European Journal of Neuroscience, Vol. 27, pp. 2380–2390, 2008 doi:10.1111/j.1460-9568.2008.06215.

Differential effects of pro-BDNF on sensory neurons after

sciatic nerve transection in neonatal rats

Yong-jun Fan,* Linda Lin-yan Wu,* Hong-yun Li, Yan-Jiang Wang and Xin-Fu Zhou
Department of Human Physiology and Centre for Neuroscience, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia

Keywords: p75NTR, proneurotrophins, sciatic nerve injury, sensory neurons, sortilin

Abstract
Brain-derived neurotrophic factor (BDNF) plays a critical role in the development of the central and peripheral nervous systems, and
also in neuronal survival after injury. The actions of BDNF are mediated by its high-affinity receptors TrkB and p75NTR. Recent
studies have shown that proneurotrophins bind p75NTR and sortilin with high affinity, and trigger apoptosis of neurons in vitro. As
proneurotrophins are a dominant form of gene products in developing and adult animals, it is imperative to understand their
physiological functions in animals. Here, we showed differential roles of proBDNF in injured and uninjured sensory neurons.
proBDNF, p75NTR and sortilin are highly expressed in dorsal root ganglia (DRG) neurons. Recombinant proBDNF induced a dose-
dependent death of PC12 cells and the death activity was completely abolished in the presence of antibodies against the prodomain
of BDNF. The exogenous proBDNF enhanced the death of axotomized sensory neurons and the neutralizing antibodies to the
prodomain or exogenous sortilin-extracellular domain-Fc fusion molecule reduced the death of axotomized sensory neurons.
Interestingly, the treatment of neutralizing antibody in vivo increased the number of sensory neurons in the contralateral DRG. We
conclude that proBDNF may induce the death of axotomized sensory neurons and suppress neuronal addition in the intact DRG in
neonatal rats, and the suppression of endogenous proBDNF may protect neurons after neurotrauma.

Introduction
Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin
family, which plays a role in the survival and differentiation of
neurons during development (Henderson et al., 1993; Barde, 1994).
The requirement of BDNF signalling at the early stage of development
of murine sensory neurons was demonstrated by the loss of
approximately 30% of lumbar dorsal root ganglia (DRG) neurons in
animals with deletion of the gene (Jones et al., 1994). In addition to
regulating peripheral nerve development, BDNF also plays an
important role in regulating sensory neuron survival after neuron
axotomy (Zhou et al., 2005). BDNF expression was switched between
small and large axotomized neurons, and the anterograde transport of
BDNF was increased after peripheral nerve injury (Tonra et al., 1998;
Li et al., 1999; Zhou et al., 1999). The actions of BDNF are mediated
by its high-affinity receptor tropomyosin-related kinase B (TrkB) and
p75NTR, a member of the tumour necrosis factor receptor superfamily
(Huang & Reichardt, 2003). The relative expression levels of TrkB
and p75NTR by neurons determine life or death of developing and
injured neurons (Casaccia-Bonnefil et al., 1999). Our previous studies
demonstrated that BDNF regulates the survival of axotomized sensory
neurons depending on the expression levels of p75NTR and TrkB
(Zhou et al., 2005). BDNF prevented loss of axotomized sensory
neurons in DRG in vivo where TrkB was reduced after nerve injury,
whereas BDNF promotes cell apoptosis of cultured neurons in vitro
where p75NTR was upregulated (Zhou et al., 2005).
Correspondence: Dr X.-F. Zhou, as above.
E-mail: Xin-fu.zhou@flinders.edu.au
*Y.-J.F. and L.L.-Y.W. contributed equally to this work.
Received 21 December 2007, revised 17 March 2008, accepted 18 March 2008
All neurotrophins are synthesized first as precursor forms (proneu-
rotrophins), which can be either secreted from cells or cleaved
intracellularly to yield C-terminal mature neurotrophin dimers (Chao
& Bothwell, 2002). Recently, these proneurotrophins were also
recognized as functional proteins that distinctly activate the p75NTR
to mediate apoptosis of neurons (Lee et al., 2001; Teng et al., 2005).
Nerve growth factor precursor (ProNGF) binds both sortilin, a type I
transmembrane protein known as neurotensin receptor-3 (Mazella
et al., 1998), and p75NTR with a high affinity, inducing apoptosis of
neonatal sympathetic neurons in vitro (Lee et al., 2001; Nykjaer et al.,
2004). Sortilin is required for the naturally occurring cell death of
retinal neurons and age-dependent degeneration of sympathetic
neurons and neuronal death after brain injury (Jansen et al., 2007).
Although proNGF can cause the death of oligodendrocytes and
neurons in the injured brain and injured spinal cord in vivo (Beattie
et al., 2002; Harrington et al., 2004), the effect of proNGF on the
survival of sympathetic neurons is controversial as studies from a
different group showed that proNGF promotes survival of sympathetic
neurons by phosphorylating TrkA but with a lower potency than
mature NGF in vitro (Fahnestock et al., 2004).
ProBDNF has a crucial role in long-term depression (LTD) in the
hippocampus enhancing NR2B-dependent LTD and NR2B-mediated
synaptic currents by activation of p75NTR (Woo et al., 2005).
ProBDNF is also recognized as being released in cultured cortical
neurons and at hippocampal synapses (Pang et al., 2004; Teng et al.,
2005), and released proBDNF from overexpressed cells can also
activate TrkB (Mowla et al., 2001; Fayard et al., 2005). In a similar
function to proNGF, proBDNF also induces neuronal apoptosis via
activation of a receptor complex of p75NTR and sortilin in cultured
sympathetic neurons in vitro (Teng et al., 2005; Kenchappa et al.,

ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd

2006). The question that arises is whether proBDNF also acts as a
death factor in vivo under physiological conditions and after nerve
injury. As sensory neurons express sortilin, p75NTR and TrkB (Zhou
et al., 2005; Arnett et al., 2007) and a high level of BDNF in neonatal
sensory neurons after sciatic nerve injury (Zhou et al., 2005), we
examined the potential role of proBDNF in the neuronal survival
in vivo, using axotomized neonatal rat sensory neurons as a model.
Materials and methods
Animals
All procedures involving animals were approved by the Animal
Welfare Committee of Flinders University, and undertaken according
to the guidelines of the National Health and Medical Research Council
of Australia. Neonatal [postnatal day 1 (P1)] Sprague–Dawley rats
were kept with their mother under standardized barrier-breeding
conditions (12 h light : dark cycle). Food and water were provided
ad libitum. Unless otherwise specified, all reagents were of analytical
grade and purchased from Sigma (St Louis, MO, USA).
Preparation of sortilin-Fc
Sortilin gene was amplified from plasmid gp95 ⁄ sortilin (from Carlos
R. Morales, McGill University, Montreal, Quebec, Canada) in pBK-
CMV vector by polymerase chain reaction (PCR) using primers to
delete the transmembrane and intracellular domain, and introduce an
XhoI site at the 3¢ terminus and BglII site at the 5¢-terminus. The
construct was then subcloned into the plasmid pcDNA3.1-Fc, which
contains the sequence encoding the Fc region of human IgG1 between
the XhoI and BglII site and bidirectionally sequenced. The confirmed
constructs (plasmid pcDNA3.1-sortilin-Fc) were transfected into CHO
cells and stable clones were generated after selection in media
containing 0.5 mg ⁄ mL G418. Medium from cells cultured in HyQ
SFM4CHO (Hyclone, USA) was harvested for purification. Protein G
Sepharose beads (50% v ⁄ v slurry) were added into the medium
and incubated overnight at 4 C and then beads were collected into
a column for chromatography. After the column was washed
with washing buffer (150 mm NaCl, 10–20 mm Tris, pH 7.5–8.5
with 0.04% azide), the Fc-tagged sortilin protein was eluted with
elution buffer (100 mm glycine-HCl, pH 2.5 with 0.04% sodium
azide). Eluted recombinant proteins were dialysed against Hank’s
balanced salt solution (HBSS; Invitrogen, CA, USA) and stored at
)80 C until use.
Preparation of proBDNF and prodomain of BDNF
The expression and purification of proBDNF and the prodomain of
BDNF were performed using Champion pET100 Directional TOPO
Expression Kit. Briefly, the full-length proBDNF and the prodomain
of BDNF were amplified by PCR from murine point mutant proBDNF
plasmid [RR (amino acids 129 and 130) to AA] as a template (from Dr
Masami Kojima, Research Institute for Cell Engineering, National
Institute of Advanced Industrial Science and Technology, Ikeda,
Osaka) using primer pair: proBDNF (forward) 5¢-TTAGCGCCGA
ACCCTCATAGA and proBDNF (reverse) 5¢-CTACCTTCCCCTTT-
TAATGGT, which could generate proBDNF furin-resistant recombi-
nant protein; the reverse primer for the prodomain was 5¢-CTAG
CGCCGAACCCTCATAGA-3¢. The fragments were cloned into
pET100 ⁄ D-TOPO directional vector according to the manufacturer’s
instructions and the sequences were confirmed by sequencing. The
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 27, 2380–2390
Differential effects of pro-BDNF on sensory neurons 2381
plasmids were transformed into E. coli BL21, and the bacterial host
was grown in 1000 mL LB media with 100 lg ⁄ mL of ampicillin as a
selective agent (300 rpm, 37 C) till the O.D. 580 reached 0.8.
Following the addition of isopropyl-b-d-thiogalactopyranoside (IPTG)
to the final concentration of 0.5 mm and an overnight incubation at
30 C, the bacteria were harvested at 11 000 g for 20 min at 4 C. The
pellet was resuspended in 40 mL of binding buffer [50 mm
K-phosphate buffer, 0.3 m NaCl, 10% glycerol, 0.005% Triton
X-100, 10 mm imidazole and 1 mm dithiothreitol (DTT), and 1 mm
phenylmethylsulphonyl fluoride (PMSF) as a protease inhibitor).
Lysozyme (Sigma) was added to the solution to a final concentration
of 0.2 mg ⁄ mL to lyse the cells and the solution was kept on ice for
25 min. The solution was then sonicated 10 times on ice during a 30-s
period at a power of 50 Watts. The solution was then centrifuged at
11 000 g for 20 min at 4 C, and the pellet was resuspended with
50 mL of buffer I (20 mm Tris pH 8.0, 0.2 m NaCl and 1%
deoxycholic acid sodium salt) and subject to agitation on ice for
30 min. The suspension was then centrifuged at 3000 g for 10 min.
For the purification of the prodomain of BDNF, the pellet was then
resuspended in 50 mL of cold buffer II (10 mm Tris pH 8.0, 1 mm
EDTA and 0.25% deoxycholic acid sodium salt). The solution was
then centrifuged at 3000 g for 10 min. The pellet was washed three
times using 40 mL of buffer II as above. After the final wash, the
proteins in the pellet were denatured and solublized in 40 mL of 8 m
urea solution. The nickel column was prepared according to manu-
facturer’s instructions. The solublized protein solution was centrifuged
at 11 000 g for 25 min at 4 C. The cleared supernatant was added to
the column. After all the supernatant passed through the column, the
column was then washed with wash buffer (8 m urea, 5 mm imidazole
and 0.5 m NaCl). The OD of the wash eluate was monitored until it
had dropped to the baseline. The elution buffer (8 m urea, 1 m
imidazole and 0.5 m NaCl) was added to the column to elute the target
proteins. The collected proteins were subjected to Coomassie Brilliant
Blue staining after separation by gel electrophoresis. The elution
solution containing proteins was diluted 10 times with refolding
solution [0.75 m l-arginine, 5 mm of GSH(R), 0.5 mm GSSH(O),
5 mm EDTA and 0.1 m Tris pH 9.5]. After refolding, the protein
solution was dialysed against 2 L of phosphate-buffered saline (PBS)
for 4 h at 4 C, followed by 5 L for 4 h and then by 10 L overnight.
The final protein concentration was tested using BCA Protein Assay
Kit (Pierce, Rockford, IL, USA).
For full-length proBDNF, supernatant lysate in binding buffer
(50 mm K-phosphate buffer, 0.3 m NaCl, 10% glycerol, 0.005%
Triton X-100, 10 mm imidazole and 1 mm DTT and 1 mm of PMSF
as a protease inhibitor) was used without urea, and the soluble protein
was affinity purified on the nickel column. The affinity-purified
recombinant proBDNF was soluble in water. All recombinant proteins
were characterized by sodium dodecyl sulphate–polyacrylamide gel
electrophoresis (SDS–PAGE) with Coomassie Brilliant Blue staining
and Western blot analysis. The gels were scanned and the percentage
of the main band was determined by ImageJ software on the optic
density value. For characterization of antibodies to the prodomain of
BDNF we also produced the prodomains of rat NGF and mouse NT3
with the same method as described above.
Generation and characterization of antiserum to proBDNF
The prodomain of BDNF (500 lg) purified from E. coli was
emulsified with Freund adjuvant complete and injected s.c. into a
sheep. Subsequently, the sheep was injected with the same antigen
every 2 weeks with incomplete Freund adjuvant. Antibodies in the
2382 Y.-J. Fan et al.
serum were monitored with ELISA assays. The sheep was bled out
1 week after the fourth injection when the antibody titres reached
1 ⁄ 100 000. The antibody was affinity purified and characterized by
Western blot, ELISA assays and bioassays.
Bioassay of proBDNF on PC12 cells and antibody
neutralization
PC12 cells were maintained in Dulbecco’s minimum essential medium
(DMEM) supplemented with 10% foetal bovine serum, 5% heat-
inactivated horse serum, 50 lg ⁄ mL streptomycin and 50 IU ⁄ mL
penicillin in a humidified atmosphere at 37 C and 5% CO2, as
described previously (Gueorguiev et al., 1999). Three days before the
experiment, the PC12 cells were plated on polylysine-coated slides,
cultured on differentiation medium (DMEM supplemented with 1%
horse serum, 0.5% foetal bovine serum, 1% glutamine, 1% penicil-
lin ⁄ streptomycin and 10 ng ⁄ mL NGF). On the day of the experiment,
triplicate cultures were rinsed five times with NGF-free medium and
treated with proBDNF with four different dilutions of proBDNF
protein (1 ng ⁄ mL; 10 ng ⁄ mL; 100 ng ⁄ mL; 1 lg ⁄ mL). For neutra-
lization experiments, PC12 cells were cultured in 100 ng of proBDNF
in the presence of different concentrations of antibodies to proBDNF
or normal sheep IgG (purified in house). After 48 h, the PC12 cells
were fixed in 4% paraformaldehyde and stained with 4¢,6-diamidino-
2-phenylindole (DAPI; Sigma, MO, USA) to visualize nuclei. As
DAPI or Hoechst 33342-labelled fragmented nuclei morphology is a
very reliable indicator of apoptosis as reported (Kelly et al., 2003), we
Fig. 1. Expression and purification of pro-brain-derived neurotrophic factor
(BDNF) and sortilin. (A) Western blot of purified proBDNF (MW 37 kDA).
Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) gel
stained with Coomassie Brilliant Blue showing the purity of the recombinant
proBDNF. (B) Western blot of purified sortilin (MW 140 kDA). SDS–PAGE
gel stained with Coomassie Brilliant Blue showing the purity of the
recombinant sortilin. (C) Pull-down assay of proBDNF by sortilin-Fc. The
assay was performed as described in the text. Lane 1: sortilin-Fc + proBDNF;
lane 2: sortilin-Fc + proBDNF + neurotensin; (3) sortilin-Fc + proB-
DNF + prodomain of BDNF; (4) proBDNF load directly; (5) sortilin + Fc
only; (6) human IgG + proBDNF; The two bands detected by antihuman IgG
in lane 6 were light and heavy chains, respectively.
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
decided to use this simple method to measure the death of PC12 cells.
The apoptotic nuclei and total number of nuclei were counted. Ten
randomly selected fields were counted for each culture condition and
the experiments were repeated three times. The percentage of
apoptotic cells was calculated against the total number of nuclei
counted. As there was a variation among different assays, we corrected
the variation using the control value as 100% each time and the data
were calculated against the control.
In a separate experiment, the cell toxicity on PC12 cells caused by
proBDNF was tested by 3-[4,5-dimethylthizaol-2-yl]-2,5-diphenyl
tetrazolium bromide (MTT) assay. Briefly, PC12 cells were plated in
triplicate in each condition at a number of 5 · 104 cells ⁄ well in 96-
well plates in 100 lL of media. After overnight incubation, the cells
were rinsed with serum-free DMEM media. ProBDNF was dissolved
in this serum-free media and added to the cells in a serial dilution
(0 ng ⁄ mL, 0.1 ng ⁄ mL, 0.3 ng ⁄ mL, 1.0 ng ⁄ mL, 3.0 ng ⁄ mL,
10.0 ng ⁄ mL). The treated cells were incubated for 20 h at 37 C in
5% CO2. The MTT (Sigma) reagent was reconstituted in PBS to
5 mg ⁄ mL, and the solubilization solution was 10% SDS in 0.01 m
HCl. Ten microlitres of MTT-labelling reagent was added to each well,
and the plate was incubated at 37 C for 4 h. One-hundred microlitres
of solubilization solution was added to each well, and the plate was
incubated overnight at 37 C. The absorbance of the samples was
measured at 563 nm (EIA reader, Bio-Rad). The data were plotted by
using the OD of control wells as 100% survival.
Pull-down assay of proBDNF by sortilin-Fc
To test the binding ability of proBDNF to its receptor sortilin, a pull-
down assay was performed using a recombinant sortilin-Fc fusion
protein expressed in CHO cells. In reaction mixtures of 0.5 mL PBS
containing 0.5 mg bovine serum albumin (BSA) or protein A agarose
beads, the following agents are added: (i) 1 lg sortilin-Fc +
0.5 lg proBDNF; (ii) 1 lg sortilin-Fc + 0.5 lg proBDNF + 50 lg
neurotensin; (iii) 1 lg sortilin-Fc + 0.5 lg proBDNF + 50 lg prodo-
main of BDNF; (iv) 1 lg sortilin-Fc; (v) 0.5 lg proBDNF only; (vi)
human IgG (1 lg, purified in house) + 0.5 lg proBDNF. After
incubation at 4 C for 2 h, the samples were briefly centrifuged and
protein A beads were washed three times and boiled in loading buffer
to release the bound proteins. The proteins were loaded on to SDS–
PAGE (12%), and pulled-down proteins were transferred onto
nitrocellulose paper (Amersham Biosciences) and probed with anti-
bodies to human immunoglobulin (Sigma) and to proBDNF.
Immunohistochemistry of p75NTR and sortilin
For sortilin and p75NTR immunohistochemistry, neonatal rats (P1;
n ¼ 5) or neonatal rats 24 h after sciatic nerve cut (n ¼ 5) were
perfused with 50% Histochoice (AMRESCO, Solon, OH, USA)
containing 2% paraformaldehyde and postfixed in the same fixative
containing 30% sucrose overnight. DRGs dissected from perfused
animals were sectioned on a cryostat microtome at 20 lm. Sections on
glass slides were blocked in 1% blocking solution (Roche, Switzer-
land) and incubated overnight in rabbit polyclonal antibodies against
sortilin at 2 lg ⁄ mL (Abcam), and monoclonal antibody against
p75NTR (MC192) at 1 lg ⁄ mL (Chemicon, CA, USA). After
extensive washing in PBS containing 0.1% Tween 20 (PBST), the
sections were incubated with Cy3-linked or Cy2-linked secondary
antibodies (Vector Laboratories, Burlingame, CA, USA) followed by
DAPI staining for nuclei. After rinsing in PBS, slides were observed
under fluorescence microscopy.
European Journal of Neuroscience, 27, 2380–2390

Fig. 2. Characterization of sheep antibody to the recombinant prodomain
brain-derived neurotrophic factor (BDNF). (A) Using sheep anti-prodomain
BDNF antibody to probe prodomain BDNF (lane 1), mature BDNF (lane 2)
and proBDNF (lane 3). (B) ELISA assay confirmed that the sheep anti-
prodomain antibody binds to prodomain BDNF in a dose-dependent manner.
(C) The sheep anti-prodomain BDNF antibody did not recognize the
recombinant prodomain NT-3 or prodomain NGF.
Western blot of proBDNF, p75NTR and sortilin
The P1 neonatal rat DRGs were homogenized in RIPA buffer (1%
NP-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 m NaCl, 0.01 m
sodium phosphate, pH 7.2, 1% Trasylol). The samples were centri-
fuged at 14 000 r.p.m. for 15 min at 4 C and the supernatant
collected for 10% SDS–PAGE. After SDS–PAGE, the proteins were
transferred to a nitrocellulose membrane. The membranes were probed
with rabbit anti-proBDNF antibody at 1 lg ⁄ mL, monoclonal antibody
against p75NTR (MC192, 2 lg ⁄ mL) and rabbit antisortilin (Abcam,
2 lg ⁄ mL), respectively. After further incubation in horseradish
peroxidase (HRP)-labelled rabbit anti-sheep antibody (1 : 2000) and
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 27, 2380–2390
Differential effects of pro-BDNF on sensory neurons 2383
Fig. 3. Bioassay of pro-brain-derived neurotrophic factor (BDNF) in
PC12 cells and antibody neutralization. (A–D) Double-labelling of
p75NTR and sortilin on PC12 cells (A) anti-p75NTR; (B) antisorti-
lin; (C) DAPI; (D) merged. (E) DAPI labelling of control PC12 cells. (F)
DAPI labelling of apoptotic PC12 cells (the white arrows point out the
apoptotic cells). (G) Recombinant proBDNF induces apoptosis in PC12 cells.
In the presence of serial concentrations of proBDNF from 1 ng ⁄ mL to
1 lg ⁄ mL for 48 h, the number of PC12 cells undergoing cell death was
assessed by DAPI labelling. (H) Affinity-purified antibody to proBDNF
neutralized the effect of proBDNF. The antibodies against proBDNF at serial
concentrations from 2 ng ⁄ mL to 2 lg ⁄ mL were added into culture medium
prior to proBDNF administration. PC: proBDNF administration only; NC:
nothing administration. Data are presented as mean ± SEM (*P < 0.05;
**P < 0.01).
HRP-labelled rabbit anti-mouse antibody for 1 h at room temperature,
the membranes were developed by ECL.
Sciatic nerve transection and treatment
For anaesthesia, neonatal rats at P1 were wrapped with a piece of cloth
and put on ice. After 2–3 min, the mice were put on an ice-cold plate.
When the rat was motionless, a 1-cm-long incision was made in the
left thigh and the left sciatic nerve was exposed and transected.
Immediately after the transection, the gel foam containing proBDNF
or sortin-Fc (10 lg) was placed on the sciatic nerve lesion site and the
wound closed by sutures. BSA was used as a negative control.
2384 Y.-J. Fan et al.

the proBDNF function and the relationship between proBDNF and

Fig. 4. Effects of pro-brain-derived neurotrophic factor (BDNF) on the
survival of PC12 cells. PC12 cells were cultured in the presence of different
concentrations of proBDNF as indicated. The survival of PC12 cells was
assessed by the MTT method. The survival rate decreases with the increase in
proBDNF concentration. *P < 0.05 compared with the control.
Animals were kept on a warm blanket for recovery and then returned
to their mothers. In another group, the neonates were injected
subcutaneously (s.c.) with antiserum to proBDNF or normal sheep
serum (NSS; 5 mL ⁄ kg weight, twice a week), and NSS immediately
after the transection. On Day 8 after the sciatic nerve injury, the rats
were killed with an overdose of sodium pentobarbitone and perfused
with modified Zamboni’s fixative containing 4% paraformaldehyde.
L4 and L5 DRGs were dissected for stereological cell-counting
studies.
sortilin, we cloned proBDNF to pET100 ⁄ D-TOPO and sortilin to
pcDNA3.1-Fc. ProBDNF was expressed in E. coli, purified and
characterized. The recombinant extracellular domain sortilin protein
fused with Fc was expressed in mammalian cells, purified and
characterized. The purities of recombinant purified proteins are shown
Fig. 1A and B. Coomassie Brilliant Blue staining showed that
proBDNF was over 85% pure and sortilin-Fc was 90% pure. The
recombinant proBDNF and sortilin were recognized by specific
antibodies against the prodomain of BDNF and sortilin (Fig. 1A
and B).
To see whether the expressed proBDNF could bind sortilin, we
performed a binding assay using a pull-down approach. As shown in
Fig. 1C, proBDNF bound sortilin-Fc. The binding was competitively
suppressed by a high concentration of neurotensin or the prodomain of
BDNF, suggesting the interaction between proBDNF and sortilin is
reversible and the interaction between proBDNF and sortilin-Fc
resembles that on cell surface in vivo. Using human immunoglobulin
G as a control, we did not detect proBDNF in the sample, suggesting the
binding of proBDNF to sortilin is specific and the human Fc fragment
did not bind proBDNF (Fig. 1C), but human IgG was detected at 25 and
50 kDa as light and heavy chains, respectively (Fig. 1C).
Characterization of sheep antibody to the recombinant
prodomain of BDNF
For the characterization of antibodies to the prodomain, the
recombinant protein was conjugated to Sepharose B. Sheep antiserum
to the prodomain was passed through the column and binding
antibodies were eluded from the column. As shown in Fig. 2A, the
antibody specifically recognized the prodomain and full-length
proBDNF but not mature BDNF. ELISA assay confirmed that the
antibody binds to the prodomain of BDNF in a dose-dependent
manner (Fig. 2B). The antibody did not recognize the recombinant
prodomain of NGF or the prodomain of NT-3 (Fig. 2C).

Bioassay of proBDNF in PC12 cells and antibody

Stereological counting of total numbers of neurons in DRGs
The fixation and sectioning of dissected L5 DRG in neonatal rats were
conducted as described (Zhou et al., 2005).
Statistics
Data are presented as mean ± SEM. Statistical significances were
calculated using Student’s t-test for unpaired samples. Statistically
significant difference was set at P < 0.05.
Results
Expression of proBDNF and sortilin recombinant protein
Recently, it has been demonstrated that proBDNF-induced apoptosis is
mediated by p75NTR and sortilin (Teng et al., 2005). To investigate
neutralization
The interaction of proBDNF with both p75NTR and sortilin is
required to induce neuronal apoptosis (Teng et al., 2005). To examine
the bioactivity of recombinant proBDNF, co-expression of p75NTR
and sortilin in PC12 cells was assessed with immunofluorescence
labelling. The results illustrate that both p75NTR (Fig. 3A) and sortilin
(Fig. 3B) were co-localized in PC12 cells (Fig. 3C and D). In the
presence of proBDNF for 48 h, the number of cells undergoing death
was significantly increased in a dose-dependent manner (Fig. 3G),
with an IC50 of 5–10 ng ⁄ mL. Using a different approach to detect
survival of PC12 cells, the MTT assay, we showed that proBDNF
caused a dose-dependent decrease in cell viability (Fig. 4).
To further demonstrate biological activity of proBDNF and the
biological activity of the antibodies against the prodomain, the
affinity-purified antibody against the prodomain was added together
with proBDNF. The cell death-inducing effects of proBDNF were

Fig. 5. Co-localization of p75NTR and sortilin in subpopulation of neurons in normal neonatal rat DRG (A–H) and DRG (J–M) 24 h after sciatic nerve cut. (A–D)
The representative low-magnification microphotographs for p75NTR (red), sortilin (green), DAPI nuclear counterstaining (blue), and the merged images,
respectively. The boxed region in (D) was enlarged as panel pictures, as in (E–H). For better demonstration of co-localization of p75 and sortilin, the boxed region
in (H) was further selected to take high-power confocal microimage as shown in (I), the typical double-labelling neurons were marked by thick arrowheads, and the
single-labelling neurons were also categorized into expressing p75NTR-only (arrows) and expressing sortilin-only (thin arrowheads). (J), (K), (L) and (M) Pho-
tographs of a DRG section stained for p75NTR, sortilin, DAPI and merged, respectively, from a rat with sciatic nerve cut. Scale bars: 50 lm (D, H, I).
(N) The quantitative data of single- and ⁄ or double-labelling neurons in DRG of normal P1 rat and DRG of rats with sciatic nerve injury.

ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 27, 2380–2390
 Differential effects of pro-BDNF on sensory neurons 2385

ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 27, 2380–2390
2386 Y.-J. Fan et al.
Fig. 6. Western blot analysis of p75NTR, sortilin and proBDNF on DRG
homogenate. (A) p75NTR; (B) sortilin; (C) proBDNF; lane 1: brain; lane 2:
DRG.
Fig. 7. Exogenous proBDNF reduced the number of sensory neurons in L5
DRG after sciatic nerve transection. The sciatic nerve was cut on P1 and
recombinant proBDNF trapped into gel foams were placed in the injury sites
(n ¼ 6). In control group, the gel foams containing BSA were placed in the
injury sites or NSS was injected s.c. after sciatic nerve transaction (n ¼ 10).
Total numbers of sensory neurons in bilateral DRG were counted stereolog-
ically. cl, contralateral DRG; ip, ipsilateral DRG. Data are presented as
mean ± SEM. **P < 0.01 compared with the contralateral DRGs from the
same animal. ##P < 0.01 compared with ipsilateral DRGs of control group.
abolished by different concentrations of antibody to proBDNF
(Fig. 3H). The level of apoptosis was reduced in the presence of the
antibodies to proBDNF in a dose-dependent manner (Fig. 3H). These
results suggest that recombinant proBDNF has the biological activity
of inducing the death of PC12 cells in vitro. These results indicate that
the antibody to the prodomain is biologically active and can be used to
investigate physiological functions of proBDNF in vivo.
Expression of proBDNF, p75NTR and sortilin in DRG
To test the function of proBDNF in sensory neurons in vivo, we firstly
investigated the expression of proBDNF receptors (p75NTR and
sortilin). p75NTR-immunoreactivity is mainly present in small- and
medium-sized sensory neurons, non-neuronal cells and occasionally in
axons (Fig. 5A). Sortilin is expressed in most neuronal cells (Fig. 5B,
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
Fig. 8. proBDNF antiserum increases the survival of sensory neurons in L5
DRG after sciatic nerve transaction. The sciatic nerve was cut on P1 and the
antiserum to proBDNF (n ¼ 6) was injected s.c. In control group, the gel foams
containing BSA were placed in the injury sites or NSS was injected s.c. after
sciatic nerve transaction (n ¼ 10). Total number of sensory neurons in bilateral
were counted stereologically. cl, contralateral DRG; ip, ipsilateral DRG. Data
are presented as mean ± SEM. **P < 0.01 compared with the contralateral
DRGs from the same animals. ##P < 0.01 compared with ipsilateral DRGs of
control group. $$P < 0.01 compared with the contralateral DRGs of the control
group.
Fig. 9. Effects of sortilin-Fc on axotomized sensory neurons. The sciatic
nerve was cut on P1 and recombinant sortilin-Fc trapped into gel foams were
placed in the injury sites (n ¼ 6). In control group, the gel foams containing
BSA were placed in the injury sites (n ¼ 10). Total numbers of sensory neurons
in DRG were counted stereologically. cl, contralateral DRG; ip, ipsilateral
DRG. **P < 0.01, compared with the contralateral DRGs from the same
animals. ##P < 0.05 compared with ipsilateral DRG of the control group.
F and M), and it is also co-localized with p75NTR (orange ⁄ yellow) in
a subpopulation of small neurons (Fig. 5D, H and M). Some sortilin-
positive neurons did not express p75NTR (thin arrows, Fig. 5M).
Some p75NTR-positive neurons did not express sortilin (thick arrows.
Fig. 5M). Sortilin is also expressed in non-neuronal cells, possibly
Schwann cells and satellite glia. Confocal microscopic examination
showed that sortilin is present in vesicular structures and displayed
punctate staining in both neurons and non-neuronal cells (Fig. 5M).
Statistical analysis indicated that about 60% of neurons were p75NTR
positive, 70% of neurons were positive for sortilin, and 30% of
neurons were positive for both p75NTR and sortilin (Fig. 5N).
Sciatic nerve lesion did not cause significant changes in the
expression pattern of sortilin in DRG neurons (Fig. 5I–L). Twenty-
European Journal of Neuroscience, 27, 2380–2390

four hours following axotomy, the percentage of neurons expressing
p75NTR was reduced from 60% to 45%, and the percentage of
neurons expressing sortilin slightly increased (Fig. 5N). As a result,
the percentage of co-localization of these two receptors increased
slightly but without statistical significance (Fig. 5N).
Western blot further confirmed the expression of sortilin, p75NTR
and proBDNF in rat DRG (Fig. 6). A single band of 110 kDa
corresponding to size of sortilin was detected by rabbit antibody to
sortilin (Fig. 6B). A single band of 75 kDa corresponding to p75NTR
was detected by the specific MC192 antibody (Fig. 6A). Consistent
with our previous studies (Zhou et al., 2004), a specific band at about
32 kDa in DRG and brain homogenates was recognized by proBDNF
antibody (Fig. 6C).
Exogenous proBDNF reduces the survival of sensory
neurons in vivo
To examine the effects of proBDNF in sensory neurons in vivo,
proBDNF was point-mutated to generate furin-resistant proBDNF
(Mowla et al., 2001). The dried gel foam containing recombinant
proBDNF was placed at the injury stump of sciatic nerve transected in
P1 neonatal rats. Seven days after the treatment, the L5 DRGs were
dissected and the numbers of the sensory neurons were stereologically
counted. Results show that the average total number of neurons in
ipsilateral L5 DRGs was significantly reduced compared with the
contralateral side in both control and proBDNF-treated groups
(Fig. 7). The total numbers of neurons in ipsilateral and contralateral
DRGs of control group (treated with BSA or NSS were 7297 ± 1271
and 13536 ± 1570, while the total number of neurons in ipsilateral and
the contralateral DRG treated with proBDNF were 4961 ± 1154 and
12024 ± 1739, respectively. The number of neurons in the ipsilateral
DRGs treated with proBDNF was significantly lower than ipsilateral
DRGs of the control group, suggesting that exogenous proBDNF
exaggerated the loss of sensory neurons induced by axotomy.
ProBDNF antiserum reduces cell death of axotomized
sensory neuron in vivo
To investigate the function of endogenous proBDNF on the cell
survival of sensory neuron after nerve lesion, the specific serum against
proBDNF was injected s.c. after sciatic nerve transection. The total
numbers of neurons in ipsilateral and contralateral DRGs of control
group (treated with BSA or NSS) were 7297 ± 1271 and
13536 ± 1570, while the average total numbers of neurons in ipsilateral
and the contralateral DRGs treated with the antiserum to the prodomain
of BDNF were 11297 ± 613 and 17952 ± 2156, respectively (Fig. 8).
The number of neurons in the ipsilateral DRGs treated with proBDNF
antiserum significantly diminished the loss of sensory neurons after the
sciatic nerve transection, compared with the control group, suggesting
that the neutralization of endogenous proBDNF could rescue the loss of
sensory neurons induced by axotomy. Interestingly, the anti-proBDNF
treatment in neonatal rats increased the total number of sensory neurons
in the contralateral DRGs. Compared with the contralateral DRG
treated with proBDNF antibody, the number of neurons in the
ipsilateral DRG was still significantly reduced (Fig. 8).
Sortilin-Fc protein increases the survival of axotomized
DRG neuron
To further examine whether the apoptosis of sensory neurons after nerve
transection is mediated by sortilin, the recombinant extracellular domain
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 27, 2380–2390
Differential effects of pro-BDNF on sensory neurons 2387
of sortilin-Fc was stored in gel foam and released persistently at the
injury sites of sciatic nerve transection of P1 neonatal rats. Seven days
after the administration of sortilin protein, the average total numbers of
the ipsilateral and contralateral DRG neurons were 10394 ± 1452 and
12542 ± 1519, respectively. In control group, the average numbers of
the ipsilateral and contralateral DRG were 7297 ± 1271 and
13536 ± 1570. Apparently, the sortilin-Fc significantly reduced the
loss of neurons in L5 DRGs after sciatic nerve transection (Fig. 9). No
significant difference in the number of neurons was found between
ipsilateral and contralateral DRG treated with sortilin-Fc.
Discussion
In the present study, we have shown that recombinant proBDNF
induces a dose-dependent increase in PC12 cell death, which is
abolished in the presence of a neutralizing antibody to the prodomain.
The biological activity of proBDNF was also demonstrated in vivo in
our previous studies (Wang et al., 2006) by injecting into the sciatic
nerve, suggesting that proBDNF was internalized by sensory neurons,
transported anterogradely and retrogradely in the axons. With the
biologically active proBDNF and the neutralizing antibodies we have
illustrated that exogenous proBDNF induces an increase in the loss of
sensory neurons after axotomy in neonatal rats and that the treatment
of neonatal rats with the prodomain antibody prevents the loss of
axotomized neurons. We further showed that the soluble sortilin
protein applied in vivo in the axotomized axons prevents the loss of
axotomized sensory neurons. Our data suggest that endogenous
proBDNF has a physiological function of inducing the death of
axotomized neurons.
All neurotrophins are initially synthesized as precursor proteins
containing a signal peptide with glycosylation sites and pairs of
dibasic amino acids that are recognized by processing enzymes
(Hempstead, 2006). Mature BDNF is derived from proBDNF that is
cleaved by the serine protease furin or other members of the
prohormone convertase family (Mowla et al., 2001) and released in
an activity-dependent manner (Goodman et al., 1996; Balkowiec &
Katz, 2002). Mature BDNF is essential for cell survival during the
development of a subpopulation of sensory neurons (Altar et al., 1997;
Conner et al., 1997) and axotomized sensory neurons after the
peripheral nerve injury (Zhou et al., 2005). Mature BNDF is derived
from both neuronal targets such as skin and visceral organs (Erickson
et al., 1996; EIShamy & Ernfors, 1997), and sensory neurons by
autocrine secretion mechanism (Acheson et al., 1995). Sensory
neurons highly express BDNF during development (Schecterson &
Bothwell, 1992), and the expression continues in adults (Zhou &
Rush, 1996; Zhou et al., 2005). This pattern of autocrine secretion of
mature BDNF strongly suggests that proBDNF may also be expressed,
transported and secreted by the sensory neurons in DRG (Wang et al.,
2006). Whether proBDNF is released is controversial. Previous studies
showed that proBDNF can be released from transfected and overex-
pressed cells (Mowla et al., 1999), and from CNS neurons (Traficante
et al., 2007). However, a recent study showed that constitutively
expressed proBDNF is rapidly converted within CNS neurons into
mature BDNF, which is released by excitatory activity, whereas only
10% of proBDNF is released (Matsumoto et al., 2008). In the previous
study (Zhou et al., 2004), we showed that proBDNF is expressed by
sensory neurons in rat DRG and was axonally transported in both
directions by sensory neurons (Wang et al., 2006). In the present
study, we showed that proBDNF in DRG neurons can be detected by
Western blot (Zhou et al., 2004), consistent with previous studies that
secreted proBDNF dimer is about 60 kDa and glycosylated in
2388 Y.-J. Fan et al.
mammalian cells (Kolbeck et al., 1994; Heymach & Shooter, 1995).
P75NTR and sortilin, the functional receptors of proBDNF, are
co-localized in sensory neuron in DRG, indicating that proBDNF
may be an alternative ligand to modulate cell survival and cell death
in DRG.
The prodomain of BDNF is highly conserved between species
(Kolbeck et al., 1994; Heymach & Shooter, 1995). The prodomain is
required for correct folding and intracellular trafficking of BDNF
(Egan et al., 2003; Chen et al., 2004). The Val66Met mutation in the
prodomain causes dysfunction of BDNF transport, reduction in
hippocampal volume and impaired episodic learning (Egan et al.,
2003; Hariri et al., 2003). The mutation also caused a number of
neurological disorders (Egan et al., 2003; Hariri et al., 2003; Bath &
Lee, 2006; Numata et al., 2006; Hashimoto, 2007). It was reported
that proBDNF is a potential proapoptotic factor, inducing sympathetic
ganglion neuron apoptosis in vitro (Teng et al., 2005). In the present
study, we confirmed that proBDNF induced cell death of cultured
PC12 cells. These cells express p75NTR and sortilin on their cell
surface. This effect was blocked by the antibody to the prodomain,
suggesting the apoptotic effect may be mainly mediated via the
prodomain of proBDNF.
In neonatal rats, transection of sciatic nerve caused a loss of about
50% neurons in the L5 DRG, and mature BDNF is required for the
survival of a population of these neurons (Zhou et al., 2005).
ProBDNF with point mutations within the cleavage site trapped in
gelatin sponge further reduced the survival of sensory neuron. It is
plausible that exogenous proBDNF activates p75NTR and sortilin to
affect the cell death. Labelled exogenous proBDNF injected into the
sciatic nerve can also be transported anterogradely and retrogradely
(Wang et al., 2006). ProBDNF immunoreactivity accumulates in the
proximal and distal segments of crushed sciatic nerve, suggesting that
endogenous proBDNF can also be secreted and transported antero-
gradely and retrogradely (Wang et al., 2006). To further investigate
whether endogenous proBDNF elicits cell death after lesion, the
antiserum against the prodomain was injected after axotomy. The loss
of sensory neurons in the ipsilateral DRG decreased significantly by
the antibody to proBDNF, indicating that endogenous proBDNF is
responsible for the apoptosis of sensory neuron after the transection.
These results also suggest that proBDNF is likely released and exerts
its physiological functions in vivo. As proBDNF is also highly
expressed in adult DRG neurons (Zhou et al., 2004) and axotomy of
sensory neurons in the adult rat results in upregulation of BDNF (Zhou
et al., 1999), it is likely that proBDNF also plays a role in the death of
axotomized sensory neurons observed in the adult (Hu & McLachlan,
2002).
We showed that about one-third of sensory neurons highly express
both p75NTR and sortilin. It is likely that this population of sensory
neurons is more sensitive to proBDNF. However, we found that both
p75NTR and sortilin were mainly localized to intracellular organelles
but not on the cell surface (Fig. 5). How proBDNF activates sortilin
and p75NTR, triggering the death of these neurons is not clear. It is
possible that autocrine-released proBDNF stimulates translocation of
intracellular p75NTR and sortilin to lipid rafts or clathrin-coated pits,
as seen in neurons stimulated by NGF (Deinhardt et al., 2007). It has
been reported that the induction of apoptosis of sympathetic neuron
and glia by proBDNF is dependent on the p75NTR and sortilin
co-receptors (Teng et al., 2005). Moreover, proBDNF is at least
10–20 times more effective at inducing apoptosis compared with
mature BDNF (Teng et al., 2005; Kenchappa et al., 2006). It was
predicted that the access of proBDNF to its physiological targets must
be strictly regulated (Teng et al., 2005). Their results showed that
soluble sortilin can form a high-affinity complex with proBDNF, and
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
this preformed complex is ineffective in eliciting death of cells
co-expressing p75NTR and sortilin (Teng et al., 2005). To investigate
the role of endogenous proneurotrophins in sensory neurons in DRG
after axotomy, exogenous sortilin-Fc fusion molecule trapped in
gelatin sponge was placed into the injury site. Our data illustrate that
the exogenous sortilin-Fc could also increase the number of neurons
by about 42% compared with BSA ⁄ NSS treatment in DRG after
lesion. As sortilin is also the receptor for proNGF, which signals the
death of p75NTR-expressing neurons (Nykjaer et al., 2004; Volosin
et al., 2006; Domeniconi et al., 2007), the protection of dying
sensory neuron by soluble sortilin may be due to the formation of
complexes with endogenous proBDNF or proNGF. Exogenous
sortilin-Fc may prevent the interaction of endogenous proBDNF or
proNGF with their receptors on sensory neurons, block the retrograde
transport and restrict the biological activity in vivo. As the NGF gene
is upregulated in Schwann cells after sciatic nerve lesion (Frostick
et al., 1998; Agthong et al., 2006), proNGF released from the
Schwann cells at the lesion site may also induce the death of
axotomized sensory neurons.
Previous studies from our group showed that mature BDNF reduces
the rate of death of sensory neurons in DRG after peripheral nerve
injury (Zhou et al., 2005), while this current study demonstrates that
proBDNF increases cell death of axotomized neurons. These findings
support the hypothesis that the balance between cell death and cell
survival may be determined by the ratio of proBDNF and mature
BDNF in DRG after lesion. This balance may also be determined by
expression levels of their receptors TrkB, p75NTR and sortilin after
axotomy. Our previous studies showed that the expression of TrkB and
p75NTR was downregulated after axotomy in neonatal rats (Zhou
et al., 2005), whereas we did not find a significant change in the
sortilin expression in sensory neurons in the present study. These
studies suggest that the expression levels of p75NTR and TrkB but not
sortilin could determine the life or death of these neurons. Indeed, our
previous study and current study showed that exogenous recombinant
mature BDNF, proBDNF or their respective neutralizing antibodies
either rescued or exaggerated the death of axotomized sensory neurons
(Zhou et al., 2005). The prediction of this balance model has also been
demonstrated by the differential effect of proNGF and mature NGF.
ProNGF preferentially activates p75NTR to elicit apoptosis, while
mature NGF preferentially activates TrkA receptor, which negates this
proapoptotic effect (Yoon et al., 1998; Lee et al., 2001). The diversity
of neurotrophin functions may be modulated by the regulated release
of precursors vs mature proteins in the nervous system, or by the
activity of proteases processing these precursors (Bruno & Cuello,
2006). In addition, our data also suggest that the death of sensory
neurons after axotomy is not only due to the lack of neurotrophic
factors from their targets but also due to positive death signals released
from neurons and injured nerves. Our study suggests that the
administration of specific antibodies to the prodomain of neurotro-
phins or soluble extracellular domain of sortilin may have a
therapeutic potential of rescuing apoptotic neurons after neurotrauma
or other pathological conditions.
In the present study, we found that the number of DRG neurons was
significantly increased in the contralateral uninjured DRG. This result
was surprising, as sensory neurons in the DRG are well developed
after birth and the critical period of naturally occurring neuronal death
is during the embryonic stage in rodents (Snider, 1994; Ma et al.,
1999). It is unlikely that this effect was due to the suppression of the
programmed cell death or increase in neuroblast proliferation by the
antibody. Recently, we showed that neuronal addition in the trigeminal
ganglia is the natural process during postnatal maturation with age,
and neural crest precursors are present in adult DRG and develop into
European Journal of Neuroscience, 27, 2380–2390

mature sensory neurons in vivo and in vitro (Lagares et al., 2007; Li
et al., 2007). It is likely that the increased number of sensory neurons
in the contralateral DRG was due to the increased differentiation or
proliferation of neural crest precursors induced by suppression of
endogenous proBDNF. Thus, our data suggest that endogenous
proBDNF may suppress neuronal differentiation or maturation of
sensory neuron precursors. This interesting phenomenon requires
further characterization in our future studies.
Acknowledgements
We wish to thank Dr Carlos Morales for providing sortilin plasmids, Dr M.
Kojima for the mutated proBDNF plasmid, Ms Jinxian Mi, Ms Jinhua Zhong
and Xiao-Hui Guo for technical assistance, and Dr Damien Keating and Tony
Pollard for critical reading for the manuscript. This work was supported by
NHMRC grants (375109 and 375110).
Abbreviations
BDNF, brain-derived neurotrophic factor; BSA, bovine serum albumin; DAPI,
4¢,6-diamidino-2-phenylindole; DMEM, Dulbecco’s minimum essential med-
ium; DRG, dorsal root ganglia; DTT, dithiothreitol; HRP, horseradish
peroxidase; LTD, long-term depression; MTT, 3-[4,5-dimethylthizaol-2-yl]-
2,5-diphenyl tetrazolium bromide; NGF, nerve growth factor; NSS, normal
sheep serum; P, postnatal day; PBS, phosphate-buffered saline; PCR,
polymerase chain reaction; PMSF, phenylmethylsulphonyl fluoride; SDS–
PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis; TrkB,
tropomyosin-related kinase B.
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