<p>A) Accumulation of autophagosomes was assessed by determining the levels of the autophagic marker protein microtubule-associated protein 1 light chain 3 -II (LC3-II) in wild-type or <i>nclf</i> total mouse brain extracts at 54 weeks of age by western blotting. B) Densitometric quantification of LC3-II levels normalized to tubulin as a loading control revealed enhanced autophagosome numbers with increasing age. Data are presented as mean ±SD, n = 3 per age. Wild-type values were set to 1. C) Double-membrane autophagic vacuoles (marked by arrows) were also found in hippocampal neurons from <i>nclf</i> mice cultured for 14 days. D) For quantification of autophagic vacuoles, pictures were taken from 37 randomly selected wild-type and <i>nclf</i> neurons of two different preparations. The number of autophagic vacuoles related to the cytoplasmic area was determined. Data are presented as mean ± S.E.M. Scale bars: 1 µm.</p
