We have demonstrated that in the GAA KO mice there is a progressive accumulation of autophagic material in multiple type II-rich muscles. The process appears to begin with the formation of a number of autophagosomes, usually in the core of the fiber. At a later stage, the same area becomes enlarged and filled with vesicles of the autophagic and endocytic pathways. Eventually, in older mice, the vesicular structures appear to be lost, leaving massive lakes of what we assume to be glycogen and the remnants of membranes. These data, therefore, challenge the current view that enlargement of glycogen-filled lysosomes leading to lysosomal rupture represents the only pathogenic mechanism of muscle damage.
The question remains as to what triggers the autophagy in type II KO fibers. Nutrient limitation is a powerful inducer of autophagy, and the autophagic response in skeletal muscle has been shown to be fiber-type specific; it is rapid and intense in type II- rich muscle, but moderate in type I-rich muscle [23
]. We have previously suggested that a local nutritional stress in a segment of a fiber may be such a trigger [19
]. Another condition that may induce autophagy is oxidative stress (reviewed in [24
]). Based on experimental data showing reparative autophagocytosis in fibroblasts exposed to mild oxidative stress [25
], Kiffin R. et al. hypothesized that moderate stress, affecting only a small number of lysosomes, may activate autophagy to sequester the leaking lysosomes [26
]. A similar sequence of events could take place at the beginning stages of Pompe disease; the autophagy might be up-regulated to remove small damaged lysosomes. Our observation of autophagic vacuoles containing lysosomes without clear membrane boundaries in young KO supports such a possibility.
Regardless of whether oxidative stress contributes to the initial stage of the disease, its role is evident at the later stages by the increased formation of lipofuscin. Near exclusive concentration of lipofuscin in the autophagic areas in the KO indicates that the process is disease-related. Lipofuscin, an autofluorescent material composed of oxidatively modified molecules, normally accumulates in lysosomes of postmitotic cells during aging. However, abnormal increase of lipofuscin is associated with oxidative stress [24
]. Enhanced deposition of lipofuscin and large areas of centrally located cellular debris were observed by Hesselink R. et al. [29
] in muscles of another mouse model of Pompe disease (AGLU-/-
]; these inclusions most likely represent the autophagic area. The time course and the extent of the changes in the AGLU-/-
mice, however, are quite different from those seen in the model reported here. One explanation for the discrepancy is that the disease is milder in AGLU-/-
mice. A more likely explanation is different methods used to assess morphology.
The dysregulation of the autophagic pathway, which converges with the endocytic pathway at the late endosomes, may lead to the mis-targeting of the endocytosed therapeutic enzyme. We addressed the issue by using a unique experimental system - analysis of endocytosis in live myofibers - which has been used only once before to characterize the endocytic compartments in normal fully differentiated rat myofibers from flexor digitorum brevis muscle [31
]. We have presented here experimental evidence that the endocytosed therapeutic enzyme (and dextran) in the KO fibers accumulates along the length of the fibers, primarily in the vesicular compartments of the autophagic areas. Some of these compartments represent autophagosomes, late endosomes, intermediate autophagosomes, and possibly damaged nonfunctional lysosomes unable to degrade the autophagocytosed material. These structures, which include both intralysosomal (lipofuscin) as well as extralysosomal undigested material could be called biological garbage [24
]. The recombinant enzyme, trapped in these areas, is wasted since it is diverted from glycogen-filled lysosomes in the rest of the fiber, but is unable to resolve the autophagic build-up [19
], which continues to expand as the disease progresses.
Furthermore, processing of the rhGAA precursor to fully active mature forms seems to be altered in the diseased fibers. Cleavage of the precursor results in the activation of the enzyme, and it is the mature forms that are most active toward the natural substrate glycogen [10
]. The conversion of the precursor to the 95 kDa intermediate most likely occurs in the late endocytic compartment. The subsequent conversion to the mature forms requires both proteases and glycosidases and is thought to occur in the lysosomes [10
]. In the myofibers, processing of the 95 kDa intermediate to the 76 kDa mature form seems to be stalled, resulting in a shift in the ratio of these two forms toward the intermediate with a lower affinity for glycogen. Additionally, only a small part of the mature form detected in muscle biopsies after ERT actually reaches the myofibers. Thus, the mature GAA in non-muscle components of a muscle biopsy contributes significantly to the overall level of enzyme detected after ERT. This finding should be considered when muscle biopsies are used to monitor the efficacy of ERT.
A number of factors make skeletal muscle a challenging target for ERT, among them are its sheer mass, the low density of CI-MPR [32
], and the fact that the vast majority of the administered enzyme is delivered to the liver [17
]. Type II fibers in the KO are at an even greater disadvantage since they have lower receptor density and lower levels of trafficking proteins compared to type I fibers [19
]. Mis-targeting of rhGAA in type II fibers exacerbates the problem. When the enzyme does reach the lysosomes, it seems to work well. However, the reversal of pathology in Pompe skeletal muscle requires more than clearance of lysosomal glycogen. Furthermore, assuming that similar autophagic changes occur in Pompe patients, the current therapy should be considered for pre-symptomatic late onset patients before autophagic buildup occurs.