Using genetic mouse models to study the biology and pathology of autophagy in the central nervous system

Methods Enzymol. 2009:453:159-80. doi: 10.1016/S0076-6879(08)04008-1.

Abstract

Autophagy is a cellular self-eating process that plays an important role in neuroprotection as well as neuronal injury and death. The detailed pathway of autophagy in these two opposing functions remains to be elucidated. Neurons are highly specialized, postmitotic cells that are typically composed of a soma (cell body), a dendritic tree, and an axon. Here, we describe methods for studying autophagy in the central nervous system (CNS). The first involves the use of recently developed transgenic mice expressing the fluorescent autophagosome marker, GFP-LC3. Although CNS neurons show little evidence for the presence of GFP-LC3-containing puncta under normal conditions, under pathological conditions such neurons exhibit many GFP-LC3 puncta. The onset and density of GFP-LC3 puncta have been found to vary significantly in the subcompartments of the affected neurons. These studies suggest that autophagy is distinctly regulated in CNS neurons and that neuronal autophagy can be highly compartmentalized. While transgenic mice expressing GFP-LC3 are a valuable tool for assessing autophagic activity in the CNS, caution needs to be taken when interpreting results solely based on the presence of GFP-LC3 puncta. Therefore, traditional ultrastructural analysis using electron microscopy remains an important tool for studying autophagosomes in vivo. Additional reporters of autophagy are constantly being sought. For example, recently a selective substrate of autophagy p62/SQSTM1 has been shown to be specifically regulated by autophagic activity. Therefore, p62/SQSTM1 protein levels can be used as an additional reporter for autophagic activity.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Autophagy / physiology*
  • Central Nervous System / cytology
  • Central Nervous System / metabolism*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Immunohistochemistry
  • Mice
  • Microscopy, Immunoelectron
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism
  • Models, Animal
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • Map1lc3b protein, mouse
  • Microtubule-Associated Proteins
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins