Munc18 and Munc13 regulate early neurite outgrowth

Background information. During development, growth cones of outgrowing neurons express proteins involved in vesicular secretion, such as SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins, Munc13 and Munc18. Vesicles are known to fuse in growth cones prior to synapse formation, which may contribute to outgrowth

Integrin Signaling Switches the Cytoskeletal and Exocytic Machinery that Drives Neuritogenesis

Neurons establish their unique morphology by elaborating multiple neurites that subsequently form axons and dendrites. Neurite initiation entails significant surface area expansion, necessitating addition to the plasma membrane. We report that regulated membrane delivery coordinated with the actin cytoskeleton is crucial for neuritogenesis, and identify two independent pathways that use distinct exocytic and cytoskeletal machinery to drive neuritogenesis. One pathway employs Ena/VASP-regulated actin dynamics coordinated with VAMP2-mediated exocytosis, and involves a novel role for Ena/VASP in exocytosis. A second mechanism occurs in the presence of laminin through integrin-dependent activation of FAK and src, and utilizes coordinated activity of the Arp2/3 complex and VAMP7-mediated exocytosis. We conclude that neuritogenesis can be driven by two distinct pathways that differentially coordinate cytoskeletal dynamics and exocytosis. These regulated changes and coordination of cytoskeletal and exocytic machinery may be utilized in other physiological contexts involving cell motility and morphogenesis.Jane Coffin Childs Memorial Fund for Medical ResearchNational Institutes of Health (U.S.) (NIH grant GM68678)Stanley Medical Research Institut

Clostridial Neurotoxins: Mechanism of SNARE Cleavage and Outlook on Potential Substrate Specificity Reengineering

The clostridial neurotoxin family consists of tetanus neurotoxin and seven distinct botulinum neurotoxins which cause the diseases tetanus and botulism. The extreme potency of these toxins primarily relies not only on their ability to specifically enter motoneurons but also on the activity their catalytic domains display inside presynaptic motoneuronal terminals. Subsequent to neurotoxin binding and endocytosis the catalytic domains become translocated across endosomal membranes and proteolyze unique peptide bonds of one of three soluble N-ethylmaleimide-sensitive fusion protein attachment receptors (SNAREs), vesicle associated membrane protein/synaptobrevin, synaptosome associated protein of 25 kDa, or syntaxin. As these substrate proteins are core components of the vesicular membrane fusion apparatus, cleavage of any of the substrate molecules results in the blockade of neurotransmitter release. This review summarizes the present knowledge about the molecular basis of the specific substrate recognition and cleavage mechanism and assesses the feasibility of reengineering catalytic domains to hydrolyze non-substrate members of the three SNARE families in order to expand the therapeutic application of botulinum neurotoxins

Differential distribution of stathmin and SCG10 in developing neurons in culture.

The neuron-specific protein SCG10 and the ubiquitous protein stathmin are two members of a family of microtubule-destabilizing factors that may regulate microtubule dynamics in response to extracellular signals. To gain insight into the function of these proteins in the nervous system, we have compared their intracellular distribution in cortical neurons developing in culture. We have used double-immunofluorescence microscopy with specific antibodies for stathmin and SCG10 in combination with antibodies for axonal, microtubule, and synaptic marker proteins. Stathmin and SCG10 were coexpressed in individual neurons. While both proteins were highly expressed in developing cultures during differentiation, their subcellular localization was strikingly different. Stathmin showed a cytosolic distribution, mainly in cell bodies, whereas SCG10 strongly labeled the growth cones of axons and dendrites. During neurite outgrowth, SCG10 appeared as a single concentrated spot in a region of the growth cone where the microtubules are known to be particularly dynamic. Disassembly of labile microtubules by nocodazole caused a dispersal of the SCG10 staining into punctate structures, indicating that its subcellular localization is microtubule-dependent. Upon maturation and synapse formation, the levels of both stathmin and SCG10 decreased to become undetectable. These observations demonstrate that the expression of both proteins is associated with neurite outgrowth and suggest that they perform their roles in this process in distinct subcellular compartments