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Reactivation of autophagy by spermidine ameliorates the myopathic defects of collagen VI- mice.

Chrisam M, Pirozzi M, Castagnaro S, Blaauw B, Polishchuck R, Cecconi F, Grumati P, Bonaldo P - Autophagy (2015)

Bottom Line: Autophagy is a self-degradative process responsible for the clearance of damaged or unnecessary cellular components.We have previously found that persistence of dysfunctional organelles due to autophagy failure is a key event in the pathogenesis of COL6/collagen VI-related myopathies, and have demonstrated that reactivation of a proper autophagic flux rescues the muscle defects of Col6a1- (col6a1(-/-)) mice.The beneficial effects of spermidine, together with its being easy to administer and the lack of overt side effects, open the field for the design of novel nutraceutical strategies for the treatment of muscle diseases characterized by autophagy impairment.

View Article: PubMed Central - PubMed

Affiliation: a Department of Molecular Medicine ; University of Padova ; Padova , Italy.

ABSTRACT
Autophagy is a self-degradative process responsible for the clearance of damaged or unnecessary cellular components. We have previously found that persistence of dysfunctional organelles due to autophagy failure is a key event in the pathogenesis of COL6/collagen VI-related myopathies, and have demonstrated that reactivation of a proper autophagic flux rescues the muscle defects of Col6a1- (col6a1(-/-)) mice. Here we show that treatment with spermidine, a naturally occurring nontoxic autophagy inducer, is beneficial for col6a1(-/-) mice. Systemic administration of spermidine in col6a1(-/-) mice reactivated autophagy in a dose-dependent manner, leading to a concurrent amelioration of the histological and ultrastructural muscle defects. The beneficial effects of spermidine, together with its being easy to administer and the lack of overt side effects, open the field for the design of novel nutraceutical strategies for the treatment of muscle diseases characterized by autophagy impairment.

No MeSH data available.


Related in: MedlinePlus

Spermidine induces autophagy in wild-type and COL6-deficient tibialis anterior muscles. (A) Detection of fluorescent puncta in cross sections of tibialis anterior muscles from untreated (Ctrl) and per os spermidine-treated (Spd PO) wild-type and col6a1−/− mice bearing a GFP-LC3 reporter construct. Mice were analyzed under fed conditions or after 24 h fasting. White arrowheads point at some GFP-LC3-positive puncta. Nuclei were stained with Hoechst. Scale bar: 25 μm. (B, C) Western blot analysis of LC3B lipidation in protein extracts from tibialis anterior muscles of untreated (–) and per os spermidine-treated (+) wild-type and col6a1−/− mice. Densitometric quantifications of LC3B-II vs LC3B-I ratio, as determined by at least 3 independent western blot experiments, are shown in the lower panels (n = 3 or 4; *, P< 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant). (D) Detection of fluorescent puncta in cross sections of tibialis anterior muscles from untreated (Ctrl) and i.p. spermidine-treated (Spd IP) wild-type and col6a1−/− mice. Mice were analyzed under fed conditions or after 24 h fasting. White arrowheads point at some GFP-LC3-positive puncta. Nuclei were stained with Hoechst. Scale bar: 25 μm. (E, F) Western blot analysis of LC3B lipidation in protein extracts from tibialis anterior muscles of untreated (–) and i.p. spermidine-treated (+) wild-type and col6a1−/− mice. Densitometric quantifications of LC3B-II vs LC3B-I ratio, as determined by at least 3 independent western blot experiments, are shown in the lower panels (n = 3 or 4; *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant). 24 h, twenty-four hour-long starvation; Ctrl, untreated control condition; Spd IP, spermidine i.p.; Spd PO, spermidine per os; WT, wild-type.
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f0001: Spermidine induces autophagy in wild-type and COL6-deficient tibialis anterior muscles. (A) Detection of fluorescent puncta in cross sections of tibialis anterior muscles from untreated (Ctrl) and per os spermidine-treated (Spd PO) wild-type and col6a1−/− mice bearing a GFP-LC3 reporter construct. Mice were analyzed under fed conditions or after 24 h fasting. White arrowheads point at some GFP-LC3-positive puncta. Nuclei were stained with Hoechst. Scale bar: 25 μm. (B, C) Western blot analysis of LC3B lipidation in protein extracts from tibialis anterior muscles of untreated (–) and per os spermidine-treated (+) wild-type and col6a1−/− mice. Densitometric quantifications of LC3B-II vs LC3B-I ratio, as determined by at least 3 independent western blot experiments, are shown in the lower panels (n = 3 or 4; *, P< 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant). (D) Detection of fluorescent puncta in cross sections of tibialis anterior muscles from untreated (Ctrl) and i.p. spermidine-treated (Spd IP) wild-type and col6a1−/− mice. Mice were analyzed under fed conditions or after 24 h fasting. White arrowheads point at some GFP-LC3-positive puncta. Nuclei were stained with Hoechst. Scale bar: 25 μm. (E, F) Western blot analysis of LC3B lipidation in protein extracts from tibialis anterior muscles of untreated (–) and i.p. spermidine-treated (+) wild-type and col6a1−/− mice. Densitometric quantifications of LC3B-II vs LC3B-I ratio, as determined by at least 3 independent western blot experiments, are shown in the lower panels (n = 3 or 4; *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant). 24 h, twenty-four hour-long starvation; Ctrl, untreated control condition; Spd IP, spermidine i.p.; Spd PO, spermidine per os; WT, wild-type.

Mentions: Per os administration of spermidine at both 3 mM and 30 mM was effective in stimulating autophagy in skeletal muscles. Fluorescence microscope analysis of tibialis anterior showed several GFP-LC3 puncta in wild-type animals after 30 d spermidine treatment (Fig. 1A). Autophagy induction was further confirmed by immunoblotting analysis of cleavage and lipidation of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β), quantified both as LC3B-II/-I and LC3B-II/GAPDH ratios, which was increased at both low- and high-dose spermidine treatment (Fig. 1B and C; Fig. S1A and B). Of note, these treatments did not exert any overt change in the response to 24 h starvation in wild-type mice (Fig. 1A-C; Fig. S1A and B). In agreement with previous work,8col6a1−/− tibialis anterior displayed a markedly decreased autophagy in the 24-h fasting condition, when compared to the corresponding wild-type muscles. Interestingly, spermidine administration induced autophagy in col6a1−/− muscles. Indeed, fluorescence microscopy showed the presence of GFP-LC3 puncta in tibialis anterior of spermidine-treated col6a1−/− animals, which were clearly evident in mice treated with 30 mM spermidine and subjected to 24-h starvation (Fig. 1A). Immunoblotting analysis of tibialis anterior extracts confirmed a significant increase of LC3B-II/-I and LC3B-II/GAPDH ratios in fasted col6a1−/− mice treated with 30 mM spermidine, when compared to fasted col6a1−/− mice without spermidine, whereas no significant change in LC3B-II/-I was detected in col6a1−/− mice maintained under fed conditions or treated with the lower dose of spermidine (Fig. 1B and C; Fig. S2A and B). Interestingly, the LC3B-II/GAPDH ratio was instead significantly increased in fed col6a1−/− mice following a low dose per os treatment (Fig. S1A). In order to further clarify the status of the autophagy machinery in the skeletal muscle of fed wild-type and col6a1−/− mice subjected to per os spermidine treatment at different doses, we analyzed by immunoblotting the protein levels of RAB7, a small Ras-like GTPase with an essential role in lysosomal biogenesis and in the late phases of endosome and autophagosome fusion with lysosomes,12-13 and GABARAP, a yeast Atg8 ortholog playing a key role in the final stages of autophagosome biogenesis.14 Both the lipidated form of GABARAP (GABARAP-II) and RAB7 were increased in a dose-dependent fashion following spermidine treatment not only in wild-type but also in col6a1−/− mice. These data indicate an increased rate of autophagosome and autolysosome formation, respectively, and are consistent with autophagy induction in fed animals of both genotypes (Fig. S2A-C). Altogether, these findings indicate that spermidine supplementation in drinking water for 30 d, promptly induces autophagy in wild-type muscles and also affects the autophagic process of col6a1−/− muscles.Figure 1 (see previous page).


Reactivation of autophagy by spermidine ameliorates the myopathic defects of collagen VI- mice.

Chrisam M, Pirozzi M, Castagnaro S, Blaauw B, Polishchuck R, Cecconi F, Grumati P, Bonaldo P - Autophagy (2015)

Spermidine induces autophagy in wild-type and COL6-deficient tibialis anterior muscles. (A) Detection of fluorescent puncta in cross sections of tibialis anterior muscles from untreated (Ctrl) and per os spermidine-treated (Spd PO) wild-type and col6a1−/− mice bearing a GFP-LC3 reporter construct. Mice were analyzed under fed conditions or after 24 h fasting. White arrowheads point at some GFP-LC3-positive puncta. Nuclei were stained with Hoechst. Scale bar: 25 μm. (B, C) Western blot analysis of LC3B lipidation in protein extracts from tibialis anterior muscles of untreated (–) and per os spermidine-treated (+) wild-type and col6a1−/− mice. Densitometric quantifications of LC3B-II vs LC3B-I ratio, as determined by at least 3 independent western blot experiments, are shown in the lower panels (n = 3 or 4; *, P< 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant). (D) Detection of fluorescent puncta in cross sections of tibialis anterior muscles from untreated (Ctrl) and i.p. spermidine-treated (Spd IP) wild-type and col6a1−/− mice. Mice were analyzed under fed conditions or after 24 h fasting. White arrowheads point at some GFP-LC3-positive puncta. Nuclei were stained with Hoechst. Scale bar: 25 μm. (E, F) Western blot analysis of LC3B lipidation in protein extracts from tibialis anterior muscles of untreated (–) and i.p. spermidine-treated (+) wild-type and col6a1−/− mice. Densitometric quantifications of LC3B-II vs LC3B-I ratio, as determined by at least 3 independent western blot experiments, are shown in the lower panels (n = 3 or 4; *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant). 24 h, twenty-four hour-long starvation; Ctrl, untreated control condition; Spd IP, spermidine i.p.; Spd PO, spermidine per os; WT, wild-type.
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Related In: Results  -  Collection

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f0001: Spermidine induces autophagy in wild-type and COL6-deficient tibialis anterior muscles. (A) Detection of fluorescent puncta in cross sections of tibialis anterior muscles from untreated (Ctrl) and per os spermidine-treated (Spd PO) wild-type and col6a1−/− mice bearing a GFP-LC3 reporter construct. Mice were analyzed under fed conditions or after 24 h fasting. White arrowheads point at some GFP-LC3-positive puncta. Nuclei were stained with Hoechst. Scale bar: 25 μm. (B, C) Western blot analysis of LC3B lipidation in protein extracts from tibialis anterior muscles of untreated (–) and per os spermidine-treated (+) wild-type and col6a1−/− mice. Densitometric quantifications of LC3B-II vs LC3B-I ratio, as determined by at least 3 independent western blot experiments, are shown in the lower panels (n = 3 or 4; *, P< 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant). (D) Detection of fluorescent puncta in cross sections of tibialis anterior muscles from untreated (Ctrl) and i.p. spermidine-treated (Spd IP) wild-type and col6a1−/− mice. Mice were analyzed under fed conditions or after 24 h fasting. White arrowheads point at some GFP-LC3-positive puncta. Nuclei were stained with Hoechst. Scale bar: 25 μm. (E, F) Western blot analysis of LC3B lipidation in protein extracts from tibialis anterior muscles of untreated (–) and i.p. spermidine-treated (+) wild-type and col6a1−/− mice. Densitometric quantifications of LC3B-II vs LC3B-I ratio, as determined by at least 3 independent western blot experiments, are shown in the lower panels (n = 3 or 4; *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant). 24 h, twenty-four hour-long starvation; Ctrl, untreated control condition; Spd IP, spermidine i.p.; Spd PO, spermidine per os; WT, wild-type.
Mentions: Per os administration of spermidine at both 3 mM and 30 mM was effective in stimulating autophagy in skeletal muscles. Fluorescence microscope analysis of tibialis anterior showed several GFP-LC3 puncta in wild-type animals after 30 d spermidine treatment (Fig. 1A). Autophagy induction was further confirmed by immunoblotting analysis of cleavage and lipidation of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β), quantified both as LC3B-II/-I and LC3B-II/GAPDH ratios, which was increased at both low- and high-dose spermidine treatment (Fig. 1B and C; Fig. S1A and B). Of note, these treatments did not exert any overt change in the response to 24 h starvation in wild-type mice (Fig. 1A-C; Fig. S1A and B). In agreement with previous work,8col6a1−/− tibialis anterior displayed a markedly decreased autophagy in the 24-h fasting condition, when compared to the corresponding wild-type muscles. Interestingly, spermidine administration induced autophagy in col6a1−/− muscles. Indeed, fluorescence microscopy showed the presence of GFP-LC3 puncta in tibialis anterior of spermidine-treated col6a1−/− animals, which were clearly evident in mice treated with 30 mM spermidine and subjected to 24-h starvation (Fig. 1A). Immunoblotting analysis of tibialis anterior extracts confirmed a significant increase of LC3B-II/-I and LC3B-II/GAPDH ratios in fasted col6a1−/− mice treated with 30 mM spermidine, when compared to fasted col6a1−/− mice without spermidine, whereas no significant change in LC3B-II/-I was detected in col6a1−/− mice maintained under fed conditions or treated with the lower dose of spermidine (Fig. 1B and C; Fig. S2A and B). Interestingly, the LC3B-II/GAPDH ratio was instead significantly increased in fed col6a1−/− mice following a low dose per os treatment (Fig. S1A). In order to further clarify the status of the autophagy machinery in the skeletal muscle of fed wild-type and col6a1−/− mice subjected to per os spermidine treatment at different doses, we analyzed by immunoblotting the protein levels of RAB7, a small Ras-like GTPase with an essential role in lysosomal biogenesis and in the late phases of endosome and autophagosome fusion with lysosomes,12-13 and GABARAP, a yeast Atg8 ortholog playing a key role in the final stages of autophagosome biogenesis.14 Both the lipidated form of GABARAP (GABARAP-II) and RAB7 were increased in a dose-dependent fashion following spermidine treatment not only in wild-type but also in col6a1−/− mice. These data indicate an increased rate of autophagosome and autolysosome formation, respectively, and are consistent with autophagy induction in fed animals of both genotypes (Fig. S2A-C). Altogether, these findings indicate that spermidine supplementation in drinking water for 30 d, promptly induces autophagy in wild-type muscles and also affects the autophagic process of col6a1−/− muscles.Figure 1 (see previous page).

Bottom Line: Autophagy is a self-degradative process responsible for the clearance of damaged or unnecessary cellular components.We have previously found that persistence of dysfunctional organelles due to autophagy failure is a key event in the pathogenesis of COL6/collagen VI-related myopathies, and have demonstrated that reactivation of a proper autophagic flux rescues the muscle defects of Col6a1- (col6a1(-/-)) mice.The beneficial effects of spermidine, together with its being easy to administer and the lack of overt side effects, open the field for the design of novel nutraceutical strategies for the treatment of muscle diseases characterized by autophagy impairment.

View Article: PubMed Central - PubMed

Affiliation: a Department of Molecular Medicine ; University of Padova ; Padova , Italy.

ABSTRACT
Autophagy is a self-degradative process responsible for the clearance of damaged or unnecessary cellular components. We have previously found that persistence of dysfunctional organelles due to autophagy failure is a key event in the pathogenesis of COL6/collagen VI-related myopathies, and have demonstrated that reactivation of a proper autophagic flux rescues the muscle defects of Col6a1- (col6a1(-/-)) mice. Here we show that treatment with spermidine, a naturally occurring nontoxic autophagy inducer, is beneficial for col6a1(-/-) mice. Systemic administration of spermidine in col6a1(-/-) mice reactivated autophagy in a dose-dependent manner, leading to a concurrent amelioration of the histological and ultrastructural muscle defects. The beneficial effects of spermidine, together with its being easy to administer and the lack of overt side effects, open the field for the design of novel nutraceutical strategies for the treatment of muscle diseases characterized by autophagy impairment.

No MeSH data available.


Related in: MedlinePlus