Abstract
The discovery that impaired autophagy is linked to a wide variety of prominent diseases including cancer and neurodegeneration has led to an explosion of research in this area. Methodologies that allow investigators to observe and quantify the autophagic process will clearly advance knowledge of how this process contributes to the pathophysiology of many clinical disorders. The recent identification of essential autophagy genes in higher eukaryotes has made it possible to analyze autophagy in mammalian cells that express autophagy proteins tagged with fluorescent markers. This chapter describes such methods using primary cultured neurons that undergo up-regulation of autophagy when trophic factors are removed from their medium. The prolonged up-regulated autophagy, in turn, contributes to the death of these neurons, thus providing a model to examine the relationship between enhanced autophagy and cell death. Neurons are isolated from the cerebellum of postnatal day 7 rat pups and cultured in the presence of trophic factors and depolarizing concentrations of potassium. Once established, the neurons are transfected with an adeno-viral vector expressing MAP1-LC3 with red fluorescent protein (RFP). MAP1-LC3 is the mammalian homolog of the yeast autophagosomal marker Atg8 and when tagged to GFP or RFP, it is the most widely used marker for autophagosomes. Once expression is stable, autophagy is induced by removing trophic factors. At various time points after inducing autophagy, the neurons are stained with LysoSensor Green (a pH-dependent lysosome marker) and Hoechst (a DNA marker) and subjected to live-cell imaging. In some cases, time-lapse imaging is used to examine the stepwise process of autophagy in live neurons.