Figure 2.

Oxidative stress induces autophagy/mitophagy in neurons. Cortical neurons were exposed to H₂O₂ for 6 h. (A) Electron microscopy revealed numerous autophagosomes in the cell body (b, d), neurites (e) and neurite terminals (f, arrow) in H₂O₂-treated neurons, compared to a relative paucity of autophagosomes in control neurons (a). Black arrows indicate autophagosomes and red arrows indicate mitochondria. Large autophagosomes contained membranous structures and dense aggregates (g), autolysosomes (h), and degenerated mitochondria (mitophagy) (i, arrows). The remaining mitochondria in H₂O₂-treated neurons had degenerative features including swelling, enlarged matrix and disrupted cristae (j, arrows). These electron micrographs are representative of ultrastructural features observed in more than 60 neurons examined from at least 3 separate cultures for each treatment condition. (B) Immunoblot revealed slightly increased levels of the LC3-II/LC3-I ratio in cells exposed to oxidative insults at 6 h, while at 18 h the levels of LC3-I were reduced resulting in an elevated LC3-II/LC3-I ratio. The LC3-I level and the LC3-II/LC3-I ratio were maintained in NAM-treated neurons exposed to H₂O₂. (C) Results of densitometric analysis of immunoblots. Values are the mean ± SD of determinations made on samples from 3 different experiments (samples from 6-7 cultures/experiment were pooled); values were normalized to the β-actin band.