sign in sign up
<<
Home , Endogenous agonist, Pleiotrophin, TLR3

The Microtubule Regulator Stathmin Is an Endogenous for TLR3 Malika Bsibsi, Jeffrey J. Bajramovic, Mario H. J. Vogt, Eveline van Duijvenvoorden, Aabed Baghat, Carla This information is current as Persoon-Deen, Frans Tielen, Richard Verbeek, Inge of October 1, 2021. Huitinga, Bernhard Ryffel, Alexander Kros, Wouter H. Gerritsen, Sandra Amor and Johannes M. van Noort J Immunol 2010; 184:6929-6937; Prepublished online 12 May 2010; doi: 10.4049/jimmunol.0902419 Downloaded from http://www.jimmunol.org/content/184/12/6929

References This article cites 33 articles, 12 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/184/12/6929.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on October 1, 2021 • Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Microtubule Regulator Stathmin Is an Endogenous Protein Agonist for TLR3

Malika Bsibsi,*,† Jeffrey J. Bajramovic,‡ Mario H. J. Vogt,*,x Eveline van Duijvenvoorden,* Aabed Baghat,* Carla Persoon-Deen,* Frans Tielen,* Richard Verbeek,* Inge Huitinga,{ Bernhard Ryffel,‖ Alexander Kros,# Wouter H. Gerritsen,** Sandra Amor,**,†† and Johannes M. van Noort*,†

TLR3 recognizes dsRNAs and is considered of key importance to antiviral host-defense responses. TLR3 also triggers neuro- protective responses in astrocytes and controls the growth of axons and neuronal progenitor cells, suggesting additional roles for TLR3-mediated signaling in the CNS. This prompted us to search for alternative, CNS-borne protein for TLR3. A genome-scale functional screening of a transcript library from tumors revealed that the microtubule regulator stathmin is an activator of TLR3-dependent signaling in astrocytes, inducing the same set of neuroprotective factors as the known TLR3 Downloaded from agonist polyinosinic:polycytidylic acid. This activity of stathmin crucially depends on a long, negatively charged a helix in the protein. Colocalization of stathmin with TLR3 on astrocytes, microglia, and neurons in multiple sclerosis-affected human brain indicates that as an endogenous TLR3 agonist, stathmin may fulfill previously unsuspected regulatory roles during inflammation and repair in the adult CNS. The Journal of Immunology, 2010, 184: 6929–6937.

oll-like receptors are key components of the innate immune development of host-defense responses against viral infection. Re- http://www.jimmunol.org/ system that detect microbial infection, trigger innate host- cent evidence indicates that TLR3-mediated signaling involves T defense responses, and control adaptive immune responses additional, still unknown upstream events in the signaling pathway (1, 2). In addition to recognizing a range of pathogen-associated (9). Particularly intriguing are recent observations that disruption of molecules, several TLRs sense endogenous ligands that emerge membrane microdomains reduces the cellular response mediated by upon tissue damage or inflammation. These include breakdown TLR3 (10). This strongly suggest that still unknown surface inter- products of the extracellular matrix (3) and inducible molecules, actions influence binding and subsequent signaling by TLR3. such as CD14 and heat-shock (4, 5). The only known li- Although the role of TLR3 in mediating antiviral responses is gands for TLR3 are dsRNAs, derived from viral, mammalian, or supported by a wealth of evidence, several findings indicate that synthetic origin (6–8). Different from other TLR family members, TLR3-mediated signaling can play additional roles, especially in the by guest on October 1, 2021 TLR3 cannot activate MyD88-dependent signaling pathways (1, 2). CNS. TLR3 is selectively and strongly induced in astrocytes by a Instead, TLR3 signals through the Toll/IL-1 (TIR) domain- variety of stressors, including noninfectious ones, and it triggers containing adaptor , which activates IkB kinase-ε and TANK- production of a range of immunoregulatory mediators and factors binding kinase 1 and, ultimately, IFN regulatory factors 3 and 7. that counteract gliosis and promote neuronal survival, , Also, NF-kB and MAPKs are activated by TLR3 (1, 2). Con- and remyelination (11). In addition, TLR3 is expressed in neurons sequently, TLR3 mediates the production of type I IFNs, IFN-in- (12) and oligodendrocytes (13) in which it regulates growth and ducible products, and other inflammatory mediators crucial for the differentiation. Neuronal TLR3 is a major negative regulator of the growth of axons and dendrites, promotes dendrite branching, and controls proliferation of embryonic neural progenitor cells (14, 15). *Department of Biomedical Research, TNO Quality of Life; †Delta Crystallon; Together, these observations indicate that at least in some cell #Department of Soft Matter Chemistry, Leiden University, Leiden; ‡Alternatives Unit, types and under some conditions, TLR3 may play a role other than Biomedical Primate Research Centre, Rijswijk; xDepartment of and Immu- nology and **Department of Pathology, VU University Medical Center; {Netherlands Brain controlling antiviral host-defense responses. Considering, in par- Bank, Amsterdam, The Netherlands; ‖Institut Transgenose, Centre National de la Recherche ticular, the emerging role of TLR3 in astrocyte-mediated neuro- †† Scientifique, Orleans, France; and Center, Queen Mary University of Lon- protection and in morphogenesis in the CNS, we hypothesized that don, London, United Kingdom for such alternative, noninfectious roles of TLR3, alternative Received for publication July 24, 2009. Accepted for publication April 6, 2010. endogenous agonists might exist. To explore this idea, we used This work was supported by The Netherlands Foundation for Multiple Sclerosis cDNA from brain tumors as a source of endogenous CNS-borne Research. proteins to screen for such alternative TLR3 agonists. By using The sequences presented in this article have been submitted to ArrayExpress under accession number E-TABM-851. primary human astrocytes as initial reporter cells, we identified the Address correspondence and reprint requests to Dr. Johannes M. van Noort, Delta microtubule regulator stathmin as an agonist for TLR3-dependent Crystallon BV, Zernikedreef 9, 2333 CK Leiden, The Netherlands. E-mail address: signaling in human astrocytes and microglia. [email protected] Abbreviations used in this paper: CD, circular dichroism; MS, multiple sclerosis; poly(I:C), polyinosinic:polycytidylic acid; RB3, stathmin-like protein B3; SCG10, Materials and Methods superior cervical ganglion 10 protein; SCLIP, SCG10-like protein; siRNA, small Isolation of human astrocytes and microglia interfering RNA; TFA, trifluoroacetic acid; TFE, 29,29,29-trifluoroethanol; TIR, Toll/IL-1 receptor. Fresh post mortem human brain tissue samples were used for the isolation of primary human glial cells. Brain materials were collected after review and Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 approval of the protocol by a local ethical committee. Adult human www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902419 6930 STATHMIN IS A TLR3 AGONIST astrocytes and microglia from subcortical white matter, as well as murine Treatment with RNAse III was performed using 350 U/ml enzyme in astrocytes from TLR3-deficient mice or control Biozzi ABH mice, were 10 mM Tris HCl (pH 7.9), 50 mM NaCl, 10 mM MgCl2, and 1 mM DTT isolated and cultured as previously described (13). Final astrocyte and for 4 or 8 h at 37˚C. Digestions were terminated by freezing the samples microglia cultures were judged to be .99% pure by staining for glial fi- and storing them at 220˚C until testing their ability to induce CXCL8 brillary acidic protein (astrocyte marker) and CD68 (microglia marker). release in astrocytes, as described above. Genome-scale screening of a cDNA library derived from cDNA array and Ab profiling human brain tumor cells Analysis of the mRNA profile of astrocytes stimulated with TLR agonists Plasmid pools were prepared from a cDNA library derived from pooled was performed using Clontech Atlas arrays (BD Clontech, Palo Alto, CA), human brain tumors (meningioma, oligodendroglioma, astrocytoma [grades as previously described (16). Hybridization signals were calculated as the II and IV] and medulloblastoma [Invitrogen, Carlsbad, CA]) and transfected mean of duplicate measurements, corrected for background intensity and into COS-7 cells, as previously described in detail (4). To screen for po- divided by the mean signal for all housekeeping reference . The tential TLR-activating proteins, COS-7 culture supernatants collected after relative levels of expression and their ratios shown in Fig. 3 and Table I are 48 h were added to astrocyte cultures, and CXCL8 accumulation in the representative of two independent experiments. Ab array profiling of cy- astrocyte culture medium was evaluated after 48 h by ELISA (Sanquin, tokine and chemokine production was performed using Raybio arrays, Amsterdam, The Netherlands). Sequencing of relevant plasmids was per- according to the manufacturer’s instructions (Raybiotech, Norcross, GA). formed by BaseClear (Leiden, The Netherlands). These experiments were performed three times, using freshly isolated microglia from different donors. Production of recombinant human stathmin The transcript profiling data presented in this article have been deposited in the public database ArrayExpress (www.ebi.ac.uk/microarray-as/ae/) Commercially available recombinant human stathmin (Calbiochem, San under accession code E-TABM-851. Diego, CA) was used in the experiments in Fig. 1. In all other experiments, we used recombinant human stathmin produced in-house. Stathmin was Staining of multiple sclerosis-affected human brain tissue amplified from the astrocytoma cell line U373 cDNA using the forward Downloaded from primer 59-CATATGCCATGGCTTCTTCTGATATCCAGGTG-39 and re- Human subcortical white matter or gray matter, containing chronic active verse primer 59-TCTAGATGGATCCTTAGTCAGCTTCAGTCTCGTC-39 multiple sclerosis (MS) lesions, or noninflamed normal-appearing tissue and cloned into a TOPO vector following the manufacturer’s protocol (In- was stained for TLR3 (mouse anti-human TLR3 clone 3.7; eBioscience, San vitrogen). After subcloning, stathmin was purified from recombinant Diego, CA) and stathmin (rabbit anti-stathmin; Calbiochem, Darmstadt, Escherichia coli bacteria by disruption of cells, anion exchange chroma- Germany), treated with secondary Abs (goat anti-mouse Alexa-Fluor 594 tography, and, finally, reversed-phase HPLC. To eliminate endotoxins, the and goat anti-rabbit Alexa-Fluor 488; both from Invitrogen), and examined purified protein preparation was passed over MustangE membranes (Pall, for colocalization of both markers by confocal laser-scanning fluorescence http://www.jimmunol.org/ East Hills, NY). To obtain active stathmin, the a helical content was maxi- microscopy. Images for both reporters were collected simultaneously. mized by incubating the purified protein for 1 h at 37˚C in PBS supplemented Identification of the different neural cell types was based on their mor- with 5% (v/v) 29,29,29-trifluoroethanol (TFE) and subsequent dialysis phology, and additional staining data from adjacent serial sections were against three changes of PBS over a period of 48 h at 4˚C. The protein characterized using cell-specific markers. The data in Fig. 8 are repre- preparation obtained was stored at 280˚C. Purity and identity were verified sentative of 10 samples of MS lesions from five patients and three samples by SDS-PAGE and Western blotting. Adequate elimination of bacterial of normal-appearing white matter. contaminants was verified by testing the preparation for activation of TLR2- Statistical analysis or TLR4/MD2-expresssing HEK293 reporter cells (see below). Statistical analysis was performed using an SPSS statistical package Response profiling by transfected HEK293 cells (Chicago, IL). A p value , 0.5 was considered statistically significant. Human endothelial (HEK293) cells transfected with TLR2, TLR3, by guest on October 1, 2021 or TLR4/MD2 (InvivoGen, San Diego CA) were cotransfected using Results Polyfect (Qiagen Benelux, Venlo, The Netherlands) with a reporter vector Identification of stathmin as an activator of human astrocytes expressing luciferase under the control of an NF-kB–responsive promoter (pNifty2-luc; InvivoGen). Stably transfected clones were selected and used by genome-scale functional screening of brain tumor-derived in bioassays. Cells were plated in flat-bottom 96-wells plates at a density of proteins 3 5 1 10 cells/well and incubated with the various test ligands for 16 h at Given the intriguing roles of TLR3, especially in the CNS, we 37˚C. Subsequently, cells were lysed in Steady Glo luciferase buffer (Promega Benelux, Leiden, The Netherlands), and bioluminescence was chose a cDNA library derived from pooled human brain tumors as a measured using a Packard 9600 Topcount Microplate Scintillation & source of potential protein agonist sequences. As a first step, Luminescence Counter (Packard Instrument, Meriden, CT). As a positive previously described in more detail (4), COS-7 cells were sepa- control for NF-kB–mediated activation (i.e., the presence of the pNifty2- rately transfected with 768 plasmid pools, each containing ∼50 luc vector), 25 ng/ml TNF-a (PeproTech, London, U.K.) was used (data not shown). TLR-specific ligands (all from InvivoGen) were used as different sequences from the cDNA library. Next, COS-7 culture positive controls for TLR-mediated activation. supernatants collected 48 h after transfection were separately transferred to cultures of human astrocytes, in which the accu- Knockdown of TLR3 in human astrocytes by small interfering mulation of CXCL8 was evaluated after 48 h. CXCL8 was chosen RNA treatment as a reporter chemokine for astrocyte activation based on our Transfection of human astrocytes with small interfering RNA (siRNA) was previous studies (11, 16). Although not a specific reporter for any performed using Lipofectamine 2000 (Invitrogen). Sequences used were: 1) individual TLR family member, or even for TLRs as such, CXCL8 sense sequence: 59-GAAGCUAUGUUUGGAAUUAUU-39, antisense se- quence: 59-pUAAUUCCAAACAUAGCUUCUU-39; 2) sense sequence: is a highly sensitive initial astrocyte marker for activation of NF- 59-GAUCAUCGAUUUAGGAUUGUU-39, antisense sequence: 59-pCAA kB, which occurs in astrocytes upon TLR3 signaling. Although UCCUAAAUCGAUGAUCUU-39; and 3) sense sequence: 59-GAAGAGG most COS-7 culture supernatants did not dramatically influence AAUGUUUAAAUCUU-39, antisense sequence: 59-pGAUUUAAACAUU CXCL8 production by astrocytes, one of the supernatants induced CCUCUUCUU-39. Levels of TLR3-encoding transcripts were determined marked CXCL8 release by human astrocytes compared with by real-time PCR and compared with levels of transcripts encoding b-actin, as previously described (11). background (Fig. 1A). This active plasmid pool was subcloned, and individual plasmid sequences were again used for COS-7 cell Digestion with trypsin and RNAse III transfection and subsequent astrocyte screening. Of 96 individual To verify the nature of the active components in stathmin and polyinosinic: plasmids derived from the active pool, two were found to encode polycytidylic acid [poly(I:C)], both preparations were digested with trypsin proteins that stimulated CXCL8 accumulation to levels of 160 and (Sigma-Aldrich, Zwijndrecht, The Netherlands) or the dsRNA-cleaving 210 ng/ml, whereas in all other cultures CXCL8 levels remained enzyme RNAse III (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands). Treatment with trypsin was performed at an enzyme/substrate around background levels of 2–3 ng/ml (Fig. 1B). Analysis of one ratio of 1:100 in 20 mM Tris-HCl (pH 7.6) for 15 or 30 min at 37˚C. of the active plasmids (indicated by the open arrow in Fig. 1B) The Journal of Immunology 6931 Downloaded from http://www.jimmunol.org/

FIGURE 1. Identification of stathmin as an activator of human adult astrocytes. A screen for TLR agonist potential was performed using a collection of sequences contained in a cDNA library from brain tumor cells (4). In a first selection round, 768 plasmid pools, each containing ∼50 library sequences, were transfected into COS-7 production cells. After 48 h, culture supernatants were supplied to human astrocytes which, after an additional 48 h, were tested in triplicate for secretion of CXCL8 (A). The most active pool (arrow) was subcloned, and individual clones were tested in a second selection round (B). Two clones emerged that apparently encoded active proteins. The positive clone F5 (black arrow in B) contained the sequence 44–139 of human stathmin (C). The other positive clone (white arrow in B) encoded soluble CD14, a TLR2 agonist (4). Guided by the sequence of clone F5, full-length recombinant human stathmin was tested and found to dose-dependently induce elevated CXCL8 release in human astrocytes up to levels similar to those induced by 50 mg/ml poly(I:C) (D). Data in D are average results (6 SD) for triplicate measurements, using recombinant stathmin from a commercial source. by guest on October 1, 2021 yielded the sequence of soluble CD14. Subsequent analysis al- involved. In addition to TLR3, astrocytes express TLR1, TLR2, and lowed us to define it as a selective TLR2 agonist (4). The sequence TLR4 (11), and other receptors could also be involved in mounting a of the second plasmid was found to encode residues cellular response to stathmin. Therefore, we examined the response 44–149 of human stathmin (Fig. 1C). to stathmin of astrocytes isolated from wild-type or TLR3-deficient Prompted by this finding, we tested whether a commercial mice. In this case, IL-6 release was used as a marker for TLR- preparation of human stathmin could also activate human astro- mediated activation on murine astrocytes. A marked dose-dependent cytes. This recombinant stathmin indeed led to markedly enhanced induction of IL-6 by stathmin was found in all cultures of wild-type CXCL8 release by human astrocytes in a dose-dependent manner murine astrocytes (Fig. 2A), whereas such a response was com- (Fig. 1D). Most likely, therefore, full-length stathmin and the pletely absent from astrocytes isolated from TLR3-deficient mice truncated C-terminal fragment of stathmin, which emerged from (Fig. 2B). This response profile paralleled that of poly(I:C), which the above screen, share a similar activity. When added at concen- was equally unable to induce any response in TLR3-deficient as- trations of 300 mg/ml, recombinant full-length stathmin promoted trocytes. This result also rules out any contribution to the poly(I:C)- the release of CXCL8 to levels similar to those found in response to induced response in astrocytes by alternative receptors for dsRNA, 50 mg/ml poly(I:C) (Fig. 1D). To verify that this response was not such as RIG-I or mda-5. If such receptors had contributed to the due to endotoxin contamination of the recombinant preparation, astrocyte response against poly(I:C), some of that response should responses by HEK293 cells transfected with TLR2 or the combi- have persisted in cells from which only TLR3 was eliminated. nation of TLR4 and MD2 were tested. Neither of these reporter cell Astrocytes from wild-type or TLR3-deficient mice responded lines showed any response to the recombinant stathmin prepara- equally well to LPS, confirming adequate astrocyte viability and tion, while showing the expected response to the appropriate TLR-mediated IL-6 release potential in all cultures. These findings control agonists. Preparations of recombinant stathmin that were demonstrated that the astrocyte response to stathmin, like that to subsequently prepared in-house for the remainder of this study poly(I:C), critically depends on the presence of TLR3. were also consistently found to be free of functional endotoxins To confirm this, human adult astrocytes were stimulated with and other bacterial contaminants (see below). stathmin or poly(I:C) following transfection with different siRNAs to selectively silence TLR3 expression. First, we verified that The stathmin-induced response of astrocytes is TLR3 siRNA-mediated silencing of TLR3 expression was effective when dependent TLR3-encoding mRNA is induced at the same time, as is the case Although the above data indicate a clear astrocyte response to upon activation of astrocytes with poly(I:C) (11) or, indeed, stathmin, they do not immediately clarify the nature of the receptor(s) stathmin itself. As shown in Fig. 2C, both stimuli induced TLR3 6932 STATHMIN IS A TLR3 AGONIST

FIGURE 2. Stathmin-induced activation of as- trocytes is TLR3 dependent. The release of IL-6 by astrocytes isolated from wild-type mice (A) or TLR3- deficient mice (B) after stimulation with human re- combinant stathmin, 50 mg/ml poly(I:C), or 200 ng/ml ultrapure LPS completely abrogated the stathmin- induced response in the absence of TLR3. In human astrocytes, this was confirmed by siRNA-mediated knockdown of TLR3-encoding mRNA (C), which was accompanied by an almost complete loss of re- sponsiveness to stathmin and poly(I:C) (D). Data are average results (6 SD) for triplicate measurements; they are representative of two independent experiments using recombinant stathmin produced in-house.

expression in the absence of any siRNA, but silencing with any undetectable. Given this critical role of TLR3 in the astrocyte re- Downloaded from one of the three siRNA variants consistently resulted in a decrease sponse to stathmin and poly(I:C), transcript analysis would be (by .90%) in TLR3-encoding mRNA expression. In parallel, a expected to reveal the same intracellular response profile. There- strong reduction was found in CXCL8 levels that were induced by fore, astrocytes were stimulated for 48 h with 50 mg/ml stathmin or either stathmin or poly(I:C) in siRNA-treated human astrocytes poly(I:C), and transcript profiling was performed using a cDNA over a period of 48 h (Fig. 2D). In all cases, levels of CXCL8 were array containing probes for 268 , chemokines, growth reduced to around background levels. The almost complete ab- factors, and their receptors, the prime domain of functional medi- http://www.jimmunol.org/ rogation of TLR3 mRNA expression and CXCL8 release after ators produced by astrocytes. We demonstrated the validity of these treating astrocytes with siRNAs confirms that stathmin-mediated arrays for astrocyte-response profiling in previous studies (11, 16). cellular activation is crucially dependent on TLR3 in human as- As illustrated in Fig. 4A and 4B, and summarized in more detail trocytes as well. in Table I, the stimulation of astrocytes with either agonist led to To verify that the CXCL8-inducing activity of the stathmin induction of the same range of mediators and at very similar levels. preparation is fully mediated by a protein component, rather than any In contrast, stimulation of astrocytes via TLR4 using ultrapure LPS RNA contaminant, stathmin was treated with trypsin or the dsRNA- induced a very different response profile, confirming the selectivity cleaving enzyme RNAse III. As shown in Fig. 3, tryptic digestion of of the response profiles to stathmin or poly(I:C) (Table I). stathmin led to the disappearance of astrocyte-activating potential, In line with previous data (11), stathmin and poly(I:C) induced a by guest on October 1, 2021 whereas treatment with RNAse III had no effect. The result was variety of factors involved in neuroprotection and repair, including the opposite for the agonist poly(I:C). LIF, b nerve , metallothionin-III, fibroblast growth factors, GTPase-activating protein-43, , vascular Stathmin and poly(I:C) induce the same set of transcripts in endothelial growth factor, brain-derived neurotrophic factor, astrocytes pleiotrophin, glial maturation factor, and -like growth factor The above findings clarify that stathmin- and poly(I:C)-induced 1. Previously, we demonstrated the ability of this mixture of cytokine responses in murine and human astrocytes are dependent mediators to significantly improve survival of human neurons in on the presence of TLR3. As clarified above, any possible response organotypic brain slice cultures (11). Stathmin also induced mediated by alternative dsRNA receptors, such as intracellular type A receptor 2, as well as B1 and B3, bidirectional helicases (2), which would have persisted in TLR3-deficient as- signaling molecules controlling axonal growth, synapse formation, trocytes or after siRNA-mediated TLR3 silencing is essentially and astrocyte reactivity. Among the cytokines induced were the immunoregulatory factors IL-10 and -11, IFN-g antagonist, and IFN-b. No significant induction of TNF-a was observed. Che- mokines induced included CCL2, CCL3, CCL5, CXCL2, CXCL6, and CXCL8; the latter confirmed the suitability of CXCL8 as an initial marker for TLR3-mediated activation of astrocytes. No was markedly suppressed by stathmin or poly(I:C). Im- portantly, the Pearson correlation coefficient, which expresses the extent of similarity between the two datasets (Fig. 4C), reached +0.951, confirming that the functional response at this level to a 50-mg/ml dose of stathmin or poly(I:C) is essentially identical (p , 1025). Again, these results provided strong evidence for the notion that stathmin, like poly(I:C), activates TLR3-dependent signaling in astrocytes. FIGURE 3. Trypsin, but not RNAse III, destroys the ability of stathmin to activate astrocytes. Treatment of stathmin with trypsin, but not RNase The a helical structure of stathmin is required for TLR3 III, rapidly led to a complete loss of its ability to activate astrocytes. It was agonist activity the other way around for the agonist poly(I:C). Data represent average results (6 SD) for triplicate measurements using stathmin produced in- In the course of our experiments, we noted marked differences in house; they are representative of two independent experiments. the dose-response profiles of different preparations of recombinant The Journal of Immunology 6933

FIGURE 4. Gene profiling of astrocytes stimulated with recombinant human stathmin or poly(I:C). Human astrocytes were stimulated for 48 h with 50 mg/ml recombinant human stathmin (A)or50mg/ml poly(I:C) (B), and transcript levels for 268 cytokines, chemokines, growth factors, and their receptors Downloaded from were assessed by microarray profiling. A and B provide an overview of all relative transcript signals in either case compared with untreated parallel cultures of the same astrocyte isolate. C, Direct comparison of transcripts found after stathmin stimulation with those found after poly(I:C) stimulation, illustrating that levels of all transcripts were very similar, aggregating along the diagonal. Statistical analysis of the data indicated that the Pearson correlation co- efficient (r) reached a value of +0.951, indicative of essentially identical datasets (p , 1025). Transcript levels were determined in duplicate, using stathmin prepared in-house.

stathmin. Initial experiments using a commercial preparation of stathmin preparations tested were otherwise identical in purity and http://www.jimmunol.org/ stathmin suggested that the agonist would be markedly less effi- concentration and were contained in the exact same solvent fol- cient on a weight basis than poly(I:C) in activating astrocytes (Fig. lowing dialysis, we concluded that the a helix of stathmin is of 1). However, material subsequently produced in-house was as critical importance to its ability to activate TLR3. To further effective as poly(I:C), on a weight basis, in inducing cellular re- support this notion, stathmin completely lost its TLR3-agonist sponses (Fig. 4, Table I). Because the purities of the different activity after full denaturation as a consequence of exposure to stathmin preparations were comparable, and all were free from organic solvents at pH 2 during reversed-phase HPLC, followed bacterial contaminants, this raised the possibility that additional by lyophilization (Fig. 5B). This finding confirms the absence of factors involving, for example, secondary structure, affected the interference by bacterial contaminants, whose activity would not by guest on October 1, 2021 TLR3-activating activity of stathmin. One striking feature of be affected by these conditions. stathmin is its ability to fold into a very long a helix that may contain up to 94 aa (17). This helix contains no less that 24 Stathmin also activates intracellular TLR3 in microglia negatively charged amino acids, mostly glutamic acid residues. We next tested whether stathmin also induces TLR3-mediated Therefore, we examined whether the a helical content of signaling in human microglia. As opposed to astrocytes, which stathmin would affect its ability to activate TLR3-mediated as- express TLR3 primarily on their surface, microglia express TLR3 trocyte responses. Purified human recombinant stathmin was exclusively in intracellular vesicles (13). Given the apparently subjected to conditions that differentially affect helical content. critical role of the a helical conformation of stathmin, uptake and One part of the protein preparation was incubated in PBS con- acidification of stathmin in the endosomal pathway of microglia taining 5% of the helix-promoting solvent TFE (18). The other could conceivably affect its ability to activate TLR3. However, as part was kept in water acidified with 5% of the helix-breaking shown in Fig. 6, stathmin also retains TLR3-activating potential solvent trifluoroacetic acid (TFA). After exposure to either of when taken up by microglia. Examination of microglial responses these conditions, stathmin was extensively dialyzed over 48 h should take into account that cultured, but otherwise untreated, against several changes of PBS and subsequently analyzed by microglia already secrete significant amounts of certain mediators. near-UV circular dichroism (CD) spectroscopy. The TFE-treated In our cultures, freshly isolated, but otherwise untreated, microglia sample contained 37% a helix on average, whereas the TFA- grown with GM-CSF produced large amounts of IL-6 and several treated sample produced a CD spectrum indicative of only 12% chemokines, including CXCL8, CCL2, CCL3, CCL4, and CCL8. helix content (Fig. 5A). No other changes in the protein prepara- Several of these mediators were present at such high levels that tions (e.g., solubility or aggregated state) were observed. As a any additional increase by subsequent treatment with a TLR ag- reference sample for CD measurements, part of the stathmin onist probably would be limited for this reason alone. This is preparation was also dissolved in a solution of 50% TFE in PBS particularly likely to apply to IL-6, CXCL8, and CCL2. Despite immediately prior to CD spectroscopy. This sample produced a this, exposure of microglia to stathmin led to marked induction of spectrum indicative of 98% a helical content (Fig. 5A), confirming IL-13, CCL5 (RANTES), IL-1b, IL-6 soluble receptor, TNF-a, the extreme a helical propensity of stathmin. and several other mediators that are consistent with TLR-mediated In parallel, human adult astrocytes were stimulated with different signaling. Again, the response by microglia to stathmin was amounts of either dialyzed protein preparation, and CXCL8 release similar to that induced by poly(I:C) on a weight basis, albeit not was monitored after 48 h. As shown in Fig. 5B, the TFE-treated as identical as was found in astrocytes. Compared with stathmin, stathmin preparation that contained three times more a helix poly(I:C) induced a very similar set of factors but markedly higher triggered CXCL8 release ∼3-fold more effectively on a weight levels of CXCL10 and lower levels of CCL1 and IL-1a (Fig. 6). basis than did the TFA-treated preparation. Because the two These differences between stathmin- and poly(I:C)-induced responses 6934 STATHMIN IS A TLR3 AGONIST

Table I. Transcript levels in astrocytes stimulated with stathmin, poly(I:C) or LPS, relative to untreated cells

Gene Name and Functional Classification Stathmin Poly(I:C) LPS Regeneration. growth and proliferation LIF 5.26 5.85 — b (b NGF) 5.26 4.91 — Growth inhibitory factor; metallothionein-III (MT-III) 5.25 6.67 — Ephrin type-A receptor 2 (EPHT1) 5.06 5.06 — Insulin-like growth factor binding protein 1 (IGFBP1) 4.73 5.49 — Insulin-like growth factor binding protein 3 (IGFBP3) 4.41 5.60 — 7 (FGF7) 4.01 3.67 — Neuromodulin (GAP-43) 3.55 3.57 — Insulin-like growth factor binding protein 6 (IGFBP6) 3.50 3.66 — -3/4 (NT-3/NT-4) 3.34 5.00 — Brain-derived neurotrophic factor (BDNF) 3.13 3.15 — Platelet-derived growth factor A subunit (PDGF1) 2.99 3.00 — Fibroblast growth factor 5 precursor (FGF5) 2.95 3.02 — Vascular endothelial growth factor C (VEGF-C) 2.68 3.09 — (EPHB1) 2.58 3.87 — Neurogenic locus notch protein 5.56 3.05 — wnt-8B 2.38 2.13 — Glial maturation factor b (GMF-b) 2.29 3.62 — Pleiotrophin (neurite growth promoting factor 1; PTN) 2.27 3.28 — Downloaded from Ephrin B3 (EPHB3) 2.05 2.00 — Insulin-like growth factor 1A (IGF1) 2.05 2.98 — Intracellular signaling IFN-a–inducible protein (G1P3) 5.51 4.71 — IFN regulatory factor 1 (IRF1) 2.96 3.87 — Cytokines and chemokines

MIP-1a (CCL3) 12.82 15.28 2.1 http://www.jimmunol.org/ IL-8 (CXCL8) 7.04 10.56 4.8 IL-6 6.15 7.16 — IL-10 4.86 7.25 — IL-11 3.85 3.22 — IFN-g antagonist 3.49 4.36 — IFN-b 3.30 6.26 — RANTES (CCL5) 3.03 4.14 — Monocyte chemotactic protein 1 precursor (CCL2) 2.96 2.93 — MIP-2a (CXCL2) 2.41 2.47 4.1 Granulocyte chemotactic protein 2 (CXCL6) 2.41 2.99 2.1 Receptors by guest on October 1, 2021 TNFR superfamily member 5 (CD40) 5.98 5.92 — Related to receptor (RYK) 4.96 2.15 — High-affinity nerve 3.68 4.08 — G-CSFR precursor (CD114) 2.75 4.55 2.4 IL-12R 2.65 2.33 — receptor 2.49 2.76 — Macrophage inflammatory protein 1 a receptor (CCR1) 2.46 2.94 — IL-4Ra subunit (CD124) 2.45 2.26 — GM-CSFRa 2.34 3.20 — IL-2Rb subunit (CD122) 2.25 3.35 — Only induction factors $2.0 are included. in microglia likely reflect the influence of additional intracellular Although this confirmed that cellular responsiveness to our receptors for poly(I:C) in these cells, such as RIG-I, mda-5, and stathmin preparations was not attributable to bacterial con- other helicases that are not functional in astrocytes. taminants, the most obvious of which will trigger a response by TLR2- or TLR4-expressing HEK cells, the consistent lack of re- TLR3-transfected HEK293 cells fail to respond to stathmin sponsiveness to stathmin by TLR3 transfectants was unexpected. The above data provided compelling evidence for the ability of In view of recent evidence that TLR3-mediated signaling likely stathmin to activate astrocytes and microglia. The lack of any depends on several unknown factors other than TLR3 alone, in- response by TLR3-deficient astrocytes or astrocytes subjected to cluding additional unknown surface factors (9, 10), we concluded selective TLR3 knockdown, as well as the congruity in astrocyte that HEK293 cells probably lack such factors, whereas they are transcript responses to stathmin and poly(I:C), support the notion present on astrocytes and microglia. that this stathmin-induced response is TLR3 dependent. Therefore, we expected to be able to confirm the agonist activity of stathmin In vivo interactions between stathmin and TLR3 using TLR3-transfected HEK293 cells, a routine approach to Experimental verification of functional stathmin–TLR3 inter- screen for TLR ligands. Surprisingly, however, TLR3-expressing actions in vivo by straightforward knockout approaches is ham- HEK cells failed to respond to stathmin, despite being fully pered by the multiple biological roles and interaction partners of responsive to poly(I:C). As shown in Fig. 7, HEK cells transfected either molecule and by the fact that mammals possess four with TLR2 or TLR4 did not show any response to stathmin, while stathmin homologs that share the active a helical segment of mounting a significant response to appropriate control ligands. stathmin. Yet, given that TLR3 and stathmin are induced during The Journal of Immunology 6935

FIGURE 5. TLR3 agonist activity of stathmin relies on its a helix. A, Recombinant human stathmin was exposed to helix-promoting 5% TFE or to helix- breaking 5% TFA in PBS, extensively dialyzed against fresh PBS, and subjected to CD spectroscopy to assess helical content. The TFE-treated preparation (thick line) contained 37% helix, whereas the TFA-treated sample (thin line) contained only 12% helix. As a control, stathmin was also subjected to CD spectroscopy in the presence of 50% TFE to illustrate its potential to form up to 98% helix (dotted line). B, When tested for the ability to trigger CXCL8 release by human astrocytes, the TFE-treated sample (d) was about three times more effective in this respect on a weight basis than the TFA-treated sample (n). As a comparison, purified and lyophilized stathmin without any pretreatment to restore the conformation of helical segments remained fully inactive Downloaded from (:). Data are the average of duplicate measurements and are representative of three independent experiments using stathmin produced in-house. neuroinflammation (13, 19, 20), we examined whether they co- astrocytes. Although oligodendrocytes in MS lesions displayed localized under such conditions in the human CNS. Upon ex- marked surface expression of TLR3, as well as intracellular ex- amination of chronic active MS lesions, widespread expression of pression of stathmin, colocalization of both proteins on the surface TLR3 was found, primarily inside microglia and on the surface of of or inside oligodendrocytes was only rarely observed. http://www.jimmunol.org/ astrocytes and their processes, as well as on neurons and oligo- dendrocytes. Stathmin was expressed at comparable levels in all Discussion cell types. This localization of either protein is consistent with The main conclusion from this study is that stathmin, a protein that earlier reports (13, 19, 21). Previously, we reported that TLR3 is has long been known as a ubiquitously expressed regulator of undetectable by immunohistochemistry in healthy control microtubule formation, is able to activate TLR3-dependent sig- or noninflamed normal-appearing white matter of MS patients naling in astrocytes and microglia. Several key observations sup- (13); our current data, as illustrated in Fig. 8, confirmed this. port this notion. The stathmin-induced CXCL8 reporter response is However, TLR3 is clearly upregulated in chronic active MS le- completely abrogated in TLR3-deficient murine astrocytes and in by guest on October 1, 2021 sions. Under these conditions, colocalization of TLR3 and stath- human astrocytes subjected to TLR3 knockdown. In both cases, min was mainly observed on the surface of astrocytes and neurons the elimination of stathmin-induced responses paralleled the and inside small intracellular vesicular structures of microglia and elimination of poly(I:C)-induced ones. Transcript profiling of

FIGURE 6. Cytokine and chemokine responses in human microglia to stathmin or poly(I:C). Human microglia were stimulated with stathmin (50 mg/ml) or poly(I:C) (50 mg/ml) for 24 h, and culture super- natants were examined for the presence of 40 cyto- kines and chemokines using Ab arrays. The histograms show the levels of each mediator relative to those found in untreated parallel cultures kept for the same time without any stimulus. The mediators are arranged according to increasing induction fac- tors in response to stathmin. Results are the average data (6 SD) from three experiments, each performed with freshly isolated microglia from a different donor and using stathmin prepared in-house. 6936 STATHMIN IS A TLR3 AGONIST

which disruption of such domains reduced poly(I:C)-mediated signaling in human PBMCs (10). Evidence is rapidly accumulating that TLR-dependent responses are generally controlled by multiple receptor/coreceptor interactions of previously unsuspected com- plexity (22, 23). It seems likely that this also applies to TLR3- mediated responses to stathmin, and it may explain why the ex- pression of only TLR3 itself by HEK293 cells might not be suffi- cient to confer stathmin responsiveness. In addition to astrocytes and microglia, we found that human monocyte-derived dendritic cells were responsive to stathmin, as evidenced by marked secre- tion of CXCL8 in response to the protein (data not shown). Non- responsiveness to stathmin in HEK293 cells might be related to FIGURE 7. Stathmin failed to activate HEK293 cells transfected with their endothelial origin, a trait by which they differ from apparently TLR2, TLR3, or TLR4. HEK293 reporter cells were transfected with each responsive myelomonocytic or fibroblast-like cells. of the three TLRs as described in Materials and Methods and tested for Stathmin has been well characterized as a catalyst of the NF-kB–mediated responses to stathmin or the control agonists macro- reversible process of microtubule catastrophe and repolymerization phage-activating lipopeptide 2 kDa (TLR2), poly(I:C) (TLR3), or ultrapure (24, 25). As such, stathmin is widely expressed in many cells, LPS (TLR4). The data reveal the absence of any response to stathmin in all especially in those engaging in proliferation and/or migration. three cases. Data represent average results (6 SD) for triplicate meas- Intriguingly, however, the strikingly dominant expression of urements and are representative of two independent experiments using stathmin in mammalian brains and spinal cord, and especially in Downloaded from stathmin prepared in-house. fetal brains (26), suggests a special relevance to the CNS, un- explained by its traditional and ubiquitous role as a regulator of stathmin- or poly(I:C)-induced astrocyte responses, using cDNA microtubules. This applies even more strongly to the three other arrays to monitor 268 cytokines, chemokines, and growth factors, family members of stathmin, the homologs superior cervical revealed essentially identical activation pathways. The notion that ganglion 10 protein (SCG10; stathmin-2), SCG10-like protein stathmin may act as a TLR3 agonist in vivo is supported by the (SCLIP; stathmin-3), and RB3 (stathmin-4), which are highly http://www.jimmunol.org/ finding that, at least during the neuroinflammatory process of MS, conserved among vertebrates. For example, expression levels of stathmin and TLR3 colocalize on and inside a variety of neural SCLIP and RB3 in the CNS are $100 times greater than in other cells, including astrocytes, microglia, and neurons. human tissues (26). After brain trauma, stathmin levels increase Yet, TLR3 alone is apparently not sufficient to confer cellular (19, 20), as they do during axonal and dendritic growth (21, 27). responsiveness to stathmin, as clarified by the lack of any response in The particular significance of stathmins to the CNS is further HEK293–TLR3 transfectants. Therefore, stathmin-induced cel- supported by the observations that stathmin-deficient mice show lular activation most likely requires additional receptor compo- defects in innate and acquired fear (28) and selectively develop nents, possibly surface coreceptors, that are absent from HEK293 axonopathy at advanced age (29). Drosophila flies in which the by guest on October 1, 2021 cells. That TLR3-dependent signaling requires additional recep- stathmin gene has been knocked down by siRNAs develop tor components was suggested by a detailed analysis of TLR3- widespread defects in the only (30). All of these signaling pathways. As explained by Helmy et al. (9), dynamic observations point to a special role of stathmins in nervous system simulation of TLR3 signaling strongly suggests the existence of development and are difficult to rationalize in the context of the still-unknown intermediary steps in the cascade of signaling events, ubiquitous role of stathmins in microtubule rearrangement alone. notably upstream of TLR3 dimerization. That such still-missing It seems likely that all four stathmin family members share the elements likely include additional receptor components in surface ability to activate TLR3. The helix-forming sequence 44–139 of membrane microdomains also emerged from a recent study in stathmin shares between 92% and 95% and

FIGURE 8. Stathmin and TLR3 colocalize in MS lesions. Chronic active MS lesions were stained for TLR3 (red) and stathmin (green) and examined by confocal laser-scanning microscopy (original magnifi- cation 320). The top and middle panels in the right column show extensive colocalization of the two pro- teins in inflamed areas (highlighted by the arrows), primarily involving astrocytes and microglia and only rarely oligodendrocytes. The lower panel in the right column shows patchy or vesicular colocalization of TLR3 and stathmin that was also observed on neurons in gray matter lesions. Control H&E staining, an iso- type control for the TLR3 Ab and a representative merged stathmin/TLR3 stain of noninflamed normal- appearing white matter are shown in the left column. The Journal of Immunology 6937 between 67% and 75% amino acid identity with the other three 7. Ranjith-Kumar, C. T., W. Miller, J. Xiong, W. K. Russell, R. Lamb, J. Santos, K. E. Duffy, L. Cleveland, M. Park, K. Bhardwaj, et al. 2007. Biochemical and family members SCG10, SCLIP, and RB3. However, the exact functional analyses of the human Toll-like receptor 3 ectodomain. J. Biol. Chem. mechanism via which stathmins might activate TLR3 in vivo re- 282: 7668–7678. mains to be established. Other family members of stathmin are 8. Pirher, N., K. Ivicak, J. Pohar, M. Bencina, and R. Jerala. 2008. A second binding site for double-stranded RNA in TLR3 and consequences for plausible in vivo ligands, much more so than stathmin itself, best activation. Nat. Struct. Mol. Biol. 15: 761–763. known as a cytosolic protein. SCG10, SCLIP, and RB3 do not 9. Helmy, M., J. Gohda, J.-I. Inoue, M. Tomita, M. Tsuchiya, and K. Selvarajoo. differ from stathmin in their a helical properties, but they do differ 2009. Predicting novel features of toll-like receptor 3 signaling in macrophages. PLoS ONE 4: e4661. in having an extended N-terminal sequence. Acylation of this 10. Parker, L. C., E. C. Prestwich, J. R. Ward, E. Smythe, A. Berry, M. Triantafilou, sequence targets these stathmins to Golgi vesicles and to addi- K. Triantafilou, and I. Sabroe. 2008. A phosphatidylserine species inhibits a tional vesicular neuronal structures that are found along neurite range of TLR- but not IL-1b-induced inflammatory responses by disruption of membrane microdomains. J. Immunol. 181: 5606–5617. shafts and in growth cones (31, 32). These vesicular structures in 11. Bsibsi, M., C. Persoon-Deen, R. W. Verwer, S. Meeuwsen, R. Ravid, and neurons are very similar to those reported to contain TLR3 (14). J. M. Van Noort. 2006. Toll-like receptor 3 on adult human astrocytes triggers production of neuroprotective mediators. Glia 53: 688–695. Although the roles of neuronal TLR3 and stathmins are not fully 12. Lafon, M., F. Megret, M. Lafage, and C. Prehaud. 2006. The innate immune understood and are probably complex, striking similarities are facet of brain: human neurons express TLR-3 and sense viral dsRNA. J. Mol. starting to emerge. Both can inhibit the growth of neuronal pre- Neurosci. 29: 185–194. 13. Bsibsi, M., R. Ravid, D. Gveric, and J. M. van Noort. 2002. Broad expression of cursors, axons, and dendrites, and both can promote dendrite Toll-like receptors in the human central nervous system. J. Neuropathol. Exp. branching (14, 15, 32). Therefore, direct stathmin–TLR3 inter- Neurol. 61: 1013–1021. actions on or inside neurons, as revealed by our present data (Fig. 14. Cameron, J. S., L. Alexopoulou, J. A. Sloane, A. B. DiBernardo, Y. Ma, B. Kosaras, R. Flavell, S. M. Strittmatter, J. Volpe, R. Sidman, and T. Vartanian. 8), might underlie this functional congruence between the two 2007. Toll-like receptor 3 is a potent negative regulator of axonal growth in Downloaded from proteins. Other ways for stathmins to contact TLR3 in the CNS mammals. J. Neurosci. 27: 13033–13041. may be envisaged as well. Because stathmin can be found in 15. Lathia, J. D., E. Okun, S. C. Tang, K. Griffioen, A. Cheng, M. R. Mughal, G. Laryea, P. K. Selvaraj, C. ffrench-Constant, T. Magnus, et al. 2008. Toll-like myelin sheaths (19, 33) and is even upregulated during neuro- receptor 3 is a negative regulator of embryonic neural progenitor cell pro- inflammation (19, 20), it is likely to become ingested by phag- liferation. J. Neurosci. 28: 13978–13984. ocytic microglia that emerge in response to infection or trauma. 16. Meeuwsen, S., C. Persoon-Deen, M. Bsibsi, R. Ravid, and J. M. van Noort. 2003. Cytokine, chemokine and growth factor gene profiling of cultured human as- Myelin phagocytosis would readily allow stathmin to directly trocytes after exposure to proinflammatory stimuli. Glia 43: 243–253. contact endosomal TLR3 in microglia, a scenario supported by the 17. Wallon, G., J. Rappsilber, M. Mann, and L. Serrano. 2000. Model for stathmin/ http://www.jimmunol.org/ OP18 binding to tubulin. EMBO J. 19: 213–222. present immunohistochemical findings (Fig. 8). Finally, alter- 18. Roccatano, D., G. Colombo, M. Fioroni, and A. E. Mark. 2002. Mechanism by native splicing products might play a role. Preliminary data sug- which 2,2,2-trifluoroethanol/water mixtures stabilize secondary-structure for- gested that truncated forms of stathmin that contain the helix- mation in peptides: a molecular dynamics study. Proc. Natl. Acad. Sci. USA 99: 12179–12184. forming C-terminal sequence 44–149, but lack the N terminus, can 19. Liu, A., C. Stadelmann, M. Moscarello, W. Bruck, A. Sobel, F. G. Mastronardi, be secreted by mammalian cells, while retaining their ability to and P. Casaccia-Bonnefil. 2005. Expression of stathmin, a developmentally activate TLR3. This observation suggests that alternative splicing controlled cytoskeleton-regulating molecule, in demyelinating disorders. J. Neurosci. 25: 737–747. of stathmin genes could lead to N-truncated, naturally secreted 20. Chabas, D., S. E. Baranzini, D. Mitchell, C. C. Bernard, S. R. Rittling, forms that could conceivably activate TLR3 on other cells. D. T. Denhardt, R. A. Sobel, C. Lock, M. Karpuj, R. Pedotti, et al. 2001. The by guest on October 1, 2021 Clearly, clarification of the exact physiological mechanisms via influence of the proinflammatory cytokine, osteopontin, on autoimmune de- myelinating disease. Science 294: 1731–1735. which stathmin and/or its family members contact and activate 21. Gavet, O., S. El Messari, S. Ozon, and A. Sobel. 2002. Regulation and sub- TLR3 require more detailed studies. cellular localization of the microtubule-destabilizing stathmin family phospho- proteins in cortical neurons. J. Neurosci. Res. 68: 535–550. In conclusion, our data provide evidence for the ability of 22. Ishii, K. J., C. Coban, and S. Akira. 2005. Manifold mechanisms of Toll-like stathmins to activate TLR3-dependent cellular responses. Also, in receptor-ligand recognition. J. Clin. Immunol. 25: 511–521. the context of other data, our findings suggest that the functional 23. Amiel, E., A. Alonso, S. Uematsu, S. Akira, M. E. Poynter, and B. Berwin. 2009. Pivotal Advance: Toll-like receptor regulation of scavenger receptor-A-mediated link between stathmins and TLR3 is likely to be of particular phagocytosis. J. Leukoc. Biol. 85: 595–605. relevance to development and repair in the CNS. 24. Cassimeris, L. 2002. The oncoprotein 18/stathmin family of microtubule de- stabilizers. Curr. Opin. Cell Biol. 14: 18–24. 25. Curmi, P. A., O. Gavet, E. Charbaut, S. Ozon, S. Lachkar-Colmerauer, Acknowledgments V. Manceau, S. Siavoshian, A. Maucuer, and A. Sobel. 1999. Stathmin and its Human post mortem brain tissue was obtained through the Netherlands phosphoprotein family: general properties, biochemical and functional inter- Brain Bank. We thank L. van Straalen and E. Zuiderwijk-Sick for technical action with tubulin. Cell Struct. Funct. 24: 345–357. 26. Bie`che, I., A. Maucuer, I. Laurendeau, S. Lachkar, A. J. Spano, A. Frankfurter, support. P. Le´vy, V. Manceau, A. Sobel, M. Vidaud, and P. A. Curmi. 2003. Expression of stathmin family genes in human tissues: non-neural-restricted expression for Disclosures SCLIP. Genomics 81: 400–410. 27. Pellier-Monnin, V., L. Astic, S. Bichet, B. M. Riederer, and G. Grenningloh. The authors have no financial conflicts of interest. 2001. Expression of SCG10 and stathmin proteins in the rat olfactory system during development and axonal regeneration. J. Comp. Neurol. 433: 239–254. 28. Shumyatsky, G. P., G. Malleret, R. M. Shin, S. Takizawa, K. Tully, E. Tsvetkov, References S. S. Zakharenko, J. Joseph, S. Vronskaya, D. Yin, et al. 2005. stathmin, a gene 1. O’Neill, L. A., and A. G. Bowie. 2007. The family of five: TIR-domain-containing enriched in the amygdala, controls both learned and innate fear. Cell 123: 697–709. adaptors in Toll-like receptor signalling. Nat. Rev. Immunol. 7: 353–364. 29. Liedtke, W., E. E. Leman, R. E. Fyffe, C. S. Raine, and U. K. Schubart. 2002. 2. Kawai, T., and S. Akira. 2008. Toll-like receptor and RIG-I-like receptor sig- Stathmin-deficient mice develop an age-dependent axonopathy of the central and naling. Ann. N. Y. Acad. Sci. 1143: 1–20. peripheral nervous systems. Am. J. Pathol. 160: 469–480. 3. Taylor, K. R., J. M. Trowbridge, J. A. Rudisill, C. C. Termeer, J. C. Simon, and 30. Ozon, S., A. Guichet, O. Gavet, S. Roth, and A. Sobel. 2002. Drosophila R. L. Gallo. 2004. Hyaluronan fragments stimulate endothelial recognition of stathmin: a microtubule-destabilizing factor involved in nervous system for- injury through TLR4. J. Biol. Chem. 279: 17079–17084. mation. Mol. Biol. Cell 13: 698–710. 4. Bsibsi, M., J. J. Bajramovic, E. Van Duijvenvoorden, C. Persoon, R. Ravid, 31. Chauvin, S., F. E. Poulain, S. Ozon, and A. Sobel. 2008. Palmitoylation of J. M. Van Noort, and M. H. J. Vogt. 2007. Identification of soluble CD14 as an stathmin family proteins domain A controls Golgi versus mitochondrial sub- endogenous agonist for Toll-like receptor 2 on human astrocytes by genome- cellular targeting. Biol. Cell 100: 577–589. scale functional screening of glial cell derived proteins. Glia 55: 473–482. 32. Poulain, F. E., S. Chauvin, R. Wehrle´, M. Desclaux, J. Mallet, G. Vodjdani, 5. Calderwood, S. K., S. S. Mambula, and P. J. Gray, Jr. 2007. Extracellular heat I. Dusart, and A. Sobel. 2008. SCLIP is crucial for the formation and develop- shock proteins in cell signaling and immunity. Ann. N. Y. Acad. Sci. 1113: 28–39. ment of the Purkinje cell dendritic arbor. J. Neurosci. 28: 7387–7398. 6. Bell, J. K., J. Askins, P. R. Hall, D. R. Davies, and D. M. Segal. 2006. The 33. Southwood, C., C. He, J. Garbern, J. Kamholz, E. Arroyo, and A. Gow. 2004. dsRNA binding site of human Toll-like receptor 3. Proc. Natl. Acad. Sci. USA CNS myelin paranodes require Nkx6-2 homeoprotein transcriptional activity for 103: 8792–8797. normal structure. J. Neurosci. 24: 11215–11225.