+TIPS AND SYNAPSES · Role of microtubule plus-end tracking proteins in synaptogenesis

Final Activity Report Summary - +TIPS AND SYNAPSES (Role of microtubule plus-end tracking proteins in synaptogenesis)

Microtubules (MTs) are essential components of the neuronal cytoskeleton. MTs are reorganised in response to extracellular signals during neurite outgrowth and synapse formation/stability. 'Plus-end tracking'? proteins, or +TIPs, specifically associate with the distal end of MTs. Certain +TIPs, like Cytoplasmic-linker associated proteins or CLASPs, stabilise specific subsets of MTs upon reception of signalling cues in particular cell regions. Furthermore, CLASP2 is predominantly expressed in brain, with the CLASP2 isoform appearing to be brain-specific. CLASP is highly enriched in growth cones. CLASPs are therefore good candidates as local subcellular regulators of the 'fine-tuning' of MT dynamics during neurite extension and synapse formation and stabilisation. Therefore, we focused our investigation in the elucidation of the role of CLASPs in neuritogenesis and synaptogenesis. We also addressed how distinct signalling pathways, involving the kinase GSK-3, lead to CLASP-mediated local rearrangements of MTs in neuronal cells. For these purposes, we have used primary hippocampal neurons from CLASP2 knockout mice as well as neuroblastoma cell lines. Immunofluorescence studies show that CLASP2 deficient neurons develop axons earlier than wild-type littermate neurons. CLASP2-/- neurons develop longer axons and dendrites, and the axons are more branched than in wilr-type neurons. CLASP2 deficient neurons form synapses in culture; however, preliminary data indicate that CLASP2-/- neurons present fewer synapses than wild-type neurons. Taken together these data suggest that CLASP2 might play a role in neurite extension and branching and in synapse formation.By the use of standard immunofluorescence, immunofluorescence time-lapse microscopy and FRAP techniques, we have shown that CLASP1 and CLASP2 proteins are differentially regulated by GSK-3-mediated phosphorylation in N1E-115 neuroblastoma cells. GSK-3 inhibition by different compounds (Lithium, SB21673) leads to a prominent increase of CLASP2 binding to MT-plus ends, not so marked in CLASP1. Insulin and IGF-1 signal through PI3-Kinase/PKB leading to GSK-3 inhibition. In N1E-115 cells, both growth factors lead to lamellipodia formation and CLASP increased binding to MT-plus ends, which are relocalised to the lamellipodia. Based on pharmacological treatments with different inhibitors, Insulin/IGF-1 increase in CLASP binding to MTs is mediated by PI3-K signalling but is independent on the integrity of actin cytoskeleton. In summary our data suggest that the function of CLASP proteins regulated to different extents downstream of the Insulin-IGF-1/PI3-K/PKB/GSK-3 signalling pathways in neuroblastoma cells.

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