Moss-Derived Human Recombinant GAA Provides an Optimized Enzyme Uptake in Differentiated Human Muscle Cells of Pompe Disease.

Pompe disease is an autosomal recessive lysosomal storage disorder (LSD) caused by deficiency of lysosomal acid alpha-glucosidase (GAA). The result of the GAA deficiency is a ubiquitous lysosomal and non-lysosomal accumulation of glycogen. The most affected tissues are heart, skeletal muscle, liver, and the nervous system. Replacement therapy with the currently approved enzyme relies on M6P-mediated endocytosis. However, therapeutic outcomes still leave room for improvement, especially with regard to skeletal muscles. We tested the uptake, activity, and effect on glucose metabolism of a non-phosphorylated recombinant human GAA produced in moss (moss-GAA). Three variants of moss-GAA differing in glycosylation pattern have been analyzed: two with terminal mannose residues in a paucimannosidic (Man3) or high-mannose (Man 5) configuration and one with terminal N-acetylglucosamine residues (GnGn). Compared to alglucosidase alfa the moss-GAA GnGn variant showed increased uptake in differentiated myotubes. Moreover, incubation of immortalized muscle cells of Gaa-/- mice with moss-GAA GnGn led to similarly efficient clearance of accumulated glycogen as with alglucosidase alfa. These initial data suggest that M6P-residues might not always be necessary for the cellular uptake in enzyme replacement therapy (ERT) and indicate the potential of moss-GAA GnGn as novel alternative drug for targeting skeletal muscle in Pompe patients.

Assessing metabolic profiles in human myoblasts from patients with late-onset Pompe disease.

BACKGROUND: Pompe disease is a neuromuscular disease caused by a deficiency of lysosomal acid alpha-glucosidase (GAA) which degrades glycogen, resulting in progressive accumulation of lysosomal glycogen, lysosomal swelling and rupture. In addition, mitochondrial abnormalities have been frequently observed in muscle biopsy specimens of Pompe patients. Enzyme replacement therapy (ERT) using alglucosidase alfa, a recombinant human GAA, is so far the only available therapy. We evaluated glycolysis and basal respiration in primary human myoblasts from patients with Pompe disease and in mouse myoblasts from GAA knockout mice before and after alglucosidase alfa treatment. METHODS: We tested patient-derived primary human myoblasts and immortalized GAA-/- mouse myoblasts for GAA activity, glycolytic activity, and mitochondrial respiration before and after alglucosidase alfa treatment using enzyme activity assays and SeaHorse measurements. RESULTS: A significant reduction in glycolysis (30%) and in mitochondrial respiration (50%) was observed in both, human and mouse GAA-deficient myoblasts. Treatment with alglucosidase alfa resulted in partial recovery of both metabolic pathways with some variability in human myoblasts. CONCLUSIONS: Future assessments of treatment efficacy should include screening for the metabolic effects on both glycolysis and mitochondrial respiration in order to obtain a better read-out of the cellular energy metabolism.

Myotonic Dystrophy-A Progeroid Disease?

Myotonic dystrophies (DM) are slowly progressing multisystemic disorders caused by repeat expansions in the DMPK or CNBP genes. The multisystemic involvement in DM patients often reflects the appearance of accelerated aging. This is partly due to visible features such as cataracts, muscle weakness, and frontal baldness, but there are also less obvious features like cardiac arrhythmia, diabetes or hypogammaglobulinemia. These aging features suggest the hypothesis that DM could be a segmental progeroid disease. To identify the molecular cause of this characteristic appearance of accelerated aging we compare clinical features of DM to "typical" segmental progeroid disorders caused by mutations in DNA repair or nuclear envelope proteins. Furthermore, we characterize if this premature aging effect is also reflected on the cellular level in DM and investigate overlaps with "classical" progeroid disorders. To investigate the molecular similarities at the cellular level we use primary DM and control cell lines. This analysis reveals many similarities to progeroid syndromes linked to the nuclear envelope. Our comparison on both clinical and molecular levels argues for qualification of DM as a segmental progeroid disorder.

Nuclear Envelope Transmembrane Proteins in Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder with predominant myotonia and muscular dystrophy which is caused by CTG-repeat expansions in the DMPK gene. These repeat expansions are transcribed and the resulting mRNA accumulates RNA-binding proteins involved in splicing, resulting in a general splicing defect. We observed nuclear envelope (NE) alterations in DM1 primary myoblasts. These included invaginations of the NE as well as an altered composition of the nuclear lamina. Specifically, we investigated NE transmembrane proteins (NETs) in DM1 primary myoblasts, staining to determine if their distribution was altered compared to controls and if this could contribute to these structural defects. We also tested the expression of these NETs in muscle and how localization changes in the DM1 primary myoblasts undergoing differentiation in vitro to myotubes. We found no changes in the localization of the tested NETs, but most tended to exhibit reduced expression with increasing DMPK-repeat length. Nonetheless, the DM1 patient expression range was within the expression range of the controls. Additionally, we found a down-regulation of the possible nesprin 1 giant isoform in DM1 primary myoblasts which could contribute to the increased NE invaginations. Thus, nesprin 1 may be an interesting target for further investigation in DM1 disease pathology.