(A) Western blot analyses showing a rapid and sustained decrease in mTOR-mediated phosphorylation of p-p70 immunoreactivity relative to total p70 immunoreactivity after rapamycin (10nM) addition to the medium for 1-24 hr. (B) Densitometric analyses of gels in (A) (n = 6, Mean ± S.E.M.): ratios of immunoreactive p-p70 relative to total p70 in rapamycin-treated neurons are expressed as a percentage of the untreated control value for each time point (***, p<0.001). (C) Representative blot of LC3-I and LC3-II immunoreactivity in neurons treated with rapamycin (10nM) for 1 hr, 6 hr, and 24 hr. (D) Graph depicting changes in LC3-I, LC3-II and tubulin levels in neurons treated with rapamycin 1 hr, 6 hr and 24 hr. (* p<0.05). (E) Immunostaining for LC3 distribution in control cortical neurons and rapamycin-treated (10nM, 24 hr) neurons (F). Arrows indicate punctate LC3 representative of AV membranes. Scale bars = 5μm. (G, H, and I) Live imaging of BODIPY-pepstatin-FL in DsRed-LC3 transfected neurons. DsRed-LC3 is primarily cytosolic in normal cells (G, left panel) but relocated into vesicles after treatment with rapamycin (10nM) for 6 hr (H, left panels). Rapamycin-induced AVs contain lysosomal enzymes: (H and inset, I), illustrate varying degrees to which autolysosomes have matured to lysosomes. Background BODIPY-pepstatin-FL labeling indicates lysosomal compartments in neighboring untransfected neurons. Scale bars (G, H, I) = 10 microns.

Five-day-old primary cortical neurons displayed few AVs in their cell bodies (A, scale bar = 2μm) and neurites (B). Appearance of large clear autophagic fusion compartments in the cell body after rapamycin (10nM) treatment for 1 hr (C). Electron dense sequestered material was observed in neurites after rapamycin (10nM) treatment for 1 hr (D). Prolonged activation of autophagy with rapamycin (10nM, 24 hr) revealed multiple autophagic compartments in both cell bodies (E) and neurites (F). Ultrastructural localization of cathepsin D in control (G) and rapamycin (10nM, 6 hr) treated neurons by silver-enhanced immunogold labeling with cathepsin D antibody (H). In control neurons, cathepsin D is abundant in lysosomes with relatively little electron dense content (arrows). Most AVs in rapamycin-treated neurons are decorated with silver enhanced immunogold (arrows) indicating the presence of cathepsin D. Scale bars = 500nm except as otherwise noted.

(A) Immunoblot analyses of phospho-p70 and total p70 after leupeptin (20μM), pepstatin (20μM), or both (LP) were added to the medium for 24 hr. (B) Densitometric analyses of gels in (A) (n = 5): ratios of phospho-p70 relative to total p70 are expressed as a percentage of the untreated control value. (C) Immunoblot analyses of LC3-I and LC3-II after neurons were exposed to protease inhibitors for 24 hr. (D) Densitometric analyses of LC3-I, LC3-II, and tubulin immunoreactivity in neurons after protease inhibitor treatment (n = 5, **p< 0.01). (E) Live-image of DsRed-LC3 transfected neurons treated with leupeptin (20μM) for 24hr loaded with BODIPY-pepstatin-FL. Vesicular LC3 localizes within BODIPY-pepstatin-positive compartments: lack of degradation results in a strong LC3 signal (E, right panel inset). Neurons treated with leupeptin and pepstatin for 24hr immunostained for LC3 show a similar pattern of punctate LC3 accumulation (E, left panel inset). (F) GFP-Endomarker transfected neurons immunostained with LC3 (red) and cathepsin D (blue). Under normal conditions, LC3 and GFP-Endo occasionally colocalize (F, inset of middle panel), whereas endosomes and cathepsin D do not (F, inset of left panel). Co-treatment with leupeptin and pepstatin causes accumulation of LC3- and GFP-Endomarker-positive amphisomes (G, middle panel, arrows), as well as GFP-Endomarker and cathepsin D-positive endosomes and lysosomes.

After 6 hr, numbers of mainly double-membrane-limited autophagosomes containing amorphous electron dense material are seen in the cell bodies of some neurons (A) and in neurites (B), which was more evident after 24 hr in all neurons (C). Undegraded organellar material was also present in AVs within the neurites of neurons after 24hr (D). AV morphologies were similar in neurons treated with leupeptin plus pepstatin (data not shown). (E) Silver enhanced immunogold ultra-structural localization of Cathepsin D in AVs (arrowheads) in neurons treated with leupeptin (24 hr, 20μM). Scale bars = 500nm.

(A) Western blot analyses of p-p70 and total p70 in neurons grown in Earle's Balanced Salt Solution (EBSS) in the absence (no inhibitor - NI) or presence of pepstatin (20μM), leupeptin (20μM), or both (LP) for 24 hr. (B) Densitometric analyses of multiple gels in (A) (n = 5): ratios of p-p70 and p70 immunoreactivity are expressed as a percentage of the untreated control value shown at 100%, p < 0.0001 for 1hr, p < 0.001 for 6hr and p < 0.05 for 24hr treatments in EBSS culture media (C) Representative immunoblots of LC3-I and LC3-II immunoreactivity in neurons grown in EBSS in the absence or presence of protease inhibitors for 24 hr. (D) Densitometric analyses of LC3-I and LC3-II levels after neurons grown in EBSS in the absence or presence of protease inhibitors for 24 hr. In B and D, densitometric values are expressed as ratios of immunoreactivity levels after each inhibitor treatment relative to the corresponding ratio for untreated control neurons grown in NBM/B27. (E) Ultrastructure of AVs in neuronal perikaryon after growth in EBSS for 24 hr (F) EBSS with leupeptin (20μM) and pepstatin (20μM) for 6hr (scale bar = 500nm). (G) Cell body and (H) neurite of neurons cultured in EBSS with leupeptin (20μM) and pepstatin (20μM) for 24hr. Scale bars = 500nm.

(A) Western blot analyses of p-p70 and total p70 after exposure of neurons to vinblastine (10μM) for 1-24 hr. (B) Densitometric analyses of immunoblots in (A) (n = 6): ratios of p-p70 relative to total p70 are expressed as percentages of the control value from each set of treatments, *p<0.05, **p<0.01, ***p<0.001. (C) Representative immunoblots of LC3-I and LC3-II in neurons exposed to vinblastine (10μM) for 1, 6, and 24 hr. (D) Densitometry of LC3-I, LC3-II and tubulin levels after neurons exposed to vinblastine (10μM) for 1, 6, and 24 hr (n = 6, *p <0.05, **p<0.01). (E-H) Immunostaining of primary neurons stained with TUJ-1 antibody for neuron-specific beta-III-tubulin. Tubulin remains intact when autophagy is induced (F) or lysosomal proteolysis is inhibited by leupeptin (20μM) (G), but is disrupted after 24 hr treatment with vinblastine (10μM) (H). Scale bars = 5μm. (I and J). Live image of BODIPY-pepstatin-FL in DsRed-LC3 transfected neurons after treatment with vinblastine (10μM). Autophagosomes that have not fused with lysosomes are the predominant AVs accumulating after 1hr of vinblastine (I, right panel), (J) After 6hr of vinblastine, LC3 mostly colocalizes with BODIPY-pepstatin vesicles indicating that fusion between autophagosomes and lysosomes occurs despite impairment in microtubule-dependent lysosome transport. (L) Quantification of vesicular profiles in DsRed-LC3 transfected cells loaded with BODIPY-pepstatin. The average number of each vesicle type is shown as a percentage of the total number: red = autophagosome (AP), orange = autolysosome (AL), yellow = lysosomes with minimal traces of DsRed-LC3, green = lysosomes. In untreated neurons transiently expressing DsRed-LC3 loaded with BODIPY-pepstatin, most vesicles (∼80%) are lysosomes. Autophagosomes are efficiently fused with lysosomes after rapamycin since the proportion of autophagosomes remains very low, and only autolysosomes are increased. Between 1-6hr vinblastine treatment, autophagosome maturation also occurs indicated by the increased proportion of autolysosomes.

Vinblastine treatment caused accumulation of predominantly double-membrane-limited AVs containing organelle structures with largely undegraded material (arrows) in the cell body (A) and neurites (A, inset) after 1hr. These structures contained undetectable levels of cathepsin D by silver-enhanced immunogold labeling (C). After 6 hr of vinblastine treatment, double-membrane-limited AVs contained more amorphous electron-dense material and cathepsin D (D) reflecting partial digestion of autophagosomal contents in cell bodies (B, arrows) and neurites (B, inset). Most AVs at 6 hr after vinblastine were decorated by silver-enhanced immunogold-conjugated cathepsin D antibodies. Scale bars = 500nm.

In the brains of patients with Alzheimer's Disease (A, B) or the brain of a PS/APP mouse model of AD (C), dystrophic neurites in cerebral cortex contain robust accumulations of single- and double- membrane limited AVs with electron-dense content, which contain cathepsin D (B). Panels D-L represent the internal morphology of primary neurons during different autophagy modulating conditions. Panels D-E depict conditions where autophagy is strongly induced: cell body (D, F) and neurite (E) of rapamycin (10nM, 24hr) treated neurons; cell body (G) of EBSS-starved neurons (24hr). Panel H depicts autophagosomes in perikarya after 1 hr vinblastine treatment where autophagosome fusion with lysosomes are severely impaired. Panels I-L depict conditions involving impaired AV clearance illustrating AV morphologies resembling those in AD brain: cell body of vinblastine (10μM) treated neurons for 6hr (I, J); cell body (K) of EBSS + pepstatin (20μM, 24hr) treated neurons; cell body (L) of leupeptin (20μM, 24hr) treated neurons. Scale bars = 500nm.