T H E J O U R N A L O F C E L L B I O L O G Y
© The Rockefeller University Press $30.00
The Journal of Cell Biology, Vol. 181, No. 1, April 7, 2008 37–41
Correspondence to Philip Van Damme: email@example.com
Abbreviations used in this paper: ALS, amyotrophic lateral sclerosis; AM,
acetyoxymethyl; CSF, cerebrospinal fl uid; FTLD, frontotemporal lobe dementia;
GFAP, glial fi brillary acidic protein; GRN, granulin; NF-H, neurofi lament heavy
chain; PGRN, progranulin; SLPI, secreted leucocyte protease inhibitor.
Progranulin (PGRN) is a glycosylated protein released by a
variety of cells. It contains a signal peptide and 7.5 tandem
repeats of highly conserved granulin motifs each with 12 cys-
teine residues ( Bateman et al., 1990 ; Shoyab et al., 1990 ;
Bhandari et al., 1992 ; Plowman et al., 1992 ; He and Bateman,
2003 ). PGRN is widely expressed ( Bhandari et al., 1993 ; Daniel
et al., 2000 ) and has been implicated in many processes, such
as development, tumor proliferation, wound healing, and
infl ammation ( Bateman and Bennett, 1998 ; He and Bateman,
2003 ; Ahmed et al., 2007 ). In peripheral tissues, extracellular
proteases, such as elastase, were shown to be able to cleave
PGRN into several GRNs (GRN A – F and paragranulin), which
probably have separate functions ( Zhu et al., 2002 ; He and
Bateman, 2003 ). In models of wound healing, secreted leuko-
cyte protease inhibitor (SLPI) prevents PGRN processing
through inhibition of elastase enzymatic activity and by binding
PGRN and thus sequestering it from elastase ( Zhu et al., 2002 ;
He and Bateman, 2003 ).
Little is known about the role of PGRN in the central
nervous system. PGRN is widely expressed during early
neural development ( Daniel et al., 2003 ) but later on its ex-
pression becomes restricted to defi ned neuronal populations,
such as cortical and hippocampal pyramidal neurons and Pur-
kinje cells ( Daniel et al., 2000 ). It has been implicated in the
sexual differentiation of the brain ( Suzuki and Nishiahara,
2002 ). PGRN is up-regulated in activated microglial cells
( Baker and Manuelidis, 2003 ; Baker et al., 2006 ; Mackenzie
et al., 2006 ; Mukherjee et al., 2006 ) but not in astrocytes
Recently the interest in PGRN was raised because of the
discovery of null mutations in the PGRN gene as a common
cause of autosomal dominant tau-negative frontotemporal lobe
dementia (FTLD; Baker et al., 2006 ; Cruts et al., 2006 ; Gass
et al., 2006 ; Mukherjee et al., 2006 ; Pickering-Brown et al.,
2006 ; Bronner et al., 2007 ; van der Zee et al., 2007 ). Further-
more, null mutations were also found in apparently sporadic
patients ( Le Ber et al., 2007 ). The majority of FTLD-causing
mutations in PGRN are predicted to cause functional null alleles
with premature termination of the coding sequence followed
by nonsense-mediated decay of the mutant mRNA ( Baker et al.,
2006 ; Cruts et al., 2006 ). Therefore, haploinsuffi ciency with
over, missense changes in PGRN were identifi ed in patients
with motor neuron degeneration, a condition that is re-
lated to FTLD. Most mutations identifi ed in patients with
FTLD until now have been null mutations. However, it re-
mains unknown whether PGRN protein levels are reduced
in the central nervous system from such patients. The ef-
ecently, mutations in the progranulin ( PGRN ) gene
were found to cause familial and apparently spo-
radic frontotemporal lobe dementia (FTLD). More-
fects of PGRN on neurons also remain to be established.
We report that PGRN levels are reduced in the cerebro-
spinal fl uid from FTLD patients carrying a PGRN mutation.
We observe that PGRN and GRN E (one of the proteolytic
fragments of PGRN) promote neuronal survival and en-
hance neurite outgrowth in cultured neurons. These results
demonstrate that PGRN/GRN is a neurotrophic factor
with activities that may be involved in the development of
the nervous system and in neurodegeneration.
Progranulin functions as a neurotrophic factor
to regulate neurite outgrowth and enhance
Philip Van Damme , 1,2,3 Annelies Van Hoecke , 1,3 Diether Lambrechts , 3,4 Peter Vanacker , 1,2 Elke Bogaert , 1,3
John van Swieten , 5 Peter Carmeliet , 3,4 Ludo Van Den Bosch , 1,3 and Wim Robberecht 1,2,3
1 Laboratory of Neurobiology, 2 Department of Neurology, 3 Department for Transgene Technology and Gene Therapy, and 4 Center for Transgene Technology
and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Katholieke Universiteit Leuven, Campus Gasthuisberg, 3000 Leuven, Belgium
5 Department of Neurology, Erasmus MC University Medical Center, Rotterdam, Netherlands
JCB • VOLUME 181 • NUMBER 1 • 2008 38
brain neuroblastoma) cells ( Daniel et al., 2000 ) and to inhibit
apoptosis of tumor cells ( He et al., 2002 ; Tangkeangsirisin et al.,
2004 ), the effects of PGRN on neurons have not been estab-
lished. To explore the neurotrophic potential of PGRN, we stud-
ied the effects of recombinant GRN E (aa 494 – 594 of PGRN,
a functional proteolytic fragment of PGRN) and full-length
PGRN (only lacking the first 18 aa, which code for the signal
peptide) in monocultures of rat motor and cortical neurons.
To reduce potential confounding effects from other growth
factors, neurons were treated with GRN E or PGRN in serum-
free medium with no other recombinant growth factors added.
Motor neurons isolated from the ventral spinal cord were seeded
at low density allowing reliable quantifi cation of survival by
counting experiments ( Van Den Bosch et al., 2004 ; Van Damme
et al., 2007 ). GRN E was added to these cultures on day 1 in
culture and was found to support neuronal survival in a dose-
dependent manner ( Fig. 2 A ). This neurotrophic effect was
still visible after 1 wk, a time point at which only a limited
number of neurons in these serum-free and neurotrophin-free
cultures survive ( Fig. 2 B ). The maximal dose of GRN E
increased neuronal survival by 64% on day 2 and by 101%
on day 6.
reduced PGRN-induced neuronal survival is thought to cause
neurodegeneration. Missense mutations in PGRN have been
identifi ed as well. They were found in some patients with
FTLD, with or without amyotrophic lateral sclerosis (ALS;
Spina et al., 2007 ; van der Zee et al., 2007 ), and in rare ALS
patients ( Schymick et al., 2007 ; Sleegers et al., 2008 ). The patho-
genetic nature of these missense mutations remains to be
demonstrated. It was recently shown that several of these
missense mutations reduce the release of PGRN and thus
also give rise to insuffi cient availability of PGRN ( Shankaran
et al., 2008 ).
Whether PGRN levels are indeed reduced in the central
nervous system of patients with null mutations requires confi rma-
tion and whether PGRN can directly affect neurons has not
yet been shown. We therefore measured PGRN levels in the
cerebrospinal fl uid (CSF) from patients with PGRN mutations
and controls and explored the potential neurotrophic effects of
PGRN. The effects of exogenous PGRN and one of its proteo-
lytic fragments (GRN E) were studied in cortical neurons and
spinal motor neurons, the two types of neurons relevant for
FTLD and ALS.
Results and discussion
PGRN protein levels in the CSF from
patients and controls
To measure PGRN protein levels, we developed an ELISA as-
say using a monoclonal PGRN antibody to coat 96-well plates,
a polyclonal biotinylated PGRN antibody to detect the signal,
and human recombinant PGRN as standard ( Fig. 1 A ). PGRN
protein was detectable in CSF and was measured in the CSF
from three patients carrying the Ser82fs mutation in PGRN and
from 24 controls. The optical densities were 0.08 ± 0.04 and
0.23 ± 0.02 for patients and controls, respectively (P = 0.004).
This corresponded to 2.2 ± 1.0 and 6.2 ± 0.6 ng/ml in patients
and controls, respectively (P = 0.04; Fig. 1 B ). The clearly
reduced levels in patients with the Ser82fs mutation are in
agreement with the predicted nonsense-mediated mRNA decay
caused by the premature translation termination, resulting in re-
duced protein levels.
Effect of PGRN on neuronal survival
Although PGRN was shown to induce proliferation of PC-12
(rat adrenal gland pheochromocytoma) and SK-N-DZ (human
Figure 1. Measurement of PGRN levels in the CSF by ELISA. (A) Example
of standard curve of recombinant PGRN (R 2 = 0.99). (B) Mean PGRN
levels in controls ( n = 24) and patients with a Ser82fs mutation ( n = 3;
*, P = 0.04). Error bars show mean ± SEM.
Figure 2. PGRN/GRN improves neuronal survival. (A) Effect of GRN E
on survival of motor neurons on day 2 in culture normalized to survival
on day 1 ( n = 5 – 11; R 2 = 0.99). (B) Effect of GRN E on the survival of
motor neurons at different time points in culture ( n = 5 – 11; *, P < 0.03).
(C) Fluorescence (fl uorescein counts/1,000) emitted by cortical neurons
seeded in different densities after loading with calcein-AM on day 2 in
culture ( n = 5; R 2 = 0.99; inset shows neurons loaded with calcein; Bar,
50 μ m). (D) Effect of GRN E on fl uorescence of cortical neurons on day 2
in culture normalized to untreated control ( n = 8 – 15; R 2 = 0.98). (E) Effect
of PGRN on survival of motor neurons ( n = 6 – 10; R 2 = 0.95). (F) Effect of
PGRN on survival of cortical neurons ( n = 9; R 2 = 0.95). Error bars show
mean ± SEM.
39 PROGRANULIN IS A NEUROTROPHIC FACTOR • VAN DAMME ET AL.
survival and axonal outgrowth, respectively, supports the hypoth-
esis that different GRNs and their precursors may have distinct
biological functions ( Zhu et al., 2002 ).
Recently, PGRN-deficient mice have been generated
( Kayasuga et al., 2007 ). These mice develop normally and do
not show behavioral or motor abnormalities until 11 wk of age.
However, it remains to be seen whether these animals develop
axonal loss or neurodegeneration as they age.
Our results demonstrate that PGRN/GRN is a neurotrophic
factor that enhances neuronal survival and axonal outgrowth.
It supports the hypothesis that a relative lack of PGRN in pa-
tients with PGRN mutations may alter the integrity of neurites
and lead to neurodegeneration. Mechanisms other than the lack
of neurotrophic effect resulting from PGRN shortage may play
a role in PGRN-associated neurodegeneration as well. In a
recent study, PGRN knockdown was found to induce caspase-
dependent cleavage of TDP-43 with accumulation of TDP-43
fragments ( Zhang et al., 2007 ), similar to what is seen in FTLD
and ALS ( Neumann et al., 2006 ).
Although insuffi cient trophic support for neurons has been
an appealing hypothesis for the pathogenesis of neurodegenera-
tive disorders and the premise for many therapeutic strategies
( Vande Velde and Cleveland, 2005 ), PGRN is the only neuro-
trophic factor identifi ed to cause human disease through null
mutations and haploinsuffi ciency. Therefore, it is warranted
to investigate the therapeutic potential of PGRN for neuro-
de generative disorders.
Similar results were obtained in cortical neurons. To quan-
tify survival of cortical cultures, a calcein-acetyoxymethyl (AM)
assay was used ( Bozyczko-Coyne et al., 1993 ; Lin et al., 2001 ).
This assay proved to reliably refl ect the number of living cells
in culture, as shown in Fig. 2 C . To assure that the signal ob-
tained was in the linear part of the assay, 10,000 cells per
well were seeded for survival experiments. GRN E improved
survival of cortical neurons by 41.4% ( Fig. 2 D ), confi rming
that the GRN E proteolytic fragment of PGRN has neuro-
The full-length precursor, PGRN, exerted similar neuro tro-
phic properties, both in motor and cortical neurons ( Fig. 2, E and F ).
The maximal effect observed was a 38.5 and 22.0% increase in
neuronal survival for motor and cortical neurons, respectively.
The effects of GRN E and PGRN on motor and cortical
neurons were dose-dependent and the ED 50 (0.1 – 3 ng/ml) ap-
proximated the ED 50 of the proliferative effects on cell lines
reported previously ( Zhou et al., 1993 ).
In wound healing, SLPI was shown to block PGRN pro-
cessing both by inhibiting the proteolytic activity of elastase
and by direct binding to PGRN ( Zhu et al., 2002 ). We there-
fore investigated the effect of SLPI on the effects of PGRN
in neuronal cultures and found that coadministration of SLPI
abolished the neurotrophic effects of PGRN on both motor
neurons and cortical neurons ( Fig. 3, A and B ). This suggests
that proteolysis of PGRN is needed for it to have neurotrophic
effects. Alternatively, because of its dual actions, SLPI may
scavenge PGRN and thus keep PGRN from exerting its neuro-
Effect of PGRN on neurite outgrowth
To study the effect of PGRN/GRN on neurite outgrowth, motor
and cortical neuronal cultures were treated with GRN E or
PGRN on day 0 and cells were stained for neurofi lament heavy
chain (NF-H) after 24 h. This allowed us to quantify the size of
the soma and the length of neurites present ( Fig. 4, A – D ). GRN E
and PGRN had no effect on the size of the cell soma. Both
molecules increased the maximal neurite length, an effect that
was especially apparent after the treatment of cells with full-
length PGRN ( Fig. 4, E and F ). Again, the neurite outgrowth –
stimulating effect of PGRN was inhibited by SLPI ( Fig. 4 F ).
From these experiments, it is clear that neurite length is
mainly affected by PGRN and, to a lesser extent, by GRN E,
whereas the opposite was true for the effect on neuronal sur-
vival. This differential effect of GRN E and PGRN on neuronal
Figure 3. Inhibition of PGRN by SLPI abolishes its neurotrophic properties.
(A and B) Effect of SLPI (ng/ml) on the neurotrophic properties of PGRN
(ng/ml) in motor neurons (A; n = 3 – 4; *, P < 0.001) and cortical neurons
(B; n = 6; *, P < 0.01). Error bars show mean ± SEM.
Figure 4. Effect of PGRN/GRN on soma size and neurite outgrowth.
(A – D) NF-H staining of motor neuron (A and B) and cortical culture
(C and D) treated with GRN E (B), PGRN (D), or control (A and C; Bar,
50 μ m). (E and F) Effect of 100 ng/ml GRN E or PGRN (in the absence
and presence of 100 ng/ml SLPI) on soma size (E) and maximal neu-
rite length (F; n = 109 – 476; *, signifi cantly different from control [P <
0.001]; #, signifi cantly different from GRN E [P < 0.03]). Error bars
show mean ± SEM.
JCB • VOLUME 181 • NUMBER 1 • 2008 40
more than 100 neurons per experimental condition were measured on the
obtained digital pictures using Lucia imaging software (version 4.60;
Materials and statistics
Media and additives were obtained from Invitrogen. All other chemicals
were obtained from Sigma-Aldrich. Partial recombinant human PGRN (aa
494 – 594, coding for GRN E and fl anking regions) was obtained from Ab-
nova Corporation. Full-length recombinant human PGRN (aa 18 – 593) was
obtained from R & D Systems. NF-H and GFAP antibodies were obtained
from Sigma-Aldrich. NeuN antibody was obtained from Millipore and anti-
oligodendrocyte marker 04 was obtained from R & D Systems. Alexa-
labeled secondary antibodies were obtained from Invitrogen.
Mean data are shown as mean ± SEM and Student ’ s t tests were
used to calculate signifi cance. When more than two groups were com-
pared, a one-way analysis of variance with Tukey-Kramer multiple compar-
isons was used.
We thank W. Scheveneels and N. Hersmus for their excellent technical assistance.
This work was supported by grants from the Fund for Scientifi c Research
Flanders, the University of Leuven, the Belgian government (Interuniversity At-
traction Poles, program P6/43 of the Belgian Federal Science Policy Offi ce),
the Stem Cell Institute Leuven, and the ALS Association. E. Bogaert, P. Van
Damme, and D. Lambrechts are supported by the Fund for Scientifi c Research
Flanders. W. Robberecht is supported through the E. von Behring Chair for
Neuromuscular and Neurodegenerative Disorders.
Submitted: 10 December 2007
Accepted: 7 March 2008
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Materials and methods
CSF samples from three FTLD patients caused by null mutations in PGRN
and 24 controls were obtained after informed consent and approval by the
local ethical committee. The PGRN levels were measured by ELISA. To do
so, 96-well plates were coated with monoclonal anti-PGRN antibody (R & D
Systems) blocked with 1% BSA before samples and standard (recombinant
human PGRN; R & D Systems) were loaded. Afterward, bound PGRN was
detected using biotinylated polyclonal anti-PGRN antibody (R & D Systems),
avidin – biotin – peroxidase complex, and ortho-phenylenediamine. Absor-
bance was measured at 490 nm. Western blots stained with the antibodies
used in this assay showed that only recombinant PGRN was recognized,
not recombinant GRN E or PGRN cleavage products obtained by elastase
treatment (1 U/ml for 30 min at 25 ° C; Athens Research & Technology).
Monocultures of cortical and motor neurons were prepared from 14-d-old
Wistar rat embryos as previously described ( Van Damme et al., 2003,
2007 ; Van Den Bosch et al., 2004 ) and plated in culture dishes (40,000
cells per dish for spinal cultures) or 96-well plates (10,000 cells per well)
coated with poly- L -ornithine and laminin. Ventral spinal cords were dis-
sected from rat embryos and dissociated enzymatically and mechanically.
A motor neuron – enriched neuronal population was obtained by centrifuga-
tion on a 6.5% Optiprep cushion. For cortical cultures, the cortex was dis-
sociated by trypsinization and trituration but the density centrifugation was
omitted. The culture medium consisted of L15 supplemented with 0.2%
sodium bicarbonate, 3.6 mg/ml glucose, 20 nM progesterone, 5 μ g/ml
insulin, 0.1 mM putrescine, 0.1 mg/ml conalbumin, 30 nM sodium selenite,
100 U/ml penicillin, 100 μ g/ml streptomycin, 5% chick embryo extract,
2% and horse serum. No horse serum was added in the serum-free me-
dium, which still contained chick embryo extract. Cultures were kept in a
7% CO 2 humidifi ed incubator at 37 ° C. Most cells in culture stained posi-
tive for NF-H and neuron-specifi c nuclear protein (NeuN) on day 1 after
seeding. Few cells stained positive for the astroglial marker glial fi brillary
acidic protein (GFAP) or the oligodendroglial marker O4.
The survival of monocultures of motor neurons was quantifi ed by direct
counting of unfi xed cells under phase contrast, as previously described
( Van Den Bosch et al., 2004 ). Neurons in a marked area of 1 cm 2 were
counted before (on day 1 after seeding) application of PGRN/GRN or
control. After counting on day 1, the medium was replaced by serum-free
medium. Because partial recombinant human PGRN (coding for GRN E)
was dissolved in buffer containing reduced glutathione, this was added to
the corresponding controls as well. The relative survival was calculated as
the number of cells on day 2 or 6 in culture divided by the number of cells
on day 1.
Survival of cortical neurons was quantifi ed using a calcein assay
( Bozyczko-Coyne et al., 1993 ; Lin et al., 2001 ). On day 1 in culture, neu-
ronal cultures seeded in 96-well plates were treated with PGRN/GRN or
control buffer. Neuronal survival was quantifi ed 24 h later. On day 2 in
culture, cells were loaded with 2 μ M calcein-AM in the culture medium for
1 h at 37 ° C. After replacement of the medium with PBS, fl uorescence was
measured on a microplate reader (excitation, 480 nm; emission, 535 nm).
After subtraction of background signal from wells without cells, the values
obtained were normalized to untreated control wells. For every condition,
the mean of at least fi ve wells was taken (considered as n = 1).
Immunostainings were performed as previously described ( Van Damme
et al., 2007 ). Cells were fi xed for 20 min using 4% PFA. Cultures were
incubated with NF-H antibody (1:400; rabbit polyclonal), NeuN antibody
(1:1,000; mouse monoclonal), GFAP antibody (1:1,000; mouse mono-
clonal), and O4 (1:500; mouse monoclonal) overnight. The appropriate
Alexa 555 - and Alexa 488 -labeled antibodies (1:500) were used to develop
Quantifi cation of neurite length
Motor and cortical neuronal cultures were exposed to recombinant PGRN/
GRN in serum-free medium immediately after seeding for 24 h and stained
for NF-H on day 1 in culture. Photographs of representative areas of the
culture were taken on an upright microscope (DMRB; Leica) at a magnifi ca-
tion of 10 (0.3 NA; PL Fluotar; Leica) at room temperature using a camera
(DXM 1200; Nikon). The size of the cell soma and the longest neurite of
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