Motohiro Nozumi

Identification of functional marker proteins in the mammalian growth cone

Identification of proteins in the mammalian growth cone has the potential to advance our understanding of this critical regulator of neuronal growth and formation of neural circuit; however, to date, only one growth cone marker protein, GAP-43, has been reported. Here, we successfully used a proteomic approach to identify 945 proteins present in developing rat forebrain growth cones, including highly abundant, membrane-associated and actin-associated proteins. Almost 100 of the proteins appear to be highly enriched in the growth cone, as determined by quantitative immunostaining, and for 17 proteins, the results of RNAi suggest a role in axon growth. Most of the proteins we identified have not previously been implicated in axon growth and thus their identification presents a significant step forward, providing marker proteins and candidate neuronal growth-associated proteins.

Expression and function of neuronal growth-associated proteins (nGAPs) in PC12 cells

The growth cone plays crucial roles in neural wiring, synapse formation, and axonal regeneration. Continuous rearrangement of cytoskeletal elements and targeting of transported vesicles to the plasma membrane are essential to growth cone motility; however, the proteins directly involved in these processes and their specific functions are not well established. We recently identified 17 proteins as functional marker proteins of the mammalian growth cone and as neuronal growth-associated proteins in rat cortical neurons (nGAPs; Nozumi et al., 2009). To determine whether these 17 proteins are growth cone markers in other neuronal cell types, we examined their expression and function in PC12D cells. We found that all 17 nGAPs were highly concentrated in the growth cones of PC12D cells, and that knockdown of all of them by RNAi reduced or inhibited neurite outgrowth, indicating that all of the 17 nGAPs may be general growth cone markers. Among them, eight proteins were shown to regulate the amount of F-actin in PC12D growth cones. Two of these nGAP that are cytoskeletal proteins, Cap1 and Sept2, increased the mean growth cone area and the mean neurite length by regulating the amount of F-actin; Sept2 also induced filopodial growth. Taken together, our data suggested that some of the nGAPs were generalized markers of the growth cone in multiple neuronal cell types and some of them, such as Cap1 and Sept2, regulated growth cone morphology through rearrangement of F-actin and thereby controlled neurite outgrowth.

Extracellular Signals Induce Glycoprotein M6a Clustering of Lipid Rafts and Associated Signaling Molecules

Lipid raft domains, where sphingolipids and cholesterol are enriched, concentrate signaling molecules. To examine how signaling protein complexes are clustered in rafts, we focused on the functions of glycoprotein M6a (GPM6a), which is expressed at a high concentration in developing mouse neurons. Using imaging of lipid rafts, we found that GPM6a congregated in rafts in a GPM6a palmitoylation-dependent manner, thereby contributing to lipid raft clustering. In addition, we found that signaling proteins downstream of GPM6a, such as Rufy3, Rap2, and Tiam2/STEF, accumulated in lipid rafts in a GPM6a-dependent manner and were essential for laminin-dependent polarity during neurite formation in neuronal development. In utero RNAi targeting of GPM6a resulted in abnormally polarized neurons with multiple neurites. These results demonstrate that GPM6a induces the clustering of lipid rafts, which supports the raft aggregation of its associated downstream molecules for acceleration of neuronal polarity determination. Therefore, GPM6a acts as a signal transducer that responds to extracellular signals. SIGNIFICANCE STATEMENT Lipid raft domains, where sphingolipids and cholesterol are enriched, concentrate signaling molecules. We focused on glycoprotein M6a (GPM6a), which is expressed at a high concentration in developing neurons. Using imaging of lipid rafts, we found that GPM6a congregated in rafts in a palmitoylation-dependent manner, thereby contributing to lipid raft clustering. In addition, we found that signaling proteins downstream of GPM6a accumulated in lipid rafts in a GPM6a-dependent manner and were essential for laminin-dependent polarity during neurite formation. In utero RNAi targeting of GPM6a resulted in abnormally polarized neurons with multiple neurites. These results demonstrate that GPM6a induces the clustering of lipid rafts, which supports the raft aggregation of its associated downstream molecules for acceleration of polarity determination. Therefore, GPM6a acts as a signal transducer that responds to extracellular signals.

A stochastic model of neuronal growth cone guidance regulated by multiple sensors.

Neuronal growth cones migrate directionally under the control of axon guidance molecules, thereby forming synapses in the developing brain. The signal transduction system by which a growth cone detects surrounding guidance molecules, analyzes the detected signals, and then determines the overall behavior remains undetermined. In this study, we describe a novel stochastic model of this behavior that utilizes multiple sensors on filopodia to respond to guidance molecules. Overall growth cone behavior is determined by using only the concentration gradients of guidance molecules in the immediate vicinity of each sensor. The detected signal at each sensor, which is treated as a vector quantity, is sent to the growth cone center and then integrated to determine axonal growth in the next step by means of a simple vector operation. We compared the results of computer simulations of axonal growth with observations of actual axonal growth from co-culture experiments using olfactory bulb and septum. The probabilistic distributions of axonal growth generated by the computer simulation were consistent with those obtained from the culture experiments, indicating that our model accurately simulates growth cone behavior. We believe that this model will be useful for elucidating the as yet unknown mechanisms responsible for axonal growth in vivo.

A novel method for RNA interference in neurons using enhanced green fluorescent protein (EGFP)-transgenic rats.

RNA interference (RNAi) is the simplest way of examining gene function by inhibiting expression. However, due to the low rate of introducing short interfering RNA (siRNA) into neurons, it is difficult to discriminate into which neurons that have been successfully introduced. Here, we used neurons from transgenic rats expressing enhanced green fluorescent protein (EGFP), and we simultaneously applied small interfering RNAs (siRNAs) against EGFP and a targeted gene to the EGFP-expressing neurons. EGFP fluorescence and immunoreactivity of the protein were then assessed by immunofluorescence microscopy. Quantitative analysis of the immunofluorescence confirmed that loss of EGFP closely correlates with loss of the target protein. These results indicate that this method can be used in a wider range of the neuroscientific research, especially in genome-wide studies.

Vesicular movements in the growth cone

ABSTRACT Growth cones, which are the highly motile tips of extending neuronal processes in developing neurons, have many vesicles. These vesicles are likely essential for the membrane expansion that is required for nerve growth, and probably coordinate with rearrangement of the cytoskeletons. Such mechanisms are poorly understood from molecular and cell biological aspects. Recently, we used superresolution microscopic approaches and described new mechanisms that are involved in the interaction between the vesicles and F‐actin in the leading edge of the peripheral domain. Vesicles mainly accumulate in the central domain of growth cones. However, the dynamics of vesicles in each domain, for example, clathrin dependency, are totally distinct from each other. Here, we discuss the diversity of the dynamics of vesicular and related proteins that play different roles in nerve growth. HighlightsWe provide an overview of the vesicles enriched in growth cones.These vesicles are involved in axon growth and interact with the cytoskeleton.Vesicles in the P‐domain, but not the C‐domain, are linked to F‐actin.Superresolution microscopy reveals a new mechanism for vesicles in the leading edge.

Growth Cone Phosphoproteomics Reveals that GAP-43 Phosphorylated by JNK Is a Marker of Axon Growth and Regeneration

Summary Neuronal growth cones are essential for nerve growth and regeneration, as well as for the formation and rearrangement of the neural network. To elucidate phosphorylation-dependent signaling pathways and establish useful molecular markers for axon growth and regeneration, we performed a phosphoproteomics study of mammalian growth cones, which identified >30,000 phosphopeptides of ∼1,200 proteins. The phosphorylation sites were highly proline directed and primarily MAPK dependent, owing to the activation of JNK, suggesting that proteins that undergo proline-directed phosphorylation mediate nerve growth in the mammalian brain. Bioinformatics analysis revealed that phosphoproteins were enriched in microtubules and the cortical cytoskeleton. The most frequently phosphorylated site was S96 of GAP-43 (growth-associated protein 43-kDa), a vertebrate-specific protein involved in axon growth. This previously uncharacterized phosphorylation site was JNK dependent. S96 phosphorylation was specifically detected in growing and regenerating axons as the most frequent target of JNK signaling; thus it represents a promising new molecular marker for mammalian axonal growth and regeneration.