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J Biol Chem. 2009 Jan 23;284(4):2363-73. doi: 10.1074/jbc.M806088200. Epub 2008 Nov 18.

Nutrient deprivation induces neuronal autophagy and implicates reduced insulin signaling in neuroprotective autophagy activation.

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Department of Laboratory Medicine, Center for Neurogenentics and Neurotherapeutics, University of Washington Medical Center, Seattle, WA 98195, USA.


Although autophagy maintains normal neural function by degrading misfolded proteins, little is known about how neurons activate this integral response. Furthermore, classical methods of autophagy induction used with nonneural cells, such as starvation, simply result in neuron death. To study neuronal autophagy, we cultured primary cortical neurons from transgenic mice that ubiquitously express green fluorescent protein-tagged LC3 and monitored LC3-I to LC3-II conversion by immunohistochemistry and immunoblotting. Evaluation of different culture media led us to discover that culturing primary neurons in Dulbecco's modified Eagle's medium without B27 supplementation robustly activates autophagy. We validated this nutrient-limited media approach for inducing autophagy by showing that 3-methyl-adenine treatment and Atg5 RNA interference knockdown each inhibits LC3-I to LC3-II conversion. Evaluation of B27 supplement components yielded insulin as the factor whose absence induced autophagy in primary neurons, and this activation was mammalian target of rapamycin-dependent. When we tested if nutrient-limited media could protect neurons expressing polyglutamine-expanded proteins against cell death, we observed a strong protective effect, probably due to autophagy activation. Our results indicate that nutrient deprivation can be used to understand the regulatory basis of neuronal autophagy and implicate diminished insulin signaling in the activation of neuronal autophagy.

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