Pompe disease: from pathophysiology to therapy and back again

Abstract

Pompe disease is a lysosomal storage disorder in which acid alpha-glucosidase (GAA) is deficient or absent. Deficiency of this lysosomal enzyme results in progressive expansion of glycogen-filled lysosomes in multiple tissues, with cardiac and skeletal muscle being the most severely affected. The clinical spectrum ranges from fatal hypertrophic cardiomyopathy and skeletal muscle myopathy in infants to relatively attenuated forms, which manifest as a progressive myopathy without cardiac involvement. The currently available enzyme replacement therapy (ERT) proved to be successful in reversing cardiac but not skeletal muscle abnormalities. Although the overall understanding of the disease has progressed, the pathophysiology of muscle damage remains poorly understood. Lysosomal enlargement/rupture has long been considered a mechanism of relentless muscle damage in Pompe disease. In past years, it became clear that this simple view of the pathology is inadequate; the pathological cascade involves dysfunctional autophagy, a major lysosome-dependent intracellular degradative pathway. The autophagic process in Pompe skeletal muscle is affected at the termination stage-impaired autophagosomal-lysosomal fusion. Yet another abnormality in the diseased muscle is the accelerated production of large, unrelated to ageing, lipofuscin deposits-a marker of cellular oxidative damage and a sign of mitochondrial dysfunction. The massive autophagic buildup and lipofuscin inclusions appear to cause a greater effect on muscle architecture than the enlarged lysosomes outside the autophagic regions. Furthermore, the dysfunctional autophagy affects the trafficking of the replacement enzyme and interferes with its delivery to the lysosomes. Several new therapeutic approaches have been tested in Pompe mouse models: substrate reduction therapy, lysosomal exocytosis following the overexpression of transcription factor EB and a closely related but distinct factor E3, and genetic manipulation of autophagy.

Keywords: Pompe disease; autophagy; enzyme replacement therapy; lipofuscin; lysosome.

Figure 1

In vitro model of Pompe disease replicates lysosomal, but not autophagic pathology. The images show wild type (WT) and GAA-KO (KO) myotubes. Immortalized myoblasts were used for the differentiation into multinucleated myotubes. WT myoblasts were derived from mice generated by crosses of GFP-LC3:WT to Immortomice; GAA-KO myoblasts were derived from mice generated by crosses of GFP-LC3:GAA-KO to Immortomice. LC3 (green) is a specific autophagosomal marker. No autophagosomal accumulation is seen in the GAA-KO cells. Myotubes were fixed and stained for lysosomal marker LAMP1 (red). Enlarged lysosomes are seen in the GAA-KO, but not in control WT muscle cells. Bars: 10 μm.

Figure 2

Electron microscopy images show the presence and the extent (A) of autophagic buildup in skeletal muscle from a 5 month-old GAA-KO mouse. Larger magnification images (B and C) show classical double membrane autophagosomes with undigested cytosolic material (black arrows) or glycogen particles (arrowhead), multimembrane structures (most prominent in C), multivesicular body (white arrow), electron dense material (asterisk), as well other cellular debris of unknown origin. Bars: 0.5 μm.

Figure 3

Confocal microscopy images of fibers from GFP-LC3:WT (A) and GFP-LC3:GAA-KO (B) mice. In these strains autophagosomes are labeled with green fluorescent protein. Accumulation of autophagosomes (green) can be seen in live unstained muscle fibers derived from GAA-KO, but not from WT mice. Muscle was transfected in vivo by electroporation with mCherry-LAMP1 to visualize lysosomes (red); fibers were isolated 6 days after the procedure. (A) Autophagosomes are not detectable in the control muscle. (B) This still image shows autophagic buildup in the core of a fiber from the diseased muscle. Time-lapse microscopy of mCherry-LAMP1 transfected fibers demonstrated that there is little, if any, fusion between lysosomes and autophagosomes in the areas of autophagic buildup. Bar: 10 μm.

Figure 4

Autofluorescent lipofuscin inclusions in a muscle biopsy from a patient (NBSL9a) with a childhood form of Pompe disease. The patient was diagnosed during a family study and began therapy at 7 years of age. The biopsy was taken prior to the initiation of ERT. Bar: 10 μm.