Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2008 Dec 15;17(24):3897-908.
doi: 10.1093/hmg/ddn292. Epub 2008 Sep 9.

Suppression of autophagy in skeletal muscle uncovers the accumulation of ubiquitinated proteins and their potential role in muscle damage in Pompe disease

Nina Raben  1 , Victoria Hill, Lauren Shea, Shoichi Takikita, Rebecca Baum, Noboru Mizushima, Evelyn Ralston, Paul Plotz
Affiliations
Comparative Study

Suppression of autophagy in skeletal muscle uncovers the accumulation of ubiquitinated proteins and their potential role in muscle damage in Pompe disease

Nina Raben et al. Hum Mol Genet. .

Abstract

The role of autophagy, a catabolic lysosome-dependent pathway, has recently been recognized in a variety of disorders, including Pompe disease, the genetic deficiency of the glycogen-degrading lysosomal enzyme acid-alpha glucosidase. Accumulation of lysosomal glycogen, presumably transported from the cytoplasm by the autophagic pathway, occurs in multiple tissues, but pathology is most severe in skeletal and cardiac muscle. Skeletal muscle pathology also involves massive autophagic buildup in the core of myofibers. To determine if glycogen reaches the lysosome via autophagy and to ascertain whether autophagic buildup in Pompe disease is a consequence of induction of autophagy and/or reduced turnover due to defective fusion with lysosomes, we generated muscle-specific autophagy-deficient Pompe mice. We have demonstrated that autophagy is not required for glycogen transport to lysosomes in skeletal muscle. We have also found that Pompe disease involves induction of autophagy but manifests as a functional deficiency of autophagy because of impaired autophagosomal-lysosomal fusion. As a result, autophagic substrates, including potentially toxic aggregate-prone ubiquitinated proteins, accumulate in Pompe myofibers and may cause profound muscle damage.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Generation and characterization of muscle-specific AD-GAA KO mice. (A) Western blotting of protein lysates from gastrocnemius muscle of 3-month-old mice with LC3 antibody. Lane 1: WT; Lane 2: GAA KO; Lane 3: AD-GAA KO. The absence of LC3-II in AD-GAA KO indicates an efficient suppression of autophagy (dozens of mice were analyzed for the suppression of autophagy). (B) PAS-stained sections of gastrocnemius muscle from 5-month-old WT, GAA KO and AD-GAA KO; PAS-positive material (small dots) is seen in both GAA KO and AD-GAA KO. Centrally located ‘holes’ found in the GAA KO, but not in the AD-GAA KO fibers represent areas of autophagic buildup (arrows). Magnification: 335×. A 4.5-month-old AD-GAA KO mouse shows profound muscle wasting.
Figure 2.
Figure 2.
Buildup of late endocytic vesicles in fast (psoas) fibers from AD-GAA KO. (A) Single fiber stained for LAMP-1 (LP). Arrowhead points to an enlarged lysosome. Bar: 10 µm. (B) Single fiber stained for CI-MPR (MPR) and LAMP-1. Bar: 10 µm. (C and D) Electron microscopy shows intermyofibrillar accumulations of enlarged glycogen-filled lysosomes (arrowhead), clusters of vesicles and inclusions (*). These inclusions likely represent polyubiquitinated protein aggregates (see text and Fig. 4). Some vesicles have the typical morphology of multi-vesicular bodies (right panel in D). Bars: 500 nm.
Figure 3.
Figure 3.
Accumulation of Ub-proteins in fast (gastrocnemius) muscle of GAA KO and AD-GAA KO. Muscle lysates, prepared as detergent (Triton X-100)-soluble and -non-soluble fractions, were analyzed by immunoblotting with anti-ubiquitin (FK2) antibody. (A) Muscle samples were taken from ∼4-month-old WT, GAA KO and AD-GAA KO mice. High molecular mass Ub-proteins are marked by a bracket. Data shown are representative of at least four independent experiments. (B) Age-dependent accumulation of soluble and non-soluble Ub-proteins in muscle from young (6-week-old; I) and old (10-month-old; II) GAA KO. For each time point and each genotype, three mice were used.
Figure 4.
Figure 4.
Distribution of Ub-proteins in fast (psoas) muscle of GAA KO and AD-GAA KO. Left top panel: GAA KO; single fiber stained for ubiquitin (Ub) and LAMP-1 (LP) showing the confinement of Ub-positive structures to the area of autophagic buildup (arrow); in contrast, LAMP-1 staining is present throughout the fiber—in the autophagic area and on the enlarged lysosomes (arrowhead). Bar: 10 µm. Right panel: single fiber stained for Ub and LC3 showing the presence of Ub-positive structures within LC3-positive autophagosomes. Bar: 10 µm. The EM panel shows autophagic buildup. Bar: 2 µm. Left panel: AD-GAA KO; single fiber stained for Ub and LAMP-1 showing Ub-positive structures (arrow) dispersed in a linear array throughout the fiber in a repeating pattern of inclusions flanking lysosomes (arrowhead). Bar: 10 µm. EM (left bottom panel) shows glycogen-filled lysosomes (arrowheads) and an electron-light, irregularly shaped structure (arrow), which likely represents a Ub-positive inclusion. Bar: 500 nm. At higher magnification (right EM panels) the filamentous inclusions (arrows) appear very different from the membrane-bound lysosomal glycogen (arrowhead). Bars: 500 nm (top); 100 nm (bottom).
Figure 5.
Figure 5.
Distribution of P62/SQSTM1 protein in fast (psoas) muscle of GAA KO and AD-GAA KO. Single fibers stained for P62 and for LAMP-1 (LP). P62 protein accumulates in the area of autophagic buildup in muscle from GAA KO. In AD-GAA KO P62-positive structures are dispersed throughout the fiber [this pattern was observed in profoundly atrophic fibers (∼50% of the total); the remaining fibers showed no staining for P62]. The recording parameters were optimized for each genotype. The WT P62 image was recorded at a higher gain than the other two images to ensure the absence of cytoplasmic staining. Bar: 10 µm.
Figure 6.
Figure 6.
(A) Basal level of autophagy in gastrocnemius and soleus muscles from WT and GAA KO. Western blotting of protein lysates with LC3 antibody shows a dramatic increase in the levels of both LC3-I and LC3-II in GAA KO. At least five WT and five GAA KO animals were used for these experiments. (B) Western blotting of protein lysates from soleus (slow) muscle with FK2 antibody. No difference in the levels of Ub-proteins is observed among the three genotypes in 4-month-old mice. Data shown are representative of at least three independent experiments.
Figure 7.
Figure 7.
Accumulation of Ub-proteins and P62 in AD-WT gastrocnemius muscle. Single fibers stained for Ub and LAMP-1 (LP) show a typical Ub pattern (A) and a rare pattern of large deposits (C) of ubiquitin-positive material. The overall level of Ub-positive staining is much lower than in the AD-GAA KO shown in Figure 4. The WT Ub image (D) was recorded at a higher gain than the AD-WT images (A and C) to ensure the absence of cytoplasmic staining. (B) EM shows a filamentous inclusion, which resembles those shown in Figure 4. Bars: 500 nm; 100 nm (inset). (E) Single fiber stained for P62 and LAMP-1 showing the similarity between P62 and Ub staining patterns. Bars: 10 µm.
Figure 8.
Figure 8.
Altered distribution of microtubules and lysosomes in AD-WT muscle. Single fibers from fast WT and AD-WT (gastrocnemius) muscle stained for alpha-tubulin (α tub) and LAMP-1 (LP) showing the realignment of microtubules and lysosomes in AD-WT. Arrowhead points to a string of microtubules and lysosomes. Bar: 10 µm. EM of fast (psoas) muscle from AD-WT shows vesicular structures in the intermyofibrillar space. The vesicles likely represent lysosomes. Bar: 1 µm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Levine B., Klionsky D.J. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev. Cell. 2004;6:463–477. - PubMed
    1. Mizushima N., Levine B., Cuervo A.M., Klionsky D.J. Autophagy fights disease through cellular self-digestion. Nature. 2008;451:1069–1075. - PMC - PubMed
    1. Klionsky D.J. Autophagy: from phenomenology to molecular understanding in less than a decade. Nat. Rev. Mol. Cell Biol. 2007;8:931–937. - PubMed
    1. Kiselyov K., Jennigs J.J., Jr, Rbaibi Y., Chu C.T. Autophagy, mitochondria and cell death in lysosomal storage diseases. Autophagy. 2007;3:259–262. - PMC - PubMed
    1. Settembre C., Fraldi A., Jahreiss L., Spampanato C., Venturi C., Medina D., de Pablo R., Tacchetti C., Rubinsztein D.C., Ballabio A. A block of autophagy in lysosomal storage disorders. Hum. Mol. Genet. 2008;17:119–129. - PubMed

Publication types

MeSH terms