L1CAM increases MAP2 expression via the MAPK pathway to promote neurite outgrowth

Molecular and Cellular Neuroscience 50 (2012) 169–178

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L1CAM increases MAP2 expression via the MAPK pathway to promote neurite outgrowth Gunnar Heiko Dirk Poplawski a, b, Ann-Kathrin Tranziska a, Iryna Leshchyns'ka a, c, Ingo Dunya Meier a, Thomas Streichert d, Vladimir Sytnyk a, c, Melitta Schachner a, e, f,⁎ a

Zentrum für Molekulare Neurobiologie Hamburg, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia d Institut für Klinische Chemie, Zentrum für Diagnostik, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany e Center for Neuroscience, Shantou University Medical College, 22 Xin Ling Road, Shantou 515041, PR China f Keck Center for Collaborative Neuroscience and Department of Cell Biology and Neuroscience, Rutgers University, 604 Allison Road, Piscataway, NJ 08854, USA b c

a r t i c l e

i n f o

Article history: Received 7 July 2011 Revised 21 February 2012 Accepted 29 March 2012 Available online 6 April 2012 Keywords: Cell adhesion molecule L1CAM MAP2 MAPK Microarray Neurite outgrowth

a b s t r a c t The neural cell adhesion molecule L1 (L1CAM) promotes neurite outgrowth via mechanisms that are not completely understood, but are known to involve the cytoskeleton. Here, we show that L1 binds directly to the microtubule associated protein 2c (MAP2c). This isoform of MAP2 is predominantly expressed in developing neurons. We found that the mRNA and protein levels of MAP2c, but not of MAP2a/b, are reduced in brains of young adult L1-deficient transgenic mice. We show via ELISA, that MAP2c, but not MAP2a/b, binds directly to the intracellular domain of L1. Remarkably, all these MAP2 isoforms co-immunoprecipitate with L1, suggesting that MAP2a/b associates with L1 via intermediate binding partners. The expression levels of MAP2a/b/c correlate with those of L1 in different brain regions of early postnatal mice, while expression levels of heat shock cognate protein 70 (Hsc70) or actin do not. L1 enhances the expression of MAP2a/b/c in cultured hippocampal neurons depending on activation of the mitogen-activated protein kinase (MAPK) pathway. Deficiency in both L1 and MAP2a/b/c expression results in reduced neurite outgrowth in vitro. We propose that the L1-triggered increase in MAP2a/b/c expression is required to promote neurite outgrowth. © 2012 Elsevier Inc. All rights reserved.

Introduction Neurite outgrowth and retraction are among the basic functions of a neuron not only during ontogenetic development, but also in the adult nervous system that is under constraint to change synaptic activity during learning and memory as well as regeneration after injury. Adhesion molecules perform these functions by manifold mechanisms and cell surface receptors of the immunoglobulin (Ig) superfamily were the first to be shown to engage in such activities mediating neurite outgrowth in vitro and in vivo. The signaling mechanisms triggered by these receptors include direct activation of src-family members and regulation of downstream MAPK pathways, and proteolytic release of the intracellular domain of, for instance, the neural cell adhesion molecule L1, followed by translocation to the nucleus to potentially regulate gene expression (Loers et al., 2005; Riedle et al., 2009). In addition,

Abbreviations: MAP, microtubule associated protein; MAPK, mitogen-activated protein kinase; Hsc70, heat shock cognate protein 70; Grb2a, growth factor receptor binding protein 2a. ⁎ Corresponding author at: Center for Neuroscience, Shantou University Medical College, 22 Xin Ling Road, Shantou, 515041, PR China. Fax: + 86 754 8890 0236. E-mail address: [email protected] (M. Schachner). 1044-7431/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2012.03.010

several Ig-superfamily receptors interact directly with cytoskeletal components, such as ankyrin, actin and the moesin family (Buhusi et al., 2008; Dickson et al., 2002; Mintz et al., 2003; Nishimura et al., 2003) in the case of L1, and β1-spectrin in the case of the neural cell adhesion molecule (NCAM) (Leshchyns'ka et al., 2003). These cytoskeletal components can associate directly and indirectly with each other throughout development. In adulthood, IgCAMs influence axonal and dendritic morphology and extent of synaptic contacts in response to electrical activity and activation and de-activation of synapses. Furthermore, some members of the Ig-superfamily enhance synaptic vesicle recycling and increase neurotransmitter receptor levels within the synaptic membrane (Andreyeva et al., 2010; Leshchyns'ka et al., 2006; Sytnyk et al., 2006), thus requiring a finely tuned interaction of cell surface molecules with the cytoskeletal network. In vitro studies showed that neurite outgrowth and survival of primary hippocampal neurons is stimulated by substrate-coated and soluble L1, L1-derived fragments and peptides as well as epitope-specific L1 antibodies that trigger the neuro-proactive functions of L1 (Dong et al., 2003; Schuch et al., 1989). L1 can bind in a homophilic manner to itself or in a heterophilic manner to other Ig-CAMs, such as NCAM (Grumet and Edelman, 1988; Lemmon et al., 1989) to promote neurite outgrowth in vitro. In addition, L1 interacts with several receptor molecules, such as diverse forms of

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integrins (Blaess et al., 1998; Castellani et al., 2002; Felding-Habermann et al., 1997; Ruppert et al., 1995; Silletti et al., 2000), contactin-1 (De Angelis et al., 1999; Perrin et al., 2001), axonin (Kuhn et al., 1991), basic FGFR (Williams et al., 1994), neuropilin-1 (Castellani et al., 2002) and the extracellular matrix molecules phosphacan (Milev et al., 1994; Sakurai et al., 1996), neurocan (Friedlander et al., 1994) and laminin (Grumet et al., 1993) which regulate neurite outgrowth and synapse formation. In the present study, we were interested whether in addition to ankyrin and actin (Herron et al., 2009), other components of the cytoskeleton would interact with L1 and trigger its ability to promote neurite outgrowth. We investigated the connection between L1 and microtubuleassociated protein-2 (MAP2), since microarray gene chip analyses of young adult L1-deficient mouse brains showed reduced MAP2 mRNA levels when compared to wild-type littermates. Isoform-specific, quantitative real-time PCR analysis showed that Map2c is specifically reduced in L1 deficient brains, while MAP2a/b mRNA levels remain unaltered when compared to wild-type littermates. This observation suggested a direct and/or indirect relationship between L1 and MAP2c. MAPs play crucial roles in neuritogenesis and growth cone advancement by directly altering microtubule dynamics via regulation of their stability (Drewes et al., 1998; Riederer, 2007). MAP2 expression is essential for neurite initiation in cultured cerebellar neurons (Caceres et al., 1992) and MAP2c induces neurite formation in Neuro-2a neuroblastoma cells (Dehmelt et al., 2003). These findings underline the importance of microtubule dynamics during early processes of neuritogenesis. MAPs are known to enhance the association of microtubules by regulating the distances between microtubule polymers (Sanchez et al., 2000). The differential expression of MAP isoforms in a timely and spatially regulated manner plays a crucial role during ontogenetic development in regulating neurite outgrowth and polarization (Gonzalez-Billault et al., 2002). The present study was based on the hypothesis that, in order to initiate and maintain enhanced neurite outgrowth, L1 regulates the expression of cytoskeleton-interacting proteins and thereby influences the role of the cytoskeleton during neurite elongation. Following the observation that mRNA and protein levels of MAP2c, but not MAP2a/b, are reduced in the brain of transgenic mice deficient in L1, it seemed pertinent to investigate the functional relationship between L1 and the microtubule scaffold with the view that L1mediated neurite outgrowth depends on MAP2. Here, we show that MAP2c interacts directly with the intracellular domain of L1, while MAP2a/b appear to be only indirectly associated with L1. Interestingly, protein levels of MAP2 are up-regulated upon activation of L1 transmembrane signaling. This up-regulation is blocked by inhibition of the src family kinases and MAPK kinase, which are involved in L1mediated signaling (Herron et al., 2009). MAP2 is directly involved in L1-stimulated neurite outgrowth, since down-regulation of MAP2 expression abolishes the increase in L1-stimulated neurite outgrowth. Results MAP2 levels are reduced in L1-deficient brains To analyze whether L1 regulates the expression of proteins that may be required to maintain L1-mediated functions such as neurite outgrowth, we first utilized microarray gene chip analyses to compare mRNA levels of approximately 12,000 genes in the brains of young adult male wild-type (L1+/y) and L1− deficient (L1−/y) mice. These analyses showed that mRNA levels of several genes were significantly deregulated in L1−/y brains. Of these genes, mRNA levels of microtubule-associated protein 2 (MAP2) were most prominently down-regulated and approximately two-fold lower in L1−/y brains than in brains of L1+/y littermates (Fig. 1A). mRNA levels of several other cytoskeletal proteins including actin, tubulin, MAP1B, MAP4, MAP6 and tau were not altered in L1−/y mice, indicating that MAP2 may be specifically regulated by L1 expression (Fig. 1E). MAP2 has

been shown to be directly involved in the regulation of neurite outgrowth (Harada et al., 2002; Teng et al., 2001) and as such could be considered a likely candidate to be involved in neurite outgrowth mediated by L1. Thus, our work focused on the relationship between L1 and MAP2. To confirm and extend the gene chip data, we compared MAP2 mRNA levels from the brains of L1+/y and L1−/y littermates by quantitative PCR using primers recognizing cDNAs of MAP2a/b and MAP2c

Fig. 1. L1 regulates MAP2 expression. A: The graph shows MAP2 mRNA levels in the brains of adult L1+/y and L1−/y mice determined by microarray analysis. Values obtained for two pairs of L1+/y and L1−/y littermates are shown. In each pair, MAP2 mRNA levels in L1+/y mice were set to 1. B: The graph shows mRNA levels of MAP2a/b and MAP2c in newborn L1+/y and L1−/y mice, as determined by RT-PCR analysis. The values obtained for L1+/y mice were set to 1. Six pairs of littermates were analyzed in each group. Mean values ± SEM are shown. *p b 0.05, paired t-test. C: Brain homogenates of L1+/y and L1−/y mice were probed by Western blot analysis with antibodies against MAP2 or CHL1 used as a loading control. Both, MAP2a/b (upper band) and MAP2c (lower band) were detected with MAP2 antibodies. Note the reduced levels of MAP2c and unchanged levels of CHL1 (see also E) and MAP2a/b in adult L1−/y brain homogenates. D: The graph shows the quantification of the Western blots in C with MAP2 values normalized to CHL1 levels. The values obtained for L1+/y mice were set to 1. Three pairs of L1+/y and L1−/y littermates were analyzed. Mean values ± SEM are shown. *p b 0.05, paired t-test. E: The expression level of mRNAs of cytoskeletal proteins in L1−/− mice. mRNA expression of indicated cytoskeletal proteins was measured by microarray. Expression levels in L1+/+ mice were set to 100%. The expression of Tuba-rs1, Tuba3a, Mtap1b is below the cutoff and therefore not shown. Two independent samples were analyzed per genotype. CHL1 is listed since it was used as a loading control in C.

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mice L1 is associated with the NMDA receptor (Husi et al., 2000), which may link it to MAP2 (Buddle et al., 2003). To determine whether L1 interacts directly with the isoforms of MAP2, we analyzed whether the putative binding partners would interact directly, using purified recombinant proteins in an ELISA. MAP2c-GST, but not MAP2a/b-GST, bound to the substrate-coated intracellular domain of L1 (L1-ICD) in a concentration-dependent manner (Fig. 2C). We thus conclude that L1 binds directly to MAP2c, while interacting with MAP2a/b via intermediate binding partners. In later developmental stages of neurons, MAP2 is predominantly expressed in dendrites and L1 in axons. However in immature neurons, L1 and MAP2 are expressed in the neuronal cell body and in all neurites (Fig. 2A). This co-expression in cultured hippocampal neurons suggests a functional interaction of both proteins during early processes of neuritogenesis. Thus we used the early hippocampal neuronal cultures and brains of young mice to further investigate the relationship between MAP2 and L1.

individually. This analysis showed that MAP2c mRNA levels were approximately 2-fold lower in L1−/y brains than in L1+/y brains (Fig. 1B). In contrast, the levels of MAP2a/b mRNA were not changed in L1−/y versus L1+/y brains (Fig. 1B). We next compared MAP2 protein expression in L1−/y and L1+/y brains. Western blot analysis showed that MAP2c protein levels were approximately 2-fold lower in L1−/y brain homogenates than in those from L1+/y brain homogenates, whereas MAP2a/b levels were not different between the genotypes (Figs. 1C,D). MAP2 expression levels were normalized to the levels of CHL1, whose expression was found to be unaltered in the gene expression analysis. MAP2c interacts directly with L1 We have previously observed that the neural cell adhesion molecule NCAM binds directly to and regulates the expression of the membranecytoskeleton linker protein β1-spectrin (Leshchyns'ka et al., 2003). This prompted us to analyze whether L1, which regulates the expression of MAP2c, also associates with MAP2c. Co-labeling of 1-day-old cultured hippocampal neurons by indirect immunofluorescence with antibodies against L1 and MAP2 showed that distributions of these proteins partially overlapped along neurites and in growth cones of neurons (Fig. 2A). Western blot analysis of L1-immunoprecipitates from the brain homogenates of 2-day-old mice showed that MAP2c coimmunoprecipitates with L1 (Fig. 2B), indicating that these two proteins form a complex. Interestingly, no interaction between L1 and MAP2a/b was detected in the brain homogenates of 2-day-old mice (Fig. 2B). In the case of 2-month-old mice, both MAP2c and MAP2a/b co-immunoprecipitated with L1 from brain homogenates (Fig. 2B). Co-immunoprecipitation of two proteins does not necessarily mean that these proteins interact directly with one another since they can be linked via intermediate binding partners (Leshchyns'ka et al., 2003; Sytnyk et al., 2006). For example, in brains of adult

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Expression of MAP2 correlates with the expression of L1 in different brain regions To further investigate the connection between MAP2 and L1 expression, we analyzed MAP2 and L1 protein levels in different brain regions of postnatal day 7 (P7) wild-type mice. L1 protein levels varied significantly between brain regions, with the highest levels observed in brain stem and thalamus, and the lowest levels in the olfactory bulb (Fig. 3). The specificity of the L1 antibody has been shown previously via Western blot analysis of brain homogenates from the L1-deficient mice which do not show any L1-protein expression (Dahme et al., 1997; Law et al., 2003). A similar expression pattern among brain regions was observed for MAP2, with a correlation coefficient of 0.8 between L1 and all MAP2-isoform levels combined (Fig. 3). In contrast, the expression of actin and Hsc70 protein levels did not correlate with

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Fig. 2. L1 interacts directly with MAP2c. A: Representative example of a one-day-old cultured L1+/y hippocampal neuron labeled with antibodies against L1 and MAP2. Note that in the immature neuron, L1 and MAP2 are co-expressed and partially co-localize in putative dendrites (arrowheads) and axon (arrow). Scale bar 20 μm. B: L1 immunoprecipitates from brain lysates from the 2-day-old (P2) or adult mice were subjected to Western lot analysis with antibodies against MAP2. Note, that only the MAP2c isoform coimmunoprecipitated with L1 in brain homogenates of 2-day-old mice, while all MAP2 isoforms co-immunoprecipitated with L1 in adult mice. Mock immunoprecipitation with non-specific rabbit IgG was used as control. C: The interaction between the intracellular domain of substrate-coated L1 and GST-tagged MAP2a/b and MAP2c applied at the indicated concentrations was tested via ELISA. MAP2c, but not MAP2a/b binds to the intracellular domain of L1 in a concentration-dependent manner.

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Fig. 3. MAP2 protein levels correlate with L1 levels in different brain regions. A: Cerebellum (cer.), olfactory bulb (ol. bulb), brain stem (br. stem), thalamus (thal.), hippocampus (hip.) and pons were isolated from the brains of wild type-mice and probed by Western blot analysis with the antibodies against L1, MAP2, Hsc70 or actin. Note similar patterns of L1 and MAP2a/b and MAP2c protein level distributions in different brain regions. B: Examples of linear regression graphs comparing distributions of L1 and MAP2, L1 and Hsc70 or L1 and actin between different brain regions are shown. C: The diagram shows mean correlation coefficients (r) comparing expression levels of MAP2 and L1, Hsc70 and L1 or actin and L1 in different brain regions. In B and C, MAP2a/b and MAP2c signals were pooled and designated MAP2. The correlation between levels of L1 and MAP2 is significantly higher than between levels of L1 and Hsc70 or L1 and actin. Mean values ± SEM from 3 independent experiments are shown. *p b 0.05, paired t-test.

L1 protein levels in different brain regions showing a correlation coefficient of 0.4 (Fig. 3).

L1 enhances MAP2 expression via MAP kinase signaling Since MAP2 expression was down-regulated in the brain of L1−/y mice, we sought to obtain insights into the mechanisms by which L1 regulates MAP2 expression by investigating whether activation of L1 functions would promote MAP2 expression. L1 can be activated in its neurite outgrowth promoting functions by direct interaction with antibodies against an epitope at the transition between the second and third fibronectin type III homologous repeat in the extracellular domain of the molecule (Appel et al., 1995; Schuch et al., 1989). When these antibodies are substrate-coated, they trigger L1-mediated signaling mechanisms (Appel et al., 1995; Dong et al., 2003; Von Bohlen Und Halbach et al., 1992). We found that MAP2 protein levels in hippocampal neurons were increased 3-fold when cells were cultured for two days on substrate-coated polyclonal L1 antibodies compared to IgG controls (Fig. 4), suggesting that the activation of L1-mediated signaling promotes MAP2 expression. This increase in MAP2 protein levels exceeded the increase in neurite outgrowth triggered by L1 activation (Fig. 5C). In addition, the levels of βIII-tubulin did not alter significantly when neurons were stimulated with L1 (Fig. 4A). Thus, we conclude that increased MAP2 levels are due to increased MAP2 protein production and not indirectly related to increased neurite length. Interestingly, L1 activation promoted expression of all MAP2-isoforms (Fig. 4).

The effect of L1 activation on MAP2 protein expression was inhibited when neurons were maintained in the presence of inhibitors of the down-stream kinases activated by L1. Western blot analysis showed that application of the src family kinase inhibitor PP2 resulted in a partial, but statistically significant inhibition of L1-stimulated expression of MAP2 isoforms when normalized to the levels of a housekeeping protein Hsc70 in cell lysates (Fig. 4). Similar effects were observed when neurons were incubated with a PD98059 inhibitor, an inhibitor of MAP kinase (MAPK) kinase, MEK1 (Fig. 4), which inhibits the Ras/Raf/ MEK/ERK cascade in a variety of cell types (Alessi et al., 1995). These results indicate that both the src and MAPK pathways are required for the L1-dependent increase in MAP2 expression.

Deficiency in L1 and MAP2 results in reduced neurite outgrowth To analyze whether MAP2 plays a role in L1-mediated neurite outgrowth, cultured hippocampal neurons were co-transfected with MAP2 siRNA and GFP-expressing plasmid. Forty-eight hours after transfection, the neurite lengths of these neurons were compared to the neurite lengths of neurons co-transfected with control siRNA and GFP. Although the MAP2 siRNA used was not MAP2-isoform specific, MAP2c should primarily be affected, since it is the major MAP2-isoform expressed in neurons at this developmental stage (Crandall and Fischer, 1989) (Figs. 2B and 4A). MAP2 levels were reduced by 80% in MAP2 siRNA transfected neurons compared to control siRNA transfected or non-transfected neurons, as measured by quantitative indirect immunofluorescence

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significantly reduced in L1-deficient neurons (Fig. 6A). Since NCAM interacts with L1 in a heterophilic-cis, but not trans-interaction (Kadmon et al., 1990), L1 is most likely not responsible for a direct trans-interaction with NCAM to stimulate neurite outgrowth. It is thus likely that reduced MAP2 levels in the L1-deficient neurons are responsible for the lack of neurite outgrowth stimulation by NCAMFc (see also Figs. 5B, C). We further compared the effect of laminin stimulation on neurite outgrowth of L1−/y versus L1+/y hippocampal neurons. L1−/y neurons showed a reduced response to laminin stimulation compared to L1+/y neurons (Fig. 6B), similar to that seen in L1-Fc stimulated neurons (Fig. 6B). Discussion

Fig. 4. Activation of the MAP kinase pathway is required for L1-mediated MAP2 expression. Cultured hippocampal neurons were maintained on substrate-coated polyclonal antibodies against L1 (L1 pAb) or non-specific rabbit immunoglobulins (IgG) in the absence or presence of src protein tyrosine kinase family inhibitor PP2 (50 nM) or MAPK kinase inhibitor PD98059 (10 μM). A: Lysates of the cells were probed by Western Blot with antibodies against MAP2 or Hsc70 used as a loading control or βIII-tubulin. The amount of βIII-tubulin is not increased by L1 pAb stimulation. B: The graph shows quantification of the Western blots in A. Both MAP2a/b and MAP2c signals were combined for quantification. MAP2 signal was normalized to the Hsc70 levels and was set to 1 in the non-specific IgG treated group. Note that loading was not equal in different lanes as shown by different Hsc70 levels. Mean values ± SEM from three independent experiments are shown. *p b 0.05, paired t-test.

labeling of neurons with anti-MAP2 antibodies detecting all isoforms (Fig. 5A). To verify whether L1 mediated neurite outgrowth depended on MAP2, L1-Fc was applied to the culture medium 24 h after transfection and present for another 24 h. When applied in this manner, L1-Fc binds homophilically to L1 and heterophilically to other binding partners (e.g. integrins and fibroblast growth factor beta receptor) (Doherty et al., 1995; Kiefel et al., 2010, 2011; Meiri et al., 1998; Nishimune et al., 2005; Williams et al., 1994) at the neuronal cell surface and promotes neurite outgrowth. As expected, in control siRNA-transfected neurons, application of L1-Fc increased the length of neurites when compared to Fc-treated neurons (Figs. 5B, C). In contrast, transfection with MAP2 siRNA inhibited the L1-Fc-induced increase in neurite lengths (Figs. 5B, C), emphasizing the importance of MAP2 in L1-mediated neurite outgrowth. Interestingly, MAP2 siRNA-transfected neurons also did not respond to NCAM-Fc (Figs. 5B, C), which promoted neurite outgrowth in control siRNA-transfected neurons (Figs. 5B, C). This implies that MAP2 is necessary for neurite outgrowth stimulated not only by L1, but also by NCAM. Further evidence for this view was obtained by comparing stimulation of NCAM-mediated neurite outgrowth of L1−/y versus L1+/y hippocampal neurons (Fig. 6A) after 24 h in culture. As expected, L1-Fc treatment did not increase neurite outgrowth of L1-deficient neurons (Fig. 6A) since the homophilic L1-trans interactions are abolished. Furthermore, NCAM-Fc-mediated neurite outgrowth was

In the present study, we identified MAP2c as a novel cytoskeletal interacting partner of the intracellular domain of the neural cell adhesion molecule L1. This direct interaction provides a link between extracellular L1 stimulation and intracellular cytoskeletal rearrangements, which are required for neurite outgrowth. The application of L1 antibodies or L1-Fc triggers the L1-mediated neurite outgrowth response via activation of downstream signaling pathways (Dong et al., 2003; Maness and Schachner, 2007; Maretzky et al., 2005; Schuch et al., 1989). Since this homophilic triggering interaction leads to enhanced MAP2c expression via the src and MAPK pathways, we infer that MAP2c is necessary to promote the increased neurite outgrowth seen in L1-stimulated neurons in vitro. This view is supported by the correlated expression of L1 and MAP2 in different brain regions at early postnatal stages. We further showed that both L1 and MAP2 are necessary to stimulate L1-induced neurite outgrowth in hippocampal neurons in vitro and that MAP2c mRNA and protein levels are reduced in L1deficient mice in vivo. Taken together these findings indicate that MAP2c is a novel and important player in L1-mediated regulation of neuritogenesis. MAP2c interacts directly with L1 Using immunoprecipitation and ELISA with the purified partner molecules, we show for the first time not only an association, but also a direct interaction, respectively, of the intracellular domain of L1 with MAP2c. So far, L1 has been shown to interact with the actin cytoskeleton via ankyrin, but a direct interaction with the microtubulerelated cytoskeleton has not been implicated. Since MAP2c has been shown to induce neurite formation in N2a neuroblastoma cells (Dehmelt et al., 2003), it is likely that L1 recruits MAP2c to areas of active neurite elongation to facilitate increased neurite outgrowth. It is surprising that the intracellular domain of L1 interacts directly only with MAP2c and not MAP2a/b. The amino acid sequence of MAP2a/b is almost identical to MAP2c, except for the projection domain between the PKA-binding and the microtubule-binding domain in MAP2a/b. This projection domain is responsible for MAP2a/b's function to crosslink microtubules (Sanchez et al., 2000). Since there is no direct interaction of MAP2a/b with L1, the binding domain for L1 on MAP2c is likely to be found between the PKA-binding and microtubule-binding domain, the sequence of which is interrupted in MAP2a/b. L1 modulates MAP2 expression We showed in this study that mRNA and protein expression levels of MAP2c are reduced in L1-deficient mouse brains. In addition, we observed that a specific L1 antibody triggering L1-mediated neurite outgrowth increased the expression levels of all MAP2 isoforms (MAP2a/b/c). Because L1 regulates the activity of transcription factors, such as Sox10 (Wallace et al., 2010) and NF-kappaB (Kiefel et al., 2010, 2011), we suggest that L1 enhances transcription of MAP2 mRNA possibly even via these factors. As our data indicate, L1 stimulation of MAP2

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Fig. 5. Reduced levels of MAP2 inhibit the neurite outgrowth promoting effects of L1-Fc and NCAM-Fc. A: L1+/y cultured hippocampal neurons co-transfected with GFP together with the control siRNA or MAP2 siRNA (arrows) and labeled with antibodies against MAP2. Note reduced MAP2 labeling in MAP2 siRNA transfected neurons. Scale bar 20 μm. B, C: L1+/y cultured hippocampal neurons co-transfected with GFP together with the control siRNA or MAP2 siRNA were incubated with L1-Fc or NCAM-Fc. Representative images of transfected neurons are shown in B. Scale bar 20 μm. Lengths of the longest neurites were measured and are shown on the graph in C (mean values ± SEM, n > 60 neurons, *p b 0.05, t-test). Neurite lengths were normalized to the mean neurite length in neurons transfected with control siRNA and treated with Fc. L1-Fc and NCAM-Fc increased neurite lengths in control siRNA co-transfected neurons. L1-Fc and NCAM-Fc stimulated neurite outgrowth was inhibited in neurons co-transfected with MAP2 siRNA. Neurite outgrowth was assed 48 h after plating. Fc-constructs were present for 24 h.

expression is mediated by the non-receptor tyrosine kinase src and MAPK pathways which are the cognate signaling pathways of activated L1 and which lead to neuritogenesis (Ignelzi et al., 1994; Maness et al.,

1996). Interestingly, L1 interacts directly with src which in turn interacts directly with MAP2 (Lim and Halpain, 2000). Since src kinase activity is necessary to increase MAP2c expression and is also required for L1-

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Fig. 6. L1 deficiency inhibits the neurite outgrowth promoting effects of L1-Fc, NCAMFc, and laminin. A, B: Lengths of the longest neurites were measured in cultured hippocampal neurons derived from L1+/y and L1−/y mice that were either not treated or treated with L1-Fc or NCAM-Fc in A or L1-Fc or laminin in B. NCAM-Fc, L1-Fc and laminin increase neurite lengths in L1+/y but not in L1−/y neurons. Mean values ± SEM are shown (n > 200 neurons, *pb 0.05, t-test). Neurite lengths were normalized to the mean neurite length in L1+/y neurons treated with Fc. Neurite outgrowth was assed 24 h after plating. Fc-constructs were present for 24 h.

mediated neurite outgrowth, it is likely that a tight control of MAP2 expression contributes to L1-triggered stimulation of neurite outgrowth and is regulated in a feedback loop that comprises L1, src and MAP2c. L1 protein expression levels correlate with MAP2 protein expression levels in early postnatal mouse brains MAP2 expression levels parallel those of L1 in many brain regions in 7-day-old mice. At this stage, L1, similar to MAP2c, is initially more highly expressed in post-mitotic neurons at the time of neurite outgrowth initiation, but reduced between the second and third postnatal weeks, when most of the synaptic connections are established. In the adult, MAP2c, like L1, is expressed at high levels only in regions of postnatal neuritogenesis, suggesting that both molecules have important roles in post-developmental synaptogenesis and synaptic plasticity. It is interesting in this respect that stimulation of the NMDA glutamate receptor implicated in learning and memory as well as long-term potentiation (Grover and Yan, 1999; Harney et al., 2006) in vivo induces a neuronal activity-dependent phosphorylation of MAP2c (Sanchez et al., 1997). The cytoplasmic calcium levels required for this phosphorylation are regulated not only by neurotransmitter receptor-driven calcium influx, but also by L1-driven elevation of intracellular calcium levels (Ooashi and Kamiguchi, 2009). This would lead to a scenario where L1 not only regulates the expression of MAP2c, but also regulates synaptic plasticity in the adult via activity-induced phosphorylation of the NMDA glutamate receptor. Deficiency in L1 and MAP2 results in reduced neurite outgrowth As expected, L1-deficient neurons do not respond to L1 to stimulate neurite outgrowth, because the predominant interaction partner

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to induce outgrowth, namely L1, is absent. L1-triggered neurite outgrowth is mediated by MAP2, since neurite outgrowth is reduced by MAP2 siRNA gene silencing. Interestingly, reduced MAP2 levels also abolished NCAM-triggered neurite outgrowth, indicating that MAP2 acts as a more general mediator of neuritogenesis. Additionally, L1deficient neurons not only fail to respond to L1-triggered neurite outgrowth, but also fail to respond to NCAM-triggered neurite outgrowth. Since NCAM and L1 interact in a cis-manner (Kadmon et al., 1990), this lack of responsiveness could be related to the lack of cisinteractions between L1 and NCAM on the neuronal cell surface. It is also conceivable that reduced MAP2 levels, as a consequence of L1 deficiency, result in inhibition of NCAM-triggered neurite outgrowth. In agreement with this idea, laminin-dependent neurite outgrowth is also reduced in L1-deficient neurons, suggesting that L1 deficiency results in overall reduced ability of neurons to maintain high neurite outgrowth rates possibly related to reduced levels of MAP2. Taken together, these results indicate the importance of a regulated MAP2 expression for neurite outgrowth in vitro. Since the different isoforms of MAP2 (MAP2a/b/c) are generated by alternative splicing, it is not possible to individually silence MAP2c. The knock-down of MAP2 mRNA in these experiments should, however, affect mostly MAP2c expression, since the MAP2 isoform-specific expression is developmentally regulated and MAP2c is the prominent isoform at the age of the mice that were taken for preparation of the neuronal cell cultures. We therefore cannot rule out that MAP2a/b knock-down affects neurite outgrowth in vitro. It is important in this context that MAP2c interacts with L1 directly, but MAP2a/b only via secondary binding partners.

Concluding remarks In addition to binding and stabilizing microtubule proteins, MAP2c functions as a scaffold protein, adding another and novel dimension to its role in the context of L1-triggered neurite outgrowth. MAP2c contains 11 highly conserved PxxP motifs, which are known as SH3 binding domains. These domains interact not only with the non-receptor tyrosine kinase src, but also with the adaptor protein Grb2a (growth factor receptor binding protein 2) (Lim and Halpain, 2000). It is conceivable that other SH3-domain containing proteins besides src could interact with MAP2c and thus influence the intracellular milieu for neurite outgrowth and likely also other neurotrophic functions. Additionally, MAP2c could act as a transporter protein for src, Grb2 and possibly other proteins to direct their localization to the L1-enriched periphery of neurites, where they are most needed for modulation of growth cone motility and neurite outgrowth. This is true not only during ontogenetic development, but probably also during alterations of calciummediated synaptic activities in adulthood (Dityatev et al., 2008). Grb2 and MAPK kinase have also been shown to be involved in the signaling cascade of another transmembrane receptor with neurite growth promoting functions namely the receptor protein tyrosine phosphatase κ (RPTPκ). RPTPκ stimulates neurite outgrowth of cultured cerebellar granular neurons which is greatly dependent on Grb2/MAPK signaling (Drosopoulos et al., 1999). Similar to our findings, MAPc might be also involved in the regulation of the RPTPκ mediated neurite outgrowth stimulation. It is noteworthy in this context that MAP2 turnover is necessary for fear-dependent learning (Fanara et al., 2010) and that L1 expression is upregulated following fear-related memory formation (Merino et al., 2000). Since the expression of L1 and MAP2 is interrelated and functionally connected they would also control some aspects of synaptic plasticity. Further studies will have to investigate the link between L1-driven elevation of intracellular calcium levels leading to MAP2c phosphorylation, NMDA glutamate receptor activation, and their combined roles in memory formation and in recovery from nervous system trauma.

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Experimental methods

Quantitative real-time PCR

Antibodies and inhibitors

Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions and followed by RNA-purification using the RNeasy Mini Kit (Qiagen). The RNA was reverse-transcribed with SuperScript II RNase H Reverse Transcriptase (Invitrogen) using random hexamer primers. The qRT-PCR was performed using the following primers: for MAP2a/b forward: 5′-GATCAAGCCTCCACCAAAGAACTG-3′, and reverse: 5′-CTTTCCCAGCATCTACATTCA-3′; for MAP2c/d forward: 5′-AGTGGAGGAAGCAGCAAGTGGTGACT-3′, and reverse: 5′-GAGGAGGGAGGATGGAGGAAGGTCT-3′. The obtained signal was normalized to the hypoxanthine guanine phosphoribosyl transferase (HPRT) mRNA levels detected with the following primers: forward: 5′-GTTCTTTGCTGACCTGCTGGA-3′ and reverse: 5′-TCCCCCGTTGACTGATCATT-3′.

Rabbit polyclonal antibody against the extracellular domain of L1 (Appel et al., 1995; Richard et al., 2005) was used as described. Mouse monoclonal antibody against MAP2, rabbit polyclonal antibody against actin and non-specific rabbit IgG were obtained from Sigma-Aldrich (St. Louis, MO, USA). Goat polyclonal antibody against Hsc70 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies against rabbit and mouse Ig coupled to horse radish peroxidase (HRP) and human Fc were obtained from Dianova (Hamburg, Germany). Src family protein tyrosine kinase inhibitor PP2 and MAPK kinase inhibitor PD98059 were obtained from Calbiochem (San Diego, CA, USA). Animals

Co-immunoprecipitation

L1 expressing (wild-type, L1+/y) and L1-deficient (L1−/y) male littermates obtained from heterozygous breeding were used for all experiments. L1-deficient mice were generated by insertion of a tetracycline-controlled transactivator into the second exon of the L1 gene (Loers et al., 2005).

Homogenates from adult mouse brains were prepared in 50 mM Tris–HCl buffer, pH 7.5, containing 1 mM of CaCl2, 1 mM MgCl2, and 1 mM NaHCO3. Samples containing 1 mg of total protein were lysed for 40 min at 4 °C with lysis buffer, pH 7.5, containing 50 mM Tris– HCl, 150 mM NaCl, 1% (vol/vol) Nonidet P-40, 1 mM Na2P2O7, 1 mM NaF, 1 mM EDTA, 2 mM NaVO4, 0.1 mM PMSF and Complete Protease Inhibitor Cocktail (Roche Diagnostics, Basel, Switzerland), and centrifuged for 15 min at 20,000 g at 4 °C. Supernatants were cleared with protein A/G-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (3 h at 4 °C) and incubated with specific antibodies or non-specific control Ig (overnight, 4 °C), followed by precipitation with protein A/G-agarose beads (1 h, 4 °C). The beads were washed 3 times with lysis buffer, 2 times with PBS and boiled in Laemmli buffer. Eluted material was then used for Western blot analysis.

DNA and siRNA The GFP plasmid was purchased from Clontech (Palo Alto, CA, USA). Control and MAP2 siRNAs were synthesized at Qiagen (Hilden, Germany). Microarray analysis Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Further purification of RNA was performed with the RNeasy Mini Kit (Qiagen). Total RNA concentration was determined using a spectrophotometer at 260 nm and 280 nm wavelength. To check RNA integrity, total RNA was separated on a 1% (wt/vol) agarose gel and the intensity ratio of 28S and 18S ribosomal RNA bands was assessed after ethidium bromide staining. Procedures for cDNA synthesis, cRNA in vitro transcription, labeling and hybridization were carried out according to the manufacturer's protocol (Affymetrix, Santa Clara, CA, USA). All experiments were performed using Affymetrix mouse genome GeneChip U74A version 2_A. In brief, 15 μg of total RNA was reverse transcribed into cDNA using the SuperScript First Strand Synthesis System for RT-PCR (Invitrogen) with an HPLC-purified T7-(dT)24 primer. Synthesis of biotin-labeled cRNA was carried out using the ENZO RNA transcript labeling kit (ENZO, Farmingdale, NY, USA). For hybridization, 15 μg of fragmented cRNA was incubated with the chip in 200 μl of hybridization solution in a Hybridization Oven 640 (Affymetrix) at 45 °C for 16 h. GeneChips were then washed and stained using the Affymetrix Fluidics Station according to the GeneChip Expression Analysis Technical Manual. Microarrays were scanned with the Hewlett-Packard-Agilent GeneChip scanner (Agilent, Santa Clara, CA, USA), and the signals were processed using the GeneChip expression analysis algorithm v.2 (Affymetrix). To compare samples and experiments, the trimmed mean signal of each array was scaled to a target intensity of 100. Absolute and comparison analyses were performed with Affymetrix MAS 5.0 and DMT software using default parameters. To assist in the identification of genes that were found to be positively or negatively regulated in their expression, we selected genes that were increased or decreased in L1−/y mice by at least a factor of 1.7 when compared to the L1+/y mice. Annotations were further analyzed with interactive query analysis at www.affymetrix.com. Pathways and other functional groupings of genes were evaluated for differential regulation using the visualization tool GenMAPP (UCSF, San Francisco, CA, USA) as described previously (Bonner et al., 2003; Doniger et al., 2003).

Western blot analysis Proteins were separated on a 6–10% (vol/vol) SDS-PAGE gel and electroblotted onto Nitrocellulose Transfer Membrane (PROTRAN; Schleicher & Schuell, Dassel, Germany) for 3 h at 250 mA. Immunoblots were incubated with appropriate primary antibodies (MAP2 1 μg/ml, βIII-tubulin 0.5 μg/ml, CHL1 1 μg/ml, L1 1 μg/ml, Hsc70 0.1 μg/ml, actin 0.1 μg/ml) followed by incubation with HRP-labeled secondary antibodies and visualized using Super Signal West Pico reagents (Pierce, Rockford, IL, USA) or ECL reagent (Amersham Bioscience, Buckinghamshire, UK) on BIOMAX films (Sigma). Molecular weight markers were obtained from Bio-Rad (Hercules, CA, USA). All preparations of proteins were performed three times and at least two Western blots were performed on individual protein samples (n ≥ 6). The quantification of chemiluminescence was performed using TINA 2.09 software (University of Manchester, UK) or Scion Image for Windows (National Institute of Standards and Technology, Gaithersburg, MD, USA)

ELISA Proteins were purified via their His-tag (for the intracellular domain of L1 (L1-ICD)) or GST-tag (MAP2a/b and MAP2c). Ten micrograms per milliliter L1-ICD was coated onto a 96-well ELISA plate (Nunc, Langenselbold, Germany) overnight at 4 °C. The next day the GST-tagged proteins or GST as negative control were added in the following concentrations (in μg/ml): 100, 50, 25, 12.5, 6.25, 3.125 and 1.5625. The ELISA plates were incubated with anti-GST antibody (Pharmacia Biotech, GE Healthcare Biosciences, Pittsburgh, PA, USA) followed by incubation with HRP-labeled secondary antibody. The ABTS absorbance was determined via a μQuant ELISA-reader (BIOTEK, Winooski, VT, USA).

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Culture and transfection of hippocampal neurons Cultures of hippocampal neurons were prepared from 1- to 3-dayold mice. For neurite outgrowth experiments, neurons were grown in 10% (vol/vol) horse serum on glass coverslips coated with poly-Llysine (100 μg/ml) (Leshchyns'ka et al., 2003). Six hours after plating, neurons were co-transfected with siRNA and GFP (used as a marker of transfected neurons) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Human Fc (7 μg/ml), L1-Fc (7 μg/ml), or NCAM-Fc (7 μg/ml) was applied to the neurons in culture medium for 24 h. When non-transfected neurons were analyzed, these reagents were applied immediately after plating. When cotransfected neurons were analyzed, Fc-fusion proteins and Fc as a negative control were applied 24 h after transfection. Neurite outgrowth was assayed as described (Santuccione et al., 2005). For biochemical experiments, neurons were maintained in 24 well plates (Falkon, BD Biosciences, Franklin Lakes, NJ, USA) coated with poly-Llysine (100 μg/ml) (Sigma), in conjunction with polyclonal antibodies against L1 (10 μg/ml) or non-specific rabbit IgG (10 μg/ml). Neurons were treated with 10 μM PD98059, an inhibitor of MAPK kinase (Alessi et al., 1995; Loers et al., 2005), and 50 nM PP2, a src family kinase inhibitor (Loers et al., 2005). Immunofluorescence labeling Immunolabeling was performed as described previously (Chernyshova et al., 2011). All steps were performed at room temperature and all antibodies were applied in 0.1% bovine serum albumin (BSA) in PBS. Neurons were fixed for 15 min in 4% formaldehyde in PBS, washed with PBS, permeabilized with 0.25% Triton X-100 in PBS for 5 min, blocked with 1% BSA in PBS for 20 min, and treated with primary antibodies for 2 h followed by corresponding secondary antibodies applied for 45 min. Coverslips were embedded in AquaPoly/Mount (Polysciences). Images were acquired at room temperature using a confocal laser scanning microscope Nikon C1si, NIS elements software, and 60× objective with numerical aperture 1.4 (all from Nikon). Acknowledgments We thank Peggy Putthoff, Achim Dahlmann and Eva Kronberg for genotyping and animal care and Yong Wee Wong for discussions. We further thank Andrew Matus for the supply of materials. This work was supported by Zonta Club Hamburg-Alster (Iryna Leshchyns'ka) and the Deutsche Forschungsgemeinschaft (Ann-Kathrin Tranziska). Melitta Schachner is supported by the New Jersey Commission for Spinal Cord Research and is a consultant at the Center for Neuroscience at Shantou University Medical College. References Alessi, D.R., Cuenda, A., Cohen, P., Dudley, D.T., Saltiel, A.R., 1995. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J. Biol. Chem. 270, 27489–27494. Andreyeva, A., Leshchyns'ka, I., Knepper, M., Betzel, C., Redecke, L., Sytnyk, V., Schachner, M., 2010. CHL1 is a selective organizer of the presynaptic machinery chaperoning the SNARE complex. PLoS One 5, e12018. Appel, F., Holm, J., Conscience, J.F., von Bohlen und Halbach, F., Faissner, A., James, P., Schachner, M., 1995. Identification of the border between fibronectin type III homologous repeats 2 and 3 of the neural cell adhesion molecule L1 as a neurite outgrowth promoting and signal transducing domain. J. Neurobiol. 28, 297–312. Blaess, S., Kammerer, R.A., Hall, H., 1998. Structural analysis of the sixth immunoglobulin-like domain of mouse neural cell adhesion molecule L1 and its interactions with alpha(v)beta3, alpha(IIb)beta3, and alpha5beta1 integrins. J. Neurochem. 71, 2615–2625. Bonner, A.E., Lemon, W.J., You, M., 2003. Gene expression signatures identify novel regulatory pathways during murine lung development: implications for lung tumorigenesis. J. Med. Genet. 40, 408–417. Buddle, M., Eberhardt, E., Ciminello, L.H., Levin, T., Wing, R., DiPasquale, K., RaleySusman, K.M., 2003. Microtubule-associated protein 2 (MAP2) associates with

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