Progranulin regulates neuronal outgrowth independent of Sortilin

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Title:
Progranulin regulates neuronal outgrowth independent of Sortilin
Physical Description:
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Language:
English
Creator:
Gass, Jennifer
Lee, Wing C.
Cook, Casey
Finch, Nicole
Stetler, Caroline
Jansen-West, Karen
Lewis, Jada
Link, Christopher D.
Rademakers, Rosa
Nykjaer, Anders
Petrucelli, Leonard
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BioMed Central
Publication Date:

Notes

Abstract:
Background: Progranulin (PGRN), a widely secreted growth factor, is involved in multiple biological functions, and mutations located within the PGRN gene (GRN) are a major cause of frontotemporal lobar degeneration with TDP- 43-positive inclusions (FLTD-TDP). In light of recent reports suggesting PGRN functions as a protective neurotrophic factor and that sortilin (SORT1) is a neuronal receptor for PGRN, we used a Sort1-deficient (Sort1−/−) murine primary hippocampal neuron model to investigate whether PGRN’s neurotrophic effects are dependent on SORT1. We sought to elucidate this relationship to determine what role SORT1, as a regulator of PGRN levels, plays in modulating PGRN’s neurotrophic effects. Results: As the first group to evaluate the effect of PGRN loss in Grn knockout primary neuronal cultures, we show neurite outgrowth and branching are significantly decreased in Grn−/− neurons compared to wild-type (WT) neurons. More importantly, we also demonstrate that PGRN overexpression can rescue this phenotype. However, the recovery in outgrowth is not observed following treatment with recombinant PGRN harboring missense mutations p.C139R, p.P248L or p.R432C, indicating that these mutations adversely affect the neurotrophic properties of PGRN. In addition, we also present evidence that cleavage of full-length PGRN into granulin peptides is required for increased neuronal outgrowth, suggesting that the neurotrophic functions of PGRN are contained within certain granulins. To further characterize the mechanism by which PGRN impacts neuronal morphology, we assessed the involvement of SORT1. We demonstrate that PGRN induced-outgrowth occurs in the absence of SORT1 in Sort1−/− cultures. Conclusion: We demonstrate that loss of PGRN impairs proper neurite outgrowth and branching, and that exogenous PGRN alleviates this impairment. Furthermore, we determined that exogenous PGRN induces outgrowth independent of SORT1, suggesting another receptor(s) is involved in PGRN induced neuronal outgrowth. Keywords: Progranulin, Sortilin, Neuronal outgrowth, Frontotemporal lobar degeneration, Neurotrophic factor
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Gass et al. Molecular Neurodegeneration 2012, 7:33 http://www.molecularneurodegeneration.com/content/7/1/33; Pgs.1-13
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doi:10.1186/1750-1326-7-33 Cite this article as: Gass et al.: Progranulin regulates neuronal outgrowth independent of Sortilin. Molecular Neurodegeneration 2012 7:33.

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Gass et al. Molecular Neurodegeneration 2012, 7:33
http://www.molecularneurodegeneration.com/content/7/1/33


MOLECULAR
NEURODEGENERATION


Progranulin regulates neuronal outgrowth

independent of Sortilin

Jennifer Gass', Wing C Lee', Casey Cook', Nicole Finch', Caroline Stetler', Karen Jansen-West', Jada LewisI'2,
Christopher D Link3, Rosa Rademakers Anders Nykjar4 and Leonard Petrucelli*


Abstract
Background: Progranulin (PGRN), a widely secreted growth factor, is involved in multiple biological functions, and
mutations located within the PGRN gene (GRN) are a major cause of frontotemporal lobar degeneration with TDP
43-positive inclusions (FLTD-TDP). In light of recent reports suggesting PGRN functions as a protective neurotrophic
factor and that sortilin (SORT1) is a neuronal receptor for PGRN, we used a Sort -deficient (Sortl ) marine primary
hippocampal neuron model to investigate whether PGRN's neurotrophic effects are dependent on SORT1. We
sought to elucidate this relationship to determine what role SORT1, as a regulator of PGRN levels, plays in
modulating PGRN's neurotrophic effects.
Results: As the first group to evaluate the effect of PGRN loss in Gm knockout primary neuronal cultures, we show
neurite outgrowth and branching are significantly decreased in Grnm neurons compared to wild-type (WT)
neurons. More importantly, we also demonstrate that PGRN overexpression can rescue this phenotype. However,
the recovery in outgrowth is not observed following treatment with recombinant PGRN harboring missense
mutations p.C139R, p.P248L or p.R432C, indicating that these mutations adversely affect the neurotrophic properties
of PGRN. In addition, we also present evidence that cleavage of full-length PGRN into granulin peptides is required
for increased neuronal outgrowth, suggesting that the neurotrophic functions of PGRN are contained within certain
granulins. To further characterize the mechanism by which PGRN impacts neuronal morphology, we assessed the
involvement of SORT1. We demonstrate that PGRN induced-outgrowth occurs in the absence of SORT1 in Sortl-
cultures.
Conclusion: We demonstrate that loss of PGRN impairs proper neurite outgrowth and branching, and that
exogenous PGRN alleviates this impairment. Furthermore, we determined that exogenous PGRN induces outgrowth
independent of SORT1, suggesting another receptor(s) is involved in PGRN induced neuronal outgrowth.
Keywords: Progranulin, Sortilin, Neuronal outgrowth, Frontotemporal lobar degeneration, Neurotrophic factor


Background
Frontotemporal lobar degeneration (FTLD) is the second-
most common presenile dementia after Alzheimer's dis-
ease (AD) [1]. FTLD is neuropathologically characterized
by predominant atrophy of the frontal and temporal lobes
and the presence of proteinaceous inclusions in neurons
and glial cells [2]. FTLD cases are divided into two main
pathological subgroups at autopsy: those with tau-positive
inclusions (FTLD-tau), and those with TDP-43-positive
inclusions (FTLD-TDP) [3].

Correspondence petrucelli leonard@mayo edu
Department of Neuroscence, Mayo Clinic College of Medicine, 4500
San Pablo Road, Jacksonville, Florida 32224, USA
Full list of author information is available at the end of the article


In 2006, mutations located within GRN, were described
as a major cause of FTLD-TDP [4,5]. Pathogenic muta-
tions in GRN mainly include frameshift, splice site and
nonsense mutations that produce a premature termin-
ation of the coding sequence and consequently lead to
degradation of mutant mRNA via nonsense-mediated
decay [4-6]. As a result, pathogenic mutations are thought
to cause disease by GRN haploinsufficiency. With the ex-
ception of the pathogenic missense mutation p.A9D,
which prevents PGRN secretion, it is not known whether
missense mutations in GRN influence PGRN's neuro-
trophic properties [6,7].
PGRN is a widely expressed secreted growth factor
involved in development, wound repair and inflammation


2012 Gass et al., Iicensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Biole ied Central Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.






Gass et al Molecular Neurodegeneration 2012, 7:33
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[8,9]. Recent studies suggest that PGRN functions as a
protective neurotrophic factor; however, its role in the
CNS is not well understood [10-13]. The mechanisms
underlying its effects have not been fully determined, nor
is it known how missense mutations in GRN influence its
neurotrophic properties.
The protein contains seven-and-a-half tandem repeats
of a 12-cysteine granulin motif separated by interlinked
spacer regions that are termed granulins [14]. In the
periphery, full-length PGRN is proteolytically cleaved
into mature granulins via proteases [15,16]. Secretary
leukocyte protease inhibitor (SLPI), a serine protease in-
hibitor, prevents PGRN cleavage by binding to PGRN
cleavage sites or to elastase and inhibiting its cleavage
activity [16].
Recently, two PGRN receptors have been identified:
sortilin 1 (SORT1) and tumor necorosis factor receptor
(TNFR) [17,18]. The neuronal PGRN receptor, SORT1,
has been shown to regulate the extracellular levels of
PGRN [17,19]. SORT1, a vacuolar protein sorting 10
protein domain receptor, is also involved in endocytosis
and transport of proteins to the trans-golgi network and
S...I. .,!!I. I ..... [20]. The C-terminal portion of
PGRN binds to SORT1 causing immediate endocytosis
of PGRN to the lysosome. Due to its interactions with
SORT1, we sought to determine whether PGRN utilizes
this receptor to modulate neuronal morphology. With
regard to the loss of PGRN leading to neurodegenera-
tion, we investigated the physiological impact/conse-
quences of loss of PGRN in primary hippocampal
neuronal cultures.


Results
Loss of PGRN alters neuronal morphology
GRN i,:.:.1,,- -., .. trr...-..... due to mutations in GRN cause
FTLD-TDP [2]. To evaluate the impact of PGRN defi-
ciency on neuron morphology, primary hippocampal
cultures from WT and Grn mice were prepared.
Western blot analysis confirmed the complete loss of
PGRN in hippocampal cultures from Grn mice
IF.' ... 1A). In order to assess neuronal outgrowth, neu-
rons were fixed and immunostained using neuron-
specific anti-MAP2 following 10 days in vitro (DIV10).
Using confocal microscopy, we captured and analyzed
images using MetaMorph 7.1 software (Figure 1B),
which quantitatively measures total skeletonized out-
growth of individual neurons in urms and total branching
junctions of dendrites. As i! "'i'" 1 in Figure 1C-E,
Grn neurons displayed ....i ,...i reduced neurite
outgrowth (p < 0.0001) and branching (p < 0.005) com-
pared to WT neurons, while cell body size remained the
same. In all experiments, at least 100 neurons were ana-
lyzed and repeated in three independent experiments.


Page 2 of 13


Exogenous PGRN rescues neurite outgrowth and
branching in Grn-- neurons
Having established that a loss of endogenous PGRN
causes major deficits in neurite outgrowth and branch-
ing of hippocampal neurons, we then examined whether
this phenotype can be rescued by returning PGRN to
cultures. To achieve PGRN overexpression in our cul-
tures, we packaged PGRN into a rAAV1 vector (rAAV1-
PGRN). Dosing titers are expressed genomes/mL.
To determine if we could rescue the Grn-1 phenotype,
we transduced primary hippocampal neurons with in-
creasing doses of rAAV1-PGRN for 6 days to allow for
synthesis of PGRN. Under normal conditions, our WT
primary culture's Pgrn levels were around 4 ng/mL
(F .... 2A). In testing for the production and release of
PGRN into the ..I-.:i., human specific ELISA revealed a
dose-dependent increase of PGRN secreted into the
medium (Figure 2B). Neuronal morphology was then
assessed in GrnM1 and WT neurons treated with the same
increasing titers of rAAV1-PGRN. Application of rAAV1-
PGRN resulted in i. [' i :1:; ..:... .. -i increased out-
growth and branching in both Grn and WT cultures
(F- .:..- 2C-F). rAAV1-GFP transduction in cultures did
not promote outgrowth or branching, .i.- -.t'...- that the
production of PGRN induces :i1. ..-- i-, and plays a role
in neuronal -.. iI,,1. All conditions were normalized
to WT rAAV1-GFP treated cells .. i:.. that the pheno-
type was rescued at a rAAV1-PGRN titer of 101 gen-
omes/mL or 14 ng/mL within the media.

rPGRN promotes neuronal outgrowth and branching in
Grn- and WT cultures
Since determining that exogenous overexpression of
rAAV1-PGRN is able to rescue the Grn neuronal
phenotype, we purified recombinant human PGRN
(rPGRN) from media of a stable HEK 293 cell line over-
expressing PGRN. The rPGRN was then applied to WT
and Grn cultures in increasing doses. The results
from this experiment show a dose-dependent, .. .I,,i. I,,,
increase in outgrowth and branching in Grn neurons
as %.. I: as WT neurons T ...-. 3A-D). These results also
reveal that 125 nM (10 .. '1, i rPGRN is needed to res-
cue the Grn phenotype and return outgrowth and
branching to normal conditions.

Cleavage of PGRN promotes neuronal outgrowth
PGRN is secreted as a :Ill i. l. ti 88 kDa protein, which is
cleaved into mature i. :,'...!.- In fact, Western blot ana-
lysis of rPGRN treated cultures shows rPGRN cleavage
within cultures I... ..- 3E). Note that in the media,
rPGRN becomes cleaved into granulin fragments within 1
hour. To determine whether PGRN or I. .-,.. I,- are re-
sponsible for inducing neurite outgrowth, WT cultures
were treated with recombinant SLPI (rSLPI), a serine






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100-
-)
N u 75-

50-
* 0


"100-
4) -
N. = 75-
0 50-
z E


Page 3 of 13


WT Grn--
mPgrn

GAPDH


E 125-


C 125-
E 100-

zo


7 25


***


Wvv I rn-' vv I Lrn- vv I Lrn-
Figure 1 Loss of Pgrn decreases neurite outgrowth and branching in primary neurons. (A) Western blot analysis reveals endogenous levels
of Pgrn in WT and Grn / primary hippocampal cultures. (B) WT and Grn / hippocampal neurons immunostained for MAP2. (C-E). Neurite
outgrowth and branching of MAP2-immunopositive neurons was assessed using MetaMorph version 7.1 software. Note that neurite outgrowth


(C) and branching (D), but not cell body size (E), are decreased in Grn
is presented as the mean SEM. **p < 0.01; **p < 0.001.


protease inhibitor that prevents PGRN cleavage [16]. Util-
izing an elastase digest assay, we first confirmed that rSLPI
prevents processing of rPGRN into GRNs (Figure 4A).
Upon incubation of rPGRN with elastase, PGRN cleavage
occurred within 5 minutes; however, when rSLPI was
added, the cleavage of PGRN by elastase was inhibited.
We sought to examine rSLPI's effects on endogenous
mouse Pgrn levels, which had yet to be determined.
After treating WT cultures expressing endogenous Pgrn
with rSLPI, we observed reduced outgrowth (Figure 4B)
and branching (Figure 4C) similar to untreated Grn
cultures. When rSLPI was added to Grn cultures
there was no change in outgrowth or branching, illus-
trating that rSLPI is targeting PGRN and that PGRN
cleavage is required to induce outgrowth. While treat-
ment with rPGRN alone increases outgrowth, co-
treatment of rSLPI and rPGRN in WT neurons does not
increase outgrowth (Additional file 1: Figure Si).
These findings next led us to investigate the neuro-
trophic activity of specific granulins. Here, neurons were
treated with recombinant granulin protein containing
full length PGRN, and commercially available granulin E
(rGRN E) or granulin C (rGRN C) (Figure 4D). Treat-
ment with granulins E and C (125 nM/10 ug/mL) pro-
moted neuronal outgrowth very similar to PGRN
treatment (125 nM/10 ug/mL) suggesting these granulin
species harbor neurotrophic properties (Figure 4E).

C-terminal tag inhibits SORT1 uptake and PGRN
endocytosis
SORT1 was recently identified as a neuronal receptor for
PGRN; however, its role in PGRN function is not well


lippocampal neurons compared to WT hippocampal neurons. All data



understood. We decided to test recombinant PGRN
(rPGRN) on N2A cells transduced to overexpress rAAV1-
SORT1 or rAAV1-GFP (Figure 5A). Human rPGRN was
generated from an HEK293 stable cell line containing a
His-tag located at either the N-terminal (His-rPGRN) or
C-terminal (rPGRN-His) region of rPGRN. Our results in-
dicate that overexpression of SORT1 triggers increased
uptake of His-rPGRN and not rPGRN-His, suggesting the
C-terminal tag inhibits PGRN's ability to bind to SORT.
Since the binding of PGRN to SORT1 was hindered by
the C-terminal tag, we next compared the ability of His-
rPGRN and rPGRN-His to promote outgrowth and
branching in primary hippocampal cultures. We deter-
mined that both His-rPGRN and rPGRN-His were able to
equally induce outgrowth (Figure 5B).


PGRN promotes neuronal outgrowth and branching
independent of Sortilin
We previously showed that SORT1 can regulate PGRN
levels, but to elucidate the role this receptor plays in
neurotrophic morphology, we examined Sort1 hippo-
campal cultures. Western blot analysis confirmed the
complete deletion of Sorti in hippocampal cultures
(Figure 6A). Since Sortl is involved in uptake of PGRN,
we then wanted to learn whether these cultures have a
marginal increase in secreted Pgrn. ELISA data from
WT and Sort1l cultures revealed increases of Pgrn in
Sorti" cultures compared to WT (Figure 6B). On
the other hand, neuronal outgrowth studies revealed
decreased outgrowth in Sort1 neurons compared to
WT (Figure 6C).


D 125-


T







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WT Grn-


= 150*
E im




0-
rAAV1-GFP 1011 0 0 0
rAVV1-PGRN 0 109 1010 1011
WT


O 175-
*u
.150- NS
r 125-
A 100-
0 75-
50"

25-

rAAV1-GFP 1011 0 0 0
rAVV1-PGRN 0 109 1010 1011
WT


E
125*

1 100"
0D
.N 75-

SS 50.
z o
S25-
0


rAAV1-GFP 1011 1011 0 0 0 rAAV1-GFP 1011 1011 0 0 0
rAVV1-PGRN 0 0 109 1010 1011 rAVV1-PGRN 0 0 109 1010 1011
WT Grn WT Grn
Figure 2 Primary hippocampal cultures transduced with rAAV1-PGRN promotes neurite outgrowth and branching. (A) Mouse Pgrn ELI
of endogenous Pgrn levels in hippocampal primary neuronal cultures. (B) Human PGRN ELISA shows increasing levels of hPGRN generated in t
media of treated primary cultures. (C,D) Bar graph quantification of total outgrowth (C) and branching (D)of primary hippocampal neurons
transduced with increasing amounts of rAAVI-PGRN in WT cultures normalized to WT rAAV1-GFP treated cultures. (E,F) Bar graph quantification
of normalized total outgrowth (E) and branching (F)in Gm / hippocampal neurons transduced with rAAVI-PGRN normalized to WT rAAV1-GFP
treated cultures. Note that rAAV1-GFP was supplemented in rAAVI-PGRN non-treated cultures. Data is presented as the mean SEM.
*p < 0.05; *p < 0.001.


To verify whether PGRN can increase outgrowth in
the absence of SORT1 and ultimately whether SORT1 is
a neurotrophic receptor for PGRN, we treated primary
hippocampal Sort1i and WT neurons with increasing
amounts of rPGRN. We found a dose-dependent in-
crease in outgrowth occurs in Sorti neurons similar
to WT (Figure 6D, 6F). Additionally increased branching


of these neurons was also seen (Figure 6E, 6G), suggest-
ing PGRN does not use SORT1 to promote outgrowth.


Missense mutant PGRN alters neuronal morphology
While the majority of pathogenic GRN mutations lead to
the degradation of mutant mRNA and/or cause GRN
haploinsufficiency, some missense mutations have been


Page 4 of 13


Grn







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WT


WT Gmr


E rPGRN treatment 1 24 48 72 96
(hours)
83- FL
62
47

32
25


Figure 3 rPGRN promotes neurite outgrowth and branching in primary neurons. (A) (A,B) Bar graph quantificatio
outgrowth (A) and branching (B) of WT hippocampal neurons exposed to rPGRN increasing 25, 125, 625 nM doses of -
correspond to 2,10, 50 ug/mL concentrations respectively). All doses normalized to WT non-treated cultures. (C,D) Bar g
normalized total neurite outgrowth (C) and branching (D) in of Grn / hippocampal neurons exposed to rPGRN. Grn /
to WT non-treated cultures. (E) Western blot analysis reveals PGRN cleavage in Grn hippocampal cultures treated wit[
increasing time points. Note cleavage of PGRN after only one hour. Data is presented as the mean SEM. **p < 0.01.


n of total neurite
is-rPGRN (nM doses
raph quantification of
cultures were normalized
h 125 nM rPGRN-His at


reported to disrupt PGRN processing (p.C139R) and
secretion (p.R432C, p.P248L), when compared to WT
PGRN [7,21]. The p.S120Y GRN mutation has been
found in controls and believed to be non-pathogenic
[22]. To determine whether these missense mutations
also disrupt the neurotrophic function of PGRN, we gen-
erated recombinant PGRN proteins harboring the afore-
mentioned missense mutations (Figure 7A) and assessed
their effect on neurite outgrowth in Grnm neurons.
As expected, rPGRN containing p.S120Y, a mutation
observed in controls, promoted neurite outgrowth to a
similar extent as WT rPGRN (Figure 7B). In contrast,
rPGRN with p.C139R, p.R432C and p.P248L mutations


failed to stimulate neurite outgrowth (Figure 7B),
suggesting these mutations disrupt PGRN function
and may indeed be pathogenic and contribute to the
disease phenotype observed in patients carrying these
mutations.

Discussion
While loss of PGRN is a major cause of FTLD-TDP, it
remains unclear how this loss leads to neurodegeneration.
Recent studies suggest that PGRN may function as a
protective neurotrophic factor regulating neuronal survival
and outgrowth in cortical/motor primary neurons, immor-
talized cell lines, and zebra fish embryos [10,11,23-25].


Page 5 of 13


WT


WT Gm'







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Page 6 of 13


S rSLPI -
Elastase digest (min) 0 5 10 15


rSLPI + +
WT Grn--

E


WT Grn-


17 4458 113123 179 205 261 281 336 364 417 442 496518 573
PJ 2P Y F 17 2 9 ;E rPGRN

364 430
U-Y rGRN C
494
-- rGRN E


Figure 4 PGRN processing promotes neuronal outgrowth and branc
PGRN cleavage products visualized by SDS-PAGE, post-stain with sypro ru
blocks elastase-mediated rPGRN cleavage. (B,C) Bar graphs depict quantify
Gm / neurons treated with or without rSLPI. Note that in these experime
(D) Schematic representation of recombinant proteins analyzed in primary


N E) and granulin C (rGRN C) promote out(


recombinant granulin E (rGF
*n < 0.0 *** 0001


The mechanisms underlying these effects have not been
fully determined, nor is it known how GRN missense muta-
tions and SORT1, a PGRN neuronal receptor, influence
PGRN's neurotrophic properties.
In the current study, we examined the effects of
PGRN loss in primary neuronal cultures from Grn-
mice. Our data show that neuronal outgrowth and
branching in Grn- primary cultures are significantly
reduced compared to WT cultures. We also demon-
strate that PGRN promotes neurite outgrowth independ-
ent of Sortl. Such findings advance our knowledge
beyond previous studies, and strongly suggest another re-
ceptor(s) modulates PGRN-specific neuronal outgrowth
and branching.
Since the discovery of the first GRN mutations, several
groups have developed various Grn- mouse models
[26-28]. While the murine models recapitulate several
pathological features of FTLD patients, such as increased
gliosis and TDP-43 positive inclusions, no two models


WT
hing. (A) Time course analysis of rPGRN subjected to elastase digest.
by solution -1t Note that the co-treatment of recombinant SLP
cation of total neurite outgrowth (B) and branching (C) of WT and
ints no rPGRN was added, therefore rSLPI blocks endogenous Pgrn.
/ hippocampal neurons. (E) WT hippocampal cultures treated with
)wth similar to full length PGRN Data is presented as the mean+SEM.


are the same, likely due to different backgrounds strains
or strategies used to knockout Grn. For our experiments,
we used primary hippocampal cultures prepared from
Grn- mice generated by Kayasuga et al. [27], with the
knowledge that these mice display accelerated brain
aging due to increased accumulation of lipofuscin, in
addition to enhanced gliosis and neurodegeneration
when compared to WT mice [29]. These Grn- mice
also mimic behavior changes similar to FTLD patients,
including increased aggression, decreased social inter-
action and impaired learning and memory [27,30]. Even
though our mice display increased ubiquitin staining,
they are not positive for TDP-43, the neuropathological
hallmark of FTLD with GRN mutations. Yin et al devel-
oped a Grn- mouse displaying amplified inflammation
in addition to increased ubiquitin staining positive for
TDP-43 [26]. Increased TDP-43 accumulation is also
observed in Grn- neurons treated with the proteosome
inhibitor MG132, suggesting that these neurons were


- --+







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His-rPGRN
rAAV1-GFP rAAV1-SORT1


rPGRN-His
rAAV1-GFP rAAV1-SORT1 -ve


hPGRN a


SORT1 -, -, a- a


GFP

GAPDH


200-

E 175-
S 150-

S125-
100
0 i 75-
Z O
5 50
S25 I


rPGRN-His
His-rPGRN


a


- 25 125 625 -
- 25 125 625


Figure 5 SORT1 triggers endocytosis of rPGRN. (A) Western blo
presence of His-rPGRN or rPGRN-His. Note uptake of His-rPGRN into
of WT primary neuronal cultures treated with increasing 25, 125, 62
with between treatments (nM doses correspond to 2, 10, 50 uq/m


t analysis of Neuro2A cells transduced with rAAV-GFP or rAAV-SORTI in the
cell lysates and the absence of rPGRN-His uptake. (B) Bar graph quantificati
5 nM doses of His-rPGRN or rPGRN-His depict equal amounts of outgrowth
L concentrations respectively). Data is presented as the mean SEM.


more vulnerable to cellular stresses than WT [31]. GRN
haploinsufficiency cell culture models generated similar
results, where GRN knockdown using siRNA resulted in
TDP-43 cleavage; however, downregulation of GRN for
short time points does not produce TDP-43 cleavage
[32,33].
FTLD patients with GRN mutations develop severe at-
rophy of the frontal and temporal lobes of the brain
[34]. Therefore, understanding the role of PGRN in
neuronal health and function, as well as regulating levels
are important for future studies. Recent efforts using
Grnm mice have helped shed new light on the many
functions of PGRN. Petkau and colleagues determined
that Grn mice have reduced synaptic connectivity and
altered LTP, in addition to altered morphology and
reduced spine density of apical dendrites of the hippo-
campus of aged mice [28]. Additional studies in neurons
with reduced GRN using siRNA revealed decreased


neuronal arborization and synaptic density in addition to
abnormal transmission at the synapses [31]. These find-
ings suggest that loss of PGRN alters morphology of
neurons leading to synaptic changes, which ultimately
may cause neurodegeneration.
To further characterize our mice, we compared WT
and Grnm cultures and found Grnm primary hippo-
campal neurons display significantly shorter neurites and
fewer branch points compared to WT neurons. Import-
antly, this phenotype is reversed when neurons are sup-
plemented with PGRN via recombinant or viral methods.
While rAAV1-PGRN transduction leads to the intracellu-
lar expression of PGRN, ELISA-based quantification of
PGRN from media indicated that extracellular PGRN
levels increase in a dose-dependent manner following
rAAV1-PGRN transduction. Normal physiological ranges
for human PGRN are approximately 100-400 ng/mL and
8-12 ng/mL in the PNS and CNS respectively; however,


rPGRN
AAV


Page 7 of 13


_ __







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A B
WT Sort 8-
E
mPGRN a 7
c 6-
0
mSORT1 5.
*^ (0
GAPDH -t4m 4"
S3-
S2-
S1
O.
-


WT Sort-


*- 125
NS
100-







rPGRNnM 0 25 125 625
SortN

F
175-
150
125
NSor

75-

z o
50-




rPGRNnM 0 25 125 625

WT


rPGRN nM 0 25 125 625
Sort/-


I 150-
S125- s
E" 100-
0-
Z 75-
50-
25

rPGRNnM 0 25 125 625
WT


Figure 6 Pgrn induces outgrowth independent of Sortl. (A) Western blot analysis of Pgrn and Sort levels in WT and Sort1 hippocampa
cultures. (B) Mouse specific ELISA showing increasing levels of Pgrn in Sortl culture media. (C) Bar graph quantification of normalized tota
neurite outgrowth in Sortl / and WT cultures showing e I decreases in Sort / (D-G) Normalized total outgrowth (D,F) and tota
branching (E,G) in Sortl / and WT cultures with increasing amounts of His-rPGRN treatment. Data is presented as the mean+SEM.
*p < 0.05; **p < 0.01.


in our WT primary cultures we determined that normal
levels are approximately 3.5 ng/mL [32,33]. Increasing
doses of rAAV1-PGRN elevated exogenous PGRN levels
to 3-15 ng/ml. These studies demonstrate that rAAV1-
PGRN transduction resulting in 15 ng/mL PGRN within
the media, is sufficient to restore outgrowth and branch-
ing of Grnm cultures to WT conditions. We also showed
this phenotype can be rescued with the addition of
rPGRN and in fact we were able to stimulate outgrowth


and branching beyond normal conditions revealing the
effective neurotrophic abilities of PGRN. The rPGRN
doses exceed the normal range, however, it is difficult to
assess what amount of the rPGRN is truly active or
degraded as a result of protein purification or normal
degradation of the recombinant protein in cell culture
models over time.
Our results suggest that PGRN functions as a neuro-
trophic factor involved in proper neuronal morphology


Page 8 of 13


110-
100-
90-



E 0-

.
o0 40-

S20-
10-
0-


WT Sort-






Gass et al. Molecular Neurodegeneration 2012, 7:33
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C139R
S120Y P248L


FI


250
-225
E
a 200-
c 175 -
150

SNS
S= 100 Ns
Z o 75
50-
0-
2 25

recombinant a .,
protein f ,p "
Grn--


Figure 7 Specific missense
(A) Schematic representation
graph quantification of neurit


mutations lack the ability to promote outgrowth and branching in primary hippocampal neurons.
of the GRN gene and location of missense mutations investigated (p.S120Y, p.C139R, p.P248L, p.R432C). (B) Bar
e outgrowth of Grn hippocampal neurons treated with 125 nM (10 ug/mL) of wild-type rPGRN-His or missense


rPGRN-His. Data is presented as the mean +SEM. **p< 0.01; ***p< 0.001.


and function. Considering that PGRN is a secreted pro-
tein, extracellular PGRN may be responsible for initiat-
ing signaling events that promote neurite development.
For example, we demonstrate that decreased outgrowth
and branching is reversed by the supplementation of
PGRN. Given the variety of Grnm models now avail-
able, it would be of great interest to determine if model-
specific characteristics, such as synaptic abnormalities,
altered spines and dendrites, as well as, TDP-43 path-
ology can be rescued with exogenous PGRN treatment.
In the periphery, PGRN is cleaved into mature gran-
ulins by extracellular proteases [15,16], similar to other
neurotrophins, such as nerve growth factor (NGF) and
brain-derived neurotrophic factor (BDNF), which are
synthesized as precursors and proteolytically cleaved to
form mature neurotrophins [34]. Our data suggest that
rPGRN is being cleaved within primary neuronal cul-
ture media (Figure 3A), therefore we hypothesized that
cleaved PGRN, or granulins, are responsible for indu-
cing its neurotrophic properties. SLPI, a protease in-
hibitor, is known to bind PGRN or elastase to prevent
PGRN cleavage [15,16]. To determine if full length or
cleaved Pgrn is neurotrophic, we treated WT and Grn
primary neurons with and without rSLPI. Our findings
show that rSLPI is able to prevent outgrowth in WT
cultures; however, this is not seen in knockout cultures


suggesting rSLPIs specificity for Pgrn. Van Damme et al.
also reported that SLPI abolishes PGRN-enhanced sur-
vival and neurite outgrowth in cortical neurons [11].
More importantly, we show that individual granulins C
and E have the ability to promote neuronal outgrowth,
further emphasizing that PGRN cleavage is required for
neurite outgrowth and branching. While we only tested
granulins C and E, for future studies it will be important
to determine the function of physiologically relevant
granulin products, since previous reports suggest that
they may have differential effects [16].
Given that previous studies determined the C-terminus
tail of PGRN (QLL) binds to the beta-propeller of SORT1
[35], we decided to analyze SORT1 induced uptake of
rPGRN. When generating rPGRN, we tagged our peptide
using a 6 His tag at either the C-terminus (rPGRN-HIS)
or N-terminus (HIS-rPGRN) for purification. His-
rPGRN becomes endocytosed by SORT1 overexpression;
however, we believed the His tag on rPGRN-His would
prevent SORT1 from endocytosing it. Furthermore, we
wanted to determine if these two versions of rPGRN
have similar neurotrophic properties, since only His-
rPGRN interacts with SORT1. Indeed, when we
compared His-rPGRN and rPGRN-His in WT cul-
tures, we observed a very similar dose-dependent
increase in outgrowth suggesting the neurotrophic


Page 9 of 13


R432C
I






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properties are not due to the location of the His-tag
nor to SORT1.
The impact of:!.! '...!i 1 granulins' neurotrophic func-
tions and our previously mentioned data suggest that
PGRN may be acting independently of SORT1 to pro-
mote neurite outgrowth. Our Sorti'l cultures exhibit
increased levels of PGRN within the media, a finding that
is consistent with previous reports demonstrating the
ability of SORT1 to regulate PGRN levels [17 ] These
cultures also display a decrease in neuronal outgrowth,
which may be puzzling considering the increased Pgrn
levels in the media. However, other groups have also
reported this phenomenon and point to SORTI's inter-
action with tropomyosin-receptor-kinase (Trk) receptors
as the cause. Basically, it was determined that SORT1
is involved in the anterograde transport of Trk recep-
tors to nerve ..*.:i...-- where the receptors induce
trophic signaling [36]. We believe the decreases in
outgrowth in Sorti cultures cultures are caused by
the loss of trophic support resulting from Trk recep-
tor misguidance. Furthermore, we observed that by
adding increasing amounts of PGRN to Sorti lcul-
tures, outgrowth and branching increased in a dose-
dependent manner similar to WT neurons. Such
findings further :--.. I PGRN-induced ":tL.... t'l
occurs independent of SORT1.
Pinpointing exactly which receptor is responsible for
PGRN-induced neuronal outgrowth has not yet been
determined and will be the aim of future studies. Tang
et al, recently discovered PGRN also binds to TNFR
[18], and when bound to TNFR, PGRN antagonizes
and prevents downstream TNFa-mediated inflamma-
tory signaling. One could speculate that the loss or
reduced levels of PGRN long-term will enhance cell
death/pro-inflarnmatory signaling that could be detri-
mental to neuronal viability. It may be possible that
increases in PGRN mitigate the TNFa response to the
extent that we observe neurotrophic effects. In such a
case, excess PGRN may keep the TNFR signaling in
check whereas deficits in PGRN favor a TNFa-
mediated response resulting in reduced '...iL... i' and
ultimately neuronal death. Given that PGRN promotes
outgrowth independent of SORT1, our data suggest
another receptor(s) is involved in PGRN-induced neur-
onal I..i'."... i, and may be .** i I.r ,i for maintain-
ing neuronal integrity.
Most pathogenic mutations in PGRN result in a loss
of functional PGRN caused by nonsense, splice-site and
frameshift mutations, as %.- I: as by mutations that dis-
rupt proper translation and secretion [4-6]. However,
there are several missense mutations of unknown patho-
._-!..;(. Lack of functional data currently restricts our
: 1. i l". .-L regarding the possible contribution of
these variants to disease pathogenesis. To further


Page 10 of 13


elucidate the role missense mutations with undeter-
mined pathogenicity play in causing a loss of neuro-
trophic function, rPGRN containing missense mutations
were generated and analyzed in hippocampal neuronal
cultures. Among the missense mutations studied, two
(p.P248L and p.R432C) were reported to lead to
decreased secretion, and one (p.C139R) reduces PGRN
production, stability and patient plasma levels [7,25,32].
p.S120Y, is unlikely to be pathogenic since it is found in
control patients [37,38] was found to promote neurite
outgrowth similar to WT rPGRN. In contrast, rPGRN
with p.C139R, p.R432C or .. P1 1". mutations did not
stimulate neurite outgrowth, suggesting that these
mutations adversely affect PGRN function and may be
pathogenic, which would be consistent with previous
genetic studies [39,40].

Conclusions
Taken together, our findings indicate that PGRN is
required for the proper development of neurites in pri-
mary hippocampal neurons. Our results w.-2 .t neurite
... l' ..il, and branching are regulated by extracellular
cleaved ..!l! ... We determined that PGRN induces
outgrowth independent of SORT1, which indicates that
PGRN uses a different receptor to promote neuronal
morphology. While pathogenic mutations cause decreases
in PGRN levels, certain GRN missense mutations abolish
the neurotrophic properties of PGRN, providing a putative
link between these mutations and development of FTLD-
TDP.

Methods
Mice
Grn mice have been previously I i,. l [27,29].
Sortli mice were generated and previously described
[41-43]. Timed breeding cages of C57/BL/6, Grn and
Sort/ were set up for primary neuronal cultures. All
rodent specimens were obtained from animals handled
by procedures approved by the Mayo Clinic Institutional
Animal Care and Use Committee.

Primary neuronal culture
Primary neuronal cultures from hippocampi or cortex
were prepared from postnatal day 1 mouse pups and
stored at 4C in HIBERNATE" A media without calcium
T... ~V. ...'.. .1 with B27 (Invitrogen),
0.5 mM GMAX (GIBCO), and gentamicin (GIBCO).
Excised hippocampi or cortex were :'._ ..r..-1 in papain
(2 mg/mL; Fisher :. -ti(; i. triturated with a Pasteur
pipet ..I... size 0.8-1 mM), centrifuged to collect cell
pellet, and resuspended in Neurobasal A (Invitrogen),
supplemented with B27, GMAX, gentamicin, and bFGF
(Invitrogen). F .11'. 1-L determination of cell number,
neurons were plated on poly-D-lysine-coated coverslips






Gass et al Molecular Neurodegeneration 2012, 7:33
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within 24-well plates for immunocytochemical studies
(seeded at a density of 2.5x 10' i1 i .1:i'. or
seeded on poly-D-lysine coated II plates for im-
munoblotting at a seeding density of 3 x 105 cells/well.
Cultures were allowed to mature for 10 days. Media was
changed on the third and seventh days. For experiments
using recombinant protein, cells were treated on day 4
and day 7 after media change.

Recombinant PGRN production
Human p.; .m.ii (PGRN) i I i. ,i> cDNA including
the 5' signal peptide sequence was subcloned into the
Nhe I and Age I restriction sites of the pcDNA4/V5-
HisA vector such that a 6 His tag was fused at the
carboxyl-terminus of the PGRN sequence. A PGRN
stable cell line was generated by transfecting HEK293
cells with the pcDNA4/PGRN-6His DNA and missense
PGRN-6His DNA in a 6-well culture plate format.
PGRN expression plasmids were sequence verified using
ABI3730 with Big Dye chemistry f..i.... .,: manufac-
turers's protocol (Applied Biosystems, Foster City, CA,
USA). Stable transfectants were selected for 4 weeks in
complete culture medium supplemented with 400 [g/ml
zeocin. Surviving clones were individually picked,
expanded and tested for the PGRN expression. The
clone that secretes the highest level of PGRN protein
into -1. ... i was used for recombinant PGRN (rPGRN)
production. Standard Ni-NTA (Qiagen) affinity purifica-
tion procedures based on manufacturer's instruction
were ip.i,..i to purify the rPGRN protein (Lee et al.
2005).

rAAV1 production
rAAV1-PGRN, rAAV1-SORT1 and rAAV1-GFP was pre-
pared by standard methods. Briefly, AAV vectors expres-
sing PGRN, SORT1 or GFP under the control of the
cytomegalovirus enhancer/chicken P-actin promoter, a
woodchuck post-transcriptional regulatory element, and
the bovine growth hormone, poly(A), were generated by
plasmid transfection with helper plasmids in HEK293T
cells. All plasmids were sequence verified as I. -1.. I
above. Forty-eight hours after transfection, the cells were
harvested and lysed in the presence of 0.5% sodium
deoxycholate and 50 U/ml Benzonase "_-''!,: St. Louis,
MO) by freeze thawing, and the virus was isolated using
a discontinuous iodixanol gradient. The genomic titer of
each virus was determined by quantitative PCR.

Immunocytochemistry
Primary neurons on coverslips were fixed in methanol,
permeablized with 0.5% Triton-X-100 in PBS, washed,
blocked in 5% milk-PBS for 1 hour and incubated over-
night in anti-MAP2 (1:500, S;L..- "!1.;. 11 or anti-
I I.....l.... 1.. Invitrogen) diluted in 5% milk-PBS.


Page 11 of 13


Coverslips were then washed and incubated for 2 hours
in goat anti-rabbit Alexa Fluor 568 (1:1000, Molecular
Probes) or goat anti-mouse Alexa Fluor 488 (1:1000,
Molecular Probes). Coverslips were then incubated for 10
minutes in Hoescht 33258 (1:10000, i.! ; i... -.1 washed
and mounted onto microscope slides by Fluormount-G
(Southern Biotech).

Image acquisition and quantification
All images were captured using a Zeiss LSM 510 META
confocal microscope. Neuronal morphology measure-
ments were determined using MetaMorph version 7.1
. i.i ,,i. Devices, Downingtown, PA). In each experi-
ment, 50-100 neurons sampled from randomly selected
fields were analyzed and repeated in at least three inde-
pendent experiments. Statistical analysis was performed
using GraphPad Prizm 4 by comparing means of differ-
ent groups using Student's-t test or ANOVA followed by
post hoc Kruskal-Wallis test and Dunn's Multiple Corn-
parison Test. Error bars indicate *p < 0.05; **p <0.01;
**p < 0.001.

Western blot and ELISA
Cells from 6 well plates were lysed in lysis buffer consist-
ing of Co-IP buffer (50 mM Tris-HC1, pH 7.4, 1 M
NaCI, 1% Triton-X-100, 5 mM EDTA) plus 1% SDS,
PMSF, and both a protease and phosphatase inhibitor
mixture. The protein concentration of cell lysates was
measured using a BCA assay i- .. Samples were pre-
pared in Laemmli's buffer, heated for 5 min at 95C, and
equal amounts of protein were loaded into 10-well 10%
Tris-glycine gels (Novex). After transfer, blots were
blocked with 5% nonfat dry milk in TBST (TBS plus
0.1% TritonX-100) for 1 h, and then blots were incu-
bated with anti-mouse ..... I ,..,,i, (1:1000, R&D sys-
tems), anti-human i: .:.,:,,.,! I (1:1000, Invitrogen),
mouse monoclonal GAPDH ,O1.L. I; (1:10000, Biode-
sign) overnight at 4C. Membranes were washed three
times for 10 min in TBST and then incubated with don-
key anti-rabbit, anti-mouse or anti-sheep IgG conjugated
to horseradish peroxidase (1:2500; Jackson ImmunoRe-
search) for 1 hour. Membranes were washed three times
each for 10 min, and protein expression was visualized
by ECL treatment and exposure to film. Human PGRN
ELISA ,i'..-) Systems) and Mouse i L-- ELISA (Enzo
Life Sciences) was used .. .... i... to manufacture proto-
col on media ...11. I. from rAAV1-PGRN treated
cultures.

Enzymatic digestion of rPGRN by elastase
30 pg of wild-type recombinant PGRN or mutants PGRN
protein incubated on ice was mixed with reaction buffer
(100 mM Tris-HC1, 300 mM NaC1, pH 7.5) and
0.05 U/ml ip...', i., elastase (Athens Research and







Gass et al. Molecular Neurodegeneration 2012, 7:33
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Technology) in a total reaction volume of 100 p1. The
enzyme reaction was initiated by incubation at 37C.
10 p1 of the reaction mixture was sampled every 5
minutes and the reaction was immediately quenched
by adding equal amount of 2 x SDS sample buffer.
The reaction was allowed to proceed until 35 minutes
after time zero. A separate reaction with the ii..
of recombinant SLPI at 13.3 pM was also prepared
and preceded for 35 minutes at 37C. The samples
were analyzed by SDS-PAGE, post-stain with sypro
ruby solution -. .. ii' and imaged by a gel doc station
equipped with a UV-transilluminator.


Additional file


Additional file 1: Figure S1. tS PI prevents rPGRN induced outgrowth
and branching A-B Bar graph quantification of normalized rotal
outgrowth (A) and branching (B) in primary hippocampal cultures
Irealed wilh rPGRN+ iSLPi Data is presented as the mean -SEM


List of abbreviations
PGRN Human progranulin; Pgmr Mouse progranulin; GRN Human progranulin
gene name; Gmr Mouse progranulin gene name; Sortl Mouse sortilin gene;
SORT1 human sortilin 1;Sort mouse sortilin 1; FTLD FLDrontotemporal lobar
degeneration; TDP-43 Transactive response DNA-binding protein 43;
AD Alzheimer's disease; AAV Adeno-associated virus

Competing interests
The authors declare that they have no competing interests

Authors' contributions
JG carried out the primary neuronal cell culture, immunchistochemistry,
mage acquisition and quantification, designed the study and wrote the
manuscript C ee generated recombinant proteins and performed eFastase
digest CC provided intellectual input for primary neuronal cell culture and
design of study NF and RR preformed ELISA experiments CS edited the
manuscirlt CLink provided intellectual for design of study JL and AN
provided intellectua input for mice LP is the principle investigator and
designed study All authors read and approved the final manuscript

Acknowledgements
This work was supported by Mayo Clinic Foundation (LP), National Institutes
of lealth/National Institute on Aging [5R01AG026251-04(LP)], National
Institutes of iealth!Nationa institute of Neurological Disorders and Stroke
[Ro0 NS 063964-01 (L P), R01 NS077402 (LP)], R01 NS065782 (RR),
Amyolrophic Lateral Sclelosis Associahon (LP) and Departrmene of Defense
[W81XW -10-1-0512-1 and W81XW1l-09-1-0315AL093108 (LP)]

Author details
Department of Neuroscience, Mayo Clinic College of Medicine, 4500
San Pablo Road, Jacksonville, Florida 32224, USADepartment of
Neuroscience and Center for Translational Research in Neurodegenerative
Disease, University of Florida, Gainesville, FL 32610, USA Integrative
Physiology Institute for Behavioral Genetics, University of Colorado, UBC447,
Boulder, CO 80309, USA4The Lundbeck Foundation Research Center MIND,
Department of Medical Biochemistry, Aarhus University, Aarhus, Denmark

Received: 9 March 2012 Accepted: 25 June 2012
Published: 10 July 2012

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doi:1 0.1186/1750-1326-7-33
Cite this article as: Gass et al Progranulin regulates neuronal outgrowth
independent of Sortilin. Molecular Neurodegeneration 2012 7'33


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RESEARCHARTICLEOpenAccessProgranulinregulatesneuronaloutgrowth independentofSortilinJenniferGass1,WingCLee1,CaseyCook1,NicoleFinch1,CarolineStetler1,KarenJansen-West1,JadaLewis1,2, ChristopherDLink3,RosaRademakers1,AndersNykjr4andLeonardPetrucelli1*AbstractBackground: Progranulin(PGRN),awidelysecretedgrowthfactor,isinvolvedinmultiplebiologicalfunctions,and mutationslocatedwithinthePGRNgene( GRN )areamajorcauseoffrontotemporallobardegenerationwithTDP43-positiveinclusions(FLTD-TDP).InlightofrecentreportssuggestingPGRNfunctionsasaprotectiveneurotrophic factorandthatsortilin(SORT1)isaneuronalreceptorforPGRN,weusedaSort1-deficient( Sort1 / )murineprimary hippocampalneuronmodeltoinvestigatewhetherPGRN ’ sneurotrophiceffectsaredependentonSORT1.We soughttoelucidatethisrelationshiptodeterminewhatroleSORT1,asaregulatorofPGRNlevels,playsin modulatingPGRN ’ sneurotrophiceffects. Results: AsthefirstgrouptoevaluatetheeffectofPGRNlossin Grn knockoutprimaryneuronalcultures,weshow neuriteoutgrowthandbranchingaresignificantlydecreasedin Grn / neuronscomparedtowild-type(WT) neurons.Moreimportantly,wealsodemonstratethatPGRNoverexpressioncanrescuethisphenotype.However, therecoveryinoutgrowthisnotobservedfollowingtreatmentwithrecombinantPGRNharboringmissense mutationsp.C139R,p.P248Lorp.R432C,indicatingthatthesemutationsadverselyaffecttheneurotrophicproperties ofPGRN.Inaddition,wealsopresentevidencethatcleavageoffull-lengthPGRNintogranulinpeptidesisrequired forincreasedneuronaloutgrowth,suggestingthattheneurotrophicfunctionsofPGRNarecontainedwithincertain granulins.TofurthercharacterizethemechanismbywhichPGRNimpactsneuronalmorphology,weassessedthe involvementofSORT1.WedemonstratethatPGRNinduced-outgrowthoccursintheabsenceofSORT1in Sort1 / cultures. Conclusion: WedemonstratethatlossofPGRNimpairsproperneuriteoutgrowthandbranching,andthat exogenousPGRNalleviatesthisimpairment.Furthermore,wedeterminedthatexogenousPGRNinducesoutgrowth independentofSORT1,suggestinganotherreceptor(s)isinvolvedinPGRNinducedneuronaloutgrowth. Keywords: Progranulin,Sortilin,Neuronaloutgrowth,Frontotemporallobardegeneration,NeurotrophicfactorBackgroundFrontotemporallobardegeneration(FTLD)isthesecondmostcommonpreseniledementiaafterAlzheimer ’ sdisease(AD)[1].FTLDisneuropathologicallycharacterized bypredominantatrophyofthefrontalandtemporallobes andthepresenceofproteinaceousinclusionsinneurons andglialcells[2].FTLDcasesaredividedintotwomain pathologicalsubgroupsatautopsy:thosewithtau-positive inclusions(FTLD-tau),andthosewithTDP-43-positive inclusions(FTLD-TDP)[3]. In2006,mutationslocatedwithin GRN ,weredescribed asamajorcauseofFTLD-TDP[4,5].Pathogenicmutationsin GRN mainlyincludeframeshift,splicesiteand nonsensemutationsthatproduceaprematureterminationofthecodingsequenceandconsequentlyleadto degradationofmutantmRNAvianonsense-mediated decay[4-6].Asaresult,pathogenicmutationsarethought tocausediseaseby GRN haploinsufficiency.Withtheexceptionofthepathogenicmissensemutationp.A9D, whichpreventsPGRNsecretion,itisnotknownwhether missensemutationsin GRN influencePGRN ’ sneurotrophicproperties[6,7]. PGRNisawidelyexpressedsecretedgrowthfactor involvedindevelopment,woundrepairandinflammation *Correspondence: petrucelli.leonard@mayo.edu1DepartmentofNeuroscience,MayoClinicCollegeofMedicine,4500 SanPabloRoad,Jacksonville,Florida32224,USA Fulllistofauthorinformationisavailableattheendofthearticle 2012Gassetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.Gass etal.MolecularNeurodegeneration 2012, 7 :33 http://www.molecularneurodegeneration.com/content/7/1/33

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[8,9].RecentstudiessuggestthatPGRNfunctionsasa protectiveneurotrophicfactor;however,itsroleinthe CNSisnotwellunderstood[10-13].Themechanisms underlyingitseffectshavenotbeenfullydetermined,nor isitknownhowmissensemutationsin GRN influenceits neurotrophicproperties. Theproteincontainsseven-and-a-halftandemrepeats ofa12-cysteinegranulinmotifseparatedbyinterlinked spacerregionsthataretermedgranulins[14].Inthe periphery,full-lengthPGRNisproteolyticallycleaved intomaturegranulinsviaproteases[15,16].Secretory leukocyteproteaseinhibitor(SLPI),aserineproteaseinhibitor,preventsPGRNcleavagebybindingtoPGRN cleavagesitesortoelastaseandinhibitingitscleavage activity[16]. Recently,twoPGRNreceptorshavebeenidentified: sortilin1(SORT1)andtumornecorosisfactorreceptor (TNFR)[17,18].TheneuronalPGRNreceptor,SORT1, hasbeenshowntoregulatetheextracellularlevelsof PGRN[17,19].SORT1,avacuolarproteinsorting10 proteindomainreceptor,isalsoinvolvedinendocytosis andtransportofproteinstothetrans-golginetworkand endosomes/lysosomes[20].TheC-terminalportionof PGRNbindstoSORT1causingimmediateendocytosis ofPGRNtothelysosome.Duetoitsinteractionswith SORT1,wesoughttodeterminewhetherPGRNutilizes thisreceptortomodulateneuronalmorphology.With regardtothelossofPGRNleadingtoneurodegeneration,weinvestigatedthephysiologicalimpact/consequencesoflossofPGRNinprimaryhippocampal neuronalcultures.ResultsLossofPGRNaltersneuronalmorphologyGRN haploinsufficiencyduetomutationsin GRN cause FTLD-TDP[2].ToevaluatetheimpactofPGRNdeficiencyonneuronmorphology,primaryhippocampal culturesfromWTand Grn / micewereprepared. Westernblotanalysisconfirmedthecompletelossof PGRNinhippocampalculturesfrom Grn / mice (Figure1A).Inordertoassessneuronaloutgrowth,neuronswerefixedandimmunostainedusingneuronspecificanti-MAP2following10daysinvitro(DIV10). Usingconfocalmicroscopy,wecapturedandanalyzed imagesusingMetaMorph7.1software(Figure1B), whichquantitativelymeasurestotalskeletonizedoutgrowthofindividualneuronsin msandtotalbranching junctionsofdendrites.AsquantifiedinFigure1C-E, Grn / neuronsdisplayedsignificantlyreducedneurite outgrowth(p < 0.0001)andbranching(p < 0.005)comparedtoWTneurons,whilecellbodysizeremainedthe same.Inallexperiments,atleast100neuronswereanalyzedandrepeatedinthreeindependentexperiments.ExogenousPGRNrescuesneuriteoutgrowthand branchingin Grn / neuronsHavingestablishedthatalossofendogenousPGRN causesmajordeficitsinneuriteoutgrowthandbranchingofhippocampalneurons,wethenexaminedwhether thisphenotypecanberescuedbyreturningPGRNto cultures.ToachievePGRNoverexpressioninourcultures,wepackagedPGRNintoarAAV1vector(rAAV1PGRN).Dosingtitersareexpressedgenomes/mL. Todetermineifwecouldrescuethe Grn / phenotype, wetransducedprimaryhippocampalneuronswithincreasingdosesofrAAV1-PGRNfor6daystoallowfor synthesisofPGRN.Undernormalconditions,ourWT primaryculture ’ sPgrnlevelswerearound4ng/mL (Figure2A).Intestingfortheproductionandreleaseof PGRNintothemedia,humanspecificELISArevealeda dose-dependentincreaseofPGRNsecretedintothe medium(Figure2B).Neuronalmorphologywasthen assessedin Grn / andWTneuronstreatedwiththesame increasingtitersofrAAV1-PGRN.ApplicationofrAAV1PGRNresultedinstatisticallysignificantincreasedoutgrowthandbranchinginboth Grn / andWTcultures (Figure2C-F).rAAV1-GFPtransductioninculturesdid notpromoteoutgrowthorbranching,suggestingthatthe productionofPGRNinducesoutgrowthandplaysarole inneuronalmorphology.Allconditionswerenormalized toWTrAAV1-GFPtreatedcellsrevealingthatthephenotypewasrescuedatarAAV1-PGRNtiterof1011genomes/mLor14ng/mLwithinthemedia.rPGRNpromotesneuronaloutgrowthandbranchingin Grn / andWTculturesSincedeterminingthatexogenousoverexpressionof rAAV1-PGRNisabletorescuethe Grn / neuronal phenotype,wepurifiedrecombinanthumanPGRN (rPGRN)frommediaofastableHEK293celllineoverexpressingPGRN.TherPGRNwasthenappliedtoWT and Grn / culturesinincreasingdoses.Theresults fromthisexperimentshowadose-dependent,significant increaseinoutgrowthandbranchingin Grn / neurons aswellasWTneurons(Figure3A-D).Theseresultsalso revealthat125nM(10ug/mL)rPGRNisneededtorescuethe Grn / phenotypeandreturnoutgrowthand branchingtonormalconditions.CleavageofPGRNpromotesneuronaloutgrowthPGRNissecretedasafull-length88kDaprotein,whichis cleavedintomaturegranulins.Infact,WesternblotanalysisofrPGRNtreatedculturesshowsrPGRNcleavage withincultures(Figure3E).Notethatinthemedia, rPGRNbecomescleavedintogranulinfragmentswithin1 hour.TodeterminewhetherPGRNorgranulinsareresponsibleforinducingneuriteoutgrowth,WTcultures weretreatedwithrecombinantSLPI(rSLPI),aserineGass etal.MolecularNeurodegeneration 2012, 7 :33 Page2of13 http://www.molecularneurodegeneration.com/content/7/1/33

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proteaseinhibitorthatpreventsPGRNcleavage[16].Utilizinganelastasedigestassay,wefirstconfirmedthatrSLPI preventsprocessingofrPGRNintoGRNs(Figure4A). UponincubationofrPGRNwithelastase,PGRNcleavage occurredwithin5minutes;however,whenrSLPIwas added,thecleavageofPGRNbyelastasewasinhibited. WesoughttoexaminerSLPI ’ seffectsonendogenous mousePgrnlevels,whichhadyettobedetermined. AftertreatingWTculturesexpressingendogenousPgrn withrSLPI,weobservedreducedoutgrowth(Figure4B) andbranching(Figure4C)similartountreated Grn / cultures.WhenrSLPIwasaddedto Grn / cultures therewasnochangeinoutgrowthorbranching,illustratingthatrSLPIistargetingPGRNandthatPGRN cleavageisrequiredtoinduceoutgrowth.WhiletreatmentwithrPGRNaloneincreasesoutgrowth,cotreatmentofrSLPIandrPGRNinWTneuronsdoesnot increaseoutgrowth(Additionalfile1:FigureS1). Thesefindingsnextledustoinvestigatetheneurotrophicactivityofspecificgranulins.Here,neuronswere treatedwithrecombinantgranulinproteincontaining fulllengthPGRN,andcommerciallyavailablegranulinE (rGRNE)orgranulinC(rGRNC)(Figure4D).TreatmentwithgranulinsEandC(125nM/10ug/mL)promotedneuronaloutgrowthverysimilartoPGRN treatment(125nM/10ug/mL)suggestingthesegranulin speciesharborneurotrophicproperties(Figure4E).C-terminaltaginhibitsSORT1uptakeandPGRN endocytosisSORT1wasrecentlyidentifiedasaneuronalreceptorfor PGRN;however,itsroleinPGRNfunctionisnotwell understood.WedecidedtotestrecombinantPGRN (rPGRN)onN2AcellstransducedtooverexpressrAAV1SORT1orrAAV1-GFP(Figure5A).HumanrPGRNwas generatedfromanHEK293stablecelllinecontaininga His-taglocatedateithertheN-terminal(His-rPGRN)or C-terminal(rPGRN-His)regionofrPGRN.OurresultsindicatethatoverexpressionofSORT1triggersincreased uptakeofHis-rPGRNandnotrPGRN-His,suggestingthe C-terminaltaginhibitsPGRN ’ sabilitytobindtoSORT. SincethebindingofPGRNtoSORT1washinderedby theC-terminaltag,wenextcomparedtheabilityofHisrPGRNandrPGRN-Histopromoteoutgrowthand branchinginprimaryhippocampalcultures.WedeterminedthatbothHis-rPGRNandrPGRN-Hiswereableto equallyinduceoutgrowth(Figure5B).PGRNpromotesneuronaloutgrowthandbranching independentofSortilinWepreviouslyshowedthatSORT1canregulatePGRN levels,buttoelucidatetherolethisreceptorplaysin neurotrophicmorphology,weexamined Sort1 / hippocampalcultures.Westernblotanalysisconfirmedthe completedeletionofSort1inhippocampalcultures (Figure6A).SinceSort1isinvolvedinuptakeofPGRN, wethenwantedtolearnwhetherthesecultureshavea marginalincreaseinsecretedPgrn.ELISAdatafrom WTand Sort1 / culturesrevealedincreasesofPgrnin Sort1 / culturescomparedtoWT(Figure6B).On theotherhand,neuronaloutgrowthstudiesrevealed decreasedoutgrowthin Sort1 / neuronscomparedto WT(Figure6C). AWT Grn-/-CD*** **NSE B 0 25 50 75 100 125Normalized total outgrowth( m) 0 25 50 75 100 125Normalized total branching 0 25 50 75 100 125Normalized m2 ofcell bodyWT mPgrn GAPDH WTWTWT Grn-/-Grn-/-Grn-/-Grn-/Figure1 LossofPgrndecreasesneuriteoutgrowthandbranchinginprimaryneurons. ( A )Westernblotanalysisrevealsendogenouslevels ofPgrninWTand Grn / primaryhippocampalcultures.( B )WTand Grn / hippocampalneuronsimmunostainedforMAP2.( C-E ).Neurite outgrowthandbranchingofMAP2-immunopositiveneuronswasassessedusingMetaMorphversion7.1software.Notethatneuriteoutgrowth ( C )andbranching( D ),butnotcellbodysize( E ),aredecreasedin Grn / hippocampalneuronscomparedtoWThippocampalneurons.Alldata ispresentedasthemeanSEM.**p < 0.01;***p < 0.001. Gass etal.MolecularNeurodegeneration 2012, 7 :33 Page3of13 http://www.molecularneurodegeneration.com/content/7/1/33

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ToverifywhetherPGRNcanincreaseoutgrowthin theabsenceofSORT1andultimatelywhetherSORT1is aneurotrophicreceptorforPGRN,wetreatedprimary hippocampal Sort1 / andWTneuronswithincreasing amountsofrPGRN.Wefoundadose-dependentincreaseinoutgrowthoccursin Sort1 / neuronssimilar toWT(Figure6D,6F).Additionallyincreasedbranching oftheseneuronswasalsoseen(Figure6E,6G),suggestingPGRNdoesnotuseSORT1topromoteoutgrowth.MissensemutantPGRNaltersneuronalmorphologyWhilethemajorityofpathogenic GRN mutationsleadto thedegradationofmutantmRNAand/orcause GRN haploinsufficiency,somemissensemutationshavebeen A D C***NS*** *** ** Grn-/-rAVV1-PGRN 10910111010rAAV1-GFP 1011WT 000 0 Grn-/-rAVV1-PGRN rAAV1-GFP WT 0 50 100 150 200 250Normalize total outgrowth ( m) 0 25 50 75 100 125 150 175 200 225Normalized total branchingrAVV1-PGRN10910111010rAAV1-GFP10110 0 rAVV1-PGRN10910111010rAAV1-GFP101100 00 0 0 NS* ** ** *** ***F E 0 5 10 15hPGRN media l evels (ng/ml)*** Grn-/-rAAV1-PGRN109101110100 *** *** *** 0 1 2 3 4 5 mPgrn media levels (ng/ml)WT Grn-/-B 0 25 50 75 100 125Normalized total outgrowth ( m) 0 25 50 75 100 125Normalized total branching10110 109101110101011000 0 10110 WTWT *** *** Figure2 PrimaryhippocampalculturestransducedwithrAAV1-PGRNpromotesneuriteoutgrowthandbranching. ( A )MousePgrnELISA ofendogenousPgrnlevelsinhippocampalprimaryneuronalcultures.( B )HumanPGRNELISAshowsincreasinglevelsofhPGRNgeneratedinthe mediaoftreatedprimarycultures.( C,D )Bargraphquantificationoftotaloutgrowth( C )andbranching( D )ofprimaryhippocampalneurons transducedwithincreasingamountsofrAAV1-PGRNinWTculturesnormalizedtoWTrAAV1-GFPtreatedcultures.( E,F )Bargraphquantification ofnormalizedtotaloutgrowth( E )andbranching( F )in Grn / hippocampalneuronstransducedwithrAAV1-PGRNnormalizedtoWTrAAV1-GFP treatedcultures.NotethatrAAV1-GFPwassupplementedinrAAV1-PGRNnon-treatedcultures.DataispresentedasthemeanSEM. *p < 0.05;***p < 0.001. Gass etal.MolecularNeurodegeneration 2012, 7 :33 Page4of13 http://www.molecularneurodegeneration.com/content/7/1/33

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reportedtodisruptPGRNprocessing(p.C139R)and secretion(p.R432C,p.P248L),whencomparedtoWT PGRN[7,21].Thep.S120Y GRN mutationhasbeen foundincontrolsandbelievedtobenon-pathogenic [22].Todeterminewhetherthesemissensemutations alsodisrupttheneurotrophicfunctionofPGRN,wegeneratedrecombinantPGRNproteinsharboringtheaforementionedmissensemutations(Figure7A)andassessed theireffectonneuriteoutgrowthin Grn / neurons. Asexpected,rPGRNcontainingp.S120Y,amutation observedincontrols,promotedneuriteoutgrowthtoa similarextentasWTrPGRN(Figure7B).Incontrast, rPGRNwithp.C139R,p.R432Candp.P248Lmutations failedtostimulateneuriteoutgrowth(Figure7B), suggestingthesemutationsdisruptPGRNfunction andmayindeedbepathogenicandcontributetothe diseasephenotypeobservedinpatientscarryingthese mutations.DiscussionWhilelossofPGRNisamajorcauseofFTLD-TDP,it remainsunclearhowthislossleadstoneurodegeneration. RecentstudiessuggestthatPGRNmayfunctionasa protectiveneurotrophicfactorregulatingneuronalsurvival andoutgrowthincortical/m otorprimaryneurons,immortalizedcelllines,andzebr afishembryos[10,11,23-25]. ***NS NSrPGRN nM rPGRN nM 025125625025125625 Grn-/-Grn-/-AB 0 25 50 75 100 125 150 175Normalized total outgrowth( m) 0 25 50 75 100 125 150 175 200 225Normalized total branching** ** **NS NS NSrPGRN nMrPGRN nM 0 25 125 625025125625 WT WTCD E 83 62 47 32 25*** rPGRN treatment (hours) 1 24 48 72 96 0 25 50 75 100 125 150 175 200 225Normalized total outgrowth ( m) 0 25 50 75 100 125 150 175 200 225 250 275 300Normalized total branching 0 0 WTWT** ** ** *** *FL Figure3 rPGRNpromotesneuriteoutgrowthandbranchinginprimaryneurons. ( A )( A,B )Bargraphquantificationoftotalneurite outgrowth( A )andbranching( B )ofWThippocampalneuronsexposedtorPGRNincreasing25,125,625nMdosesofHis-rPGRN(nMdoses correspondto2,10,50ug/mLconcentrationsrespectively).AlldosesnormalizedtoWTnon-treatedcultures.( C,D )Bargraphquantificationof normalizedtotalneuriteoutgrowth( C )andbranching( D )inof Grn / hippocampalneuronsexposedtorPGRN. Grn / cultureswerenormalized toWTnon-treatedcultures.( E )WesternblotanalysisrevealsPGRNcleavagein Grn / hippocampalculturestreatedwith125nMrPGRN-Hisat increasingtimepoints.NotecleavageofPGRNafteronlyonehour.DataispresentedasthemeanSEM.**p < 0.01. Gass etal.MolecularNeurodegeneration 2012, 7 :33 Page5of13 http://www.molecularneurodegeneration.com/content/7/1/33

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Themechanismsunderlyingtheseeffectshavenotbeen fullydetermined,norisitknownhow GRN missensemutationsandSORT1,aPGRNneuronalreceptor,influence PGRN ’ sneurotrophicproperties. Inthecurrentstudy,weexaminedtheeffectsof PGRNlossinprimaryneuronalculturesfrom Grn / mice.Ourdatashowthatneuronaloutgrowthand branchingin Grn / primaryculturesaresignificantly reducedcomparedtoWTcultures.WealsodemonstratethatPGRNpromotesneuriteoutgrowthindependentofSort1.Suchfindingsadvanceourknowledge beyondpreviousstudies,andstronglysuggestanotherreceptor(s)modulatesPGRN-specificneuronaloutgrowth andbranching. Sincethediscoveryofthefirst GRN mutations,several groupshavedevelopedvarious Grn / mousemodels [26-28].Whilethemurinemodelsrecapitulateseveral pathologicalfeaturesofFTLDpatients,suchasincreased gliosisandTDP-43positiveinclusions,notwomodels arethesame,likelyduetodifferentbackgroundsstrains orstrategiesusedtoknockout Grn .Forourexperiments, weusedprimaryhippocampalculturespreparedfrom Grn / micegeneratedbyKayasugaetal.[27],withthe knowledgethatthesemicedisplayacceleratedbrain agingduetoincreasedaccumulationoflipofuscin,in additiontoenhancedgliosisandneurodegeneration whencomparedtoWTmice[29].These Grn / mice alsomimicbehaviorchangessimilartoFTLDpatients, includingincreasedaggression,decreasedsocialinteractionandimpairedlearningandmemory[27,30].Even thoughourmicedisplayincreasedubiquitinstaining, theyarenotpositiveforTDP-43,theneuropathological hallmarkofFTLDwithGRNmutations.Yinetaldevelopeda Grn / mousedisplayingamplifiedinflammation inadditiontoincreasedubiquitinstainingpositivefor TDP-43[26].IncreasedTDP-43accumulationisalso observedin Grn / neuronstreatedwiththeproteosome inhibitorMG132,suggestingthattheseneuronswere C AB6 Elastase digest (min) 62 49 38 28 17 140510253535 15rSLPI -----+ 0 25 50 75 100 125Normalized total outgrowth( m) 0 25 50 75 100 125Normalized total branching NS NS*** *1 593 593 174458113123179205261281336364417442496518573PGFBACDE 494593E 364 430C 0 E rPGRN rG RN rG R NC 0 25 50 75 100 125 150Total outgrowth as % ofWT WTE** rGRN C rGRN E rPGRNDrSLPI-++ WTGrn-/-rSLPI-++ WTGrn-/Figure4 PGRNprocessingpromotesneuronaloutgrowthandbranching. ( A )TimecourseanalysisofrPGRNsubjectedtoelastasedigest. PGRNcleavageproductsvisualizedbySDS-PAGE,post-stainwithsyprorubysolution(Sigma).Notethattheco-treatmentofrecombinantSLPI blockselastase-mediatedrPGRNcleavage.( B,C )Bargraphsdepictquantificationoftotalneuriteoutgrowth( B )andbranching( C )ofWTand Grn / neuronstreatedwithorwithoutrSLPI.NotethatintheseexperimentsnorPGRNwasadded,thereforerSLPIblocksendogenousPgrn. ( D )Schematicrepresentationofrecombinantproteinsanalyzedinprimaryhippocampalneurons.( E )WThippocampalculturestreatedwith recombinantgranulinE(rGRNE)andgranulinC(rGRNC)promoteoutgrowthsimilartofulllengthPGRNDataispresentedasthemeanSEM. *p < 0.05;***p < 0.001. Gass etal.MolecularNeurodegeneration 2012, 7 :33 Page6of13 http://www.molecularneurodegeneration.com/content/7/1/33

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morevulnerabletocellularstressesthanWT[31]. GRN haploinsufficiencycellculturemodelsgeneratedsimilar results,where GRN knockdownusingsiRNAresultedin TDP-43cleavage;however,downregulationof GRN for shorttimepointsdoesnotproduceTDP-43cleavage [32,33]. FTLDpatientswith GRN mutationsdevelopsevereatrophyofthefrontalandtemporallobesofthebrain [34].Therefore,understandingtheroleofPGRNin neuronalhealthandfunction,aswellasregulatinglevels areimportantforfuturestudies.Recenteffortsusing Grn / micehavehelpedshednewlightonthemany functionsofPGRN.Petkauandcolleaguesdetermined that Grn / micehavereducedsynapticconnectivityand alteredLTP,inadditiontoalteredmorphologyand reducedspinedensityofapicaldendritesofthehippocampusofagedmice[28].Additionalstudiesinneurons withreduced GRN usingsiRNArevealeddecreased neuronalarborizationandsynapticdensityinadditionto abnormaltransmissionatthesynapses[31].ThesefindingssuggestthatlossofPGRNaltersmorphologyof neuronsleadingtosynapticchanges,whichultimately maycauseneurodegeneration. Tofurthercharacterizeourmice,wecomparedWT and Grn / culturesandfound Grn / primaryhippocampalneuronsdisplaysignificantlyshorterneuritesand fewerbranchpointscomparedtoWTneurons.Importantly,thisphenotypeisreversedwhenneuronsaresupplementedwithPGRNviarecombinantorviralmethods. WhilerAAV1-PGRNtransductionleadstotheintracellularexpressionofPGRN,ELISA-basedquantificationof PGRNfrommediaindicatedthatextracellularPGRN levelsincreaseinadose-dependentmannerfollowing rAAV1-PGRNtransduction.Normalphysiologicalranges forhumanPGRNareapproximately100 – 400ng/mLand 8 – 12ng/mLinthePNSandCNSrespectively;however, hPGRN SORT1 GFP GAPDH AAV rAAV1-GFPrAAV1-GFP rAAV1-SORT1rAAV1-SORT1-ve His-rPGRN rPGRN-His rPGRN 0 25 50 75 100 125 150 175 200Normalized total outgrowth ( m)His-rPGRN rPGRN-His---25 25 125 125 625 625 NS NS NSA B WT Figure5 SORT1triggersendocytosisofrPGRN. ( A )WesternblotanalysisofNeuro2AcellstransducedwithrAAV-GFPorrAAV-SORT1inthe presenseofHis-rPGRNorrPGRN-His.NoteuptakeofHis-rPGRNintocelllysatesandtheabsenceofrPGRN-Hisuptake.( B )Bargraphquantification ofWTprimaryneuronalculturestreatedwithincreasing25,125,625nMdosesofHis-rPGRNorrPGRN-Hisdepictequalamountsofoutgrowth withbetweentreatments(nMdosescorrespondto2,10,50ug/mLconcentrationsrespectively).DataispresentedasthemeanSEM. Gass etal.MolecularNeurodegeneration 2012, 7 :33 Page7of13 http://www.molecularneurodegeneration.com/content/7/1/33

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inourWTprimarycultureswedeterminedthatnormal levelsareapproximately3.5ng/mL[32,33].Increasing dosesofrAAV1-PGRNelevatedexogenousPGRNlevels to3-15ng/ml.ThesestudiesdemonstratethatrAAV1PGRNtransductionresultingin15ng/mLPGRNwithin themedia,issufficienttorestoreoutgrowthandbranchingof Grn / culturestoWTconditions.Wealsoshowed thisphenotypecanberescuedwiththeadditionof rPGRNandinfactwewereabletostimulateoutgrowth andbranchingbeyondnormalconditionsrevealingthe effectiveneurotrophicabilitiesofPGRN.TherPGRN dosesexceedthenormalrange,however,itisdifficultto assesswhatamountoftherPGRNistrulyactiveor degradedasaresultofproteinpurificationornormal degradationoftherecombinantproteinincellculture modelsovertime. OurresultssuggestthatPGRNfunctionsasaneurotrophicfactorinvolvedinproperneuronalmorphology mPGRN mSORT1 GAPDHABC E D* 0 1 2 3 4 5 6 7 8Pgrn concentration ng/mlWT Sort-/-Normalized total outgrowth ( m) 0 25 50 75 100 125 150 175rPGRN nM025125625** **NS Sort-/0 25 50 75 100 125 150 175 200 225Normalized total branchingrPGRN nM025125625* **NS Sort-/-WT Sort-/-*Sort-/-WT 0 10 20 30 40 50 60 70 80 90 100 110Normalized total outgrowth ( m) 0 25 50 75 100 125 150 175Normalized total outgrowth ( m)*** **NS NS NSrPGRN nMrPGRN nM 02 5 125 625025125625 WT WTG F** 0 25 50 75 100 125 150 175 200 225Normalized total branching Figure6 PgrninducesoutgrowthindependentofSort1. ( A )WesternblotanalysisofPgrnandSort1levelsinWTand Sort1 / hippocampal cultures.( B )MousespecificELISAshowingincreasinglevelsofPgrnin Sort1 / culturemedia.( C )Bargraphquantificationofnormalizedtotal neuriteoutgrowthin Sort1 / andWTculturesshowingsignificantdecreasesin Sort1 / .( D-G )Normalizedtotaloutgrowth( D,F )andtotal branching( E,G )in Sort1 / andWTcultureswithincreasingamountsofHis-rPGRNtreatment.DataispresentedasthemeanSEM. *p < 0.05;**p < 0.01. Gass etal.MolecularNeurodegeneration 2012, 7 :33 Page8of13 http://www.molecularneurodegeneration.com/content/7/1/33

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andfunction.ConsideringthatPGRNisasecretedprotein,extracellularPGRNmayberesponsibleforinitiatingsignalingeventsthatpromoteneuritedevelopment. Forexample,wedemonstratethatdecreasedoutgrowth andbranchingisreversedbythesupplementationof PGRN.Giventhevarietyof Grn / modelsnowavailable,itwouldbeofgreatinteresttodetermineifmodelspecificcharacteristics,suchassynapticabnormalities, alteredspinesanddendrites,aswellas,TDP-43pathologycanberescuedwithexogenousPGRNtreatment. Intheperiphery,PGRNiscleavedintomaturegranulinsbyextracellularproteases[15,16],similartoother neurotrophins,suchasnervegrowthfactor(NGF)and brain-derivedneurotrophicfactor(BDNF),whichare synthesizedasprecursorsandproteolyticallycleavedto formmatureneurotrophins[34].Ourdatasuggestthat rPGRNisbeingcleavedwithinprimaryneuronalculturemedia(Figure3A),thereforewehypothesizedthat cleavedPGRN,orgranulins,areresponsibleforinducingitsneurotrophicproperties.SLPI,aproteaseinhibitor,isknowntobindPGRNorelastasetoprevent PGRNcleavage[15,16].Todetermineiffulllengthor cleavedPgrnisneurotrophic,wetreatedWTand Grn / primaryneuronswithandwithoutrSLPI.Ourfindings showthatrSLPIisabletopreventoutgrowthinWT cultures;however,thisisnotseeninknockoutcultures suggestingrSLPIsspecificityforPgrn.VanDammeetal. alsoreportedthatSLPIabolishesPGRN-enhancedsurvivalandneuriteoutgrowthincorticalneurons[11]. Moreimportantly,weshowthatindividualgranulinsC andEhavetheabilitytopromoteneuronaloutgrowth, furtheremphasizingthatPGRNcleavageisrequiredfor neuriteoutgrowthandbranching.Whileweonlytested granulinsCandE,forfuturestudiesitwillbeimportant todeterminethefunctionofphysiologicallyrelevant granulinproducts,sincepreviousreportssuggestthat theymayhavedifferentialeffects[16]. GiventhatpreviousstudiesdeterminedtheC-terminus tailofPGRN(QLL)bindstothebeta-propellerofSORT1 [35],wedecidedtoanalyzeSORT1induceduptakeof rPGRN.WhengeneratingrPGRN,wetaggedourpeptide usinga6HistagateithertheC-terminus(rPGRN-HIS) orN-terminus(HIS-rPGRN)forpurification.HisrPGRNbecomesendocytosedbySORT1overexpression; however,webelievedtheHistagonrPGRN-Hiswould preventSORT1fromendocytosingit.Furthermore,we wantedtodetermineifthesetwoversionsofrPGRN havesimilarneurotrophicproperties,sinceonlyHisrPGRNinteractswithSORT1.Indeed,whenwe comparedHis-rPGRNandrPGRN-HisinWTcultures,weobservedaverysimilardose-dependent increaseinoutgrowthsuggestingtheneurotrophic 12345678 9 101112133`UTRS120Y C139R P248L R432CA B***NS NS***NS rP GRN rC 139R rP248L rR43 2C rS120 Y 0 25 50 75 100 125 150 175 200 225 250Normalized total outgrowth( m)0recombinant protein Grn-/Figure7 Specificmissensemutationslacktheabilitytopromoteoutgrowthandbranchinginprimaryhippocampalneurons. ( A )Schematicrepresentationofthe GRN geneandlocationofmissensemutationsinvestigated(p.S120Y,p.C139R,p.P248L,p.R432C).( B )Bar graphquantificationofneuriteoutgrowthof Grn / hippocampalneuronstreatedwith125nM(10ug/mL)ofwild-typerPGRN-Hisormissense rPGRN-His.DataispresentedasthemeanSEM.**p < 0.01;***p < 0.001. Gass etal.MolecularNeurodegeneration 2012, 7 :33 Page9of13 http://www.molecularneurodegeneration.com/content/7/1/33

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propertiesarenotduetothelocationoftheHis-tag nortoSORT1. Theimpactofindividualgranulins ’ neurotrophicfunctionsandourpreviouslymentioneddatasuggestthat PGRNmaybeactingindependentlyofSORT1topromoteneuriteoutgrowth.Our Sort1 / culturesexhibit increasedlevelsofPGRNwithinthemedia,afindingthat isconsistentwithpreviousreportsdemonstratingthe abilityofSORT1toregulatePGRNlevels[17,35].These culturesalsodisplayadecreaseinneuronaloutgrowth, whichmaybepuzzlingconsideringtheincreasedPgrn levelsinthemedia.However,othergroupshavealso reportedthisphenomenonandpointtoSORT1 ’ sinteractionwithtropomyosin-receptor-kinase(Trk)receptors asthecause.Basically,itwasdeterminedthatSORT1 isinvolvedintheanterogradetransportofTrkreceptorstonerveendings,wherethereceptorsinduce trophicsignaling[36].Webelievethedecreasesin outgrowthin Sort1 / culturesculturesarecausedby thelossoftrophicsupportr esultingfromTrkreceptormisguidance.Furthermore,weobservedthatby addingincreasingamountsofPGRNto Sort1 / cultures,outgrowthandbranchingincreasedinadosedependentmannersimilartoWTneurons.Such findingsfurthersuggestPGRN-inducedoutgrowth occursindependentofSORT1. Pinpointingexactlywhichreceptorisresponsiblefor PGRN-inducedneuronaloutgrowthhasnotyetbeen determinedandwillbetheaimoffuturestudies.Tang etal,recentlydiscoveredPGRNalsobindstoTNFR [18],andwhenboundtoTNFR,PGRNantagonizes andpreventsdownstreamTNF -mediatedinflammatorysignaling.Onecouldspeculatethatthelossor reducedlevelsofPGRNlong-termwillenhancecell death/pro-inflammatorysignalingthatcouldbedetrimentaltoneuronalviability.Itmaybepossiblethat increasesinPGRNmitigatetheTNF responsetothe extentthatweobserveneurotrophiceffects.Insucha case,excessPGRNmaykeeptheTNFRsignalingin checkwhereasdeficitsinPGRNfavoraTNF mediatedresponseresultinginreducedoutgrowthand ultimatelyneuronaldeath.GiventhatPGRNpromotes outgrowthindependentofSORT1,ourdatasuggest anotherreceptor(s)isinvolvedinPGRN-inducedneuronaloutgrowthandmayberesponsibleformaintainingneuronalintegrity. MostpathogenicmutationsinPGRNresultinaloss offunctionalPGRNcausedbynonsense,splice-siteand frameshiftmutations,aswellasbymutationsthatdisruptpropertranslationandsecretion[4-6].However, thereareseveralmissensemutationsofunknownpathogenicity.Lackoffunctionaldatacurrentlyrestrictsour understandingregardingthepossiblecontributionof thesevariantstodiseasepathogenesis.Tofurther elucidatetherolemissensemutationswithundeterminedpathogenicityplayincausingalossofneurotrophicfunction,rPGRNcontainingmissensemutations weregeneratedandanalyzedinhippocampalneuronal cultures.Amongthemissensemutationsstudied,two (p.P248Landp.R432C)werereportedtoleadto decreasedsecretion,andone(p.C139R)reducesPGRN production,stabilityandpatientplasmalevels[7,25,32]. p.S120Y,isunlikelytobepathogenicsinceitisfoundin controlpatients[37,38]wasfoundtopromoteneurite outgrowthsimilartoWTrPGRN.Incontrast,rPGRN withp.C139R,p.R432Corp.P248Lmutationsdidnot stimulateneuriteoutgrowth,suggestingthatthese mutationsadverselyaffectPGRNfunctionandmaybe pathogenic,whichwouldbeconsistentwithprevious geneticstudies[39,40].ConclusionsTakentogether,ourfindingsindicatethatPGRNis requiredfortheproperdevelopmentofneuritesinprimaryhippocampalneurons.Ourresultssuggestneurite outgrowthandbranchingareregulatedbyextracellular cleavedgranulins.WedeterminedthatPGRNinduces outgrowthindependentofSORT1,whichindicatesthat PGRNusesadifferentreceptortopromoteneuronal morphology.Whilepathogenicmutationscausedecreases inPGRNlevels,certain GRN missensemutationsabolish theneurotrophicpropertiesofPGRN,providingaputative linkbetweenthesemutationsanddevelopmentofFTLDTDP.MethodsMiceGrn / micehavebeenpreviouslydescribed[27,29]. Sort1 / miceweregeneratedandpreviouslydescribed [41-43].TimedbreedingcagesofC57/BL/6, Grn / and Sort1 / weresetupforprimaryneuronalcultures.All rodentspecimenswereobtainedfromanimalshandled byproceduresapprovedbytheMayoClinicInstitutional AnimalCareandUseCommittee.PrimaryneuronalculturePrimaryneuronalculturesfromhippocampiorcortex werepreparedfrompostnatalday1mousepupsand storedat4CinHIBERNATE ™ Amediawithoutcalcium (BrainBits),supplementedwithB27(Invitrogen), 0.5mMGMAX(GIBCO),andgentamicin(GIBCO). Excisedhippocampiorcortexweredigestedinpapain (2mg/mL;FisherScientific),trituratedwithaPasteur pipet(boresize0.8 – 1mM),centrifugedtocollectcell pellet,andresuspendedinNeurobasalA(Invitrogen), supplementedwithB27,GMAX,gentamicin,andbFGF (Invitrogen).Followingdeterminationofcellnumber, neuronswereplatedonpoly-D-lysine-coatedcoverslipsGass etal.MolecularNeurodegeneration 2012, 7 :33 Page10of13 http://www.molecularneurodegeneration.com/content/7/1/33

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within24-wellplatesforimmunocytochemicalstudies (seededatadensityof2.5104cells/coverslip),or seededonpoly-D-lysinecoated6-wellplatesforimmunoblottingataseedingdensityof3105cells/well. Cultureswereallowedtomaturefor10days.Mediawas changedonthethirdandseventhdays.Forexperiments usingrecombinantprotein,cellsweretreatedonday4 andday7aftermediachange.RecombinantPGRNproductionHumanprogranulin(PGRN)full-lengthcDNAincluding the5 ’ signalpeptidesequencewassubclonedintothe Nhe Iand Age IrestrictionsitesofthepcDNA4/V5HisAvectorsuchthata6Histagwasfusedatthe carboxyl-terminusofthePGRNsequence.APGRN stablecelllinewasgeneratedbytransfectingHEK293 cellswiththepcDNA4/PGRN-6HisDNAandmissense PGRN-6HisDNAina6-wellcultureplateformat. PGRNexpressionplasmidsweresequenceverifiedusing ABI3730withBigDyechemistryfollowingmanufacturers ’ sprotocol(AppliedBiosystems,FosterCity,CA, USA).Stabletransfectantswereselectedfor4weeksin completeculturemediumsupplementedwith400 g/ml zeocin.Survivingcloneswereindividuallypicked, expandedandtestedforthePGRNexpression.The clonethatsecretesthehighestlevelofPGRNprotein intomediawasusedforrecombinantPGRN(rPGRN) production.StandardNi-NTA(Qiagen)affinitypurificationproceduresbasedonmanufacturer ’ sinstruction wereappliedtopurifytherPGRNprotein(Leeetal. 2005).rAAV1productionrAAV1-PGRN,rAAV1-SORT1andrAAV1-GFPwaspreparedbystandardmethods.Briefly,AAVvectorsexpressingPGRN,SORT1orGFPunderthecontrolofthe cytomegalovirusenhancer/chicken -actinpromoter,a woodchuckpost-transcriptionalregulatoryelement,and thebovinegrowthhormone,poly(A),weregeneratedby plasmidtransfectionwithhelperplasmidsinHEK293T cells.Allplasmidsweresequenceverifiedasdescribed above.Forty-eighthoursaftertransfection,thecellswere harvestedandlysedinthepresenceof0.5%sodium deoxycholateand50U/mlBenzonase(Sigma,St.Louis, MO)byfreezethawing,andtheviruswasisolatedusing adiscontinuousiodixanolgradient.Thegenomictiterof eachviruswasdeterminedbyquantitativePCR.ImmunocytochemistryPrimaryneuronsoncoverslipswerefixedinmethanol, permeablizedwith0.5%Triton-X-100inPBS,washed, blockedin5%milk-PBSfor1hourandincubatedovernightinanti-MAP2(1:500,Sigma-Aldrich)orantiprogranulin(1:500,Invitrogen)dilutedin5%milk-PBS. Coverslipswerethenwashedandincubatedfor2hours ingoatanti-rabbitAlexaFluor568(1:1000,Molecular Probes)orgoatanti-mouseAlexaFluor488(1:1000, MolecularProbes).Coverslipswerethenincubatedfor10 minutesinHoescht33258(1:10000,Invitrogen),washed andmountedontomicroscopeslidesbyFluormount-G (SouthernBiotech).ImageacquisitionandquantificationAllimageswerecapturedusingaZeissLSM510META confocalmicroscope.NeuronalmorphologymeasurementsweredeterminedusingMetaMorphversion7.1 (MolecularDevices,Downingtown,PA).Ineachexperiment,50 – 100neuronssampledfromrandomlyselected fieldswereanalyzedandrepeatedinatleastthreeindependentexperiments.Statisticalanalysiswasperformed usingGraphPadPrizm4bycomparingmeansofdifferentgroupsusing Student ’ s-t testorANOVAfollowedby posthocKruskal-WallistestandDunn'sMultipleComparisonTest.Errorbarsindicate*p < 0.05;**p < 0.01; ***p < 0.001.WesternblotandELISACellsfrom6wellplateswerelysedinlysisbufferconsistingofCo-IPbuffer(50mMTris – HCl,pH7.4,1M NaCl,1%Triton-X-100,5mMEDTA)plus1%SDS, PMSF,andbothaproteaseandphosphataseinhibitor mixture.Theproteinconcentrationofcelllysateswas measuredusingaBCAassay(Pierce).SampleswerepreparedinLaemmli ’ sbuffer,heatedfor5minat95C,and equalamountsofproteinwereloadedinto10-well10% Tris-glycinegels(Novex).Aftertransfer,blotswere blockedwith5%nonfatdrymilkinTBST(TBSplus 0.1%TritonX-100)for1h,andthenblotswereincubatedwithanti-mouseprogranulin(1:1000,R&Dsystems),anti-humanprogranulin(1:1000,Invitrogen), mousemonoclonalGAPDHantibody(1:10000,Biodesign)overnightat4C.Membraneswerewashedthree timesfor10mininTBSTandthenincubatedwithdonkeyanti-rabbit,anti-mouseoranti-sheepIgGconjugated tohorseradishperoxidase(1:2500;JacksonImmunoResearch)for1hour.Membraneswerewashedthreetimes eachfor10min,andproteinexpressionwasvisualized byECLtreatmentandexposuretofilm.HumanPGRN ELISA(R&DSystems)andMousePgrnELISA(Enzo LifeSciences)wasusedaccordingtomanufactureprotocolonmediacollectedfromrAAV1-PGRNtreated cultures.EnzymaticdigestionofrPGRNbyelastase30 gofwild-typerecombinantPGRNormutantsPGRN proteinincubatedonicewasmixedwithreactionbuffer (100mMTris – HCl,300mMNaCl,pH7.5)and 0.05U/mlpurifiedelastase(AthensResearchandGass etal.MolecularNeurodegeneration 2012, 7 :33 Page11of13 http://www.molecularneurodegeneration.com/content/7/1/33

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Technology)inatotalreactionvolumeof100 l.The enzymereactionwasinitiatedbyincubationat37C. 10 lofthereactionmixturewassampledevery5 minutesandthereactionwasimmediatelyquenched byaddingequalamountof2SDSsamplebuffer. Thereactionwasallowedtoproceeduntil35minutes aftertimezero.Aseparatereactionwiththeaddition ofrecombinantSLPIat13.3 Mwasalsoprepared andprecededfor35minutesat37C.Thesamples wereanalyzedbySDS-PAGE,post-stainwithsypro rubysolution(Sigma)andimagedbyageldocstation equippedwithaUV-transilluminator.AdditionalfileAdditionalfile1: FigureS1. rSLPIpreventsrPGRNinducedoutgrowth andbranching. A-B .Bargraphquantificationofnormalizedtotal outgrowth( A )andbranching( B )inprimaryhippocampalcultures treatedwithrPGRNrSLPI.DataispresentedasthemeanSEM. Listofabbreviations PGRN:Humanprogranulin;Pgrn:Mouseprogranulin; GRN :Humanprogranulin genename; Grn :Mouseprogranulingenename; Sort1 :Mousesortilingene; SORT1:humansortilin1;Sort1:mousesortilin1;FTLD:Frontotemporallobar degeneration;TDP-43:TransactiveresponseDNA-bindingprotein43; AD:Alzheimer ’ sdisease;AAV:Adeno-associatedvirus. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors ’ contributions JGcarriedouttheprimaryneuronalcellculture,immunohistochemistry, Imageacquisitionandquantification,designedthestudyandwrotethe manuscript.CLeegeneratedrecombinantproteinsandperformedelastase digest.CCprovidedintellectualinputforprimaryneuronalcellcultureand designofstudy.NFandRRpreformedELISAexperiments.CSeditedthe manuscript.CLinkprovidedintellectualfordesignofstudy.JLandAN providedintellectualinputformice.LPistheprincipleinvestigatorand designedstudy.Allauthorsreadandapprovedthefinalmanuscript. Acknowledgements ThisworkwassupportedbyMayoClinicFoundation(LP),NationalInstitutes ofHealth/NationalInstituteonAging[5R01AG026251-04(LP)],National InstitutesofHealth/NationalInstituteofNeurologicalDisordersandStroke [R01NS063964 – 01(LP),R01NS077402(LP)],R01NS065782(RR), AmyotrophicLateralSclerosisAssociation(LP)andDepartmentofDefense [W81XWH-10-1-0512-1andW81XWH-09-1-0315AL093108(LP)]. Authordetails1DepartmentofNeuroscience,MayoClinicCollegeofMedicine,4500 SanPabloRoad,Jacksonville,Florida32224,USA2Departmentof NeuroscienceandCenterforTranslationalResearchinNeurodegenerative Disease,UniversityofFlorida,Gainesville,FL32610,USA3Integrative PhysiologyInstituteforBehavioralGenetics,UniversityofColorado,UBC447, Boulder,CO80309,USA4TheLundbeckFoundationResearchCenterMIND, DepartmentofMedicalBiochemistry,AarhusUniversity,Aarhus,Denmark. Received:9March2012Accepted:25June2012 Published:10July2012 References1.Graff-RadfordNR,WoodruffBK: Frontotemporaldementia. 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