Everyone knows that omega-3 polyunsaturated fatty acids (PUFAs) are good for you (eat your fish!), but no one really knows why. Although dietary PUFAs of both the omega-3 and omega-6 varieties are required for proper brain development, and may even stave off Alzheimer disease, little is known about their physiological targets. So it comes as welcome news that Frederic Darios and Bazbek Davletov at the MRC Laboratory of Molecular Biology (Cambridge, U.K.) have discovered a protein effector of PUFA action. In a paper in the April 6 Nature, the pair report that the omega-6 arachidonic acid and the omega-3 docosahexaenoic acid (DHA) stimulate neurite outgrowth in PC12 cells by binding to and activating syntaxin 3, a protein required for membrane expansion at the growth cone. Their results provide the first molecular explanation of how PUFAs regulate a defined event in neuronal cells, and suggest a way to screen for new compounds that stimulate neurite extension.
To investigate how the PUFA arachidonic acid (AA) promotes neurite outgrowth, Darios and Davletov used the familiar model of NGF-stimulated PC12 cells. In these cells, arachidonic acid is produced after NGF activates phospholipase A2, and the researchers showed that directly treating cells with phospholipase A2, or arachidonic acid itself, along with NGF, increased neurite extension. Lengthening of neurites involves membrane expansion by fusion of intracellular vesicles at the growth cone, which in turn requires SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins. So to pin down the role of fatty acids in the process, Darios and Davletov knocked down syntaxin-1, a SNARE protein that is sensitive to AA. Surprisingly, the RNAi knockdown had no effect on neurite outgrowth in response to NGF or AA. Instead, the authors found a related protein, syntaxin 3, was required for the AA response.
Syntaxins are known to partner with SNAPs (synaptosomal-associated proteins) during neurite outgrowth, and the researchers demonstrated that adding NGF or arachidonic acid to cells stimulated the association of syntaxin 3 with the SNAP25 protein. They replicated this interaction in a cell-free system with purified proteins and showed that adding arachidonic acid promoted formation of a stable syntaxin 3-SNAP25 complex. The concentration of AA required (100 μM) was consistent with the level they calculated to be present in growth cones upon phospholipase A2 activation. Protein structural analysis by circular dichroism (CD) indicated that arachidonic acid altered the conformation of syntaxin 3, but not SNAP25. The full SNARE complex that mediates membrane fusion contains an additional protein, synaptobrevin 2, and the researchers showed that formation of the ternary complex required AA activation of syntaxin 3.
In neurons, phospholipids commonly have PUFA side chains other than arachidonic acid. DHA, for instance, can account for as much as 8 percent of total brain lipid. Using their in vitro assay of protein association, the authors were able to test DHA and other fatty acids for their capacity to activate syntaxin 3. While saturated and mono-unsaturated fatty acids were ineffective, omega-6 (linoleic and arachidonic) and omega-3 (linolenic and DHA) PUFAs all stimulated syntaxin 3’s interaction with SNAP25 and induced the conformational change detected by CD. In vitro activity predicted neurite outgrowth: When tested on PC12 cells (at a single concentration of 200 μM), the two omega-3s stimulated neurite outgrowth to the same extent as arachidonic acid.
“The ability of syntaxin 3 to partner with other SNAREs strictly requires omega-3 and omega-6 PUFAs, which act as if to ‘oil’ the plasma membrane fusion machinery,” the authors write. They propose that PUFA binding to syntaxin 3 releases an inhibitory conformation and exposes a SNAP25 binding site. Because syntaxin 3 expression is ubiquitous, their findings may be applicable to membrane fusion in many kinds of cells, not just neurons.
Epidemiological evidence ties adequate intake of omega-3 PUFAs to a lower risk of Alzheimer disease, while experiments with AD mice show that DHA helps maintain synapses and reduce amyloid burden (see ARF related news story; Calon et al., 2005; and Lim et al., 2005). DHA also influences the activation of AKT, an important survival pathway for neurons (see ARF related news story). Whether or how activation of syntaxin relates to the reported beneficial effects of PUFAs in AD remains to be worked out. However, the ability of omega-3 and omega-6 PUFAs, but not other fatty acids, to activate syntaxin fits with the unique role of PUFAs in brain development and neuronal regeneration. The in vitro assay for syntaxin 3 activation will be useful for identifying new and potent PUFA mimics that might enhance neuronal growth.—Pat McCaffrey
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- Calon F, Lim GP, Morihara T, Yang F, Ubeda O, Salem N, Frautschy SA, Cole GM. Dietary n-3 polyunsaturated fatty acid depletion activates caspases and decreases NMDA receptors in the brain of a transgenic mouse model of Alzheimer's disease. Eur J Neurosci. 2005 Aug;22(3):617-26. PubMed.
- Lim GP, Calon F, Morihara T, Yang F, Teter B, Ubeda O, Salem N Jr, Frautschy SA, Cole GM. A diet enriched with the omega-3 fatty acid docosahexaenoic acid reduces amyloid burden in an aged Alzheimer mouse model. J Neurosci. 2005 Mar 23;25(12):3032-40. PubMed.
- Darios F, Davletov B. Omega-3 and omega-6 fatty acids stimulate cell membrane expansion by acting on syntaxin 3. Nature. 2006 Apr 6;440(7085):813-7. PubMed.