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ophthalmoplegia. The OPA1 protein is essential for normal mitochondrial fusion. In mouse DOA (such as OPA1Q285STOP), autophagy (recycling of spent cellular components) is dysregulated in retinal ganglion cells (RGCs), and mitophagy (mitochondrial recycling) has been implicated. Quantifying mitophagy is technically demanding. The hallmarks of mitophagy are organelles called autophagosomesis, co-localisating with, and then engulfing mitochondria. We validated and used two high throughput imaging systems [Imagestream (Amnis) and InCell analyser (GE)] to quantify mitophagy in fibroblasts. Co-localisation of mitochondria and autophagosomes was increased in fibroblasts in five patients from four families with severe OPA1 phenotypes indicating increased mitophagy. ImageStream also showed that basal mitophagy was increased when control cultures were depleted of Opa1 by siRNA. Western blotting confirmed increased basal autophagy and autophagic flux in the presence of activators. Increased mitochondrial fragmentation, mitophagy and failure of mitochondrial transport may together cause local depletion of mitochondria in critical regions of the affected neurons, such as axons or synapses. To investigate mitophagy in a DOA model we crossed the OPA1Q285STOP mouse with our RedMIT/GFP-LC3 mouse harbouring red fluorescent mitochondria and green fluorescent autophagosomes. We showed increased co-localisation between mitochondria and autophagosomes in both cell types investigated, confirming increased mitophagy in this model. The role of mitophagy in mitochondrial disease remains controversial, but appears to be excessive and poorly selective in OPA1 mutants. We intend to use our model to identify drug modulators of mitophagy for treating OPA1 and other mitochondrial patients.

Limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of genetically determined muscle disorders with a primary or predominant involvement of the pelvic or shoulder girdle musculature. More than 20 genes with autosomal recessive (LGMD2A to LGMD2Q) and autosomal dominant inheritance (LGMD1A to LGMD1H) have been mapped/identified to date. Mutations are known for six among the eight mapped autosomal dominant forms: LGMD1A (myotilin), LGMD1B (lamin A/C), LGMD1C (caveolin-3), LGMD1D (desmin), LGMD1E (DNAJB6), and more recently for LGMD1F (transportin-3). Our group previously mapped the LGMD1G gene at 4q21 in a Caucasian-Brazilian family. We now mapped a Uruguayan family with patients displaying a similar LGMD1G phenotype at the same locus. Whole genome sequencing identified, in both families, mutations in the HNRPDL gene. HNRPDL is a heterogeneous ribonucleoprotein (hnRNP) family member, which participates in mRNA biogenesis and metabolism. Functional studies performed in Saccharomyces cerevisiae showed that the loss of HRP1 (yeast orthologue) had pronounced effects on both protein levels and cell localizations, and yeast proteome revealed dramatic reorganization of proteins involved in RNA processing pathways. In vivo analysis showed that hnrpdl is important for muscle development in zebrafish, causing a myopathic phenotype when knocked down. The present study presents a novel association between a muscular disorder and a RNA-related gene and reinforces the importance of RNA binding/processing proteins in muscle development and muscle disease. Understanding the role of these proteins in muscle might open new therapeutic approaches for muscular dystrophies. CEPID-FAPESP, INCT, CNPq and ABDIM/AACD.