hey mark! keep up the great work!
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tena
Frontiers June 2006
LC-PUFAs Hold Key to Neuron Growth: AA and DHA Activate Syntaxin 3
Understanding how long-chain polyunsaturated fatty acids (LC-PUFAs) work involves tracking the footprints of molecules in and out of cells. In brain neurons, high concentrations of arachidonic acid (AA) and docosahexaenoic acid (DHA), members of the omega-6 (n-6) and omega-3 (n-3) families of PUFAs, reside in cell membranes. Their presence affects the fluidity and biophysical characteristics of membranes and the ways different regions of the membrane behave. These influences, in turn, affect cell signaling, the process by which a cell translates incoming messages into action. AA and DHA are critical elements in neuronal cell signaling.
AA and DHA do much of their work after they are released from the membrane by the action of phospholipase A2. Their release is essential for the growth of neurons during development and regeneration. Recently, Frédéric Darios and Bazbek Davletov of the Medical Research Council Laboratory of Molecular Biology, Cambridge, U.K., elucidated more precisely how AA and DHA affect neuron growth. These fatty acids activate or “turn on” a plasma membrane receptor protein, syntaxin 3, a kind of docking site for intracellular transport vesicles to discharge their cargo. The classic example is the release of neurotransmitters contained in vesicles at nerve endings. Release of neurotransmitters requires fusion of the vesicle membrane with the plasma membrane so that the contents can be discharged into the synaptic space. Syntaxins, a family of transmembrane proteins rich in neuronal plasma membranes, are required for the fusion of intracellular vesicular membranes with the plasma membrane. In neurites, the growing branches of neurons, this fusion process allows the plasma membrane to grow.
As neurons develop they send out many projections or neurites, whose growth is guided by growth cones at the tips (Figure 1). Growth of these projections requires the manufacture of large amounts of new membrane, various growth factors, such as nerve growth factor, and the activation of the enzyme phospholipase A2, enriched in nerve growth cones. Darios and Davletov have shown how AA and DHA released from nerve growth cones interact with specific plasma membrane proteins to facilitate neurite growth.
Using neuroendocrine PC12 cells, a model for nerve growth, the investigators showed that nerve growth factor stimulated growth in cells with neurites and that addition of AA further stimulated cell growth (Figure 2). However, when cells lacked syntaxin 3, neurite growth ceased, regardless of the presence of these growth factors. The effect was the same in cultured hippocampal neurons, indicating that the effect was relevant to neurons and not specific to PC12 cells.
Next the investigators examined whether AA affected the ability of SNAP25 to interact with syntaxin 3. SNAP25 is another membrane protein concentrated in growth cones that complexes with syntaxin 3 and is needed for the fusion of intracellular vesicles at the membrane. After showing that syntaxin 3 interacts with SNAP25 in the growth cones of resting cells, and that the interaction is enhanced by nerve growth factor, they pre-treated the cells with AA for 30 minutes. Exposure to AA enhanced the interaction between the two proteins in both PC12 cells and hippocampal neurons, as shown by immunoprecipitation. Interaction was independent of nerve growth factor. Further, the interaction was enhanced at a plausible biological concentration of AA in the plasma membrane of about 100 M. In the absence of AA, the two proteins eluted separately by chromatography, suggesting that they did not interact.
The final test was to see whether syntaxin 3, SNAP25 and AA actually form a membrane complex (SNARE complex) with vesicular proteins. For this experiment they used synaptobrevin 2, a membrane protein commonly found in neuronal intracellular vesicles. Without AA, syntaxin 3 did not complex with the other proteins, but with AA, the triple protein complex formed (Figure 3
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