Macroautophagy is preferentially increased in A53T mutant variant mice compared with wild-type (WT) animals. A: Beclin and LC3-II levels were higher in the substantia nigra of mice expressing the A53T variant compared with WT mice (8 to 12 months of age; n = 8 per group). Overall, beclin was higher with increased α-synuclein expression, but not as significant as expression of the mutant variant (A53T), which produced significant increases in beclin expression. B: High level expressing (+) A53T mice had increased autophagy markers compared with the low level expressors (−), whereas there was no change in the levels between high and low level expressing WT mice. LC3-II (and LC3-II/tubulin) levels were significantly higher in SN of the A53T/+ mice but were relatively unchanged in all other groups when compared with nontransgenics, or compared with the cortex of the WT. All values are expressed as mean ± SEM; **P < 0.01; ***P < 0.001.

Inhibition of macroautophagy promotes the accumulation of aggregated and oligomeric α-synuclein. A: Following RNAi reduction of autophagy by targeting Atg5 or beclin for 48 hours, or addition of 10 mmol/L 3MA for 24 hours, there was a significant decrease in LC3-II/tubulin (20% to 30% of Ctrl levels for all treatments, ***P < 0.001) and decrease in beclin (only with beclin RNAi treatment). Densitometry is provided for LC3-II/tubulin levels following RNAi or 3MA treatment in the A53T cells. B: α-Synuclein oligomers were seen in the A53T cells following either Atg5 (A) or beclin (B) RNAi or 3MA treatment and levels substantially increased relative to the untreated group (C). In wild-type (WT) cells, there was an increase in a band corresponding to 4× α-synuclein in both the Atg5 and beclin RNAi treatments. A53T-expressing cells had increased levels of the 2× to 4× oligomers following beclin RNAi treatment when detected on a higher percentage gel. There was also a significant increase in higher molecular weight oligomers/aggregates at the gel interface. Densitometric analyses for A53T cells of oligomers/tubulin identify a significant increase in Atg5 and beclin RNAi, and 3MA-treated cells (***P < 0.001). C: There was increased puncta of α-synuclein (red) that was also oligomer-specific (red), as detected by the A11 antibody. Although this was evident in both WT and A53T cells, there were more α-synuclein aggregates (arrows) in the A53T cells. Scale bar = 5 μm. All values are expressed as mean ± SEM.

Macroautophagy is up-regulated in DLB. A: Homogenates of frozen human brain tissue (see Table 1) Brodmann’s area 9 (dorsolateral prefrontal cortex) from nonaffected controls (Ctrl; n = 6; 78.7 ± 4.5 years) and DLB cases (n = 6; 78.5 ± 3.7 years) were analyzed for LC3, beclin, phospho-P70S6 kinase (T389), total P70S6 kinase, phospho-Akt (T308), total Akt, and tubulin. Representative blots show three samples from each group. Overall, tubulin levels were consistent between the groups, whereas there was a significant increase in beclin (P < 0.01) and LC3 (P < 0.001) along with a significant decrease in p-P70S6k/P70S6k and p-Akt/Akt (P < 0.05) profiles between patients with nonaffected and DLB brains (B). C: Oligomeric α-syn could also be identified in the human autopsy brains, as detected by the LB509 antibody and ran alongside α-synuclein oligomers (+) to verify the MW of the nX α-synuclein species. All values are expressed as mean ± SEM; *P < 0.05; **P < 0.01. D: Densitometric analysis of oligomers in DLB and control cases indicates that there is a significant increase in oligomer formations in DLB brains (***P < 0.001).

Macroautophagy is increased in A53T expressing cells relative to the wild-type. A: Immunoblots of representative cell harvests of wild-type (WT) and A53T-mutant cells illustrate that there are higher levels of beclin and LC3-II levels in the mutant cells than the WT cells, despite similar expression levels of the respective α-synuclein protein. There was no observable difference in the phospho-P70S6 kinase, total P70S6 kinase, or tubulin levels. B: Densitometric analysis confirms that there is a significant increase in beclin (P < 0.01) and LC3-II/tubulin (P < 0.001), but not p-P70S6k/P70S6k (sample size = nine per group). C: Although there was an abundance of monomeric α- synuclein in both WT and A53T cell lines, prolonged exposure shows a slight increase in oligomeric α-synuclein in the A53T cells, and lower levels in the WT cells. Densitometric analysis did not identify a significant difference between the two groups. D: Electron microscopic images of WT and A53T cells shows the presence of AVs (arrows) and ALs in both lines, but higher prevalence in the A53T cells (Scale bar = 2 μm). E: Quantification of AVs shows that there is a threefold increase in AVs in the A53T cells, as compared with WT cells, with approximately 10% of these AVs being nascent AVs (APL - autophagasomal with double membrane), whereas all of the AVs in WT cells were ALs. Based on surface area measurements, there was a twofold increase in total # AVs, with 26% of all AVs represented by APLs that were not evident in the WT cells. All values are expressed as mean ± SEM; **P < 0.01; ***P < 0.001.

Macroautophagy is up-regulated by A53T mutant α-synuclein. A: Beclin (P < 0.05) and LC3-II (P < 0.05) levels were increased in the substantia nigra (SN) of A53T mutant α-synuclein expressing mice (12c/SKO; n = 12) as compared with age-matched controls (SKO; n = 12) (6 to 8 months old). B: Densitometric quantification of results for beclin, LC3-II/tubulin, and tubulin levels. C: A comparison between SKO and 12c/SKO mice from two regions (Cortex – no α-syn expression; SN – α-syn expression only in 12c/SKO) at 6 months and 18 months of age shows that there is an increase in beclin and LC3-II levels in the SN of 12c/SKO mice (P < 0.05 vs P < 0.01 for beclin and P < 0.001 for LC3-II/tubulin). There was no relative change in tubulin or phospho-P70S6 kinase/P70S6 kinase levels. Graphical representation of the densitometric results are shown in (D). E: Electron microscopy of the SN of the 12c/SKO and SKO mice was performed to provide definitive identification of autophagic vacuoles in the substantia nigra. AVs were not identified in 8mo SKO mice, though there were some at 18 months. In the 12c/SKO mice, AVs (arrows) were identified beginning at 8 months of age, and there was an abundance of AVs in dystrophic neuritis (right panel, second from bottom) and in axons (bottom right panel) in older animals. Scale bar for low magnification = 2 μm; scale bar for high magnification panels = 500 nm. Quantification of AVs showed that there was a twofold increase in numbers of AVs and a fivefold increase in surface area in the 12c/SKO group compared with the SKO group. All values are expressed as mean ± SEM; *P < 0.01; **P < 0.01; ***P < 0.001.

The organic pesticide rotenone impairs mitochondrial activity and prolonged exposure limits the efficacy of autophagy. A: Rotenone (5 to 50 nmol/L) induces autophagy in wild-type (WT) and (to a lesser degree) A53T α-synuclein-transfected cells within 24 hours, but chronic exposure (7 days) of rotenone, dramatically reduces the levels of LC3-II. B: At the same time, lactate dehydrogenase levels were only significantly different in 7-day treated A53T (P < 0.05) cells relative to controls, whereas cellular activity, as determined by MTT assay, was reduced by 10% in WT cells (P < 0.05) and 22% (P < 0.01) in the A53T cells after 24 hours. Following 7 days of treatment, the decrease was 32% (P < 0.01) and 70% (P < 0.001), respectively. Cytochrome C was also significantly reduced after 7 days of rotenone treatment in both cell lines, although the change was more apparent the mutant cells. Immunochemistry of cytochrome c oxidase activity in the mutant cells showed a clear decline in diaminobenzidine immunoreactivity after 7 days of rotenone treatment. This decline in metabolic levels was even greater at 7 days in the mutant cells as determined ATP levels. C: Whereas 50 nmol/L rotenone induced autophagy, as indicated by LC3-II levels, at a similar rate to 10 nmol/L rapamycin at 24 hours, prolonged exposure to this level of rotenone depressed the levels of LC3-II/tubulin. Densitometry results are shown for the samples (n = 6; **P < 0.01, ***P < 0.001). D: At 24 hours, rapamycin (added at the 20-hour point) was competent in removing most of the oligomeric α-synuclein in both the WT and A53T α-synuclein-transfected cells treated with 50 nmol/L rotenone. This clearance mechanism was not as effective after 7 days of exposure in the cell lines, though there was a minor reduction in 3× α-syn in the WT cells. Densitometry results (n = 6) are shown for levels of oligomers (*** P < 0.001). E: Confocal images of WT and A53T cells treated for 24 hours with 50 nmol/L rotenone and colabeled with LC3 (green) and α-synuclein (red). Although there was significant colocalization (yellow, white arrow) between LC3 and α-synuclein in punctate structures indicating AVs, there were also numerous green puncta of AVs without α-synuclein (green arrow) in the A53T cells, and red puncta (red arrow) that identify α-synuclein aggregates not being degraded via autophagy. All values are expressed as mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001.