoprotein E receptor 2. Simultaneousreatment with receptor-associated protein, an apolipopro-ein receptor antagonist, inhibited the neurotrophic functionf both apolipoproteins. The application of ApoD, ApoE3, andholesterol to DRG cell cultures corresponded with in-reased expression of the chemokine stromal cell-derivedactor 1 and its receptor CXC chemokine receptor 4 (CXCR4).urprisingly, the inhibition of CXCR4 by the antagonisticrug AMD3100 decreased the apolipoprotein/cholesterol de-endent neurotrophic effects. We thus assume that apoli-oprotein-induced neuritogenesis in DRG cells interferesith CXCR4 signaling, and that adipocyte-derived apoli-oproteins might be helpful in nerve repair. © 2009 IBRO.ublished by Elsevier Ltd. All rights reserved.

Correspondence to: J. Kosacka, Institute of Anatomy, University ofeipzig, Liebigstra�e 13, D-04103 Leipzig, Germany. Tel: �49-341-7-22051; fax: �49-341-97-22009.-mail address: [email protected] (J. Kosacka).bbreviations: AMD3100, 1,1’-[1,4-Phenylenebis-(methylene)]-bis-(1,4,8,1-tetraazacyclotetradecane) octahydrochloride dehydrate; ApoD,polipoprotein D; ApoE, apolipoprotein E; ApoER2, apolipoprotein Eeceptor 2; ApoE3, apolipoprotein E3; CXCR4, CXC chemokine re-eptor 4; DMEM, Dulbecco’s modified Eagle medium; DRG, dorsaloot ganglia; FCS, fetal calf serum; GAP43, growth-associated protein3; GCOS, GeneChip operating software; LDLR, low density lipopro-ein receptor; LRP1, low density lipoprotein receptor-related protein 1;F-200, neurofilament 200; NF68, neurofilament 68; PBS, phosphate-uffered saline; PNS, peripheral nervous system; PSD-95, postsynap-ic density protein 95; RAP, receptor-associated protein; RGS, regu-ator of G-protein signaling; RT, room temperature; SDF-1, stromal

oell-derived factor; SDS, sodium dodecyl sulfate; VLDLR, very low-ensity lipoprotein receptor.

306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rightoi:10.1016/j.neuroscience.2009.04.073

282

ey words: ApoD, ApoE3, cholesterol, dorsal root ganglia,eurite outgrowth, synaptogenesis.

he apolipoproteins D and E (ApoD and ApoE) transportholesterol and triglyceride in various tissues. In the CNS,polipoproteins are produced by astrocytes, oligodendro-ytes, and microglia. Apolipoprotein receptors are necessaryor the neuronal uptake of cholesterol, which is required for

embrane synthesis and maintenance. Various well-haracterized receptors such as apolipoprotein E receptor 2ApoER2), very low-density lipoprotein receptor (VLDLR), lowensity lipoprotein receptor-related protein 1 (LRP1), and mega-

in are involved in the migration, growth, and synaptic plasticity ofeurons in the developing and adult brain (Beffert et al., 2004).polipoprotein isoforms appear to exert opposite effects.poE4 decreases, and apolipoprotein E3 (ApoE3) promotes,eurite outgrowth in cultured cortical neurons (Nathan et al.,995, 2002). Increased apolipoprotein expression in the CNS

s associated with neurodegenerative diseases, includinglzheimer’s, Niemann-Pick, and Huntington’s chorea (Coylet al., 1976; Yoshida et al., 1996; Terrisse et al., 1998).

The production and function of apolipoproteins and thenalysis of their receptors have not been comprehensivelytudied in the peripheral nervous system (PNS). Apolipopro-eins appear to be locally synthesized and influential in theepair of damaged peripheral nerves. It is reported that ApoD,poE, or ApoA-I accumulates in the regenerating sciaticerve of rabbits and that ApoE promotes neurite extension inorsal root ganglion (DRG) cell cultures isolated from rabbitsBoyles et al., 1990; Popko et al., 1993; Goodrum et al.,995). The authors suggested additional apolipoproteins be-ause repair of damaged peripheral nerves has been re-orted in mice, which are gene deficient for ApoE and ApoA-IGoodrum et al., 1995). We have described that the in-reased neurite outgrowth and synaptogenesis in adipocyte/RG co-cultures depends on adipocyte-secreted angiopoi-tin-1 (Kosacka et al., 2006). In addition to angiopoietin-1, theeurotrophic/synaptogenic effect in adipocyte/DRG co-cul-

ures could be supported by the release of apolipoproteinsnd cholesterol. The rather underestimated aspect of apoli-oprotein influence in the PNS deserves further analysis withespect to graduated effects between ApoD, ApoE, and cho-esterol, as well as regarding the receptor types involved ineurite outgrowth and synaptogenesis. Such knowledgeight be helpful in the search for an improved therapy foreripheral nerve repair. Peripheral nerve lesions often cause

nvalidity of patients because of extremely slow neuronal
utgrowth. Novel neurotrophic factors may be indicated by
s reserved.

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J. Kosacka et al. / Neuroscience 162 (2009) 282–291 283

lobal gene analysis of adipocyte/DRG co-cultures com-ared to DRG cultures. Our first aim was to verify elevatedpolipoprotein mRNA levels, as well as other mRNAs of

nterest in this co-culture type. In our second aim, we vali-ated the effects of apolipoproteins and cholesterol in ratRG cell cultures.

EXPERIMENTAL PROCEDURES

RG preparation

ll animal experiments were approved by the local governmentalommittee of Animal Care and Use (reg. No. 106/07). The exper-

ments conformed to international guidelines on the ethical use ofnimals. The number of animals used and their suffering wereinimize as much as possible. Two-day-old pups of the Wistar-urt rat strain were decapitated, and lung, heart, and gut wereemoved. DRGs were isolated along both sides of the cervical,horacic, and lumbar vertebral column under sterile conditionssing a Stemi 1000 stereomicroscope (Zeiss, Jena, Germany).

RG cell cultures

RGs were collected in ice cold phosphate-buffered saline (PBS)nd incubated with 0.125% collagenase (Worthington Cell Systemsiotechnology, Lakewood, NJ, USA) dissolved in Dulbecco’s modi-ed Eagle medium (DMEM; GIBCO, Karlsruhe, Germany) at 37 °Cor 90 min. After centrifugation, resuspended cells and cell aggre-ates from six pups were pooled and plated on glass coverslips in a4-well-culture plate for immunocytochemistry, or on Thermanoxoverslips for scanning microscopy; cells were plated into three0-mm culture dishes for Western blotting (all plates from Nunc,iesbaden, Germany). Cultures were maintained in DMEM andam’s F-12 mixed (1:1) together with 15 mM Hepes, 22 mMaHCO3, 1% glucose, 1% insulin–transferrin–selenium mixture,0% fetal calf serum (FCS) and antibiotics (100 U/mL penicillin andtreptomycin; all reagents from Sigma, Deisenhofen, Germany). Cul-ures were developed in a humidified atmosphere of 5% CO2 in air at7 °C.

xperiments

n day 1 after plating, cultures were switched to serum-freeedium in the presence of 5 �g/mL ApoD (MEDAC Gesellschaft,edel, Germany), 5 �g/mL ApoE3, or 10 �g/mL cholesterol (both

rom Sigma-Aldrich, Taufkirchen, Germany) for 2 or 6 days. Un-upplemented controls were also grown for 2 or 6 days. Theedium was changed every 48 h.

In a similar experimental setup, the low density lipoproteineceptor (LDLR) family antagonist receptor-associated proteinRAP; Fitzgerald Industries International, Concord, MA, USA; 250M) or CXC chemokine receptor 4 (CXCR4) receptor antagonistMD3100 octahydrochloride (Sigma Aldrich; 1 �M) was added to

he DRG cells 1 h prior to stimulation.

dipocyte cultures and differentiation of 3T3-L1dipocytes

T3-L1 preadipocytes (American Type Culture Collection, Rock-ille, MD, USA) were plated directly into 50-mm culture dishes thatad been coated with 1% Vitrogen 100 (Collagen, Palo Alto, CA)t 37 °C for 2 h prior to use. Cultures were grown to confluence inMEM containing 25 mM glucose (DMEM-H) and 10% FCS (all

rom Sigma Aldrich).At day 2 of confluence, preadipocytes were induced with me-

ium containing 1 mM insulin, 0.4 mg/mL dexamethasone, and 0.5M methylisobutylxanthine to differentiate into adipocytes. After an
dditional 3 days, about 95% of the cells had accumulated large-
ized fat droplets as verified by phase-contrast microscopy. Three-ay-old adipocyte cultures were used for co-culture with DRG cells.

T3-L1 adipocyte/DRG co-cultures

RG cells were seeded on top of fully-differentiated adipocytes.uring the initial 24 h, co-cultures were maintained with DMEMontaining 5% FCS. After 24 h the cultures were switched to DMEMith 2% FCS for 5 or 8 days. The medium was changed every 24 h.

ranscriptome analysis using Affymetrix GeneChipystem and determination of significantly regulatedene expression

ranscriptome analysis was conducted with RNA extracts from 5-r 8-day-old 3T3-L1/DRG co-cultures and DRG cell cultures (fromhree independent experiments per condition; Affymetrix Gene-hip according to the manufacturer’s Expression Analysis techni-al manual; Santa Clara, CA, USA) using the GeneChip Ratenome RAE 230 2.0. RNA isolation and quantification wereonducted as described by Nowicki et al. (2007). The arrays werecanned using the GeneChip Scanner 3000. Scanned image filesere visually inspected for artifacts and then analyzed, each

mage being scaled to the same target value for comparisonetween chips. GeneChip operating software (GCOS) was usedo control the fluidics station and the scanner, to capture proberray date and to analyze hybridization intensity data. Defaultarameters provided in the Affymetrix data analysis softwareackage were applied for analysis.

Significantly regulated genes were extracted from the com-arison analysis conducted by GCOS. Extraction was conductedith MS Access (Microsoft Deutschland, Unterschlei�heim, Ger-any) by applying the following criteria to the data: induced genesad to fulfill the criteria of having the change call “Increase” (I)ombined with a fold change �2; in the treatment experiment, aPresent” call had to be combined with a signal �100.

mmunocytochemistry

RG cell cultures were fixed with 2% formaldehyde in 0.1 M PBSnd 5% sucrose at 37 °C for 15 min. For detection of NF or thexonal growth cones, fixed cells were incubated with mouse

able 1. Detailed list of antibodies used

ntibody Company Dilution

ouse anti-NF-200); clone N52 Sigma Aldrich 1:500ouse anti-NF68 (NF68); cloneNR4

Sigma Aldrich 1:3000

ouse anti-GAP43 Sigma Aldrich 1:1000ouse anti-synaptophysin Sigma Aldrich 1:4000ouse anti-synaptotagmin Synaptic Systems,

Göttingen, Germany1:700

ouse anti-PSD-95 Chemicon, ChandlersFord, UK

1:1000

abbit anti-CXCR4 �ProSci Incorporated,Poway, CA

1:1000

abbit anti-LDLR Biozol, Eching,Germany

3 �g/mL

oat anti-ApoER2 Santa CruzBiotechnology,Santa Cruz, CA

1:2000

oat anti-LRP1; (A-18) Santa CruzBiotechnology

1:2000

ouse anti-D-glyceraldehyde-3-phosphate dehydrogenase

Research Diagnostics,Flanders, The

1:3000

(GAPDH) Netherlands

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onoclonal antibodies against neurofilament 200 (NF-200) orrowth-associated protein 43 (GAP43) (Table 1) in PBS with 1%SA and 0.3% TX (PBS-BT) at 4 °C overnight. The antibodiesere diluted 1:500. After rinsing with buffer, CY3-conjugated goatnti-mouse IgG (Dianova, Hamburg, Germany) antibodies werepplied at room temperature (RT) for another 2 h. The coverslipsere mounted with glycerol (DAKO, Hamburg, Germany) contain-

ng DAPI 10 �g/mL (Serva, Heidelberg, Germany) for nucleartaining and DABCO 25 �g/mL (Sigma Aldrich) to prevent photo-leaching. By replacement of the primary antibody with normalouse IgG or PBS, respectively, no specific immunoreaction oc-

urred.

estern blot analysis

or immunoblot analysis, cultured DRG cells were lysed by ultra-onication in 60 mM Tris–HCl, pH 6.8, containing 2% sodiumodecyl sulfate (SDS) and 10% sucrose. Cell lysates were diluted:1 in sample buffer (250 mM Tris–HCl, pH 6.8, containing 4%DS, 10% glycerol, and 2% �-mercaptoethanol) and denatured at5 °C for 5 min. Protein concentration was assessed with the BCArotein assay (Pierbo Science, Bonn, Germany). Proteins (15g/lane) were separated by electrophoresis on a 10% SDS–olyacrylamide gel and transferred to nitrocellulose by electrob-

otting. Nonspecific binding sites were blocked with 10% nonfatilk for 45 min. The blots were then incubated with primaryntibodies (Table 1) at 4 °C overnight.

Immunoreactions were detected by incubating blots with theppropriate secondary antibody (peroxidase-conjugated anti-ouse, anti-goat, or anti-rabbit IgG, at a 1:4000 dilution; Dianova)t RT for 2 h. Peroxidase activity was visualized with an enhancedhemiluminescence kit (Amersham Pharmacia, Freiburg, Ger-any). Integrated optical densities of the immunoreactive proteinands were measured with Gel Analyzer software (Media Cyber-etics, Silver Spring, MD, USA) and normalized to GAPDH values.

canning electron microscopy

or scanning electron microscopy analysis, the DRG cell culturesreated with ApoD, ApoE3, or cholesterol, and untreated cultures,ere fixed with 2% glutaraldehyde in 0.05 M PBS containing.02% CaCl2, pH 7.2. Post-fixation was carried out using 2%smium tetroxide in 0.05 M PBS with 3% saccharose at RT for 1 h.graded alcohol series was used for dehydration, followed by

ncreasing concentrations of amyl acetate. Cells were dried by theritical point drying method with CO2 (CPD 030, Bal-Tec, Liech-enstein). The specimens were coated with palladium in a vacuumvaporator and viewed with the Zeiss scanning electron micro-

able 2. Expression of neuritogenic and synaptogenic genes inT3-L1 adipocyte-DRG co-cultures at day 5

ene title Genesymbol

UniGene ID Signal logratio day 5

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poE Apoe Rn.32,351 1.8 IpoD Apod Rn.11,339 1.5 Ihemokine(C–C motif)ligand 12

Cxcl12* Rn.54,439 3.0 I

hemokine(C–C motif)receptor 4

Cxcr4 Rn.44,431 2.8 I

egulator of G-proteinsignaling 1

Rgs1 Rn.14,787 5.1 I


Rgs5 Rn.1150 2.4 I


Rgs18 Rn.105,775 5.5 I

Cxcl12 (acronym-SDF-1; Ch-change; I-increase).

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tatistical analysis

ll data (means�SEM) referred to three independent experiments.he Western blot data were analyzed for several groups using one-ay analysis of variance (ANOVA) followed by the Newman–Keuls

est (software “SigmaStat”). For comparison of two groups, statisticalnalysis was performed with the Student’s t-test.

RESULTS

pregulation of mRNA adipokines in DRG/adipocyteo-cultures

otential neuritogenic and synaptogenic adipokines wereignificantly upregulated in DRG/adipocyte co-cultures com-ared to DRG cultures or 3T3-L1 adipocyte cell culturesTable 2). For 122 increased genes in DRG/adipocyte co-ultures compared to DRG cultures, 65 genes showedwo- to fourfold increase. ApoD depicted a 2.8-fold changend ApoE a 3.5-fold one. For 34 genes with a four- toightfold increase, the chemokine stromal cell-derived fac-or (SDF-1) and the receptor CXCR4 were upregulatedight- and 6.9-fold. The regulators of G-protein signalingRGS) met the highest mRNA increase in co-cultures. TheGS1 gene showed a 34.3-fold change and RGS18 a5.3-fold increase. Because the upregulated genes do notecessarily correspond to the protein translation, Westernlot analysis was conducted. The expression of ApoD andpoE proteins was seen in the 3T3-L1 adipocyte/DRGo-cultures and in adipocyte cultures, whereas in theRG cell cultures ApoD and ApoE proteins were absent.RG cell cultures, 3T3-L1 adipocyte/DRG co-cultures, andT3-L1 adipocyte cultures produced both SDF-1 andXCR4 receptor proteins (Fig. 1A, B).

ig. 1. Expression of ApoD, ApoE3, SDF-1, and CXCR4 proteins areevealed in 3T3-L1 adipocyte/DRG co-cultures (DRG�AD), 3T3-L1dipocyte cultures (AD) and DRG cell cultures (DRG) at day 5 (West-rn blot analysis). (A) Expression of ApoD and ApoE proteins is noted

n co-cultures and adipocytes. Apolipoproteins are absent from DRG
ell cultures. (B) Immunobands of SDF-1 and CXCR4 proteins in theifferent culture types.

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eurite outgrowth under the influence of ApoD,poE3, and cholesterol in DRG cell cultures

o determine the effects of apolipoproteins or cholesteroln neurite outgrowth, DRG cell cultures were incubatedith ApoD, ApoE3 (both 5 �g/mL), or cholesterol (10 �g/L) for 2 or 6 days. NF-200 antibody immunostaining

evealed an increased neurite network in the treatedroups at day 2, compared to the untreated control cells.e got the impression that ApoD and ApoE3 increased

eurite outgrowth, while cholesterol augmented neuriteranching (Fig. 2A). Double immunostaining for peripherinnd GAP43 showed that treatment with ApoD and ApoE3

ed to DRG neurons with strong GAP43 expression, whileholesterol application depicted only a weak co-localiza-ion of GAP43 in peripherin-positive neurons. In the controlells, GAP43 was missing from peripherin-positive neu-ons (Fig. 3).

Immunofluorescence localization findings were semi-uantitatively validated with Western blots of primary DRGell cultures. For neurite evaluation, an antibody againsteurofilament 68 (NF68) was utilized successfully. Therotein bands for NF68 and GAP43 were distinctly stron-er when treated with ApoD, ApoE3, or cholesterol com-

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ig. 2. (A) Neurite outgrowth is increased in DRG cell cultures treatedonditions for 2 days. Immunostaining with an antibody against NF-20igh in DRG cell cultures treated with ApoD, ApoE3, or cholesterol inpoE3 stimulation. (D) Similar levels of GAP43 under ApoD or ApoE3ensitometric analysis after normalization to GAPDH were from fou
* P�0.01, or *** P�0.001, according to the one-way analysis of variance togetor NF68: F�25,261; df�11; P�0.001 and for GAP43: F�928,356; df�11; P�
ared to untreated control cells. ApoE3 induced NF68xpression more effectively than ApoD or cholesterol. Bothpolipoproteins achieved similar effects on GAP43 produc-ion (Fig. 2B–D).

As consequence of apolipoprotein/cholesterol stimula-ion we also found an upregulation of the CXCR4 chemo-ine receptor and its ligand SDF-1 in treated DRG culturesompared to untreated control cultures. Both apolipopro-eins appeared to induce SDF-1 protein expression moreffectively than cholesterol. CXCR4 protein expressionas similar under apolipoprotein and cholesterol stimula-

ion (Fig. 2B).

polipoprotein- and cholesterol-dependentpregulation of synaptic proteins

e then focused on the synaptogenetic function of thepolipoprotein/cholesterol system, because DRG neuritesormed synaptic contacts in vitro (Kosacka et al., 2006).ix-day-old DRG cell cultures treated with ApoD andpoE3 produced presynaptic markers, such as synapto-hysin and synaptotagmin l, at higher levels compared toholesterol treatment or untreated DRG cell cultures (Fig.). Expression of postsynaptic density protein 95 (PSD-

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oD, ApoE3 (both 5 �g/mL), and cholesterol (10 �g/mL) in serum-free. Scale bars�50 �m. (B) Expression levels of NF68 and GAP43 aree conditions for 6 days. (C) High expression of NF68 under ApoD or

nt. Results were averaged for four independent experiments. Data ofndent experiments and are expressed as means�SEM. * P�0.05,

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5), a postsynaptic scaffolding protein, was distinctlyigher when treated with ApoD than ApoE3. Cholesterol

reatment caused a poor response of PSD-95 intensity,hich was similar to the PSD-95 band in untreated DRGell cultures. The semiquantitative evaluation confirmedigh expression levels of both presynaptic proteins in cul-

ures treated with ApoD and ApoE3, and medium to mod-rate levels in cultures treated with cholesterol. This waslso the case for the postsynaptic PSD-95 protein, when

he 6-day-old DRG cell cultures were assessed duringpoE3 and cholesterol treatment. That ApoD was more

ig. 3. (A–D, A=–D=) Peripherin (green, left column) and GAP43 (red,ight column) are co-localized in DRG neuron cultures treated withpolipoproteins and cholesterol in serum free-conditions. (A, A=)poD; (B, B=) ApoE3; (C, C=) cholesterol. (D, D=) GAP43 signal isegligible in the control. Scale bars�15 �m. For interpretation of theeferences to color in this figure legend, the reader is referred to the

eb version of this article.

nfluential than ApoE3 on synapse formation was also g

educed from findings with the scanning electron micro-cope, where true neuro-neuronal synapses were de-ected (Fig. 4E).

nhibition of the apolipoprotein and cholesterolffects by RAP and AMD3100

he neurotrophic and synaptogenic effects of ApoD,poE3, and cholesterol were validated by the additional

reatment of DRG cell cultures with RAP, which was able tolock apolipoprotein receptors. Three-day-old DRG cellultures simultaneously incubated with apolipoproteinsnd RAP decreased the levels of NF68 and GAP43 com-ared to the control, which had been treated with apoli-oprotein only. The same held true for the simultaneouspplication of cholesterol and RAP (Fig. 5A–C). Moreover,he RAP-dependent inhibition of apolipoprotein receptorsed to a statistically significant downregulation of pre- andostsynaptic proteins in six-day-old DRG cultures (Fig.D–F).

etermination of apolipoprotein receptor typesuring ApoD, ApoE3, and cholesterol treatment

o determine the type of apolipoprotein receptors involvedn neurite outgrowth, ApoER2, LDLR, and LRP1 were an-lyzed in treated and untreated 2-day-old DRG cell cul-ures by Western blotting. The ApoER2 was at similarevels in DRG cultures stimulated with ApoD, ApoE3, andholesterol, and in all cases was distinctly higher than inhe untreated control DRG cell cultures. The LDLR wasignificantly augmented in DRG cell cultures in response topoD and cholesterol treatment in comparison to ApoE3nd no application (Fig. 6). Application of apolipoproteinsnd cholesterol did not increase LRP1 production (LRP1

evels were comparable to the untreated control results).emiquantitative measurements then confirmed the de-cribed differences in the expression intensities of ApoER2nd LDR in DRG cell cultures treated with ApoD, ApoE3,nd cholesterol. To clarify the cell type with ApoER2 andDLR in the DRG cell cultures, which represented a mix-ure of neuronal and non-neuronal cells, additional studiesere conducted with the neuroblastoma cell line B104 andith the schwannoma cell line NH22. The expression lev-ls of ApoER2 and LDLR were comparable for both cellypes dependent on the manner of treatment (data nothown).

nhibition of the apolipoprotein and cholesterolffects with AMD3100

ecause apolipoprotein/cholesterol dependent SDF-1/XCR4 upregulation could be associated with the neuro-

rophic effect, the CXCR4 receptor antagonist AMD3100as added to the DRG cell cultures during the 3 days ofpoD/E3 stimulation. The rapid decrease of NF68 protein

n apolipoprotein/cholesterol-treated cultures in the pres-nce of CXCR4 antagonist supports our assumption re-
arding an apolipoprotein–chemokine relationship (Fig. 7).

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DISCUSSION

e previously demonstrated that neurite outgrowth andpregulation of synaptic proteins in 3T3-L1 adipocyte/DRGo-cultures appear to be co-stimulated by adipocyte-de-ived Ang-1 and by other adipokines (Kosacka et al.,006). For this reason, we first conducted a microarraynalysis to monitor changes in gene expression in 5-day-ld 3T3-L1 adipocyte/DRG co-cultures, DRG cell cultures,nd 3T3-L1 adipocyte cell cultures. Global gene analysisevealed mRNA upregulation of several neuritogenic andynaptogenic adipokines, including ApoD and ApoE. Theuantitative study of gene changes was not further exam-

ned by RT-PCR analysis, because we validated the po-ential involvement of ApoD and ApoE in peripheral nerveegeneration. DRG cell cultures are a model for the regen-ration of damaged peripheral neurons.

The increased expression of GAP43 and NF68 pro-eins, as demonstrated in our treated DRG cell cultures atay 6, is in line with the knowledge that both proteins aretrongly expressed during axonal growth in the developingrain and after injury to peripheral nerves (Van der Zee etl., 1989; Benowitz et al., 1997; Szpara et al., 2007; Toth

ig. 4. Presynaptic (synaptophysin, synaptotagmin) and postsynaptpoE3, or cholesterol in serum-free conditions for 6 days. (A) Similarxpression of PSD-95 only under ApoD stimulation. (C) Similar levels of densitometric analysis after normalization to GAPDH were from fo* P�0.01, or *** P�0.001, according to the one-way analysis of varianor synaptophysin: F�189,516; df�11; P�0.001; for synaptotagmin: Fepresentative Western blots. (E) Scanning electron micrographs deppoD in serum free-conditions for 6 days. Scale bar�5.5 �m.

t al., 2008). Both apolipoproteins exerted a stronger effect A

n NF68 or GAP43 protein expression than cholesterol inhe present DRG cell cultures. This was also evident by theouble immunostaining for peripherin and GAP43; choles-erol led to a weak GAP43-positive signal. The reason for

stronger neuritotrophic effect of apolipoproteins than ofholesterol could rely on the different processing of exog-nously applied ApoD/ApoE3 compared to endogenouspolipoproteins. Exogenous ApoE is found to increase thetilization of triglycerides and the cell-dependent hydroly-is of cholesteryl ester, and also augments the intracellularptake of triglyceride-rich particles, whereas the endog-nously present apolipoprotein suppresses these reac-ions (Ho et al., 2000).

GAP43 expression was decreased in the neocortexnd hippocampus of ApoE-gene deficient mice comparedo wild-type mice, and infusion of ApoE resulted in theecovery of GAP43 production (Veinbergs et al., 2001).ndogenously produced ApoD and ApoE appear to com-ensate each other, because the brains of ApoE-deficientice show increased levels of ApoD (Terrisse et al., 1999).his suggestion of compensation is given by Goodrum etl. (1995), who induced peripheral nerve repair in ApoE or

5) proteins vary in expression in DRG cultures treated with ApoD,synaptophysin (38 kDa) under ApoD or ApoE3 stimulation. (B) High

tagmin (�65 kDa) under ApoD, ApoE3 or cholesterol stimulation. Dataendent experiments and are expressed as means�SEM. * P�0.05,her with the Newman–Keuls test. The results of the variance ratio test; df�11; P�0.001 and for PSD-95: F�114,096; df�11; P�0.001. (D)e outgrowth and a synaptic contact (arrow) in DRG cells treated with

ic (PSD-9levels of

f synaptour indepce toget�19,623

poA-I deficient mice by other apolipoproteins. Our study

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rovides evidence that ApoD and ApoE3 exert a similarnfluence during DRG cell differentiation. Apolipoproteins,hich bind diverse lipids (including cholesterol), are inter-alized by a receptor-mediated process. Apolipoproteinsave an affinity to receptors of the LDLR family, which arelso expressed by neurons (Veinbergs et al., 2001; Nathant al., 2002). The associated cell signaling and the subse-uent modulation of the intracellular lipid metabolism coulde involved in neurite outgrowth (Nathan et al., 1995).ecause apolipoproteins are involved in lipid metabolismnd cell signaling, their effects on neurite growth are ex-ected to be stronger than the effect of cholesterol.

It has been postulated that ApoE and cholesterol play

ig. 5. A RAP-dependent apolipoprotein receptor blockade (250 nM) wn serum-free conditions for 6 days. The RAP blockade causes a de

estern blots. (B, C) The statistically significant decrease of neuronaroup. (E, F) The conspicuous inhibition of synaptic proteins occurs in t

o GAPDH were from three independent experiments and are expretudent’s t-test.

mportant roles in the synaptic plasticity of the CNS by n

nvolving ApoER2 (Koudinov et al., 2001; Quattrocchi etl., 2003; Petit-Turcotte et al., 2005). ApoE regulates theholesterol content of neuronal cell membranes, modu-

ates calcium homeostasis, and stabilizes the cytoskeletonVeinbergs et al., 1999). Notably, we have found thatpoD- and ApoE3-treated DRG cell cultures express high

evels of the presynaptic proteins synaptophysin and syn-aptotagmin compared to cholesterol or to the untreatedontrol. The upregulation of synaptophysin and synapto-agmin in the treated DRG cell cultures indicates that ApoDnd ApoE3 operate in synapse formation. PSD-95 wasecently found to interact with the cytoplasmic domain ofpoER2, localized in the postsynaptic density of cortical

ucted in DRG cell cultures treated with ApoD, ApoE3, and cholesterolf neuronal (A–C) and synaptic (D–F) proteins. (A, D) representativeis more efficient for the ApoD and ApoE3 group than the cholesteroland ApoE3 groups. Data of densitometric analysis after normalizationmeans�SEM. * P�0.05, ** P�0.01, or *** P�0.001, according to

as condcrease ol proteinshe ApoDssed as

eurons and regulated by ApoE (Beffert et al., 2004; Hoe

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t al., 2005). Our study showed higher levels of PSD-95rotein under the influence of ApoD than of ApoE3. More-ver, neuro-neuronal synapses were detected with thecanning electron microscope in ApoD-treated DRG cellultures. Because synaptic differentiation depends heavilyn PSD-95 recruitment (Friedman et al., 2000; Okabe etl., 2001) and in consideration of our findings, we suggest

hat ApoD is more important than ApoE3 in postsynaptic

ig. 6. Apolipoprotein receptor ApoER2 (�90 kDa) and LDLR (130Da) levels are studied in DRG cells cultures treated with ApoE3,poD, or cholesterol in serum-free medium for two days. (A) Repre-entative Western blot analysis for DRG cell cultures demonstrates thepregulation of LDLR when treated with ApoD or cholesterol com-ared to ApoE3 treatment or control. (B) Maximum LDLR levels areeached in DRG cell cultures treated with cholesterol. (C) HighpoER2 levels correlate with ApoD, ApoE3, and cholesterol treatmentompared to control. Data of densitometric analysis after normalizationo GAPDH were from three independent experiments and are ex-ressed as means�SEM. * P�0.05, ** P�0.01, or *** P�0.001,ccording to the one-way analysis of variance together with the New-an–Keuls test. The results of the variance ratio test for ApoER2:�105,408; df�11; P�0.001; for LDLR: F�15,529; df�11; P�0.001.

aturation. The application of RAP, an inhibitor of apoli- a

oprotein receptors, before ApoD and ApoE3 treatment ofRG cell cultures significantly decreased the productionf synaptophysin and PSD-95. The synaptogenic effects ofhe apolipoproteins or cholesterol might be indirect effectsaused by the high density of neurites from differentiatingeurons.

Both ApoER2 and VLDLR receptors are important dur-ng the formation of neuronal layers in the neocortex andippocampus (Trommsdorff et al., 1999). Yet the neurotro-hic effect of ApoE is also mediated by LDLR (Handel-ann et al., 1992; Veinbergs et al., 1999). We found thatDLR expression was high in DRG cell cultures after ApoDreatment, and at its maximum after cholesterol applica-ion. Comparable to the ApoD-dependent strong expres-ion of PSD-95, the ApoD-treated DRG cells displayed aigher level of LDLR compared to ApoE3. All treatedroups developed comparable and higher levels ofpoER2 than the untreated control. This finding allows us

o conclude that, in the DRG cell cultures, the receptor types regulated differently in neurons and glial cells. Yet neu-oblastoma and schwannoma cell lines have shown similarpoER2 and LDLR levels independent of the manner of

reatment. Besides internalization of lipids into neurons,ctivated ApoER2 and LDLR led to the phosphorylation ofytoplasmic adaptor proteins and the activation of PI3inase (Hiesberger et al., 1999). ApoER2- and LDLR-eficient mice have deficiencies in synaptic density andlasticity in the CNS (Weeber et al., 2002; Mulder et al.,007), indicating that ApoER2 and LDLR are likely in-olved in functions of the PNS. Blockade of apolipoproteineceptors with the RAP antagonist inhibited neurotrophicnd synaptogenic effects in DRG cell cultures.

The outcome of the global gene analysis of DRG-dipocyte co-cultures indicated a functional relationshipetween the chemokine SDF-1, its receptor CXCR4, andegulators of CXCR4 such as RGS1 and RGS18. RGS5nd RGS18 are expressed in the human brain, but theirunctions remain unknown (Larminie et al., 2004). OtherGS proteins, such as RGS2 and RGS4, are reported tolay a crucial role in neuronal development of the CNSErdely et al., 2004; Heo et al., 2006). As our mRNA dataeads us to expect, the treatment of DRG cell cultures withpoD, ApoE, and cholesterol corresponded with an in-reased expression of SDF-1 and CXCR4 proteins thatas associated with increased levels of NF68 as marker ofeurite outgrowth. Treatment with the CXCR4 antagonistMD3100 resulted in the prominent decrease of the NF68rotein in the ApoD/ApoE3- or cholesterol-treated DRGell cultures. Because SDF-1 and CXCR4 are involved inhe migration, differentiation, and proliferation of neuronsnd glial cells in the developing CNS and PNS (Bajetto etl., 1999; Chalasani et al., 2003), we currently assume thatpolipoprotein-dependent neuritogenesis in DRG cell cul-ures interferes with CXCR4 signaling. Because SDF-1 isroduced in neurons and glial cells, the neurotrophic/syn-ptogenic effect of apolipoproteins may involve the activa-ion of CXCR4-positive cells to synthesize neurotrophins,
nd structural proteins for peripheral nerve repair.

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The CXCR4-dependent effect of immune cells isidely excluded because their presence is negligible inRG cell cultures.

cknowledgments—This work was funded by the Translationalentre of Regenerative Medicine, University of Leipzig (ProjectEMOD 1101 SF). We would like to thank San Francisco Edit forditorial assistance.

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(Accepted 29 April 2009)(Available online 3 May 2009)

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Apolipoproteins D and E3 exert neurotrophic and synaptogenic effects in dorsal root ganglion cell cultures