A small molecule accelerates neuronal differentiation in the adult rat
Proc Natl Acad Sci U S A. 2010 September 21; 107(38): 16542–16547.
11/7/13 4:02 PM
PMCID: PMC2944756
Published online 2010 September 7. doi: 10.1073/pnas.1010300107
Cell Biology
A small molecule accelerates neuronal differentiation in the adult rat
Heiko Wurdak,a,1 Shoutian Zhu,a,1 Kyung Hoon Min,a,2 Lindsey Aimone,b Luke L. Lairson,a James Watson,b Gregory
Chopiuk,b James Demas,c,3 Bradley Charette,a Rajkumar Halder,a Eranthie Weerapana,a Benjamin F. Cravatt,a Hollis T.
Cline,c Eric C. Peters,b Jay Zhang,b John R. Walker,b Chunlei Wu,b Jonathan Chang,b Tove Tuntland,b Charles Y. Cho,b
and Peter G. Schultza,4
aThe Skaggs Institute of Chemical Biology and Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037;
bGenomics Institute of the Novartis Research Foundation, San Diego, CA 92121; and
cDepartment of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037
4To whom correspondence should be addressed. E-mail: schultz@scripps.edu.
Contributed by Peter G. Schultz, July 20, 2010 (sent for review June 8, 2010)
Author contributions: H.W., S.Z., L.L.L., B.C., B.F.C., T.T., C.Y.C., and P.G.S. designed research; H.W., S.Z., K.H.M., L.A., L.L.L., J.W., G.C.,
E.W., E.C.P., J.Z., and J.C. performed research; K.H.M., G.C., R.H., and H.T.C. contributed new reagents/analytic tools; H.W., S.Z., L.L.L.,
J.D., B.C., E.C.P., J.R.W., C.W., T.T., and P.G.S. analyzed data; and H.W., S.Z., and P.G.S. wrote the paper.
1H.W. and S.Z. contributed equally to this work.
2Present address: College of Pharmacy, Chung-Ang University, Dongjak-Gu, Seoul 156-756, South Korea.
3Present address: Physics Department, St. Olaf College, Northfield, MN 55057.
Copyright notice
ABSTRACT
Adult neurogenesis occurs in mammals and provides a mechanism for continuous neural plasticity in the
brain. However, little is known about the molecular mechanisms regulating hippocampal neural
progenitor cells (NPCs) and whether their fate can be pharmacologically modulated to improve neural
plasticity and regeneration. Here, we report the characterization of a small molecule (KHS101) that
selectively induces a neuronal differentiation phenotype. Mechanism of action studies revealed a link of
KHS101 to cell cycle exit and specific binding to the TACC3 protein, whose knockdown in NPCs
recapitulates the KHS101-induced phenotype. Upon systemic administration, KHS101 distributed to the
brain and resulted in a significant increase in neuronal differentiation in vivo. Our findings indicate that
KHS101 accelerates neuronal differentiation by interaction with TACC3 and may provide a basis for
pharmacological intervention directed at endogenous NPCs.
Keywords: adult neurogenesis, neural progenitor cell, Tacc3
The adult hippocampal dentate gyrus (DG) is a major neurogenic region that harbors self-renewing
neural progenitor cells (NPCs). NPCs arise within the subgranular layer (SGL) of the DG and primarily
migrate into the adjacent granule cell layer (GCL) to differentiate and mature into neurons (1). Although
adult NPCs hold substantial promise for neural repair after brain injury, their mobilization is hampered
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by the lack of knowledge about the molecular mechanisms controlling adult neurogenesis under normal
and pathophysiological conditions. Pleiotropic signaling molecules [e.g., retinoic acid (RA), Wnt, and
brain-derived growth factor (BDNF)] as well as antidepressant and anticonvulsant drugs (e.g., fluoxetin
and sodium valproate) have been implicated in regulating adult NPC proliferation, differentiation, or
survival in vivo (2–8). Therefore, it may be possible to develop neurogenic agents for pharmacological
intervention specifically directed at the endogenous NPC pool. Here, we describe the small molecule
KHS101, which specifically induces neuronal differentiation in vitro and in vivo upon systemic
administration. Additionally, mechanism of action studies revealed a critical role for transforming acidic
coiled-coil–containing protein 3 (TACC3) in the regulation of NPC maintenance and differentiation.
RESULTS
KHS101 Induces Neuronal Differentiation in Cultured NPCs. We previously reported a phenotypic screen
that identified a group of synthetic 4-aminothiazole compounds, termed neuropathiazols, which induce
neuronal differentiation of cultured rat hippocampal NPCs (9). Synthesis of several analogs of the original
neuropathiazol structure and a focused structure–activity relationship (SAR) study afforded a molecule
(KHS101) that possessed increased activity and improved pharmacokinetic properties compared with the
original compound (Fig. 1A, Fig. S1, Scheme S1, Table S1). KHS101 was further studied in vitro using
established protocols for the isolation, propagation, and differentiation of adult rat hippocampal NPCs (3,
8–10). KHS101 increased neuronal differentiation of adherently cultured rat NPCs in a dose-dependent
fashion (EC50 ~ 1 µM) as assessed by quantitative reverse transcription (RT)-PCR for the neurogenic
transcription factor NeuroD (11) and immunostaining for the panneuronal marker TuJ1 (Fig. 1 B–D, Fig.
S1). KHS101-induced neuron formation (40–60% TuJ1+ cells at 1.5–5 µM KHS101) was also observed
under neurosphere-forming conditions (12) in secondary neurospheres derived from both the
hippocampus and the subventricular zone (SVZ) of adult rats (Fig. S1). Moreover, hippocampal NPCs
treated with KHS101 and cultured adherently on microelectrode arrays for 12 d exhibited neuronal
morphologies as well as spontaneous spiking activity, hence indicating the presence of functional,
maturing neurons (Fig. S2).
KHS101 Suppresses Astrocyte Formation in Cultured NPCs. To further analyze the effect of KHS101 on
lineage-specific differentiation, we exposed rat NPCs to the astrocyte-inducing cytokine bone
morphogenetic protein 4 (BMP4), which is expressed in the adult neurogenic niches and has been
reported to inhibit hippocampal neurogenesis (13–16). As expected, BMP4 (50 ng/mL) treatment for 4 d
increased differentiation to astrocytes (from 0.5 to 10%) within NPC cultures as assessed by
immunostaining for the astrocyte marker glial fibrillary acidic protein (GFAP). Addition of 2 µM RA did
not significantly alter BMP4-induced astrogenesis. However, KHS101 (5 µM) treatment decreased BMP4induced astrocyte differentiation >4-fold, while increasing neuronal differentiation in a dose-dependent
fashion (Fig. 1 E–G). Thus, KHS101 promotes specifically neuronal differentiation and can override the
astrocyte-promoting BMP signal.
KHS101 Negatively Affects Cell Cycle Progression and Proliferation of NPCs. To elucidate genes and
pathways affected by KHS101, we carried out microarray mRNA profiling experiments in adherently
cultured rat hippocampal NPCs that were treated with KHS101, the inactive derivative KHS92, or DMSO
control. In addition to the expected up-regulation of NeuroD, pathway analysis of differentially regulated
genes revealed that KHS101 treatment primarily affects cell cycle regulatory networks in NPCs (Fig. 1H,
Fig. S3). Several factors required for cell cycle progression (e.g., cyclins) were down-regulated, whereas
microarray mRNA profiling and quantitative RT-PCR experiments showed a marked up-regulation (5http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2944756/
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fold at 1.7 µM KHS101) of the negative cell cycle regulator Cdkn1 (Fig. 1 H and I). This result is consistent
with the notion that CDKN1 (also termed p21) plays a key role in regulating the balance between NPC
proliferation and differentiation (8, 17–20). Analysis of the proliferation marker Ki67, the mitotic marker
phospho-histone H3 (P-HH3), and the NPC marker (sex-determining region Y)-box 2 (SOX2), as well as
secondary neurosphere formation assays, confirmed that in the presence of KHS101 the vast majority of
NPCs stop proliferating within 72 h, become mitotically inactive, and lose Sox2 expression, thus shifting
toward terminal differentiation (Fig. 1 J –L and Figs. S1 and S4). Interestingly, KHS101 decreased the
proliferation of rat oligodendrocyte precursor cells from the optic nerve (21), but failed to induce myelin
basic protein expression and oligodendrocyte differentiation in a phenotypic screen, suggesting that
KHS101-induced differentiation depends on the cellular context.
KHS101 Physically Interacts with TACC3 Protein. To determine the specific target of KHS101 in NPCs, we
generated a derivative of the parent compound containing the photocrosslinking benzophenone moiety
and an alkyne substituent (KHS101-BP, Fig. 2A and Scheme S5). Upon irradiation this derivative is
expected to form a covalent bond between KHS101 and its target protein, allowing isolation of protein–
compound complexes after labeling with reporter tags such as biotin-azide. NPC lysate was incubated
with KHS101-BP and proteins were separated by 2D SDS/PAGE after UV irradiation and biotin-tag
labeling. Western blot analysis identified a distinct labeled protein, whose level was significantly reduced
by competition with a 50-fold excess of free KHS101 in independent experiments (Fig. 2B). Mass
spectrometry revealed the 80-kDa protein to be TACC3 and its identity was confirmed by Western
blotting using a TACC3-specific antibody (Fig. 2C). We further confirmed direct physical interaction
between TACC3 and KHS101 in independent pulldown experiments using purified recombinant rat
TACC3 protein and a biotinylated KHS101 derivative (Fig. 2D, Table S1).
TACC3 was originally identified as a member of a protein family with a highly conserved C-terminal
coiled-coil domain and is thought to function as an important structural component of the centrosome
and mitotic spindle apparatus (22). Moreover, previous expression studies of Tacc3 as well as loss-offunction studies in mice, hematopoietic stem cells, and embryonic neural stem cells point to a role for
Tacc3 in controlling progenitor cell expansion and terminal differentiation during development (23–27).
Postnatally, TACC3 expression becomes restricted to the remaining proliferative tissues such as spleen,
thymus, gastrointestinal (GI) tract, and cerebral areas including the hippocampus (23, 26).
Knockdown of TACC3 Significantly Increases Neuronal Differentiation of NPCs. To address whether
Tacc3 is also functionally implicated in the regulation of adult NPC fate, we used RNA interference
(RNAi) to specifically down-regulate Tacc3 expression in adult rat hippocampal NPCs. Two different
shRNAs (shTacc3-1 and shTacc3-2) efficiently decreased Tacc3 RNA levels (60–90% reduction in
independent experiments) compared with the nontargeting control shRNA (shCO) as assessed by
quantitative RT-PCR and immunocytochemistry (Fig. 3 A–C, Fig. S4). Cdkn1 RNA levels markedly
increased (~4-fold, Fig. 3A) upon treatment with Tacc3-specific shRNAs, which is consistent with
KHS101-induced up-regulation of Cdkn1 and with previous reports showing that TACC3 depletion causes
CDKN1-mediated cell cycle inhibition in fibroblasts (28). Furthermore, an overt neuronal differentiation
phenotype and a concomitant reduction in NPC marker immunoreactivity (Nestin, Sox2) were observed
in NPC cultures ~4 d after treatment with Tacc3-specific shRNAs (Fig. 3 B–F); staining for TuJ1 and Ki67
confirmed a significant increase in TuJ1+ neurons (~4-fold) and decrease in proliferation (≥3-fold)
compared with shCO-expressing NPCs (Fig. 3G). In addition, Tacc3-specific shRNAs significantly
suppressed GFAP+ astrocyte formation and concomitantly promoted neuronal differentiation of NPCs
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cultured in the presence of BMP4 (Fig. 3 H–J). Thus, TACC3 is required for the maintenance of the
undifferentiated NPC state in vitro and its knockdown is sufficient to fully recapitulate the neurogenesis
and astrocyte-suppressing phenotypes induced by KHS101. Although the contribution of other KHS101
binding partners cannot be excluded, TACC3 appears to be the functionally relevant target of KHS101 in
NPCs.
KHS101 Regulates the Nuclear Localization of the Transcription Factor ARNT2. Recently, TACC3 was
proposed to regulate cell fate decisions by controlling the subcellular localization of transcription factors,
including aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) (24, 26). Interestingly, ARNT2 is
the only known TACC3-interacting protein that is almost exclusively expressed in neuronal brain cells
throughout life (29); consistent with this report ARNT2 expression was detected within the DG by
immunohistochemistry (Fig. S4). Moreover, the expression of several genes downstream of ARNT2mediated signaling was affected by KHS101 in cultured NPCs, including genes involved in arylhydrocarbon receptor signaling (Fig. S3). To further investigate the putative neurogenic mechanisms
downstream of the TACC3-KHS101 interaction, we analyzed nuclear localization of ARNT2. Consistent
with a previous report (30), transient overexpression of rat TACC3 diminished nuclear localization of
ectopically expressed rat ARNT2 in a concentration-dependent manner in 293T cells (Fig. 3K). In
contrast, KHS101 treatment led to increased nuclear but not cytoplasmic levels of ARNT2 (Fig. 3L).
Furthermore, treatment with KHS101 (5 µM, 12 h) or Tacc3-specific shRNAs increased the levels of
endogenous nuclear ARNT2 in NPCs as quantified by confocal microscopy and image analysis (
Fig. 3 M and N, and Fig. S4). Next, we tested whether ARNT2 can regulate cell fate in adult hippocampal
NPCs by transient Arnt2 overexpression. Elevated levels of ARNT2 were detected in the nuclei of NPCs
but were insufficient to induce cell cycle exit or NPC differentiation. However, ARNT2 overexpression
markedly favored neurogenesis and suppressed the astrocyte cell fate upon BMP4-induced differentiation
(Fig. S5), suggesting that ARNT2 may provide a cell-intrinsic bias toward neuronal lineage specification
of NPCs in response to glial-inducing signals.
KHS101 Significantly Increases Neuronal Differentiation in Vivo. To determine whether KHS101 can act
as a pharmacological agent to induce neuronal differentiation in vivo, we assessed the pharmacokinetic
properties of KHS101 including blood–brain barrier penetration. Oral administration in vehicle resulted
in very low systemic exposures; however, i.v. and s.c. doses of 6 mg/kg KHS101 resulted in reasonable
plasma concentrations (>1.5 µM) with a plasma half-life of 1.1–1.4 h, and a relative bioavailability of 69%
following s.c. dosing. Most importantly, the distribution of KHS101 to the brain was extensive as
demonstrated by a brain-to-plasma AUC(0–3h) ratio of ~8 (dosing: 3 mg/kg KHS101, i.v.; Fig. 4A, Table
S2). Consequently, to study the effect of KHS101 on neurogenesis we injected adult rats with vehicle (5%
EtOH in 15% Captisol) or KHS101 (s.c., 6 mg/kg, BID) for 14 d, while including a daily
bromodeoxyuridine (BrdU) regimen (200 mg/kg, intraperitoneal) for the first 7 d to label dividing NPCs
in the DG. We then examined the fate of those BrdU-labeled cells by assessing colocalization with the
astrocyte marker GFAP and the neuronal-specific nuclear protein NeuN (Fig. 4 B and C, Fig. S6).
No significant difference was detected in the percentage of BrdU/GFAP double-positive cells upon
KHS101 treatment. However, the majority of these cells (>80%) were nonstellate and located within the
SGL, therefore likely representing a NPC subtype (GFAP+ type I progenitors) (1), rather than
differentiated astrocytes (Fig. S6). Notably, we found a significant increase in the percentage of
BrdU/NeuN double-positive cells from ~20 to ~40% upon KHS101 dosing, indicating increased neuronal
differentiation. Consistent with this result, the number of Ki67- and BrdU-positive cells significantly
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decreased within the SGL, indicating reduced proliferation of NPCs in KHS101 compared with vehicletreated animals (Fig. 4 D–F and H). The remaining Ki67 and BrdU immunoreactivity within the SGL of
treated animals suggests that the KHS101 dosing regimen did not result in the exhaustion of the selfrenewing progenitor pool. Moreover, KHS101 administration did not alter Ki67 immunostaining in
nonneural TACC3-expressing tissues such as spleen and gut (Fig. S7), suggesting either a cell typespecific effect of KHS101 or different levels of exposure or clearance in different organs. Finally, we did
not observe signs of lethargy, weight loss, or other indicators of sickness in KHS101-treated animals
during the study period and apoptosis was not altered within the DG of KHS101- compared with vehicleinjected animals as assessed by cleaved caspase 3 staining. Overall, these results are consistent with our
in vitro data and indicate a significant KHS101-induced acceleration of neuronal differentiation in vivo.
DISCUSSION
Endogenous pools of NPCs are a potential source for tissue repair and regeneration in the central nervous
system (CNS). NPCs are able to self-renew throughout life in a location-specific manner and their fate
may be modulated in response to pharmacological intervention with small molecules. NPC-mediated
neurogenesis involves at least three different processes: NPC proliferation, differentiation, and the
survival of NPCs committed toward a neuronal fate. Small molecules have been shown to modulate each
of these distinct processes. For example, the antidepressant agent fluoxetin has been shown to increase
NPC proliferation in vivo (5), and the anticonvulsant drug valproate has been shown to increase neuronal
differentiation of NPCs in the adult rat dentate gyrus (3). A very recent study describes the identification
of a proneurogenic small molecule that protects newborn hippocampal neurons from apoptosis (31).
Moreover, a series of experimental small molecules that control the self-renewal and differentiation of
stem and progenitor cells in vitro have been described (3, 9, 32–36).
This study shows that small molecules such as KHS101, isolated from phenotypic screens of chemical
libraries, can be powerful tools to elucidate and modulate the biology of endogenous progenitor cells both
in vitro and in vivo. KHS101 specifically promoted neuronal differentiation of NPCs, concomitantly
suppressing proliferation. The levels of KHS101-induced NPC differentiation were qualitatively
comparable to those induced by the known neurogenic factors RA and BDNF under adherent and sphereforming conditions (Fig. S1), although the optimal concentrations varied (2–5 µM for KHS101, 2 µM for
RA). However, unlike most known neurogenic factors, KHS101 can override astrocyte-inducing cues such
as BMP4 in favor of neuronal differentiation. Affinity-based purification methodology revealed physical
interaction of KHS101 with TACC3, although binding of KHS101 to other cellular proteins cannot be
excluded. Importantly, knockdown of TACC3 caused a strong neuronal differentiation phenotype and also
blocked BMP-induced astrocyte formation in cultured NPCs in a comparable fashion to KHS101
treatment, suggesting that TACC3 is likely the most relevant biological target of KHS101. Interestingly,
KHS101 treatment and knockdown of TACC3 led to increased nuclear localization of the nervous systemspecific transcription factor ARNT2, and overexpression of ARNT2 markedly favored neuronal
differentiation over the astrocyte cell fate upon BMP-induced differentiation.
Taken together, these findings indicate that KHS101 specifically accelerates neuronal differentiation by
interaction with the TACC3 protein and support a functional link between KHS101 and the TACC3ARNT2 axis. Our findings suggest that shRNA- or KHS101-mediated interference with TACC3 accelerates
neurogenesis through negative regulation of the cell cycle and concomitant activation of a neuronal
differentiation program in NPCs. A body of literature suggests that TACC3 is playing a crucial role in
progenitor cell maintenance and is down-regulated upon differentiation (23–27, 37), although the
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molecular mechanisms downstream of TACC3 have yet to be determined. As proposed in a previous study
(24), TACC3 may sequester distinct transcription factors to the cytoplasm or to the centrosome, therefore
preventing them from binding to transcriptionally active promoters. Alternatively, TACC3 may modulate
the proteolytic turnover of its binding partners by providing a target for proteasomal degradation.
Overall, it is conceivable that the regulation of TACC3 and its downstream interaction partners strongly
depends on the cellular context (e.g., tissue type and state of the cell cycle) and that specific TACC3
binding transcription factors, such as ARNT2, may actively contribute to lineage-specific progenitor cell
differentiation as a result of TACC3 modulation.
It is a common notion that NPC proliferation increases after injury to the CNS (38). Thus, differentiationinducing agents such as KHS101 may lead to new therapeutics that act by enhancing the contribution of
newborn cells to CNS repair. Moreover, KHS101 may affect the self-renewal potential of malignant
progenitor-like cells that can fuel aggressive brain cancer such as glioblastoma multiforme and have been
shown to share several functional features as well as in vitro growth conditions with normal neural
progenitor cells (39–41). Future research will shed light on the molecular nature of the KHS101-TACC3
interaction, the cell type- and age-specific roles of TACC3, and a potential therapeutic effect of KHS101 in
animal models of neurodegeneration and CNS malignancies.
METHODS
Hippocampal NPC Culture. Rat NPCs were derived and cultured as described previously by others (10).
After hippocampal cell isolation, the number of dissociated cells was determined and ~5 × 105 cells were
plated in 60-mm uncoated plates. After overnight incubation (37 °C, 5% CO2, and 95% humidity), the
medium was changed and the cells were expanded and maintained in an undifferentiated state on
polyornithine- (10 µg/mL in water; Sigma) and laminin-coated (5 µg/mL in PBS; Invitrogen) dishes in
DMEM/F12 (Invitrogen) supplemented with N2 (Invitrogen) and basic fibroblast growth factor (bFGF,
20 ng/mL; Invitrogen). For KHS101 and shRNA-induction experiments, early passage cells (passaged no
more than six times after hippocampal isolation) were trypsinized and plated at a density of ~1,000
cells/cm2 into N2 medium (DMEM/F12 supplemented with N2) containing KHS analogs (e.g., KHS101,
KHS92, and NP; SI Text) at different concentrations (0.5–5 µM) or DMSO (0.1%), RA (1–2 µM), BDNF
(100 ng/mL), and/or BMP4 (50–100 ng/mL) for 4 d.
Real Time RT-PCR. Total RNA was purified using the RNeasy kit (Qiagen) and cDNA was produced using
the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). TaqMan probes for rat NeuroD,
Tacc3, and Gapdh gene expression assays were purchased from Applied Biosystems and used according
to the manufacturer's instructions.
Immunocytochemistry. Cells were fixed with 10% (vol/vol) formalin solution at room temperature (RT)
for 10 min, permeabilized with 0.5% (vol/vol) Triton X-100 (Sigma) in PBS for 10 min, and then blocked
with PBS containing 0.3% (vol/vol) Triton X-100, 10% FCS, 1% (wt/vol) BSA at RT for 1 h. Cells were
incubated with primary antibodies (SI Text) in a PBS solution containing 0.1% (vol/vol) Triton X-100 and
5% FCS at 4 °C overnight.
Affinity-Based Target Identification. NPC lysate was prepared by sonication in PBS and protein samples
were prepared at a concentration of 2 mg/mL. The benzophenone-KHS101 compound (KHS101-BP, 5
µM; SI Text) was added to 50 µL of the proteome reaction with and without unlabeled compound (250
µM). Irradiation was for 1 h using a hand-held UV lamp at long wavelength (365 nm), and subsequently a
copper-catalyzed azide-alkyne cycloaddition reaction was performed (SI Text). After incubation for 1 h at
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RT, proteins were precipitated using trichloroacetic acid and resuspended in isoelectric focusing sample
buffer. 2D SDS/PAGE was performed using ReadyStripe IPG stripes (Bio-Rad) following the
manufacturer's protocol.
NPC Electroporation. Electroporation of plasmid DNA (SI Text) into hippocampal NPCs was performed
using the rat neural stem cell Nucleofector Kit (Lonza) according to the manufacturer's protocol. All
shRNA and cDNA expression vectors harbored a puromycin resistance gene allowing for the selection of
cells expressing plasmid DNA.
Animal Experiments. To investigate the pharmacokinetic properties of KHS101, male Sprague–Dawley
rats were administered 3 mg/kg KHS101 i.v. or s.c. One rat was killed per time point at 5 min, 40 min, 1 h,
and 3 h after dosing, and samples of blood (100 µL) and whole brains were collected. In a separate study,
rats were administered 6 mg/kg KHS101 i.v. or s.c. Five blood samples of 100 µL each were collected
serially via a jugular vein catheter at 2 min (i.v. only), 0.5 h (s.c. only), and 1, 3, 7 and 24 h after dosing.
Plasma and homogenized whole brain samples were analyzed by liquid chromatography tandem mass
spectrometry (LC-MS/MS). To study neuronal differentiation upon KHS101 administration in vivo, adult
Fisher 344 rats (~10 wk old) received s.c. injections of 6 mg/kg KHS101 or vehicle control (5% ethanol in
15% Captisol). All rats received one daily i.p. injection of 200 mg/kg BrdU for 6 consecutive days after the
first day. After 14 d, the animals were killed and perfusion fixed, and the brains were removed and
subjected to immunohistochemical analysis (SI Text).
Detailed experimental procedures can be found in SI Text.
SUPPLEMENTARY MATERIAL
Supporting Information:
ACKNOWLEDGMENTS
We thank M. Spooner, N. Gaylord, E. Miller, R. Halder, V. Deshmukh, J. S. Lee, M. Ruiz, B. Chandler,
and C. Wright for excellent technical assistance. We thank E. Remba and V. Seely for excellent
administrative assistance. L.L.L was supported by a fellowship from the Canadian Institute for Health
Research. H.W. was funded by the European Molecular Biology Organization and American Association
for Cancer Research fellowships. P.G.S. was supported by The Skaggs Institute for Chemical Biology.
FOOTNOTES
The authors declare no conflict of interest.
Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database,
www.ncbi.nlm.nih.gov/geo (accession no. GSE23668).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1010300107//DCSupplemental.
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FIGURES AND TABLES
Fig. 1.
KHS101 specifically induces neuronal differentiation in rat NPCs. (A) Chemical structure of KHS101. (B) Real time RTPCR analysis of NPCs treated with KHS101 for 24 h showing a dose-dependent induction of NeuroD mRNA expression
normalized to the DMSO control. (C and D) TuJ1 staining (green) of DMSO- (0.1%, C) and KHS101-treated (5 µM, D)
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NPCs after a 4-d differentiation period (see also Figs. S1 and S2). (E and F) GFAP (green) and TuJ1 staining (red) of NPCs
cultured under astrocyte-inducing conditions (50 ng/mL BMP4) in presence of DMSO (0.1%, J) or KHS101 (5 µM, K). (G)
GFAP+ and TuJ1+ cell percentages show that KHS101 significantly suppresses BMP4-induced astrogenesis in NPCs, while
significantly increasing neurogenesis. (H) Ingenuity pathway analysis reveals KHS101-induced up-regulation (red) of
negative cell cycle regulators (Cdkn1 and Gadd45) and down-regulation of positive cell cycle regulators at 24 h of
treatment (e.g., Ccnb1 and Ccne1; see also Fig. S3). (I) Real time RT-PCR analysis of NPCs treated with KHS101 for 24 h
showing a dose-dependent induction of Cdkn1 mRNA expression, whereas inactive derivatives (KHS91, KHS92, and
KHS103) fail to up-regulate Cdkn1 mRNA levels normalized to DMSO. (J and K) Ki67 (green) and P-HH3 staining (red) of
DMSO- (0.1%, G) and KHS101-treated (5 µM, H) NPCs. (L) Ki67+ and P-HH3+ cell percentages indicate a significant
reduction in proliferation and mitotic activity in KHS-treated NPCs over time. (Scale bars: 20 µm.) Error bars, SDs (three
independent experiments; biological replicates in B and I); statistical significance (t test), *P < 0.05, **P < 0.01; nuclei
were visualized with DAPI (blue).
Fig. 2.
KHS101 specifically interacts with TACC3 protein. (A) Structure of the benzophenone-containing alkyne-tagged KHS101
conjugate (KHS101-BP) used for target identification. (B) Representative two-dimensional SDS/PAGE and Western
blotting of NPC cell lysates (2 mg/mL) detecting protein-KHS101-BP complexes after photocrosslinking (1 h) and biotintag labeling (25 µM biotin-azide, Left). Unlabeled KHS101 served as a competitor for specific KHS101-BP–protein binding
(50-fold excess, Right). Independent experiments identified a protein spot that was reproducibly competed by unlabeled
KHS101 (arrowheads). Mass spectrometry revealed the 80-kDa protein to be TACC3. (C) Western blot analysis of NPC
lysate using a TACC3-specific antibody confirmed TACC3 identity after pulldown with KHS101-BP. (D) Recombinant rat
TACC3 binds KHS101. Purified protein was incubated with biotinylated KHS101 (Table S1 and Schemes S3 and S4) in
presence/absence of unlabeled compound, precipitated with streptavidin-coated agarose beads, and then detected by
silver staining of SDS/PAGE gels.
Fig. 3.
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Tacc3-specific shRNA recapitulates the neurogenic effect of KHS101 in rat NPCs. (A) Representative real time RT-PCR
showing that Tacc3 RNA levels are markedly decreased in NPCs after electroporation with Tacc3-specific shRNA
constructs (shTacc3-1, shTacc3-2). Concomitantly, Cdkn1 mRNA levels are elevated compared with the nontargeting
control (shCO). (B and C) Nestin (green) and TACC3 (red) immunopositivity is observed in shCO-expressing NPCs (B,
note centrosomal TACC3 localization) and strongly reduced upon TACC3 knockdown (C) (Fig. S4). (D) Tacc3-specific
shRNA causes a TuJ1+ neuronal phenotype in NPCs. (E and F) Staining for Ki67 (red) and TuJ1 (green) in shCO- (E) and
shTacc3-expressing NPCs (F) at 4 d after shRNA electroporation. (G) TuJ1+/Ki67− and Ki67+ cell percentages indicate
significantly increased neurogenesis and significantly decreased proliferation upon Tacc3 RNAi in NPCs. (H and I) GFAP
(green) and TuJ1 (red) staining in shCO- (H) and shTacc3-expressing (I) NPCs cultured with BMP4 (50 ng/mL) for 4 d.
(J) GFAP+ and TuJ1+ cell percentages indicate that BMP4-induced astrogenesis is significantly reduced, whereas
neurogenesis is significantly increased in NPCs upon Tacc3 RNAi. (K and L) Ectopic rat Tacc3 and Arnt2 cDNA
overexpression in 293T cells and Western blot analysis. Increased expression of TACC3 is associated with decreased levels
of nuclear ARNT2 (K). KHS101 treatment (0–15 µM) elevates nuclear but not cytoplasmic ARNT2 after 24 h of exposure
(L). (M and N) Staining for ARNT2 reveals increased nuclear localization of ARNT2 in NPCs upon 5 µM KHS101
treatment for 12 h (Fig. S4). (Scale bars: 20 µm.) Error bars, SDs (three independent experiments; biological replicates in
A); statistical significance (t test), *P < 0.05, **P < 0.01; nuclei were visualized with DAPI (blue).
Fig. 4.
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KHS101 significantly increases neuronal differentiation in rats in vivo. (A) Pharmacokinetic profile of KHS101 in brain
and plasma after single administration (3 mg/kg, i.v.) to Sprague–Dawley rats. (B and C) Immunohistochemistry of BrdU
(red) and NeuN (green). White arrowheads mark BrdU-positive nuclei in the subgranular layer (SGL) of vehicle- and
KHS101-treated animals. The yellow arrowhead marks a BrdU/NeuN double-positive nucleus, indicative of neuronal
differentiation in the granule cell layer (GCL). (Scale bar: 20 µm.) (D and E) Ki67 (black arrowhead) and cleaved caspase
3 (brown arrowhead) double-stained DG sections (including the Hilus area: H) of vehicle- and KHS101-treated animals
(E). (Scale bar: 50 µm.) (F –H) Percentage of BrdU+/NeuN+ cells indicates significantly increased neurogenesis (F),
whereas the number of Ki67-positive cells (G) significantly decreases in the DG upon KHS101 dosing; the number of
cleaved caspase 3+ cells within the DG was not altered. (H) Quantification of BrdU+ cells localized in SGL, GL, and H of
vehicle- and KHS101-treated animals (≥10 sections representative for the DG were counted for each animal). Note a
significant reduction of BrdU+ cells in the SGL and a slight increase of BrdU+ cells in the GCL upon KHS101
administration. Error bars, SDs (six animals per group); statistical significance (t test), **P < 0.01.
Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here
courtesy of National Academy of Sciences
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