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Proc Natl Acad Sci U S A. 2010 September 21; 107(38): 16542–16547.Published online 2010 September 7. doi: 10.1073/pnas.1010300107

PMCID: PMC2944756

Cell Biology

A small molecule accelerates neuronal differentiation in the adult ratHeiko Wurdak, Shoutian Zhu, Kyung Hoon Min, Lindsey Aimone, Luke L. Lairson, James Watson, GregoryChopiuk, James Demas, Bradley Charette, Rajkumar Halder, Eranthie Weerapana, Benjamin F. Cravatt, Hollis T.Cline, Eric C. Peters, Jay Zhang, John R. Walker, Chunlei Wu, Jonathan Chang, Tove Tuntland, Charles Y. Cho,and Peter G. Schultz

The Skaggs Institute of Chemical Biology and Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037;Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121; andDepartment of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037To 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.

H.W. and S.Z. contributed equally to this work.Present address: College of Pharmacy, Chung-Ang University, Dongjak-Gu, Seoul 156-756, South Korea.Present 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 thebrain. However, little is known about the molecular mechanisms regulating hippocampal neuralprogenitor cells (NPCs) and whether their fate can be pharmacologically modulated to improve neuralplasticity and regeneration. Here, we report the characterization of a small molecule (KHS101) thatselectively induces a neuronal differentiation phenotype. Mechanism of action studies revealed a link ofKHS101 to cell cycle exit and specific binding to the TACC3 protein, whose knockdown in NPCsrecapitulates the KHS101-induced phenotype. Upon systemic administration, KHS101 distributed to thebrain and resulted in a significant increase in neuronal differentiation in vivo. Our findings indicate thatKHS101 accelerates neuronal differentiation by interaction with TACC3 and may provide a basis forpharmacological 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-renewingneural progenitor cells (NPCs). NPCs arise within the subgranular layer (SGL) of the DG and primarilymigrate into the adjacent granule cell layer (GCL) to differentiate and mature into neurons (1). Althoughadult NPCs hold substantial promise for neural repair after brain injury, their mobilization is hampered

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KHS101 Induces Neuronal Differentiation in Cultured NPCs.

KHS101 Suppresses Astrocyte Formation in Cultured NPCs.

KHS101 Negatively Affects Cell Cycle Progression and Proliferation of NPCs.

by the lack of knowledge about the molecular mechanisms controlling adult neurogenesis under normaland pathophysiological conditions. Pleiotropic signaling molecules [e.g., retinoic acid (RA), Wnt, andbrain-derived growth factor (BDNF)] as well as antidepressant and anticonvulsant drugs (e.g., fluoxetinand sodium valproate) have been implicated in regulating adult NPC proliferation, differentiation, orsurvival in vivo (2–8). Therefore, it may be possible to develop neurogenic agents for pharmacologicalintervention specifically directed at the endogenous NPC pool. Here, we describe the small moleculeKHS101, which specifically induces neuronal differentiation in vitro and in vivo upon systemicadministration. Additionally, mechanism of action studies revealed a critical role for transforming acidiccoiled-coil–containing protein 3 (TACC3) in the regulation of NPC maintenance and differentiation.

RESULTS

We previously reported a phenotypic screenthat identified a group of synthetic 4-aminothiazole compounds, termed neuropathiazols, which induceneuronal differentiation of cultured rat hippocampal NPCs (9). Synthesis of several analogs of the originalneuropathiazol structure and a focused structure–activity relationship (SAR) study afforded a molecule(KHS101) that possessed increased activity and improved pharmacokinetic properties compared with theoriginal compound (Fig. 1A, Fig. S1, Scheme S1, Table S1). KHS101 was further studied in vitro usingestablished 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-dependentfashion (EC ~ 1 µM) as assessed by quantitative reverse transcription (RT)-PCR for the neurogenictranscription 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 observedunder neurosphere-forming conditions (12) in secondary neurospheres derived from both thehippocampus and the subventricular zone (SVZ) of adult rats (Fig. S1). Moreover, hippocampal NPCstreated with KHS101 and cultured adherently on microelectrode arrays for 12 d exhibited neuronalmorphologies as well as spontaneous spiking activity, hence indicating the presence of functional,maturing neurons (Fig. S2).

To further analyze the effect of KHS101 onlineage-specific differentiation, we exposed rat NPCs to the astrocyte-inducing cytokine bonemorphogenetic protein 4 (BMP4), which is expressed in the adult neurogenic niches and has beenreported to inhibit hippocampal neurogenesis (13–16). As expected, BMP4 (50 ng/mL) treatment for 4 dincreased differentiation to astrocytes (from 0.5 to 10%) within NPC cultures as assessed byimmunostaining for the astrocyte marker glial fibrillary acidic protein (GFAP). Addition of 2 µM RA didnot significantly alter BMP4-induced astrogenesis. However, KHS101 (5 µM) treatment decreased BMP4-induced astrocyte differentiation >4-fold, while increasing neuronal differentiation in a dose-dependentfashion (Fig. 1 E–G). Thus, KHS101 promotes specifically neuronal differentiation and can override theastrocyte-promoting BMP signal.

To elucidate genes andpathways affected by KHS101, we carried out microarray mRNA profiling experiments in adherentlycultured rat hippocampal NPCs that were treated with KHS101, the inactive derivative KHS92, or DMSOcontrol. In addition to the expected up-regulation of NeuroD, pathway analysis of differentially regulatedgenes 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, whereasmicroarray mRNA profiling and quantitative RT-PCR experiments showed a marked up-regulation (5-

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KHS101 Physically Interacts with TACC3 Protein.

Knockdown of TACC3 Significantly Increases Neuronal Differentiation of NPCs.

fold at 1.7 µM KHS101) of the negative cell cycle regulator Cdkn1 (Fig. 1 H and I). This result is consistentwith the notion that CDKN1 (also termed p21) plays a key role in regulating the balance between NPCproliferation and differentiation (8, 17–20). Analysis of the proliferation marker Ki67, the mitotic markerphospho-histone H3 (P-HH3), and the NPC marker (sex-determining region Y)-box 2 (SOX2), as well assecondary neurosphere formation assays, confirmed that in the presence of KHS101 the vast majority ofNPCs stop proliferating within 72 h, become mitotically inactive, and lose Sox2 expression, thus shiftingtoward terminal differentiation (Fig. 1 J –L and Figs. S1 and S4). Interestingly, KHS101 decreased theproliferation of rat oligodendrocyte precursor cells from the optic nerve (21), but failed to induce myelinbasic protein expression and oligodendrocyte differentiation in a phenotypic screen, suggesting thatKHS101-induced differentiation depends on the cellular context.

To determine the specific target of KHS101 in NPCs, wegenerated a derivative of the parent compound containing the photocrosslinking benzophenone moietyand an alkyne substituent (KHS101-BP, Fig. 2A and Scheme S5). Upon irradiation this derivative isexpected 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 incubatedwith KHS101-BP and proteins were separated by 2D SDS/PAGE after UV irradiation and biotin-taglabeling. Western blot analysis identified a distinct labeled protein, whose level was significantly reducedby competition with a 50-fold excess of free KHS101 in independent experiments (Fig. 2B). Massspectrometry revealed the 80-kDa protein to be TACC3 and its identity was confirmed by Westernblotting using a TACC3-specific antibody (Fig. 2C). We further confirmed direct physical interactionbetween TACC3 and KHS101 in independent pulldown experiments using purified recombinant ratTACC3 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-terminalcoiled-coil domain and is thought to function as an important structural component of the centrosomeand mitotic spindle apparatus (22). Moreover, previous expression studies of Tacc3 as well as loss-of-function studies in mice, hematopoietic stem cells, and embryonic neural stem cells point to a role forTacc3 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).

To address whetherTacc3 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 differentshRNAs (shTacc3-1 and shTacc3-2) efficiently decreased Tacc3 RNA levels (60–90% reduction inindependent experiments) compared with the nontargeting control shRNA (shCO) as assessed byquantitative RT-PCR and immunocytochemistry (Fig. 3 A–C, Fig. S4). Cdkn1 RNA levels markedlyincreased (~4-fold, Fig. 3A) upon treatment with Tacc3-specific shRNAs, which is consistent withKHS101-induced up-regulation of Cdkn1 and with previous reports showing that TACC3 depletion causesCDKN1-mediated cell cycle inhibition in fibroblasts (28). Furthermore, an overt neuronal differentiationphenotype and a concomitant reduction in NPC marker immunoreactivity (Nestin, Sox2) were observedin NPC cultures ~4 d after treatment with Tacc3-specific shRNAs (Fig. 3 B–F); staining for TuJ1 and Ki67confirmed 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 significantlysuppressed GFAP+ astrocyte formation and concomitantly promoted neuronal differentiation of NPCs

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KHS101 Regulates the Nuclear Localization of the Transcription Factor ARNT2.

KHS101 Significantly Increases Neuronal Differentiation in Vivo.

cultured in the presence of BMP4 (Fig. 3 H–J). Thus, TACC3 is required for the maintenance of theundifferentiated NPC state in vitro and its knockdown is sufficient to fully recapitulate the neurogenesisand astrocyte-suppressing phenotypes induced by KHS101. Although the contribution of other KHS101binding partners cannot be excluded, TACC3 appears to be the functionally relevant target of KHS101 inNPCs.

Recently, TACC3 wasproposed 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 isthe only known TACC3-interacting protein that is almost exclusively expressed in neuronal brain cellsthroughout life (29); consistent with this report ARNT2 expression was detected within the DG byimmunohistochemistry (Fig. S4). Moreover, the expression of several genes downstream of ARNT2-mediated signaling was affected by KHS101 in cultured NPCs, including genes involved in aryl-hydrocarbon receptor signaling (Fig. S3). To further investigate the putative neurogenic mechanismsdownstream of the TACC3-KHS101 interaction, we analyzed nuclear localization of ARNT2. Consistentwith a previous report (30), transient overexpression of rat TACC3 diminished nuclear localization ofectopically expressed rat ARNT2 in a concentration-dependent manner in 293T cells (Fig. 3K). Incontrast, 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 ofendogenous 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 hippocampalNPCs by transient Arnt2 overexpression. Elevated levels of ARNT2 were detected in the nuclei of NPCsbut were insufficient to induce cell cycle exit or NPC differentiation. However, ARNT2 overexpressionmarkedly 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 specificationof NPCs in response to glial-inducing signals.

To determine whether KHS101 can actas a pharmacological agent to induce neuronal differentiation in vivo, we assessed the pharmacokineticproperties of KHS101 including blood–brain barrier penetration. Oral administration in vehicle resultedin very low systemic exposures; however, i.v. and s.c. doses of 6 mg/kg KHS101 resulted in reasonableplasma 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 asdemonstrated by a brain-to-plasma AUC ratio of ~8 (dosing: 3 mg/kg KHS101, i.v.; Fig. 4A, TableS2). 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 dailybromodeoxyuridine (BrdU) regimen (200 mg/kg, intraperitoneal) for the first 7 d to label dividing NPCsin the DG. We then examined the fate of those BrdU-labeled cells by assessing colocalization with theastrocyte 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 uponKHS101 treatment. However, the majority of these cells (>80%) were nonstellate and located within theSGL, therefore likely representing a NPC subtype (GFAP+ type I progenitors) (1), rather thandifferentiated astrocytes (Fig. S6). Notably, we found a significant increase in the percentage ofBrdU/NeuN double-positive cells from ~20 to ~40% upon KHS101 dosing, indicating increased neuronaldifferentiation. 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 vehicle-treated animals (Fig. 4 D–F and H). The remaining Ki67 and BrdU immunoreactivity within the SGL oftreated animals suggests that the KHS101 dosing regimen did not result in the exhaustion of the self-renewing progenitor pool. Moreover, KHS101 administration did not alter Ki67 immunostaining innonneural TACC3-expressing tissues such as spleen and gut (Fig. S7), suggesting either a cell type-specific effect of KHS101 or different levels of exposure or clearance in different organs. Finally, we didnot observe signs of lethargy, weight loss, or other indicators of sickness in KHS101-treated animalsduring the study period and apoptosis was not altered within the DG of KHS101- compared with vehicle-injected animals as assessed by cleaved caspase 3 staining. Overall, these results are consistent with ourin 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 nervoussystem (CNS). NPCs are able to self-renew throughout life in a location-specific manner and their fatemay be modulated in response to pharmacological intervention with small molecules. NPC-mediatedneurogenesis involves at least three different processes: NPC proliferation, differentiation, and thesurvival of NPCs committed toward a neuronal fate. Small molecules have been shown to modulate eachof these distinct processes. For example, the antidepressant agent fluoxetin has been shown to increaseNPC proliferation in vivo (5), and the anticonvulsant drug valproate has been shown to increase neuronaldifferentiation of NPCs in the adult rat dentate gyrus (3). A very recent study describes the identificationof 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 ofstem 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 chemicallibraries, can be powerful tools to elucidate and modulate the biology of endogenous progenitor cells bothin vitro and in vivo. KHS101 specifically promoted neuronal differentiation of NPCs, concomitantlysuppressing proliferation. The levels of KHS101-induced NPC differentiation were qualitativelycomparable to those induced by the known neurogenic factors RA and BDNF under adherent and sphere-forming conditions (Fig. S1), although the optimal concentrations varied (2–5 µM for KHS101, 2 µM forRA). However, unlike most known neurogenic factors, KHS101 can override astrocyte-inducing cues suchas BMP4 in favor of neuronal differentiation. Affinity-based purification methodology revealed physicalinteraction of KHS101 with TACC3, although binding of KHS101 to other cellular proteins cannot beexcluded. Importantly, knockdown of TACC3 caused a strong neuronal differentiation phenotype and alsoblocked BMP-induced astrocyte formation in cultured NPCs in a comparable fashion to KHS101treatment, 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 system-specific transcription factor ARNT2, and overexpression of ARNT2 markedly favored neuronaldifferentiation over the astrocyte cell fate upon BMP-induced differentiation.

Taken together, these findings indicate that KHS101 specifically accelerates neuronal differentiation byinteraction with the TACC3 protein and support a functional link between KHS101 and the TACC3-ARNT2 axis. Our findings suggest that shRNA- or KHS101-mediated interference with TACC3 acceleratesneurogenesis through negative regulation of the cell cycle and concomitant activation of a neuronaldifferentiation program in NPCs. A body of literature suggests that TACC3 is playing a crucial role inprogenitor cell maintenance and is down-regulated upon differentiation (23–27, 37), although the

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Hippocampal NPC Culture.

Real Time RT-PCR.

Immunocytochemistry.

Affinity-Based Target Identification.

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, thereforepreventing them from binding to transcriptionally active promoters. Alternatively, TACC3 may modulatethe 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 stronglydepends on the cellular context (e.g., tissue type and state of the cell cycle) and that specific TACC3binding transcription factors, such as ARNT2, may actively contribute to lineage-specific progenitor celldifferentiation as a result of TACC3 modulation.

It is a common notion that NPC proliferation increases after injury to the CNS (38). Thus, differentiation-inducing agents such as KHS101 may lead to new therapeutics that act by enhancing the contribution ofnewborn cells to CNS repair. Moreover, KHS101 may affect the self-renewal potential of malignantprogenitor-like cells that can fuel aggressive brain cancer such as glioblastoma multiforme and have beenshown to share several functional features as well as in vitro growth conditions with normal neuralprogenitor cells (39–41). Future research will shed light on the molecular nature of the KHS101-TACC3interaction, the cell type- and age-specific roles of TACC3, and a potential therapeutic effect of KHS101 inanimal models of neurodegeneration and CNS malignancies.

METHODS

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 × 10 cells wereplated in 60-mm uncoated plates. After overnight incubation (37 °C, 5% CO , and 95% humidity), themedium was changed and the cells were expanded and maintained in an undifferentiated state onpolyornithine- (10 µg/mL in water; Sigma) and laminin-coated (5 µg/mL in PBS; Invitrogen) dishes inDMEM/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 nomore than six times after hippocampal isolation) were trypsinized and plated at a density of ~1,000cells/cm 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.

Total RNA was purified using the RNeasy kit (Qiagen) and cDNA was produced usingthe 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 accordingto the manufacturer's instructions.

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 blockedwith PBS containing 0.3% (vol/vol) Triton X-100, 10% FCS, 1% (wt/vol) BSA at RT for 1 h. Cells wereincubated with primary antibodies (SI Text) in a PBS solution containing 0.1% (vol/vol) Triton X-100 and5% FCS at 4 °C overnight.

NPC lysate was prepared by sonication in PBS and protein sampleswere prepared at a concentration of 2 mg/mL. The benzophenone-KHS101 compound (KHS101-BP, 5µM; SI Text) was added to 50 µL of the proteome reaction with and without unlabeled compound (250µM). Irradiation was for 1 h using a hand-held UV lamp at long wavelength (365 nm), and subsequently acopper-catalyzed azide-alkyne cycloaddition reaction was performed (SI Text). After incubation for 1 h at

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NPC Electroporation.

Animal Experiments.

RT, proteins were precipitated using trichloroacetic acid and resuspended in isoelectric focusing samplebuffer. 2D SDS/PAGE was performed using ReadyStripe IPG stripes (Bio-Rad) following themanufacturer's protocol.

Electroporation of plasmid DNA (SI Text) into hippocampal NPCs was performedusing the rat neural stem cell Nucleofector Kit (Lonza) according to the manufacturer's protocol. AllshRNA and cDNA expression vectors harbored a puromycin resistance gene allowing for the selection ofcells expressing plasmid DNA.

To investigate the pharmacokinetic properties of KHS101, male Sprague–Dawleyrats 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 collectedserially 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 massspectrometry (LC-MS/MS). To study neuronal differentiation upon KHS101 administration in vivo, adultFisher 344 rats (~10 wk old) received s.c. injections of 6 mg/kg KHS101 or vehicle control (5% ethanol in15% Captisol). All rats received one daily i.p. injection of 200 mg/kg BrdU for 6 consecutive days after thefirst day. After 14 d, the animals were killed and perfusion fixed, and the brains were removed andsubjected to immunohistochemical analysis (SI Text).

Detailed experimental procedures can be found in SI Text.

SUPPLEMENTARY MATERIALSupporting 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 excellentadministrative assistance. L.L.L was supported by a fellowship from the Canadian Institute for HealthResearch. H.W. was funded by the European Molecular Biology Organization and American Associationfor Cancer Research fellowships. P.G.S. was supported by The Skaggs Institute for Chemical Biology.

FOOTNOTESThe 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 RT-PCR analysis of NPCs treated with KHS101 for 24 h showing a dose-dependent induction of NeuroD mRNA expressionnormalized 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 NPCscultured 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, whilesignificantly increasing neurogenesis. (H) Ingenuity pathway analysis reveals KHS101-induced up-regulation (red) ofnegative cell cycle regulators (Cdkn1 and Gadd45) and down-regulation of positive cell cycle regulators at 24 h oftreatment (e.g., Ccnb1 and Ccne1; see also Fig. S3). (I) Real time RT-PCR analysis of NPCs treated with KHS101 for 24 hshowing a dose-dependent induction of Cdkn1 mRNA expression, whereas inactive derivatives (KHS91, KHS92, andKHS103) fail to up-regulate Cdkn1 mRNA levels normalized to DMSO. (J and K) Ki67 (green) and P-HH3 staining (red) ofDMSO- (0.1%, G) and KHS101-treated (5 µM, H) NPCs. (L) Ki67+ and P-HH3+ cell percentages indicate a significantreduction in proliferation and mitotic activity in KHS-treated NPCs over time. (Scale bars: 20 µm.) Error bars, SDs (threeindependent experiments; biological replicates in B and I); statistical significance (t test), *P < 0.05, **P < 0.01; nucleiwere visualized with DAPI (blue).

Fig. 2.

KHS101 specifically interacts with TACC3 protein. (A) Structure of the benzophenone-containing alkyne-tagged KHS101conjugate (KHS101-BP) used for target identification. (B) Representative two-dimensional SDS/PAGE and Westernblotting of NPC cell lysates (2 mg/mL) detecting protein-KHS101-BP complexes after photocrosslinking (1 h) and biotin-tag 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 unlabeledKHS101 (arrowheads). Mass spectrometry revealed the 80-kDa protein to be TACC3. (C) Western blot analysis of NPClysate using a TACC3-specific antibody confirmed TACC3 identity after pulldown with KHS101-BP. (D) Recombinant ratTACC3 binds KHS101. Purified protein was incubated with biotinylated KHS101 (Table S1 and Schemes S3 and S4) inpresence/absence of unlabeled compound, precipitated with streptavidin-coated agarose beads, and then detected bysilver 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-PCRshowing that Tacc3 RNA levels are markedly decreased in NPCs after electroporation with Tacc3-specific shRNAconstructs (shTacc3-1, shTacc3-2). Concomitantly, Cdkn1 mRNA levels are elevated compared with the nontargetingcontrol (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-specificshRNA causes a TuJ1+ neuronal phenotype in NPCs. (E and F) Staining for Ki67 (red) and TuJ1 (green) in shCO- (E) andshTacc3-expressing NPCs (F) at 4 d after shRNA electroporation. (G) TuJ1+/Ki67− and Ki67+ cell percentages indicatesignificantly 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, whereasneurogenesis is significantly increased in NPCs upon Tacc3 RNAi. (K and L) Ectopic rat Tacc3 and Arnt2 cDNAoverexpression in 293T cells and Western blot analysis. Increased expression of TACC3 is associated with decreased levelsof 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 KHS101treatment for 12 h (Fig. S4). (Scale bars: 20 µm.) Error bars, SDs (three independent experiments; biological replicates inA); 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 brainand 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- andKHS101-treated animals. The yellow arrowhead marks a BrdU/NeuN double-positive nucleus, indicative of neuronaldifferentiation in the granule cell layer (GCL). (Scale bar: 20 µm.) (D and E) Ki67 (black arrowhead) and cleaved caspase3 (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 ofcleaved caspase 3+ cells within the DG was not altered. (H) Quantification of BrdU+ cells localized in SGL, GL, and H ofvehicle- and KHS101-treated animals (≥10 sections representative for the DG were counted for each animal). Note asignificant reduction of BrdU+ cells in the SGL and a slight increase of BrdU+ cells in the GCL upon KHS101administration. 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 herecourtesy of National Academy of Sciences