The genetics, biochemistry, clinical presentations, and many other facets of Pompe disease are very well described, but important questions have remained unanswered: how is glycogen delivered to the lysosomes; and what is the mechanism of the profound skeletal muscle damage. The answers to both of these questions have implications for the proper design of therapy.
Glycogen has been thought to reach the lysosomes via the autophagic pathway. This assumption is based on data showing autophagic delivery of glycogen to the lysosomes in neonatal tissues (reviewed in 31
). In this early postnatal period, when there is a high demand for glucose, ‘glycogen autophagy’ and lysosomal degradation of glycogen to glucose constitute a mechanism of supplying this sugar. It has been suggested that hepatic glycogen autophagy is a selective and highly regulated process in the newborn. Several pathways, including the cyclic AMP/cyclic AMP-dependent protein kinase and the phosphoinositides/TOR pathways are implicated in the control of this process (33
). Earlier studies suggested that autophagic degradation and rapid mobilization of glycogen not only in liver, but also in skeletal muscle, is an important source of energy in the first critical period of extra-uterine life in newborn rats (36
). However, little is known about the role of glycogen autophagy in adult animals. We have now demonstrated that the AD-GAA KO and GAA KO mice contain a comparable amount of glycogen in muscle, indicating that macroautophagy in muscle is not the major route of glycogen delivery to the lysosome in adult animals. This is true of both slow and fast muscles. Microautophagy, the direct transport of substrates to the lysosome by invagination of the lysosomal membrane, may be a plausible alternative. Thus, the findings indicate that suppression of autophagy is not an option for substrate deprivation therapy in Pompe disease.
The need to expand our understanding of the disease pathogenesis and the mechanisms of muscle damage in Pompe disease has become particularly clear in light of the limited success of the recently developed ERT in reversing pathology in skeletal muscle (12
). The long-held view of the pathogenesis is that expansion and rupture of lysosomes result in the release of glycogen and lytic enzymes into the cytoplasm, which causes a loss of myofibrils and contractile function (38
). However, studies in a mouse model of the disease (40
) have shown that the enlarged lysosomes in skeletal muscle cannot adequately account for the reduction in mechanical performance, and that the presence of large inclusions containing degraded myofibrils contributes to the impairment of muscle function (41
). In another GAA KO mouse model (17
), we showed the enormous extent of these inclusions in fast muscle fibers, their effect on muscle architecture, the problem they pose for ERT, and their considerable autophagic component, justifying the term ‘autophagic buildup’ (43
It remained unclear, however, whether the increased number of autophagosomes in fast fibers is the result of an induction of autophagy, an impairment of autophagosomal–lysosomal fusion, or a combination of both. Distinguishing between these two opposite scenarios would greatly influence the development of therapeutic approaches. We did observe induction of autophagy in both fast and slow GAA KO fibers (although more robust in fast fibers) as evidenced by increased levels of LC3-I and LC3-II compared with WT levels.
The consequences of the induction of autophagy in the diseased muscle, however, are profoundly different depending on the fiber type. Counter-intuitively, the induction of autophagy in fast GAA KO muscle is associated with a functional deficiency of autophagy. In these fibers, there is also a problem with the other end of the autophagic process, namely impaired fusion with the lysosome. Failure to fuse with lysosomes, referred to as incomplete autophagic flux (45
), results in accumulation of autophagic substrates, such as potentially toxic Ub-proteins. The accumulation of such Ub-proteins observed in GAA KO fast fibers is clear evidence of incomplete autophagic flux. This conclusion is strengthened by the finding that P62 also accumulates in these fibers. P62/SQTSM1, a scaffold protein that binds both LC3 and ubiquitinated substrates, is itself an autophagic substrate and thus accumulates only when autophagy is blocked (25
). The autophagic dead-end in muscle is in effect a functional autophagic deficiency. The deficiency is, however, local and limited to the core of the fiber, and as such is less detrimental than the complete genetic deficiency and dispersed cytoplasmic accumulation of Ub-proteins in the AD-GAA KO fast fibers. Thus, the additional burden imposed by complete autophagy deficiency in the AD-GAA KO exaggerates the problem already present in the GAA KO. The elimination of toxic Ub-proteins was traditionally assigned exclusively to the proteasomal system, but is now recognized as a critical function of constitutive autophagy in addition to its major roles in the cellular responses to starvation or stress (reviewed in 1
). This recognition was made possible by data showing accumulation of these proteins when autophagy was suppressed in liver or brain (16
Malfunctioning of the endocytic pathway that terminates at the lysosome is also likely to contribute to the pool of Ub-proteins in the GAA KO. It has been shown that Ub-proteins destined for lysosomal degradation are delivered to this organelle not only by the autophagic, but also by the endocytic pathways. For example, multiple plasma membrane proteins and cell surface receptors are mono-ubiquitinated and sorted into the lumen of multivesicular bodies for delivery to lysosomes. Lysosomal contribution to the accumulation of Ub-proteins in GAA KO is supported by the fact that complete autophagic deficiency in healthy muscle (AD-WT) is much less harmful than the complete autophagic deficiency combined with abnormal lysosomes (AD-GAA KO). Lysosomal degradation of cell-surface receptors serves as a mechanism for transient downregulation of these polypeptides (49
), and the failure to efficiently downregulate these molecules in GAA KO may result in unrestrained signaling with profound consequences. This possibility needs to be further investigated.
Muscle wasting in GAA KO mice occurs despite the absence of the transcriptional changes shown to be associated with muscle atrophy due to a variety of conditions, such as cancer cachexia, uremia, diabetes, etc. (22
). Thus, the mechanism of muscle loss in Pompe disease and possibly other metabolic myopathies is quite distinct from the ‘common atrophy program’ (22
This distinct mechanism may involve the accumulation of undegraded aggregate-prone Ub-proteins: in the GAA KO this pathology is observed before the first clinical symptoms, but expands with age, paralleling disease progression. It has been shown that cytoplasmic accumulation of large ubiquitin-containing aggregates and inclusion bodies in autophagy-deficient hepatocytes and neurons resulted in severe liver injury and neurodegeneration (16
). Progressive neuronal loss in autophagy-deficient neurons was associated with increase in size and number of these inclusions, suggesting a strong correlation between the accumulation of the Ub-proteins and disease severity. In both autophagy-deficient brain and liver, these inclusions occurred despite the apparently normal proteasome function.
The association of skeletal muscle weakness with the accumulation of high molecular weight proteins and Ub-containing protein inclusions has been shown for sporadic inclusion-body myositis and hereditary inclusion body myopathies (IBM) (57
). A similar association has been recently shown for IBM caused by mutations in valosin-containing protein p97/VCP (58
). In these cases, the accumulation of the Ub-proteins appears to be a consequence of abnormal proteasome function.
Finally, accumulation of Ub-proteins due to an autophagic block is associated with neuronal cell death in a deficiency of another lysosomal enzyme, multiple sulfatase deficiency (5
), suggesting that abnormal autophagy and toxicity from ubiquitinated material may also be a general mechanism of cellular damage not only for metabolic myopathies, but also for other lysosomal storage disorders.