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, 10 (4), 588-602

MTOR-independent, Autophagic Enhancer Trehalose Prolongs Motor Neuron Survival and Ameliorates the Autophagic Flux Defect in a Mouse Model of Amyotrophic Lateral Sclerosis

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MTOR-independent, Autophagic Enhancer Trehalose Prolongs Motor Neuron Survival and Ameliorates the Autophagic Flux Defect in a Mouse Model of Amyotrophic Lateral Sclerosis

Xiaojie Zhang et al. Autophagy.

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder caused by selective motor neuron degeneration. Abnormal protein aggregation and impaired protein degradation pathways may contribute to the disease pathogenesis. Although it has been reported that autophagy is altered in patients and animal model of ALS, little is known about the role of autophagy in motor neuron degeneration in this disease. Our previous study shows that rapamycin, an MTOR-dependent autophagic activator, accelerates disease progression in the SOD1(G93A) mouse model of ALS. In the present report, we have assessed the role of the MTOR-independent autophagic pathway in ALS by determining the effect of the MTOR-independent autophagic inducer trehalose on disease onset and progression, and on motor neuron degeneration in SOD1(G93A) mice. We have found that trehalose significantly delays disease onset prolongs life span, and reduces motor neuron loss in the spinal cord of SOD1(G93A) mice. Most importantly, we have documented that trehalose decreases SOD1 and SQSTM1/p62 aggregation, reduces ubiquitinated protein accumulation, and improves autophagic flux in the motor neurons of SOD1(G93A) mice. Moreover, we have demonstrated that trehalose can reduce skeletal muscle denervation, protect mitochondria, and inhibit the proapoptotic pathway in SOD1(G93A) mice. Collectively, our study indicated that the MTOR-independent autophagic inducer trehalose is neuroprotective in the ALS model and autophagosome-lysosome fusion is a possible therapeutic target for the treatment of ALS.

Keywords: Cu/Zn superoxide dismutase 1; amyotrophic lateral sclerosis; autophagosome-lysosome fusion; autophagy; trehalose.

Figures

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Figure 1. Effects of trehalose on the phenotype, disease onset, and life span in SOD1G93A mice. (A) The different hind limb-clasping phenotype in 120 d old SOD1G93A mice; (B) The body weight curves in the 4 mouse groups; The results of Kaplan-Meier survival analysis (SPSS 17.0) showed the probability of disease onset (C) and the probability of survival (D) in the Tg-NT, Tg-Suc, Tg-Tre mice; (E) The data of disease duration, onset, and life span in NT, Suc or Tre mice. There were 6 mice in the WT group, and 12 mice in each group of Tg-NT, Tg-Suc, and Tg-Tre. P values were analyzed by one-way ANOVA. Data are presented as mean ± SEM **P < 0.01 as compared with Tg-NT group; &&P < 0.01 as compared with Tg-Suc group.
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Figure 2. Effects of trehalose on motor neuron survival in SOD1G93A mice. (A) Representative photomicrographs of motor neurons in the anterior horn of spinal cords of 4 mouse groups by Nissl staining; (C) Representative photomicrographs of motor neurons in the anterior horn of spinal cord of 4 mouse groups by SMI-32 immunostaining. There were 3 mice in each group; asterisks mark the motor neurons. Scale bar: 100 μm; (B) The number of motor neurons in L4-5 segments by Nissl staining; (D) The mean number of SMI-32 positive motor neurons in both sides of one slice of an L4-5 segment. Data were analyzed using one-way ANOVA followed by Tukey post hoc test. All values are presented as mean ± SEM ##P < 0.001 as compared with WT mice; **P < 0.01 as compared with Tg-NT mice; &&P < 0.01 as compared with Tg-Suc mice.
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Figure 3. Effects of trehalose on skeletal muscle pathology in SOD1G93A mice. HE (A) and NADH staining (B) of gastrocnemius muscle sections in 4 mouse groups. Arrows marked the significant grouped atrophic fibers and hematoxylin inclusions. Scale bar: 100 μm; (C) The fiber area of gastrocnemius muscle in 4 mouse groups; (D) Levels of MDA in gastrocnemius muscle of 4 mouse groups. There were 3 mice in each group. Data were analyzed using one-way ANOVA followed by Tukey post hoc test. All values are presented as mean ± SEM ##P < 0.01 and #P < 0.05 as compared with WT mice; *P < 0.05 as compared with Tg-NT mice; &P < 0.05 as compared with Tg-Suc mice.
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Figure 4. Effects of trehalose on the NMJs of gastrocnemius muscle in SOD1G93A mice. NSE staining (A) and ACHE immunostaining (B) of gastrocnemius muscle sections in 4 mouse groups. Arrows marked the ACHE-positive NMJs in the muscle slides. Scale bar: 100 μm; (C) Quantitative analysis of the number of NMJs per field in 4 mouse groups; (D) Statistical analysis of the mean major diameter of NMJs in different mouse groups. There were 3 mice in each group. Data were analyzed using one-way ANOVA followed by Tukey post hoc test. Values are presented as mean ± SEM ##P < 0.01 and #P < 0.05 as compared with WT mice; **P < 0.01 as compared with Tg-NT mice; &&P < 0.01 and &P < 0.05 as compared with Tg-Suc mice.
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Figure 5. Effects of trehalose on the MTOR-independent pathway and autophagy-related proteins. Western blot analysis of protein levels of p-MTOR (A), p-AKT1 (B), and p-RPS6KB (C) in the 4 mouse groups (WT, Tg-NT, Tg-Suc, Tg-Tre). Quantitative analysis of the ratio of p-MTOR/MTOR (D), p-AKT1/AKT1 (E) and p-RPS6KB/RPS6KB (F) in the 4 mouse groups. There were 3 mice in each group; ##P < 0.01 when compared with WT mice. Western blot analysis of protein levels of LC3-II (G) and ATG5 (H) in 4 mouse groups. Quantitative analysis of LC3-II/LC3-I (I) and ATG5 (J). There were 3 mice in each group; ##P < 0.01 as compared with WT mice. (K) Immunostaining of LC3 in the motor neurons of 4 mouse groups. Scale bar: 20 μm; (L) Quantitative analysis of LC3 puncta numbers in the 4 different mouse groups. There were 3 mice in each group. Data were analyzed using one-way ANOVA followed by Tukey post hoc test. All values are presented as mean ± S.E.M, ##P < 0.01 as compared with WT mice.
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Figure 6. Effects of trehalose on SQSTM1 level and protein aggregation in SOD1G93A mice. (A) Double labeling of SQSTM1 and SOD1 in the motor neurons of WT and SOD1G93A mice. (D) Immunostaining of ubiquitin in motor neurons. Scale bar: 20 μm. Quantitative analysis of SQSTM1 density (B), SOD1 density (C) and ubiquitin density (E) in motor neurons. Western blot analysis of SQSTM1 level (F), soluble and insoluble SOD1 protein level (H) and high molecular weight bands of ubiquitinated proteins (J) in the 4 different mouse groups. Quantitative analysis of SQSTM1 protein levels (G), human soluble SOD1 and insoluble SOD1 protein (I) and ubiquitinated proteins levels (K) in the spinal cord of SOD1G93A mice. There were 3 mice in each group. Data were analyzed using one-way ANOVA followed by Tukey post hoc test. Values are presented as mean ± SEM ##P < 0.01 and #P < 0.05 as compared with WT mice; **P < 0.01 and *P < 0.05 as compared with Tg-NT mice; &&P < 0.01 and &P < 0.05 as compared with Tg-Suc mice.
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Figure 7. EM analysis of mitochondria in the motor neurons of SOD1G93A mice. Representative EM photomicrographs of mitochondrial structure in WT (A), Tg-NT (B), Tg-Suc (C) and Tg-Tre mice (D). Quantitative analysis of the number (E) and the mean diameter of mitochondria (F) in the motor neuron of 4 mouse groups. There were 3 mice in each group; asterisks marked mitochondria. Data were analyzed using one-way ANOVA followed by Tukey post hoc test. Values are presented as mean ± SEM ##P < 0.01 as compared with WT mice; *P < 0.05 as compared with Tg-NT mice; &P < 0.05 as compared with Tg-Suc mice. All scale bars: 1 μm.
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Figure 8. EM analysis of autophagosomes and autolysosomes in the motor neurons of SOD1G93A mice. Double-membrane autophagosome (A) and single-membrane autolysosomes (B and C) in motor neurons of trehalose-treated mice. Arrow marks the typical autophagosome; arrowheads mark the autolysosomes. (D) Quantitative analysis of the number of autophagosomes per field of 4 mouse groups. (E) Quantitative analysis of autophagosome/autolysosome ratios per motor neuron of the 4 different mouse groups. There were 3 mice in each group. Data were analyzed using one-way ANOVA followed by Tukey post hoc test. Values are presented as mean ± SEM ##P < 0.001 and #P < 0.01 as compared with WT mice; **P < 0.01 as compared with Tg-NT mice; &&P < 0.01 as compared with Tg-Suc mice. Scale bars: 1 μm.
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Figure 9. Effects of trehalose on proapoptotic pathway in the SOD1G93A mice. Western blot analysis of BAX (A), CYCS (B), PARP1 (E), and cleaved CASP3 (G) in the 4 different groups of mice. Quantitative analysis BAX (C), CYCS (D), PARP1 (F) in the spinal cord of the 4 different groups of mice. There were 3 mice in each group. Data were analyzed using one-way ANOVA followed by Tukey post hoc test. Values are presented as mean ± SEM ##P < 0.01 as compared with WT mice; #P < 0.05 as compared with WT mice; **P < 0.01 as compared with Tg-NT mice; *P < 0.05 as compared with Tg-NT mice; &&P < 0.01 when compared with Tg-Suc mice; &P < 0.05 when compared with Tg-Suc mice.
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Figure 10. Proposed model of the autophagic flux dysfunction and the possible roles of trehalose in the motor neurons of SOD1G93A mice. Impaired autophagosome-lysosome fusion might contribute to the degeneration of motor neurons, which leads to the abnormal high SQSTM1 level, LC3 puncta aggregation, and the increase in autophagosomes in the motor neurons of SOD1G93A mice. Rapamycin activates autophagy in an MTOR-dependent pathway and it does not affect the status of autophagic flux in the ALS mice. However, trehalose induces autophagosome formation in an MTOR-independent pathway and rescues the impaired fusion step, which results in SQSTM1 and aggregated autophagosome degradation in the motor neurons of SOD1G93A mice. AVs, autophagic vacuoles.

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