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Small-intestinal neuroendocrine tumors (SI-NETs) are defined as locally invasive only after extension to the muscularis propria. To gain further insight into the molecular mechanisms, we applied a proteomic approach to an orthotopic xenograft model to identify candidate proteins evaluable in human SI-NETs. After grafting STC-1 neuroendocrine tumor cells on the caecum of nude mice, comparative proteomic studies were performed between the pre-invasive and the invasive stages, respectively 2 and 8 weeks after grafting. We identified 24 proteins displaying at least a 1.5-fold differential expression between 2 and 8 week-stages. Most were cytoskeleton-associated proteins, among which five showed decreasing expression levels (CRMP2, TCP1e, TPM2, vimentin, desmin) and two increasing expression levels (14-3-3γ, CK8). Changes for CRMP2, TCP1e, TPM2 and 14-3-3γ were confirmed in experimental tumors and in a series of 28 human SI-NETs. In conclusion, our results underline the relevance of proteomics to identify novel biomarkers of tissue invasion.

International audience; The collapsin response-mediator proteins (CRMPs) are multifunctional proteins highly expressed during brain development but down-regulated in the adult brain. They are involved in axon guidance and neurite outgrowth signalling. Among these, the intensively studied CRMP2 has been identified as an important actor in axon outgrowth, this activity being correlated with the reorganisation of cytoskeletal proteins via the phosphorylation state of CRMP2. Another member, CRMP5, restricts the growth-promotional effects of CRMP2 by inhibiting dendrite outgrowth at early developmental stages. This inhibition occurs when CRMP5 binds to tubulin and the microtubule-associated protein MAP2, but the role of CRMP5 phosphorylation is still unknown. Here, we have studied the role of CRMP5 phosphorylation by mutational analysis. Using non-phosphorylatable truncated constructs of CRMP5 we have demonstrated that, among the four previously identified CRMP5 phosphorylation sites (T509, T514, T516 and S534), only the phosphorylation at T516 residue was needed for neurite outgrowth inhibition in PC12 cells and in cultured C57BL/6J mouse hippocampal neurons. Indeed, the expression of the CRMP5 non-phosphorylated form induced a loss of function of CRMP5 and the mutant mimicking the phosphorylated form induced the growth inhibition function seen in wildtype CRMP5. The T516 phosphorylation was achieved by the glycogen synthase kinase-3β (GSK-3β), which can phosphorylate the wildtype protein but not the non-phosphorylatable mutant. Furthermore, we have shown that T516 phosphorylation is essential for the tubulin-binding property of CRMP5. Therefore, CRMP5-induced growth inhibition is dependent on T516 phosphorylation through the GSK-3β pathway. The findings provide new insights into the mechanisms underlying neurite outgrowth.

International audience; In the central nervous system, collapsin response mediator protein 2 (CRMP2) is a transducer protein that supports the semaphorin-induced guidance of axons toward their cognate target. However, we previously showed that CRMP2 is also expressed in immune cells and plays a crucial role in T lymphocyte migration. Here we further investigated the molecular mechanisms underlying CRMP2 function in chemokine-directed T-cell motility. Examining Jurkat T-cells treated with the chemokine CXCL12, we found that 1) CXCL12 induces a dynamic re-localization of CRMP2 to uropod, the flexible structure of migrating lymphocyte, and increases its binding to the cytoskeletal protein vimentin; 2) CXCL12 decreases phosphorylation of the glycogen synthase kinase-3beta-targeted residues CRMP2-Thr-509/514; and 3) tyrosine Tyr-479 is a new phosphorylation CRMP2 residue and a target for the Src-family kinase Yes. Moreover, phospho-Tyr-479 increased under CXCL12 signaling while phospho-Thr-509/514 decreased. The functional importance of this tyrosine phosphorylation was demonstrated by Y479F mutation that strongly reduced CXCL12-mediated T-cell polarization and motility as tested in a transmigration model and on neural tissue. We propose that differential phosphorylation by glycogen synthase kinase-3beta and Yes modulates the contribution of CRMP2 to cytoskeletal reorganization during chemokine-directed T-cell migration. In addition to providing a novel mechanism for T lymphocyte motility, our findings reveal CRMP2 as a transducer of chemokine signaling.

International audience; BACKGROUND: During development, neuronal growth cones integrate diffusible and contact guidance cues that are conveyed to both actin and microtubule (MT) cytoskeletons and ensure axon outgrowth and pathfinding. Although several post-translational modifications of tubulin have been identified and despite their strong conservation among species, their physiological roles during development, especially in the nervous sytem, are still poorly understood. METHODOLOGY/FINDINGS: Here, we have dissected the role of a post-translational modification of the last amino acid of the alpha-tubulin on axonal growth by analyzing the phenotype of precerebellar neurons in Tubulin tyrosin ligase knock-out mice (TTL(-/-)) through in vivo, ex vivo and in vitro analyses. TTL(-/-) neurons are devoid of tyrosinated tubulin. Their pathway shows defects in vivo, ex vivo, in hindbrains open-book preparations or in vitro, in a collagen matrix. Their axons still orient toward tropic cues, but they emit supernumerary branches and their growth cones are enlarged and exhibit an emission of mis-oriented filopodia. Further analysis of the TTL(-/-) growth cone intracellular organization also reveals that the respective localization of actin and MT filaments is disturbed, with a decrease in the distal accumulation of Myosin IIB, as well as a concomitant Rac1 over-activation in the hindbrain. Pharmacological inhibition of Rac1 over-activation in TTL(-/-) neurons can rescue Myosin IIB localization. CONCLUSIONS/SIGNIFICANCE: In the growth cone, we propose that tubulin tyrosination takes part in the relative arrangement of actin and MT cytoskeletons, in the regulation of small GTPases activity, and consequently, in the proper morphogenesis, organization and pathfinding of the growth cone during development.

Collapsin Response Mediator Protein (CRMP)-2 is involved in T-cell polarization and migration. To address the role of CRMP2 in neuroinflammation, we analyzed its involvement in lymphocyte recruitment to the central nervous system in mouse infected with neurotropic and non-neurotropic virus strains (RABV, CDV). A sub-population of early-activated CD69+CD3+ T lymphocytes highly expressing CRMP2 (CRMP2hi) peaked in the blood, lymph nodes and brain of mice infected with neurotropic viruses, and correlated with severity of disease. They displayed high migratory properties reduced by CRMP2 blocking antibody. These data point out the potential use of CRMP2 as a peripheral indicator of neuroinflammation.

During development, microtubule-associated protein 1B (MAP1B) is one of the earliest MAPs, preferentially localized in axons and growth cones, and plays a role in axonal outgrowth. Although generally downregulated in the adult, we have shown that MAP1B is constitutively highly expressed in adult dorsal root ganglia (DRGs) and associated with central sprouting and peripheral regeneration of these neurons. Mutant mice with a complete MAP1B null allele that survive until adulthood exhibit a reduced myelin sheath diameter and conductance velocity of peripheral axons and lack of the corpus callosum. Here, to determine the function of MAP1B in axonal regeneration, we used cultures of adult DRG explants and/or dissociated neurons derived from thismap1b-/- mouse line. Whereas the overall length of regenerating neurites lacking MAP1B was similar to wild-type controls, our analysis revealed two main defects. First,map1b-/- neurites exhibited significantly (twofold) higher terminal and collateral branching. Second, the turning capacity of growth cones (i.e., “choice” of a proper orientation) was impaired. In addition, lack of MAP1B may affect the post-translational modification of tubulin polymers: quantitative analysis showed a reduced amount of acetylated microtubules within growth cones, whereas the distribution of tyrosinated or detyrosinated microtubules was normal. Both growth cone turning and axonal branch formation are known to involve local regulation of the microtubule network. Our results demonstrate that MAP1B plays a role in these processes during plastic changes in the adult. In particular, the data suggest MAP1B implication in the locally coordinated assembly of cytoskeletal components required for branching and straight directional axon growth.