Deconstructing Pompe disease by analyzing single muscle fibers: to see a world in a grain of sand...

Autophagy is a major pathway for delivery of proteins and organelles to lysosomes where they are degraded and recycled. We have previously shown excessive autophagy in a mouse model of Pompe disease (glycogen storage disease type II), a devastating myopathy caused by a deficiency of the glycogen-degrading lysosomal enzyme acid alpha-glucosidase. The autophagic buildup constituted a major pathological component in skeletal muscle and interfered with delivery of the therapeutic enzyme. To assess the role of autophagy in the pathogenesis of the human disease, we have analyzed vesicles of the lysosomal-degradative pathway in isolated single muscle fibers from Pompe patients. Human myofibers showed abundant autophagosome formation and areas of autophagic buildup of a wide range of sizes. In patients, as in the mouse model, the enormous autophagic buildup causes greater skeletal muscle damage than the enlarged, glycogenfilled lysosomes outside the autophagic regions. Clearing or preventing autophagic buildup seems, therefore, a necessary target of Pompe disease therapy.

Multiple cryptic splice sites can be activated by IDS point mutations generating misspliced transcripts.

Mutations in the gene encoding the enzyme iduronate-2-sulfatase (IDS) were reported as the cause of the X-linked recessive lysosomal disease, mucopolysaccharidosis II (MPS II). Amongst the different mutations, it emerges that nearly 10% are nucleotide substitutions causing splicing mutations. We now report the molecular characterisation of three MPS II patients with multiple aberrant transcripts due to three different point mutations. The c.418+1G>C that occurred in the invariant splice-site motif, produced only aberrantly spliced transcripts. Whilst the mutations affecting variant motifs (c.419G>T) or coding regions (c.245C>T) led to aberrantly spliced transcripts in addition to correctly spliced transcripts with the respective predicted missense mutation, p.G140V or p.A82V. A combination of experimental tests and computational approaches were used to understand the molecular basis underlying the altered transcription patterns. In addition, by using real-time reverse transcriptase polymerase chain reaction, the reduction of mRNA amount in two patients observed was likely due to nonsense-mediated mRNA decay pathway. Overall, our results further emphasised the importance of cloning and sequencing independent transcripts to reveal less abundant, aberrant products, which often could not be detected by direct sequencing. Moreover, the different splicing patterns observed in the three patients as a consequence of point mutations show how sensitive the balance is between constitutive and cryptic splice sites in the IDS gene. The generation of such diverse transcripts, together with their level of expression, could contribute to the profound phenotypic variability reported in MPS II.

Mycobacteria-containing phagosomes associate less annexins I, VI, VII and XI, but not II, concomitantly with a diminished phagolysosomal fusion.

We have studied the intracellular localization of annexins I,II, VI, VII, and XI in cells containing latex beads or Mycobacterium avium at different times after ingestion in order to establish whether a correlation existed between the association of annexins to phagosomes and phagolysosomal fusion, since the intracellular survival of mycobacteria is linked to an impairment of phagosome maturation. We demonstrate an important decrease in the levels of association of annexins I, VI, VII and XI, but not II to phagosomes containing either live or killed mycobacteria compared with phagosomes containing inert latex particles. The reduced association of annexins observed was detected only on M. avium-containing phagosomes and not in other cell membrane nor in cytosolic fractions from infected cells, and was apparent from 8 hours through to 4 days after phagocytosis. These findings add elements to the present knowledge of the phagosomal modifications that accompany the survival of intracellular pathogens, suggesting that annexins I, VI, VII, and XI play a secondary role in phagosomal fusion events while annexin II does not seem to be related to the mechanism of regulation of endolysosomal fusion.