GFP-LC3 puncta formation induced by nutrient deprivation is inhibited by 3-methyladenine. A, primary cortical neurons from GFP-LC3 transgenic mice were cultured in CM or NLM, either in the absence of 3-methyladenine (-3-MA) or the presence of 3-methyladenine (+3-MA). GFP-LC3 puncta are infrequent in CM-cultured primary cortical neurons without 3-MA (upper left) but are common in NLM-cultured primary cortical neurons in the absence of 3-MA (bottom left). Inclusion of 3-MA in the culture media does not affect GFP-LC3 puncta frequency in CM cultured primary neurons (upper right) but markedly reduces GFP-LC3 puncta formation in NLM-cultured primary cortical neurons (bottom right). Green, GFP-LC3; blue, DAPI. Scale bar, 20 μm. B, quantification of GFP-LC3 puncta formation in primary neurons grown in different culture medium conditions with or without 3-MA. Although 3-MA has no effect upon GFP-LC3 puncta formation in primary neurons grown in CM (p = 0.47, one-way ANOVA), 3-MA treatment significantly reduces GFP-LC3 puncta formation in primary neurons grown in NLM (p < 0.01, one-way ANOVA; error bars, S.E.).

Analysis of B27 supplement components indicates that insulin is responsible for nutrient deprivation induction of autophagy in primary neurons. A, the addition of CM components back to NLM represses autophagy activation. Western blot analysis of primary cortical neurons grown in CM or NLM reveals a shift from LC3-I to LC3-II under NLM conditions (upper left). We added B27 supplement to NLM or reformulated a “MIX” consisting of the aqueous, ethanolic, and fresh components of B27 supplement (Table 1). Primary neurons cultured in NLM + B27 or NLM + MIX did not display induction of autophagy, based upon LC3 Western blot analysis (upper right) and confocal imaging analysis (bottom). B, “fresh” components of B27 repress autophagy induction. Primary cortical neurons were cultured in NLM combined with either B27 MIX AQ or ET or FR. LC3 Western blot analysis (top) and confocal imaging (bottom) both reveal induction of autophagy upon the addition of aqueous or ethanolic components and repression of autophagy activation upon the addition of the fresh components. C, evaluation of individual compounds comprising the fresh components of B27. Primary cortical neurons were cultured in NLM combined individually with each fresh component: bovine serum albumin (BSA), catalase (CAT), glutathione (GLUT), insulin (INS), superoxide dismutase (SOD), transferrin (TRAN), or tri-iodo-l-thyronine (T3). Comparison of LC3 Western blot analysis of primary neurons cultured with these different medium combinations revealed that the addition of insulin to NLM diminished LC3-I to LC3-II conversion to the greatest extent. D, insulin is the principal factor in B27 responsible for neuronal autophagy induction. When we compared primary neurons cultured in CM or NLM with primary neurons cultured in NLM lacking just insulin (MIX - INS) or in NLM supplemented with just insulin, LC3 Western blot analysis (top) and confocal imaging (bottom) revealed that the absence of insulin from NLM induced autophagy, whereas the addition of insulin to NLM repressed autophagy. All immunoblots were reprobed for β-actin to ensure equivalent loading.

Polyglutamine-expanded AR induces autophagy in primary neurons. A, primary cortical neurons from GFP-LC3 transgenic mice were transfected with amino-terminally truncated AR with either 19 glutamines (trAR19Q) or 112 glutamines (trAR112Q), fused to a carboxyl-terminal HA tag. Anti-HA staining reveals expression of AR in perinuclear cytosol and neurite processes (left panels; red, HA; blue, DAPI). GFP-LC3 distribution remained diffuse in trAR16-expressing neurons, but in trAR112Q-expressing neurons, GFP-LC3 localized into discrete puncta. Merging of these images indicated that some GFP-LC3 puncta co-localized with trAR112Q aggregates (yellow arrowheads); however, most GFP-LC3 puncta were distinct from areas of AR112Q accumulation (white arrowheads). Scale bar, 20 μm. B, quantification of GFP-LC3 puncta formation in primary neurons expressing either amino-terminal truncated AR with either 19 glutamines (trAR19Q) or 112 glutamines (trAR112Q). Polyglutamine-expanded AR expression markedly increases the frequency of GFP-LC3 puncta (p < 0.01, one-way ANOVA; error bars, S.E.), indicating polyglutamine-length dependent autophagy activation.

Insulin supplementation promotes trAR112Q neurotoxicity. Primary neurons expressing GFP-tagged trAR112Q were cultured for 24 h in either CM or CM supplemented with insulin (CM + INS). When we quantified caspase-3 activation, we noted a significant increase in trAR112Q-expressing neurons cultured in CM plus insulin. Increased AR112Q neurotoxicity in primary cortical neurons cultured in CM plus insulin was accompanied by decreased MAP2 staining and increased nuclear condensation (not shown).

Autophagy is induced by nutrient deprivation in primary cortical neurons. A and B, analysis of GFP-LC3 distribution in primary cortical neurons from GFP-LC3 transgenic mice grown in different culture medium conditions. Primary neurons cultured in CM consisting of Neurobasal-A™ with B27 supplement display diffuse GFP-LC3 staining (A), but primary neurons cultured in NLM with just DMEM show prominent GFP-LC3 puncta formation (B), clearly discernible at higher magnification (arrowheads; see inset). Green, GFP-LC3; blue, DAPI. Scale bar, 20 μm. C, LC3 Western blot analysis of primary cortical neurons grown in CM and NLM. A shift in the LC3 isoform type is progressively observed in primary neurons cultured in NLM. D, quantification of GFP-LC3 puncta formation in primary neurons grown in different culture medium conditions. NLM significantly induces autophagosome formation in comparison with CM (p < 0.05, one-way ANOVA; error bars, S.E.).

GFP-LC3 puncta formation induced by nutrient deprivation is inhibited by Atg5 shRNA knockdown. A, validation of Atg5 shRNA knockdown constructs. The levels of Atg5 expression in Neuro2a cells transfected with two different Atg5 shRNA knockdown constructs are reduced to ∼37–42% of the Atg5 expression levels detected in Neuro2a cells transfected with the corresponding empty vector (p < 0.01 by one-way ANOVA). Data are based upon real time RT-PCR analysis of three independent sets of Neuro2a transfections per experiment. Error bars, S.E. B, primary cortical neurons from GFP-LC3 transgenic mice were cultured in CM or NLM and were transfected with either empty CFP vector (-sh-Atg5) or a Atg5-shRNA-CFP-tagged expression construct (+shAtg5-1 or +shAtg5-2). GFP-LC3 puncta are infrequent in CM cultured primary cortical neurons expressing CFP alone (upper left) but are common in NLM-cultured primary cortical neurons expressing CFP alone (upper right; GFP-LC3 puncta are indicated by arrows). GFP-LC3 puncta frequency in CM cultured primary neurons is unaffected by co-expression of a CFP-tagged Atg5 shRNA (middle left/lower left), but GFP-LC3 puncta formation is eliminated in NLM cultured primary cortical neurons expressing Atg5 shRNA-CFP (middle right/lower right). Green, GFP-LC3; light blue, CFP. Scale bar, 20 μm. C, quantification of GFP-LC3 puncta formation in primary neurons grown in different culture medium conditions with or without Atg5 shRNA knockdown. Although Atg5 knockdown has no significant effect upon GFP-LC3 puncta formation in primary neurons grown in CM (p > 0.05 by one-way ANOVA), Atg5 shRNA knockdown significantly reduces GFP-LC3 puncta formation in primary neurons grown in NLM (p < 0.01 for each Atg5 shRNA, one-way ANOVA; error bars, S.E.). Two different Atg5 shRNA constructs were used to rule out “off-target” effects.

Nutrient deprivation of primary neurons is accompanied by decreased activation of the Akt-mTOR signaling pathway. A, loss of Akt activation in neurons cultured in NLM. Western blot analysis of phosphorylated Akt (pAkt) and total Akt (tAkt) indicates that Akt activation is markedly decreased in primary cortical neurons cultured in NLM compared with CM. The addition of B27 supplement (B27) or the MIX to NLM yields a level of Akt activation that is comparable with CM. B, fresh components of B27 supplement yield Akt activation. When primary cortical neurons were cultured in NLM combined with either B27 MIX AQ or ET FR, the addition of the fresh components resulted in detectable Akt activation. C, evaluation of individual compounds comprising the fresh components of B27 for Akt activation. Primary cortical neurons were cultured in NLM combined individually with each fresh component: bovine serum albumin (BSA), catalase (CAT), glutathione (GLUT), insulin (INS), superoxide dismutase (SOD), transferrin (TRAN), or tri-iodo-l-thyronine (T3). Western blot analysis of phosphorylated Akt and total Akt indicates that Akt is most strongly activated in neurons cultured in NLM combined with insulin (NLM + INS). D, decreased mTOR activation in neurons cultured in NLM. Western blot analysis of phosphorylated mTOR (pMTOR) and total mTOR (tMTOR) shows that mTOR activation is decreased in primary cortical neurons cultured in NLM compared with CM. E, decreased S6 kinase activation in neurons cultured in NLM. Western blot analysis of phosphorylated S6 kinase (pP70-S6K) and total S6 kinase (pP70-S6K) reveals reduced S6 kinase activation in primary cortical neurons cultured in NLM compared with CM. F, fresh components of B27 supplement yield S6 kinase activation. When primary cortical neurons were cultured in NLM combined with either B27 MIX AQ, ET, or FR, the addition of the fresh components resulted in marked S6 kinase activation. G, evaluation of individual compounds comprising the fresh components of B27 for S6 kinase activation. Primary cortical neurons were cultured in NLM combined individually with different fresh components, as in C. Western blot analysis of phosphorylated S6 kinase (pP70-S6K) and total S6 kinase (pP70-S6K) demonstrates that S6 kinase is most strongly activated in neurons cultured in NLM combined with insulin (NLM + INS).

Nutrient deprivation prevents neuron cell death produced by expression of polyglutamine-expanded AR. A, primary neurons expressing GFP-tagged trAR112Q were cultured for 4 or 24 h in either CM or NLM and then assayed for apoptotic activation and neurotoxicity by immunostaining for caspase-3 activation and MAP2. Here we see representative low magnification images of these experiments. CM-cultured primary neuron expressing AR112Q-GFP (arrow) is positive for active caspase-3 and lacks MAP2-positive neurites. For neurons cultured in NLM for 4 h, AR112Q-GFP expression does not result in caspase-3 activation or loss of MAP2 reactivity (above left of neuron marked by an arrow). Culturing in NLM for 24 h yields even more robust protection for AR112Q-GFP-expressing neuron (arrow), since this neuron is negative for active caspase-3 but retains prominent MAP2 immunostaining. Left panels, AR112Q-GFP (fluorescein isothiocyanate; green), active caspase-3 (red), and DAPI (blue). Right panels, MAP2 (Cy5; green), active caspase-3 (red), and DAPI (blue). Scale bar, 20 μm. B, when we quantified caspase-3 activation in neurons expressing trAR112Q at 4 h post-transfection, we found that trAR112Q-expressing neurons cultured in NLM were significantly protected against apoptotic activation and neuron cell death (p < 0.01, one-way ANOVA). At 24 h post-transfection, the protective effect of culturing trAR112Q-expressing neurons in NLM was even more pronounced (p < 0.005, one-way ANOVA). Error bars, S.E.