The Journal of Neuroscience, April 11, 2012 • 32(15):5151–5164 • 5151

Development/Plasticity/Repair

Adaptor Protein LNK Is a Negative Regulator of Brain Neural

Stem Cell Proliferation after Stroke
Henrik Ahlenius,1 Karthikeyan Devaraju,2 Emanuela Monni,1 Koichi Oki,1 Somsak Wattananit,1 Vladimer Darsalia,1
Robert E. Iosif,2 Olof Torper,1 James C. Wood,2 Sebastian Braun,3 Lucas Jagemann,3 Ulrike A. Nuber,3
Elisabet Englund,5 Sten-Eirik W. Jacobsen,4 Olle Lindvall,2 and Zaal Kokaia1
1Laboratory of Neural Stem Cell Biology and Therapy, 2Laboratory of Neurogenesis and Cell Therapy, 3Stem Cell Gene Regulation Group, 4Hematopoietic

Stem Cell Laboratory, 5and Division of Neuropathology, Lund Stem Cell Center, Lund University Hospital, SE-221 84 Lund, Sweden

Ischemic stroke causes transient increase of neural stem and progenitor cell (NSPC) proliferation in the subventricular zone (SVZ), and
migration of newly formed neuroblasts toward the damaged area where they mature to striatal neurons. The molecular mechanisms
regulating this plastic response, probably involved in structural reorganization and functional recovery, are poorly understood. The
adaptor protein LNK suppresses hematopoietic stem cell self-renewal, but its presence and role in the brain are poorly understood. Here
we demonstrate that LNK is expressed in NSPCs in the adult mouse and human SVZ. Lnk ⫺/⫺ mice exhibited increased NSPC proliferation
after stroke, but not in intact brain or following status epilepticus. Deletion of Lnk caused increased NSPC proliferation while overex-
pression decreased mitotic activity of these cells in vitro. We found that Lnk expression after stroke increased in SVZ through the
transcription factors STAT1/3. LNK attenuated insulin-like growth factor 1 signaling by inhibition of AKT phosphorylation, resulting in
reduced NSPC proliferation. Our findings identify LNK as a stroke-specific, endogenous negative regulator of NSPC proliferation, and
suggest that LNK signaling is a novel mechanism influencing plastic responses in postischemic brain.

Introduction liferation in SVZ and neurogenesis (Jin et al., 2006; Martí-

Throughout adult life, neural stem and progenitor cells (NSPCs) Fàbrega s et al., 2010). Endogenous neurogenesis may be one of
in the subventricular zone (SVZ), lining the lateral ventricle, and the mechanisms underlying the spontaneous recovery after
the subgranular zone (SGZ) of the dentate gyrus proliferate and stroke, but its contribution is probably minor. The increased
generate neurons, which integrate into olfactory bulb and gran- NSPC proliferation in SVZ only lasts up to 2 weeks following the
ule cell layer, respectively (Zhao et al., 2008). Adult neurogenesis insult, even though the production of new neuroblasts and ma-
is modulated by pathological conditions, which has raised the ture neurons continues for several months (Thored et al., 2006).
possibility that it could contribute to functional recovery. Status Little is known about the endogenous mechanisms and mole-
epilepticus (SE) leads to increased NSPC proliferation and neu- cules negatively regulating NSPC proliferation in the adult brain,
rogenesis in SVZ and SGZ (Bengzon et al., 1997; Parent et al., which act to suppress the neurogenic response after stroke.
1997, 2002). Stroke, induced by middle cerebral artery occlusion The adaptor protein LNK is an intrinsic negative regulator of
(MCAO), gives rise to increased proliferation of NSPCs in SVZ hematopoietic stem cell (HSC) proliferation and expansion.
and generation of neuroblasts, which migrate toward the dam- Transgenic mice lacking Lnk exhibit increased B-cell lymphopoi-
aged striatum where they differentiate to mature striatal neurons esis and number of HSCs (Takaki et al., 2000; Buza-Vidas et al.,
(Arvidsson et al., 2002). Also in humans, stroke induces cell pro- 2006). LNK also regulates endothelial progenitor proliferation
and inhibits neovascularization (Kwon et al., 2009; Kamei et al.,
2010). Mutated Lnk was recently detected in leukemic cells in
Received Feb. 1, 2012; revised Feb. 25, 2012; accepted Feb. 29, 2012. patients with myeloproliferative neoplasms, and loss of Lnk was
Author contributions: H.A., K.D., U.A.N., O.L., and Z.K. designed research; H.A., K.D., E.M., K.O., S.W., V.D., R.E.I., found to exacerbate myeloproliferative disease (Bersenev et al.,
O.T., J.C.W., S.B., L.J., and Z.K. performed research; E.E. and S.-E.W.J. contributed unpublished reagents/analytic
tools; H.A., K.D., E.M., K.O., S.W., R.I., J.C.W., S.B., L.J., U.A.N., O.L., and Z.K. analyzed data; H.A., K.D., U.A.N., O.L., and
2010; Oh et al., 2010). LNK negatively regulates several cytokine
Z.K. wrote the paper. pathways, e.g., C-KIT (Velazquez et al., 2002) and erythropoietin
This work was supported by the Swedish Research Council, EU project LSHB-CT-2006-037526 (STEMSTROKE), EU (EPO) (Tong et al., 2005), which are important for hematopoie-
7th work program through the European Stroke Network (Grant no. 201024), AFA Foundation, and Swedish Gov- sis. C-KIT is also a chemoattractant and survival factor for NSPCs
ernment Initiative for Strategic Research Areas (StemTherapy).We thank Drs. W. Tong and H. Lodish for providing
the retroviral constructs, Dr. L. Velazquez for providing Lnk antibody, Drs. L. Aigner, S. Couillard-Despres, L. Pevny,
in the developing cortex and injured brain, while EPO promotes
and S. Kohsaka for providing transgenic mice, Zhi Ma and Teona Roschupkina for help with cell sorting, Marianne proliferation and neuronal differentiation in vitro and in SVZ
Rissler for virus production, and Camilla Ekenstierna for technical assistance. after stroke (Sun et al., 2004; Tsai et al., 2006). Whether LNK is
The authors declare no conflict of interest. present in the brain and involved in the regulation of NSPCs
Correspondence should be addressed to Dr. Zaal Kokaia, Laboratory of Neural Stem Cell Biology and Therapy,
Lund Stem Cell Center, Lund University Hospital, SE- 221 84 Lund, Sweden. E-mail: [email protected].
under normal and pathological conditions has been unknown.
DOI:10.1523/JNEUROSCI.0474-12.2012 Normal and insult-induced neurogenesis occurs in a multi-
Copyright © 2012 the authors 0270-6474/12/325151-14$15.00/0 step process starting with proliferation of NSPCs and continuing
5152 • J. Neurosci., April 11, 2012 • 32(15):5151–5164 Ahlenius et al. • LNK, Stroke and Neurogenesis

Figure 1. LNK is expressed in SVZ NSPCs in vivo and in vitro. A, RT-PCR and Q-PCR analysis of Lnk expression in tissue and neurospheres derived from mouse SVZ, and in FAC-sorted neuroblasts
(Dcx-GFP), NSPCs (Sox2-GFP), and microglia (Iba1-GFP) from SVZ and hippocampus (HPC) of reporter mice. B, Confocal photomicrographs showing LNK immunoreactivity (Figure legend continues.)
Ahlenius et al. • LNK, Stroke and Neurogenesis J. Neurosci., April 11, 2012 • 32(15):5151–5164 • 5153

with migration and maturation of newly formed neurons and parallel to the injection site, after applying conductive gel (Parker Labo-
ultimately their integration in established networks. We have ratories). A square-wave electroporator CUY21 EDIT (NepaGene) was
previously identified TNF-␣ signaling through TNF-R1 as a neg- used to deliver three 50 ms pulses of 200 V with an interval of 950 ms
ative regulator of insult-induced NSPC proliferation (Iosif et al., (Barnabé-Heider et al., 2008). After 72 h, mice were injected with bro-
modeoxyuridine (BrdU) four times with 2 h intervals, and 2 h after last
2006, 2008), establishing TNF-␣ as an important factor for the
injection mice were perfused.
neurogenic response to injury. Given the potent action of Lnk in
Fluorescence-activated cell sorting. Neurospheres were passaged and
inhibiting cytokine-induced proliferation in the hematopoietic SVZ tissue dissociated as described above. Cells were washed and resus-
system, and that several known LNK targets including EPO and pended in PBS containing 30 mM glucose and 5% FBS. Suspensions were
C-KIT have been shown to regulate NSPCs, we hypothesized that passed through a 70 ␮m filter and cells were sorted using a fluorescence-
LNK might be a negative regulator also of NSPCs. activated cell sorting (FACS) Aria (BD Biosciences). The sorting gate was
Our findings here for the first time identify LNK as a novel set around the main GFP⫹ population, at least 1 log higher than GFP⫺
endogenous negative regulator of NSPC proliferation, which controls. For BrdU incorporation and cell cycle analysis WT and Lnk ⫺/⫺
could have an important role for brain plasticity during recovery neurospheres were pulsed with BrdU and cells harvested at 3, 6, and 10 h
after stroke. and stained for BrdU and labeled with propidium iodide. Data were
acquired on an LSR II flow cytometer, with Diva software version 6.0
Materials and Methods (Becton Dickinson). Data analysis was done on FlowJo software ver-
Animals. Experimental procedures were approved by the Malmö-Lund sion 9.4.9 (TreeStar).
Ethical Committee. Generation of Lnk ⫺/⫺ mice has been described Immunocytochemistry in vitro. Neurospheres or cells attached to slides
(Takaki et al., 2000). Sox2-GFP mice were provided by Dr. L. Pevny, were fixed with 4% paraformaldehyde (PFA), preincubated in potassium
Dcx-GFP mice from Drs L. Aigner and S. Coulliard-Despres, and Iba1- PBS (KPBS) with 0.025% Triton X-100 and 5% serum for 1 h, and
GFP mice from Dr. Kohsaka. Mice were backcrossed into C57BL6 incubated with primary antibodies overnight. Primary antibodies in-
(Charles River), which were used as wild-type (WT) controls. A total of cluded rabbit anti-p-H3 (1:400; Millipore), rabbit anti-SOX2 (1:500;
10 Sox2-GFP, 12 Iba1-GFP, 10 Dcx-GFP mice of either sex, and 55 WT Millipore), and goat anti-LNK (1:100; Santa Cruz Biotechnology). After
C57BL6 and 35 Lnk ⫺/⫺ male mice were used for this study. washing, slides were incubated with goat anti-rabbit Alexa Fluor 488 or
Human tissue. Ganglionic eminence (GE) was subdissected from donkey anti rabbit Cy3 for p-H3 staining. For LNK/SOX2 double-
brains of dead aborted 6- to 9-week-old human fetuses. Tissue was ob- staining, biotinylated horse anti-goat together with donkey anti-rabbit
tained from Lund University hospital after informed consent in accor- Cy3 secondary antibodies followed by streptavidin-Alexa Fluor 488 was
dance with guidelines set by the Lund/Malmö Ethical Committee. used.
Anonymized SVZ brain samples were obtained, according to national Proliferation and survival assay. Neurospheres or sorted cells were
regulations, from autopsies of adult humans in which neuropathological plated on PDL/laminin-coated coverslips in proliferative medium. For
investigations was performed as part of clinical diagnostic routine. assessing mitosis, neurospheres or cells were stained for p-H3. The per-
Dissection and cell culture. Neurospheres were generated from SVZ of centage of p-H3⫹ cells of the total number of Hoechst⫹ cells was quan-
intact 8- to 12-week-old mice as described previously (Iosif et al., 2008). tified. Apoptosis was quantified with TUNEL staining (Roche) with
For neurosphere formation, cells were grown at clonal density (10 cells/ Hoechst counterstaining. The percentage of TUNEL⫹ cells with apopto-
␮l). Differentiation of neurospheres was performed by plating on poly- tic Hoechst morphology of the total Hoechst⫹ cells was estimated.
D-lysine (PDL)/laminin coated coverslips and culturing in medium
Induction of stroke. The MCAO filament model was used (Iosif et al.,
without growth factors but with N2 and 1% fetal bovine serum (FBS). 2008). Briefly, the left external carotid was ligated and temporary sutures
Human fetal GE was mechanically dissociated and cultured in DMEM/ were placed around the common and internal carotid arteries. An 8 – 0
F12 with N2, 2 ␮g/ml heparin, 10 ng/ml leukemia inhibitory factor monofilament (Alcon) coated with silicone was advanced through the
(Sigma), and 20 ng/ml epidermal growth factor (EGF) and basic fibro- internal carotid artery until it blocked the blood flow in the MCA. For
blast growth factor (bFGF). reperfusion, mice were re-anesthetized after 40 min of occlusion and the
Retroviral infection, transient transfection, and in vivo electroporation. filament was removed. We observed no differences between Lnk ⫺/⫺ and
Expanded neurospheres from WT mice were infected with retroviruses WT mice in initial neurological deficits, or in body weight and motor
expressing either MSCV-IRES-GFP or Lnk (MIG-Lnk) (Tong and Lo- behavior at 1 week after MCAO (Iosif et al., 2008).
dish, 2004) (provided by Drs. J. Lodish and W. Tong,) using a multiplic- Induction of SE. Mice were anesthetized and implanted with a stimu-
ity of infection of 2. Cells were allowed to grow for an additional 5 days in lating/recording electrode (Plastics One) unilaterally into ventral hip-
vitro before cell sorting. Constitutively active (CA)- or dominant- pocampal CA1–CA3 region. Ten days later, mice were subjected to
negative (DN)-STAT1 plasmids (provided by Drs. T. Ouchi and J. Dar- electrically induced SE (Iosif et al., 2006). Animals received 1 h of su-
nell, through Addgene, respectively) were transiently expressed in prathreshold stimulation consisting of 10 s trains of 1 ms biphasic square
neurospheres using lipofectamine (Invitrogen) and 48 h later expression wave pulses at a frequency of 50 Hz. Stimulation was interrupted for 1
of Lnk was assessed by Q-PCR. For in vivo transfection of SVZ cells, 6 ␮g min every 10 min to allow for electroencephalographic (EEG) recording
of CA- or DN-AKT (provided by Dr. M. Hung, through Addgene) plas- and measurement of afterdischarges (MacLab). After ending the stimu-
mids were injected into the lateral ventricle of 8- to 12-week-old WT lation, all mice exhibited self-sustained, continuous ictal EEG activity,
mice. Immediately after injection, mice were electroporated using 5 mm and associated motor behavioral convulsions (Iosif et al., 2006). Epileptic
disk electrodes, which were placed laterally on the skin covering the skull activity was arrested with pentobarbital at 2 h after stimulation offset.
BrdU administration. BrdU (50 mg/kg, i.p.), dissolved in PBS, was
4 given four times with a 2 h interval to intact mice and at 7 d after SE, and
animals were killed 2 h thereafter. Mice subjected to stroke received BrdU
(Figure legend continued.) in mouse SVZ neurospheres (NS) costained for SOX2. Double- once daily for 7 d after the insult and were killed the following day. For
positive cell depicted by arrowhead on low-magnification images is shown also at higher mag- BrdU label retention studies, mice received two daily BrdU injections
nification. C, RT-PCR analysis of LNK expression in GE tissue and NS derived from human fetal GE during day 9 and 10 after stroke and were killed 8 weeks later.
(hGE). Photomicrographs showing LNK immunoreactivity in human NS (hNS) costained for Immunohistochemistry in vivo. Animals were transcardially perfused
SOX2. Arrowhead indicates example of double-labeled cell. D, RT-PCR analysis of LNK in three with ice-cold 4% PFA. Free-floating sections (30 ␮m) were preincubated
different specimens of adult human SVZ tissue. E, Overview of adult human SVZ stained for LNK in 0.25% Triton X-100 in KPBS containing 5% donkey or goat serum for
(green) and GFAP (red). Boxed areas shown in higher magnification, top box (F–I) and lower 1 h. Sections were incubated with rat anti-BrdU (1:100; Sigma), rabbit
box (J–M) depict LNK⫹ (F, J), GFAP⫹ (G, K), LNK⫹/GFAP⫹ (H, I), and Hoechst⫹ (I, M) cells. anti-p-H3, rabbit anti-IBA1 (1:1000; Wako), goat anti-Dcx (1:400; Santa
Arrowheads indicate double-labeled cells. Scale bars: B, 50 ␮m; (in C) C–D, 20 ␮m; E, 15 ␮m, Cruz Technology), rabbit anti-GFAP (1:400; Zymed), mouse anti-Nestin
and (in M) F–M, 5 ␮m. (1:200; Millipore Bioscience Research Reagents), mouse anti-FoxJ1 (1:
5154 • J. Neurosci., April 11, 2012 • 32(15):5151–5164 Ahlenius et al. • LNK, Stroke and Neurogenesis

1000; eBiosciences), rat anti-CD31 (1:200; BD

Biosciences), or mouse anti-Sox2 (1:50; R & D
Systems) primary antibodies overnight at 4°C.
For BrdU staining, DNA was denatured in 1N
HCl for 30 min at 65°C or in 2N HCl at room
temperature for 2 h. Sections were washed and
incubated for 2 h with Cy3-conjugated donkey
anti-rat, donkey anti-rabbit (1:200; Jackson Im-
munoResearch) or Alexa-Fluor 488-conjugated
goat anti-mouse and donkey anti-goat secondary
antibodies at room temperature (1:500; Invitro-
gen). TUNEL staining was performed using the
in situ cell death detection kit (Roche) according
to manufacturer’s suggestions.
Fixed paraffin-embedded 5 ␮m human
brain sections were rehydrated and heat-
induced antigen retrieval was performed in 10
mM (pH 6) citrate buffer at 95°C for 10 min.
Slides were then blocked with 5% horse and don-
key serum for 1 h at room temperature and incu-
bated with goat anti-LNK, and mouse anti-GFAP
(1:500; DAKO) antibody overnight. After wash-
ing, slides were incubated with biotinylated horse
anti-goat and donkey anti-mouse Cy3 secondary
antibody for 2 h. Biotinylated antibodies were vi-
sualized using streptavidin-Alexa-Fluor 488 and
slides were counterstained with Hoechst.
Microscopical analysis. All assessments were
performed by a blinded observer. Immunos-
tainings were examined using fluorescence
light microscope. Cells were counted in four
evenly spaced sections throughout the SVZ.
Results are given as a mean number of cells per
section. Assessment of CD31 immunoreactiv-
ity was done using cellSens Dimension imaging
software (Olympus) in randomly chosen, fixed
size areas of SVZ and striatum in WT and
Lnk ⫺/⫺ mice subjected to MCAO. In each sec-
tion, areas of CD31 immunoreactivity were
identified using defined representative ranges
of threshold for specific signal. Using these de-
fined parameters, the images of each area were
analyzed by software, which calculated the to-
tal area covered by the specific immunoposi-
tive signal.
RNA isolation and RT-PCR. RNA was isolated
from subdissected SVZ primary tissue, ex-
panded neurospheres, or fluorescence-activated
sorted cells derived from subdissected SVZ tissue
typically pooled from 5 mice. SVZ tissue was sub-
dissected from 1 mm coronal brain sections using
microscissors and fine-point forceps. Total RNA

4
and Lnk ⫺/⫺ mice. Photomicrographs (D) and percentage of
p-H3⫹ (E) and apoptotic TUNEL⫹ (F) neurosphere cells out
of a total number of Hoechst⫹ cells. (G, H) FACS analysis of
BrdU incorporation and cell cycle in WT and Lnk ⫺/ ⫺ NSPCs.
Neurospheres were pulsed with BrdU and analyzed 3, 6, and
10 h thereafter. Histograms showing increased percentage of
BrdU⫹ cells in Lnk ⫺/⫺ (bottom) as compared with WT (top)
neurospheres (G). Bivariate (x-axis, propidium iodide; y-axis,
BrdU) cell cycle distribution analysis of WT (top) and Lnk ⫺/⫺
neurosphere cells. Density plots showing increased per-
centage of cells in S- and G2/M-phase in Lnk ⫺/⫺ (bottom)
as compared with WT (top) (H). Means ⫾S EM, n ⫽ 5 (A,
Figure 2. Deletion of Lnk leads to increased in vitro proliferation of SVZ NSPCs by shortening cell cycle duration. Number (A) and B) and 4 (E, F). *p ⬍ 0.05, Student’s unpaired t test. Scale
size (B) of primary and secondary neurospheres formed from WT and Lnk ⫺/⫺ SVZ. C, Photomicrographs of neurospheres from WT bars: C, 100 ␮m; D, 10 ␮m.
Ahlenius et al. • LNK, Stroke and Neurogenesis J. Neurosci., April 11, 2012 • 32(15):5151–5164 • 5155

(Invitrogen) were incubated (1:2) with block-

ing buffer (Lonza) containing 1 mg/ml herring
sperm DNA (Promega) and 1 mg/ml bovine
serum albumin (Sigma-Aldrich) overnight at
4°C on a rotator. For each reaction, beads in
100 ␮l of the slurry-blocking buffer mix were
separated with a DynaMag magnet (Invitro-
gen) and incubated for 20 min at room with
rabbit anti-STAT1, normal rabbit IgG (both
from Cell Signaling Technology), or H2O,
diluted in 200 ␮l Binding & Washing Buffer
(Invitrogen), and washed with Binding &
Washing Buffer. For immunoprecipitation,
100 ␮l cell lysate was diluted with 900 ␮l of
radioimmunoprecipitation assay buffer con-
taining PMSF and protease inhibitor, and in-
cubated with the prepared beads for 2 h. Beads
were washed three times with Washing buffer.
Cross-linking was reversed by resuspending
beads in 250 ␮l digestion buffer containing 10
mM Tris, 10 mM EDTA and 1% SDS, addition
of proteinase K to a concentration of 50 ␮g/ml,
and incubation for 2 h at 68°C. Ten microliters
of raw lysate were diluted in 240 ␮l digestion
buffer and treated equally to the ChIP samples,
served as input control. Finally, the material
was phenol-chloroform extracted and ethanol
precipitated. DNA was resuspended in 50 ␮l
of nuclease-free water and 2 ␮l was used as a
template for PCR. Primers are available upon
request.
Luciferase assay. The regulatory element of
Figure 3. Overexpression of Lnk leads to decreased in vitro proliferation of SVZ NSPCs. A, Relative expression of Lnk in sorted
mouse Lnk where we detected STAT1 binding,
GFP⫹ cells with or without retrovirally (RV) mediated Lnk-overexpression. Number (B) and size (C) of neurospheres formed from
2000 bp upstream and 300 bp downstream of the
sorted RV-GFP and RV-GFP-Lnk transfected cells. D, Brightfield and fluorescence photomicrographs illustrating size difference
predicted Lnk transcription start site, was cloned
between RV-GFP and RV-GFP-Lnk-transfected neurospheres. Percentage and photomicrographs of proliferating p-H3⫹ cells (E,
as WT (PGL4.10LNK) or with mutated (TCCG
F), and percentage of apoptotic TUNEL⫹ cells (G) out of the total number of Hoechst⫹ cells in sorted GFP⫹ and GFP⫹/LNK⫹
to GATC) binding sites (PGL4.10LNK-Mut) into
cells. Means ⫾ SEM, n ⫽ 5 (B, C) and 4 (E, G). *p ⬍ 0.05, Student’s unpaired t test. Scale bars: D, 50 ␮m; F, 10 ␮m.
PGL 4.10 luciferase reporter plasmid (Promega).
PGL4.10, PGL4.10LNK-Mut, or PGL 4.10LNK,
was isolated using RNAeasy (Qiagen) with DNase treatment according to alone or in combination with CA-STAT1 or
manufacturer’s instructions. RNA was reverse transcribed using oligoDT STAT3, was transiently expressed in 3T3 cells or the STAT1-deficient cell line
primers and superscript-II (Invitrogen). Q-PCR was performed with Taq- U3A (provided by Dr. G. Stark) using lipofectamine Luciferase activity was
Man universal master mix and TaqMan Gene expression assays (Applied assessed 24 h later using the dual luciferase reporter kit and a Glomax 20/20
Biosystems). One hundred nanograms of DNA (tissue) or cDNA from 150 luminometer (Promega).
cells (sorted cells) was used for each Q-PCR and all experiments were run in ELISA. Neurosphere cells were grown adherent in 96-well plates in
triplicate. cDNA input was normalized to GAPDH. Relative gene expression expansion medium until semiconfluent, when growth factors were with-
was calculated using the ⌬⌬CT method. RT-PCR primer sequences and drawn overnight. Cells were stimulated with 10 ng/ml EGF and 10 ng/ml
gene expression assay information are available upon request. bFGF, 50 ng/ml IGF1, or 5 U/ml EPO for 10 min. Levels of total and
Computational prediction of transcription factor binding sites. DNA phosphorylated ERK1/2 and AKT were analyzed using FACE in-cell
sequences for SH2B3 (LNK ) corresponding to conserved regions be- Western phospho ELISAs (Active Motif).
tween the human, mouse, rat, horse, and dog species upstream of the Statistical analysis. Comparisons were performed using Student’s un-
5⬘ UTR and including a part of the 5⬘ UTR were retrieved using the VISTA paired t test, two-way ANOVA followed by Scheffé’s post hoc test. Data
Browser (http://pipeline.lbl.gov) (Mayor et al., 2000) and the UCSC Ge- are presented as means ⫾ SEM, and differences are considered significant
nome Browser (http://genome.ucsc.edu/) (Kent et al., 2002). These at p ⬍ 0.05.
regions corresponded to the following positions in the human genome:
Human SH2B3 March 2006 chr12:110319473–110319590; chr12: Results
110320884 –110320980, chr12:110325532–110325770, and chr12: LNK is expressed by NSPCs in adult mouse and human SVZ
110327105–110328342. We applied MotifScanner 3.1.1 (http://med. LNK belongs to an adaptor protein family whose members share
kuleuven.be/lcb/toucan/help/WebServices/motifscanner.htm) (Aerts et NH2-terminal, homologous PH and SH domains, and a con-
al., 2005) for binding site prediction using matrix files from TRANSFAC and served C-terminal tyrosine phosphorylation site (Rudd, 2001).
murine and human conserved noncoding sequences as background model. Other members of the family, APS and PSM/SH2, have been
Chromatin immunoprecipitation PCR. Neurospheres were fixed with detected in rodent brain tissue (Welsh et al., 1998; Iseki et al.,
1% formaldehyde for 10 min at room temperature, and the process
2000; Yousaf et al., 2001), and Lnk has been detected in embry-
stopped by the addition of glycine at a final concentration of 0.0125 M for
5 min. Cells were washed and resuspended in 1 ml lysis buffer with onic rat cortex (Wang et al., 2011), but whether LNK is present in
protease inhibitor mixture and 1 mM serine protease inhibitor, phenyl- the mouse or human brain has been unknown. Using RT-PCR,
methylsulfonyl fluoride (PMSF; Roche). Sonication was performed with we found Lnk mRNA in mouse SVZ tissue and neurospheres
a Branson 450 probe, yielding DNA fragments with a bulk size of 50 –300 derived from SVZ (Fig. 1A). In agreement, neurosphere cells ex-
bp. The debris-cleared lysate was frozen at ⫺80°C. Magnetic Dynabeads pressing the neural stem cell marker SOX2 were immunoreactive
5156 • J. Neurosci., April 11, 2012 • 32(15):5151–5164 Ahlenius et al. • LNK, Stroke and Neurogenesis

Figure 4. Proliferation of NSPCs in SVZ is enhanced after stroke in Lnk ⫺/⫺ mice. Number and distribution of cells expressing p-H3 (A, C) and BrdU (B, D) in SVZ of intact WT and Lnk ⫺/⫺ mice
(A, B) or 7 d after stroke (C, D). Representative confocal images of BrdU⫹/Dcx⫹ (E) and BrdU⫹/Sox2⫹ (F) cells in the SVZ after stroke. Number of proliferating Dcx⫹ and Sox2⫹ cells after stroke
(G). Size and distribution of lesioned area after stroke (H). BrdU was injected 4 times 2 h apart in intact animals (A, B) or once daily following MCAO for 7 d (C–F). Ipsilateral denotes side of stroke.
Means ⫾ SEM, n ⫽ 4 and 5 for WT and Lnk ⫺/⫺ mice, respectively. *p ⬍ 0.05, t test (A, B, G, H) or two-way ANOVA with Scheffé’s post hoc test (C–D). Scale bar, 100 ␮m.
Ahlenius et al. • LNK, Stroke and Neurogenesis J. Neurosci., April 11, 2012 • 32(15):5151–5164 • 5157

for LNK protein (Fig. 1 B). To clarify the

cellular localization of LNK, we sorted
NSPCs, neuroblasts, and microglia from
SVZ and hippocampus of Sox2-GFP,
Dcx-GFP, and Iba1-GFP reporter mice,
respectively. Using RT-PCR and Q-PCR,
we detected Lnk mRNA in Sox2-, Dcx-,
and Iba1-GFP-positive and -negative cell
populations (Fig. 1 A), providing evidence
that Lnk is expressed not only in NSPCs
but in a wide variety of cells in the mouse
SVZ and hippocampus.
Next we wanted to determine whether
LNK is also expressed in the human brain.
The human fetal GE gives rise to SVZ in
the adult brain and contains NSPCs which
generate striatal neurons (Young et al.,
2007). LNK mRNA was detected in hu-
man fetal GE tissue and neurospheres
generated from this structure (Fig. 1C).
Moreover, similar to mouse SVZ-derived
neurospheres, SOX2 and LNK protein
were colocalized in cells of human fetal
GE neurospheres (Fig. 1C). We also de-
tected Lnk mRNA and protein in the SVZ
of adult human brain using PCR and
Figure 5. Proliferation of NSPCs in SGZ is enhanced after stroke in Lnk ⫺/⫺ mice. Number and distribution of cells expressing
p-H3 (A, C) and BrdU (B, D) in SVZ of SE WT and Lnk ⫺/⫺ mice (A, B) or 7 d after stroke (C, D). Means ⫾ SEM, n ⫽ 6 and 8 for WT
immunocytochemistry, respectively (Fig.
and Lnk ⫺/⫺ mice, respectively for SE (A, B) and n ⫽ 7 and 10 for WT and Lnk ⫺/⫺ mice, respectively for stroke (C, D) *p ⬍ 0.05, 1 D–M ). LNK immunoreactivity was ob-
two-way ANOVA with Scheffé’s post hoc test. Scale bar, 100 ␮m. served in GFAP⫹, putative NSPCs in
human SVZ (Fig. 1 E–M ).Together, our
findings clearly demonstrate the presence
of LNK in mouse and human brain in-
cluding the SVZ.

LNK is a negative regulator of NSPC

proliferation in vitro
The localization of LNK in the neurogenic
SVZ raised the possibility that LNK could
influence the properties of NSPCs. We hy-
pothesized that LNK regulates the prolif-
eration of NSPCs similar to what has been
described for HSCs (Buza-Vidas et al.,
2006; Seita et al., 2007). Lnk ⫺/⫺ mice have
previously been reported to exhibit no
gross anatomical differences compared
with their WT counterparts (Takaki et al.,
2000). We observed no obvious changes
in the appearance or size of the brain in
Lnk ⫺/⫺ mice, nor did we detect any ab-
normalities in the cytoarchitecture of
SVZ, SGZ, hippocampus, and striatum as
assessed by Hoechst, NeuN, Sox2, or Dcx
staining patterns. Cells from SVZ of
Lnk ⫺/⫺ mice were first expanded at clonal
density in vitro as neurospheres using epi-
dermal and basic fibroblast growth factor
(EGF and bFGF). We did not detect any
differences in the number of primary neu-
Figure 6. No difference in proliferation between WT and Lnk ⫺/⫺ SVZ NSPCs after SE. WT and Lnk ⫺/ ⫺ mice display no
differences in percentage of partial or generalized seizures during SE (A). SE-induced increase in SVZ proliferation of WT and rospheres between WT and Lnk ⫺/⫺ cells,
Lnk ⫺/⫺ mice (B). Number and distribution of cells expressing BrdU (C) and p-H3 (D) in SVZ of WT and Lnk ⫺/⫺ mice 7 d after SE. but we observed a modest 11% increase in
BrdU was administered 4 times 2 h apart and animals were perfused 2 h after last injection. Ipsilateral denotes side of stimulating the number of secondary neurospheres
electrode. Means ⫾ SEM, n ⫽ 7 and 10 for WT and Lnk ⫺/⫺ mice, respectively. p ⬎ 0.05, two-way ANOVA with Scheffé’s post hoc (Fig. 2A). The size of primary and second-
test.. ary neurospheres from Lnk ⫺/⫺ and WT
5158 • J. Neurosci., April 11, 2012 • 32(15):5151–5164 Ahlenius et al. • LNK, Stroke and Neurogenesis

mice was also similar (Fig. 2 B, C). However, with prolonged time
in culture from second passage, cells from Lnk ⫺/⫺ mice exhibited
an increased percentage of cells in mitosis as detected by p-H3
staining. The fraction of p-H3⫹ cells in neurospheres from
Lnk ⫺/⫺ mice was 181% higher as compared with that from WT
animals (Fig. 2 D, E). The increased percentage of p-H3⫹ cells
was not due to improved survival since we did not find any dif-
ference in the percentage of apoptotic TUNEL⫹ cells between
WT and Lnk ⫺/⫺ neurospheres (Fig. 2 F). To exclude the possibil-
ity that the increased number of p-H3⫹ cells was due to cell cycle
arrest in G2/M phase, we pulsed WT and Lnk ⫺/⫺ NSPCs with
BrdU and analyzed BrdU incorporation and cell cycle status by
FACS at 3, 6, and 10 h. Both WT and Lnk ⫺/⫺ NSPCs increased the
percentage of BrdU⫹ cells over time. Strikingly, in Lnk ⫺/⫺
NSPCs the percentage of BrdU⫹ cells was higher at all time
points increasing to 21.6, 33.4, and 40.6%, respectively (Fig. 2G)
as compared with 14.4, 15.2, and 18.1% in WT. The percentage of
cells in S-phase increased in WT from 16.6% to 19.2 and 21.2%
and in Lnk ⫺/⫺ from 19.9, 30.1, and 35.3% at 3, 6, and 10 h,
respectively. The number of WT cells in G0/G1 was similar (74.9,
71.0, and 72.3%) at all time points, but cells in G2/M decreased
from 1.5 and 1.4% at 3 and 6 h, respectively, to 0.5% at 10 h. In
contrast the number of Lnk ⫺/⫺ cells in G0/G1 decreased from Figure 7. Deletion of Lnk does not alter number of microglia in SVZ. A, Number of Iba1⫹
76.1% to 65.1 and 58.5% at 3, 6, and 10 h, respectively, while the microglia in intact SVZ, and ipsilateral and contralateral to the ischemic lesion or stimulating
percentage of cells in G2/M remained unchanged (1.5, 1.3, and electrode for stroke and SE, respectively, in WT and Lnk ⫺/⫺ mice. Means ⫾ SEM, n ⫽ 8 and 10
(Intact), 9 and 7 (Stroke), and 7 and 9 (SE) for WT and Lnk ⫺/⫺, respectively. No differences
1.2%) (Fig. 2 H).Together, our data indicate that Lnk ⫺/⫺ cells
between WT and Lnk ⫺/⫺ mice, p ⬎ 0.05 Student’s unpaired t test or two-way ANOVA with
proliferate faster by shortening the G0/G1 phase. This conclusion Scheffé’s post hoc test. B, Photomicrograph showing the distribution of IBA⫹ cells (IBA1 in red,
is supported by previous reports that decreasing G1 length ex- Hoechst in blue) in SVZ of intact WT and Lnk ⫺/⫺ mice. Scale bar, 30 ␮m. Str, striatum;. LV,
pands NSPC populations in the developing and adult brain lateral ventricle.
(Lange et al., 2009; Artegiani et al., 2011).
To provide further evidence for a suppressant role of LNK in
NSPC proliferation, we infected SVZ neurospheres from WT the peak of the increased proliferation, 7 d after stroke. BrdU was
mice with retroviruses to express either GFP alone or GFP to- injected once daily following MCAO. In contrast to the lack of
gether with Lnk. Five days thereafter, GFP⫹ cells were sorted and change in the intact brain, stroke increased the number of p-H3⫹
Lnk gene expression was assessed by Q-PCR, as well as neuro- cells by 60 and 63% (Fig. 4C) and BrdU⫹ cells by 32 and 37%
sphere formation, proliferation, and survival were analyzed. In (Fig. 4 D) in SVZ of Lnk ⫺/⫺ compared with WT mice, ipsilateral
the Lnk-infected cells, we detected a threefold increase in Lnk and contralateral to MCAO, respectively.
gene expression (Fig. 3A) and a dramatic, 96 and 61% decrease in We then analyzed whether NSPCs and neuroblasts, which
the number and size of neurospheres, respectively (Fig. 3B–D). In were both expressing LNK, contributed to stroke-enhanced cell
agreement, the number of cells in mitosis was 63% lower (Fig. proliferation in the SVZ of Lnk ⫺/⫺ mice. Sections were double-
3 E, F ). Lnk overexpression also influenced survival, the percent- stained for BrdU and the NSPC marker Sox2 or the neuroblast
age of TUNEL⫹ apoptotic cells being 107% higher as compared marker Dcx (Fig. 4 E, F ). Loss of Lnk increased poststroke prolif-
with that in control cells (Fig. 3G). Our findings after deletion eration of both NSPCs and neuroblasts in SVZ by 25 and 35%,
and overexpression of Lnk clearly show a role of this adaptor respectively, although in contrast to the Sox2⫹ NSPCs, the
protein as a negative regulator of in vitro proliferation of NPSCs. change in number of Dcx⫹ cells did not reach statistical signifi-
cance (Fig. 4G).
Stroke-induced proliferation of NSPCs is negatively regulated To explore the possibility that the higher cell proliferation in
by LNK SVZ caused by Lnk deletion was not confined to NSPCs but in-
Next we explored whether LNK also regulates NSPC proliferation cluded also other cell types, we analyzed the proliferation of
in the SVZ in vivo. The proliferative marker BrdU was injected FoxJ1⫹ ependymal, CD31⫹ endothelial, and Iba1⫹ microglial
four times 2 h apart in intact Lnk ⫺/⫺ mice and WT controls, and cells using BrdU injections at 1 week after MCAO. However, we
animals were killed 2 h thereafter. To reveal changes in cell cycle did not detect any colabeled cells in the SVZ (data not shown),
duration or cell division of NSPC in SVZ we analyzed BrdU in- providing evidence that at this time point after stroke, the vast
corporation in replicated DNA and p-H3 expression. No differ- majority of proliferating cells are NSPCs, and that Lnk deletion
ences in number of p-H3⫹ or BrdU⫹ cells were detected enhances the proliferation of this cell type.
between WT and Lnk ⫺/⫺ mice (Fig. 4 A, B), indicating that LNK Given the importance of the vascular niche in regulating
does not regulate proliferation in the healthy, intact SVZ in vivo. NSPCs, and the previously reported effect of LNK on endothelial
We have previously shown that stroke caused by MCAO cells and vasculature (Kwon et al., 2009; Kamei et al., 2010; Gold-
induces a transient increase of NSPC proliferation in SVZ man and Chen, 2011), we also assessed the overall staining pat-
(Arvidsson et al., 2002; Thored et al., 2006). To determine tern of CD31 in the SVZ and striatum. We measured the area
whether LNK influences NSPC proliferation in SVZ under path- covered by CD31 immunoreactivity in randomly selected areas of
ological conditions, we compared NSPC proliferation and early SVZ and striatum of WT and Lnk ⫺/ ⫺ mice at 1 week after stroke.
survival of newly formed cells in SVZ of WT and Lnk ⫺/⫺ mice at Arguing against an involvement of an altered vascular environ-
Ahlenius et al. • LNK, Stroke and Neurogenesis J. Neurosci., April 11, 2012 • 32(15):5151–5164 • 5159

renewal or mobilization of NSPCs after

stroke, we analyzed the number of BrdU
label-retaining cells, which has been used
as a marker of stem cells in SVZ (Doetsch
et al., 1999; Ferron et al., 2007). Support-
ing a specific effect of LNK on prolifera-
tion, we did not detect any difference in
the number of BrdU label-retaining cells
in SVZ at 8 weeks after BrdU injec-
tions given 1 week after stroke (data not
shown). Together, our data suggest that
LNK suppresses stroke-induced NSPC
proliferation but has minor or no effect
on self-renewal.
We then investigated if the enhance-
ment of cell proliferation caused by the
loss of LNK function could also occur in
other pathological conditions associated
with increased proliferation in SVZ. SE
gives rise to increased SVZ cell prolifera-
tion with low levels of inflammation (Iosif
et al., 2008). We induced SE for 2 h by
electrical stimulation in the hippocam-
pus. No differences in the percentage of
partial and generalized seizures were ob-
served between WT and Lnk ⫺/⫺ mice
(Fig. 5A). In agreement with our previous
findings (Iosif et al., 2006, 2008), SE in-
creased SVZ cell proliferation in both WT
and Lnk ⫺/⫺ mice (55 and 51%, respec-
tively) at 7 d after the insult as assessed by
the number of BrdU⫹ cells. However, we
did not detect any differences in SVZ cell
proliferation between WT and Lnk ⫺/⫺
mice following SE (Fig. 5 B, C).
To determine whether LNK acts as a
stroke-specific suppressor of NSPC prolifer-
ation also in the hippocampus, we counted
the number of p-H3⫹ and BrdU⫹ cells in
Figure 8. STAT1 regulates expression of Lnk in NSPCs after stroke. A, Relative gene expression of Lnk in SVZ tissue from intact, the SGZ of intact and SE- or MCAO-
stroke, and SE mice ipsilateral and contralateral to lesion and stimulating electrode, respectively. B, Occurrence of SP1, E2F1, subjected, WT and Lnk ⫺/ ⫺ mice. We did
STAT1, and STAT3 binding motifs (in bold) in mouse Lnk genomic region. C, Relative expression of transcription factor genes in SVZ not detect any differences in SGZ cell prolif-
tissue from intact mice or 7 d after stroke or SE. D, Lnk expression in neurospheres after transfection with CA- and DN-STAT1. E, eration caused by Lnk deletion in intact
Schematic representation of the murine Lnk locus with STAT1/3 binding motifs and primers used for ChIP. F, ChIP PCR analysis (data not shown) or SE animals (Fig. 6A,B).
showing binding of STAT1 to genomic region of the Lnk gene and the positive control gene Irf1. Genomic DNA immunoprecipitated
Similar to the findings in SVZ, we found
with IgG, as a negative control, or STAT1 antibodies was used for PCR with primers spanning binding motifs of STAT1 in Lnk and Irf1.
G, Relative luciferase activity in 3T3 and U3A cell lines transfected with control (pGL4.10), Lnk (pLnk), mutated Lnk (pLnk-Mut),
higher cell proliferation in SGZ following
pLnk⫹pCA-STAT1, and pLnk⫹pCA-STAT3. stroke in Lnk ⫺/ ⫺ compared with WT mice,
as evidenced by the numbers of pH-3⫹ and
BrdU⫹ cells (Fig. 6C,D, respectively).
ment in the effect on NSPC proliferation, we did not detect any We have previously shown that microglia activation can influ-
difference in the area of CD31 immunoreactivity in SVZ or stria- ence adult neurogenesis (Ekdahl et al., 2009). Because we found
tum between WT and Lnk ⫺/ ⫺ mice (data not shown). here that also Iba1⫹ microglia expressed Lnk, we speculated that
We found no difference in the area of the ischemic lesion effects of Lnk deletion on SVZ cell proliferation could potentially
between WT and Lnk ⫺/⫺ mice after stroke (Fig. 4 H). This indi- be caused by changes in microglia numbers. To test this hypoth-
cated that the effect of Lnk deletion was not mediated indirectly esis, we quantified the number of Iba⫹ cells in the SVZ. In agree-
by alteration in the size of the injury, which is known to influence ment with our previous data (Iosif et al., 2008), we found an
the magnitude of the stroke-induced neurogenic response in the increased number of Iba1⫹ microglia in SVZ after stroke but not
SVZ (Thored et al., 2006). after SE as compared with intact animals (Fig. 7 A, B). However,
Our finding that NSPCs from SVZ of intact Lnk ⫺/⫺ mice neither in intact mice nor after SE or stroke did deletion of Lnk
exhibited no increase in primary and only a modest increase in cause any changes in the number of Iba1⫹ microglia the SVZ
secondary neurosphere formation indicated that Lnk does not compared with WT mice (Fig. 7 A, B). These findings argue
affect NSPC number or self-renewal but negatively regulates pro- against the possibility that Lnk suppresses NSPC proliferation by
liferative capacity. To further investigate if LNK affects self- influencing microglia activation.
5160 • J. Neurosci., April 11, 2012 • 32(15):5151–5164 Ahlenius et al. • LNK, Stroke and Neurogenesis

Stroke upregulates Lnk expression in NSPCs through

STAT1/3 signaling
To determine how stroke regulates Lnk expression, we first mea-
sured the level of Lnk mRNA in SVZ tissue from intact mice and
at 1 week after stroke or SE using Q-PCR. Consistent with a role
of LNK in stroke-induced but not SE-induced SVZ cell prolifer-
ation, we found that Lnk expression was upregulated ipsilateral to
the damage by 70% after stroke but downregulated by 50% fol-
lowing SE (Fig. 8 A). Similar changes were detected in the con-
tralateral SVZ.
To identify transcription factors that might regulate Lnk ex-
pression after stroke we analyzed the promoter regions of Lnk for
predicted transcription factor binding motifs. A computational
search for motifs revealed the presence of SP1, E2F1, STAT1, and
STAT3 transcription factor binding sites in conserved regions of
the Lnk gene in five different species (mouse, rat, horse, dog, and
human) (Fig. 8 B). Using Q-PCR we found that Sp1 was slightly
downregulated after SE while E2f1 was slightly upregulated after
stroke. The most pronounced changes were observed for Stat1
and 3, which were upregulated 3.1- and 2.7-fold, respectively,
after stroke but were virtually unchanged after SE (Fig. 8C), in-
dicating that these transcription factors might regulate Lnk
expression in SVZ after stroke. We then transfected SVZ neuro-
spheres with CA-STAT1 or DN-STAT1. Supporting a role of
STAT1 in the regulation of Lnk, Q-PCR showed a 51% induction
of Lnk expression in CA-STAT1-transfected neurospheres, while
DN-STAT1 transfection reduced the expression by 65% (Fig.
8 D). To provide further evidence for a regulatory role of STAT
family members on Lnk gene expression, we assessed whether
STAT1 protein binds to genomic regions of the Lnk gene in SVZ-
derived NPSC. Chromatin immunoprecipitation experiments were
performed with a STAT1 antibody followed by PCR using primers
spanning putative binding motifs in the genomic sequence of Lnk
and the known STAT1 target interferon regulatory factor 1 (Irf1). In
two independent experiments, using two different primer pairs, we
consistently detected binding of STAT1 to one specific motif in the
first exon of the Lnk sequence (Fig. 8E,F).
We then cloned the regulatory elements of Lnk where we had
detected STAT1 binding into a dual luciferase reporter plasmid.
This plasmid was then transiently expressed in murine 3T3 and
human STAT1-deficient U3A cell lines. We also cotransfected the
luciferase plasmid with CA-STAT1 and CA-STAT3 plasmids and Figure 9. Stroke regulates gene expression of growth factors and their receptors in SVZ.
measured luciferase activity 24 h after transfection. These exper- Relative expression of known (A) and potential (B–D) LNK targets. Q-PCR analysis in SVZ tissue
iments revealed that the regulatory elements of Lnk were active in from intact mice, and 7 d after stroke or SE. Means ⫾ SEM, n ⫽ 3.
both mouse and human cells (Fig. 8G). Furthermore, we detected
a 1.8- and 1.2-fold induction of luciferase activity in 3T3 and U3A
cells, respectively, when the reporter plasmid was cotransfected Epo were upregulated almost fourfold after stroke but unchanged
with CA-STAT1, while CA-STAT3 increased luciferase activity after SE (Fig. 9A). C-kit and its ligand stem cell factor (Scf ) were
by 1.8- and 1.4-fold compared with reporter plasmid alone (Fig. downregulated after both insults (Fig. 9A). The receptor tyrosine
8G). In contrast plasmids with mutated STAT1 and STAT3 bind- kinases (RTKs) FgfR1, FgfR2, and EgfR were upregulated by 40%
ing sites decreased the activity by 33 and 29% in 3T3 and U3A after stroke but only slightly changed following SE (Fig. 9 B, C).
cells, respectively, as compared with the WT plasmid. Together, The ligands Egf and transforming growth factor ␣ (Tgf-␣) were
these findings show that Lnk is upregulated in SVZ after stroke downregulated after both stroke and SE. Expression of Fgf2 in the
and indicate that STAT1 and STAT3 regulate its expression in intact SVZ was below detection limit and only detected after
NSPCs. stroke (Fig. 9 B, C). We found a slight upregulation of IGF1 re-
ceptor (Igf1R) but a dramatic, 30-fold increase of its ligand (Igf1)
LNK suppresses NSPC proliferation by decreasing growth after stroke (Fig. 9D). In contrast, SE increased the expression of
factor signaling through inhibition of AKT phosphorylation Igf1 only by 30% and decreased receptor expression by 10%.
To identify mechanisms through which LNK might suppress Based on the specific alterations of gene expression induced by
stroke-induced NSPC proliferation, we first investigated using stroke, we hypothesized that LNK may act on the EPO, EGF, FGF,
Q-PCR the expression of known and potential LNK targets in and IGF1, signaling pathways.
SVZ tissue from intact mice and at 1 week after stroke or SE. Gene LNK is known to inhibit the phosphorylation cascade in RTK
expression of EpoR was marginally decreased whereas levels of signaling by direct interaction with receptors (Takaki et al., 2000;
Ahlenius et al. • LNK, Stroke and Neurogenesis J. Neurosci., April 11, 2012 • 32(15):5151–5164 • 5161

transfected with CA-AKT, while the num-

ber of BrdU⫹ and p-H3⫹ cells decreased
by 20 and 21%, respectively, in DN-AKT-
transfected SVZ (Fig. 10C).
The LNK-induced changes in growth
factor signaling could lead to alterations
in gene expression and subsequently in
cell proliferation. To explore this fur-
ther, we analyzed the expression of sev-
eral genes that have been implicated in
NSPC proliferation and maintenance in
the Lnk-overexpressing neurosphere cells.
Interestingly, we found that in parallel to
the dramatic reduction of cell prolifera-
tion, there was 81 and 68% reduction in
Hes5 and Gli1 gene expression, respec-
tively, in Lnk-overexpressing neuro-
spheres (Fig. 10 D).
Together, our findings provide evi-
dence that LNK inhibits IGF1 signaling by
attenuating phosphorylation of AKT, and
possibly also EGF/FGF signaling via
ERK1/2, and decreases gene expression of
Figure 10. LNK inhibits phosphorylation of AKT, gene expression in NSPCs, and SVZ cell proliferation. A, RT-PCR analysis of Hes5 and Gli1, resulting in reduction of
receptors for growth factors and the known LNK target EPOR in mouse SVZ neurospheres. B, Relative level of phosphorylation of
stroke-induced NSPC proliferation.
ERK1/2 or AKT in WT and Lnk ⫺/⫺ neurospheres after stimulation with EGF ⫹ FGF, IGF1, or EPO. Means ⫾ SEM, n ⫽ 5, *p ⬍ 0.05,
Student’s unpaired t test. C, Proliferation (expressed as percentage change of p-H3⫹ and BrdU⫹ cell numbers) after in vivo
transfection of SVZ cells by electroporation of CA- or DN-AKT plasmids. Means ⫾ SEM, n ⫽ 3. D, Relative gene expression in RV-GFP
Discussion
or RV-GFP-Lnk transfected neurospheres of factors involved in NSPC maintenance and proliferation. Means ⫾ SEM, n ⫽ 4. We show here for the first time that the
adaptor protein LNK is expressed and has
a functional role in the adult brain. The
Tong et al., 2005) or with downstream signaling (Bersenev et al., number of proliferating cells in the neuro-
2008). One common signaling pathway of EGF, FGF, and EPO is genic SVZ was increased after stroke but unchanged in intact
the phosphorylation of ERK1/2 through RAS and RAF kinases brain and following SE in Lnk ⫺/⫺ mice. Thus, LNK suppresses
(Subramaniam and Unsicker, 2006). IGF1 signaling, on the other NSPC proliferation specifically after one brain disorder, stroke,
hand, leads to AKT phosphorylation by PI3K (Ye and D’Ercole, but not after another one, SE, despite both pathological condi-
2006), and AKT is involved in maintenance of neural precursor tions giving rise to increased cell proliferation. The previously
cells (Oishi et al., 2009). We hypothesized that LNK inhibits unknown action of LNK as a suppressant of brain NSPC prolif-
phosphorylation of ERK1/2 and AKT downstream of EGF, FGF, eration is in line with its function in the hematopoietic system,
EPO, and IGF1 receptors. To test this idea, we first showed expres- where LNK is a negative regulator of B-cell progenitor (Takaki et
sion of these receptors in neurosphere cells (Fig. 10A). Next we an- al., 2003) and HSC proliferation (Velazquez et al., 2002) and
alyzed, using ELISA, the degree of phosphorylation of ERK1/2 and self-renewal (Buza-Vidas et al., 2006; Seita et al., 2007). LNK also
AKT in neurosphere cells from SVZ of Lnk ⫺/⫺ and WT mice in inhibits the malignant expansion and transformation of HSC and
response to EGF/FGF, IGF1, and EPO stimulation. We found a progenitor cells (Bersenev et al., 2010), and mutations in the Lnk
modest increase (16%) of ERK1/2 phosphorylation following EGF/ gene are associated with myeloproliferative neoplasms in patients
FGF stimulation whereas IGF1 gave rise to a more marked increase (Oh et al., 2010). Finally, loss of Lnk increases proliferation of
of AKT phosphorylation (by 32%) in Lnk ⫺/⫺ neurospheres com- endothelial cells and angiogenesis (Kwon et al., 2009; Kamei et al.,
pared with WT controls (Fig. 10B). In contrast, ERK1/2 phosphor- 2010).
ylation by EPO stimulation was not altered. The functional consequences of the LNK-mediated suppres-
To provide proof-of-principle for the role of AKT as a regula- sion of poststroke NSPC proliferation in SVZ remain to be ex-
tor of SVZ proliferation after stroke, we injected CA-AKT or plored. It should be emphasized, though, that there are several
DN-AKT plasmids into the lateral ventricle of WT mice and used mechanisms which may contribute to recovery in the poststroke
in vivo electroporation to transfect the SVZ. Electroporation is a brain. These mechanisms include plastic changes in surviving
safe and efficient method to target both quiescent ependymal neurons and neurons on the contralateral side, redistribution
and actively dividing NSPCs without affecting proliferation of brain representation, release of growth factors and anti-
(Barnabé-Heider et al., 2008, Carlén et al., 2009, Nomura et al., inflammatory factors from immune cells, synaptogenesis and
2010). To control for transfection efficiency, CA-AKT or DN- changes in synaptic strength, and changes in dendritic arboriza-
AKT plasmids were coinjected with red fluorescent protein or tion and spines, as well as generation of new neurons, glial, and
GFP plasmids. No differences in efficiency between injections endothelial cells (Moskowitz et al., 2010). Based on its expression
were detected (data not shown). After 3 d, animals were injected in several different cell types, it is conceivable that LNK, in addi-
with BrdU, and SVZ proliferation was assessed using BrdU and tion to its effect on NSPC proliferation in the SVZ, can influence
p-H3 immunohistochemistry. Consistent with a regulatory role poststroke functional and structural reorganization through sev-
of AKT on NSPC proliferation, we found 14 and 17% increase in eral of these mechanisms. We describe here a very specific effect
the number of BrdU⫹ and p-H3⫹ cells, respectively, in SVZ of LNK in regulating NSPC proliferation after stroke through
5162 • J. Neurosci., April 11, 2012 • 32(15):5151–5164 Ahlenius et al. • LNK, Stroke and Neurogenesis

alteration of IGF1-mediated Akt phosphorylation and gene ex- stroke in rats (Thored et al., 2009) and has been reported to be a
pression of stem cell regulators. However, this is most likely not mediator of the increased NSPC proliferation after stroke (Yan et
the only way that LNK may influence stroke-induced neurogen- al., 2006). The low levels of both Lnk and growth factors in SVZ of
esis. LNK is also known to affect endothelial cell proliferation and intact and SE animals probably explain the lack of change in
angiogenesis, to inhibit several cytokine signaling pathways in the NSPC proliferation under these conditions in the Lnk ⫺/⫺ mice.
hematopoietic system, and to exacerbate myeloproliferative dis- In contrast, we detected increased proliferation in the neuro-
ease. Furthermore, LNK is expressed in many cell types other sphere cultures from Lnk ⫺/⫺ mice. This in vitro environment is
than NSPCs. Therefore, it is conceivable that LNK acts not only more likely to mimic the SVZ niche after stroke with high levels of
on NSPC proliferation but also can influence stroke-induced growth factor signaling, which could explain why LNK has an
neurogenesis through other mechanisms. It is possible that LNK effect in vitro but not in vivo under basal conditions. LNK is
acts on a global level by fine-tuning the response to growth factor
known to inhibit RTK signaling by interacting either directly with
or cytokine stimulation.
the growth factor receptors or with downstream kinases, result-
We obtained no evidence for differences between WT and
ing in decreased phosphorylation (Takaki et al., 2000; Tong et al.,
Lnk ⫺/ ⫺ mice in endothelial cell proliferation or vascularization
in SVZ after stroke. However, due to the importance of the vas- 2005; Bersenev et al., 2008). We found that Lnk deletion in NSPCs
cular niche for NSPCs and the previously reported influence of gave rise to an increase in the phosphorylation of ERK1/2 in
LNK on endothelial cells, one cannot completely exclude that response to EGF receptor and FGF receptor stimulation and a
discrete changes in the vascular niche in Lnk ⫺/ ⫺ mice might more pronounced increase in the phosphorylation of AKT fol-
contribute to the NSPC proliferative effect observed here. lowing IGF1 receptor stimulation. We also showed that changes
The action of LNK as a negative regulator of NSPC prolifera- in AKT activation induced by transfection of CA-AKT or DN-
tion in the SVZ after stroke is most likely exerted in the NSPCs AKT plasmids into SVZ gave rise to increased and decreased
themselves and not indirectly through other cell types. First, we NSPC proliferation, respectively. Consistent with our data, LNK
found that the NSPCs express LNK. Second, although our data inhibits AKT and ERK1/2 signaling in hematopoietic stem/pro-
indicated that also microglia express Lnk, we obtained no in vivo genitor and endothelial cells, respectively (Tong et al., 2005; Wan
evidence that deletion of Lnk influenced the activation of SVZ et al., 2006; Seita et al., 2007). Thus, LNK can suppress NSPC
microglia. Third, our in vitro data clearly showed that altering the proliferation following stroke by inhibiting phosphorylation of
levels of Lnk in NSPCs, in the absence of immune cells and mi- kinases downstream of growth factor receptors, in particular
croglia, gave rise to the expected changes of cell proliferation. IGF1R. Hypothetically, the reduction of ERK1/2 and AKT signal-
We observed that the proliferation of NSPCs in SVZ of intact ing could lead to the decreased gene expression of Hes and Gli1,
and SE-subjected animals was not influenced by the deletion of which are involved in NSPC proliferation (Cayuso et al., 2006;
Lnk in the transgenic mice. In contrast, we detected increased Stecca and Ruiz i Altaba, 2009; Gregory et al., 2010). In support of
proliferation in neurosphere cultures from Lnk ⫺/ ⫺ mice as well this hypothesis, ERK and AKT activation positively regulates Gli
as in the NSPCs in SVZ of animals subjected to stroke. This expression in chicken neural explants and 3T3 cells (Riobó et al.,
discrepancy in the effect of LNK deficiency is most probably due 2006). The most prominent pathway discovered here is IGF1-
to the low levels of growth factors in SVZ of intact WT and
AKT signaling. However, given the broad spectrum of growth
SE-subjected animals and the high levels of growth factors in
factors and cytokine pathways known to be influenced by LNK, it
neurosphere culture medium and in the SVZ after stroke. Thus,
is possible that LNK also inhibits other pathways in NSPCs fol-
LNK is known to inhibit RTK signaling by interacting either di-
rectly with the growth factor receptors or with downstream ki- lowing stroke.
nases, resulting in decreased phosphorylation (Takaki et al., 2000; Here we have for the first time demonstrated the presence of
Tong et al., 2005; Bersenev et al., 2008). LNK in the adult brain and identified LNK signaling as a negative
The suppressant effect of LNK on NSPC proliferation was regulator of NSPC proliferation. This mechanism has so far been
specific for stroke and not observed in intact or SE animals and, shown to operate only after stroke, i.e., in response to a severe
accordingly, Lnk was upregulated after stroke but downregulated brain injury. Since stroke gives rise to NSPC proliferation in the
after SE. This finding is the first evidence that the expression of human brain (Martí-Fàbregas et al., 2010), and we found LNK to
Lnk is regulated in the adult brain. Our findings indicate that be expressed in human NSPCs, it is conceivable that LNK acts to
STAT1 and STAT3 induce the elevated Lnk expression in SVZ suppress the enhanced NSPC proliferation not only in rodents
NSPCs after stroke. We found that Stat1 was upregulated follow- but also in patients after stroke. Our findings raise the possibility
ing stroke but not SE in NSPCs, consistent with its previously that LNK action is a key player for cellular plasticity and a poten-
reported activation in neurons following ischemia (Takagi et al., tial novel therapeutic target in the postischemic brain.
2002). Furthermore, STAT3 has been shown to be activated in the
peri-infarct areas following ischemia, a process that was inhibited
by blocking IL-6 signaling (Yamashita et al., 2005). Stroke in- References
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