FHL1 reduces dystrophy in transgenic mice overexpressing FSHD muscular dystrophy region gene 1 (FRG1)

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disease with no effective treatment. The genetic cause of FSHD is complex and the primary pathogenic insult underlying the muscle disease is unknown. Several disease candidate genes have been proposed including DUX4 and FRG1. Expression analysis studies of FSHD report the deregulation of genes which mediate myoblast differentiation and fusion. Transgenic mice overexpressing FRG1 recapitulate the FSHD muscular dystrophy phenotype. Our current study selectively examines how increased expression of FRG1 may contribute to myoblast differentiation defects. We generated stable C2C12 cell lines overexpressing FRG1, which exhibited a myoblast fusion defect upon differentiation. To determine if myoblast fusion defects contribute to the FRG1 mouse dystrophic phenotype, this strain was crossed with skeletal muscle specific FHL1-transgenic mice. We previously reported that FHL1 promotes myoblast fusion in vitro and FHL1-transgenic mice develop skeletal muscle hypertrophy. In the current study, FRG1 mice overexpressing FHL1 showed an improvement in the dystrophic phenotype, including a reduced spinal kyphosis, increased muscle mass and myofiber size, and decreased muscle fibrosis. FHL1 expression in FRG1 mice, did not alter satellite cell number or activation, but enhanced myoblast fusion. Primary myoblasts isolated from FRG1 mice showed a myoblast fusion defect that was rescued by FHL1 expression. Therefore, increased FRG1 expression may contribute to a muscular dystrophy phenotype resembling FSHD by impairing myoblast fusion, a defect that can be rescued by enhanced myoblast fusion via expression of FHL1

Methanosarcina acetivorans C2A Topoisomerase IIIα, an Archaeal Enzyme with Promiscuity in Divalent Cation Dependence

Topoisomerases play a fundamental role in genome stability, DNA replication and repair. As a result, topoisomerases have served as therapeutic targets of interest in Eukarya and Bacteria, two of the three domains of life. Since members of Archaea, the third domain of life, have not been implicated in any diseased state to-date, there is a paucity of data on archaeal topoisomerases. Here we report Methanosarcina acetivorans TopoIIIα (MacTopoIIIα) as the first biochemically characterized mesophilic archaeal topoisomerase. Maximal activity for MacTopoIIIα was elicited at 30–35°C and 100 mM NaCl. As little as 10 fmol of the enzyme initiated DNA relaxation, and NaCl concentrations above 250 mM inhibited this activity. The present study also provides the first evidence that a type IA Topoisomerase has activity in the presence of all divalent cations tested (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+ and Cd2+). Activity profiles were, however, specific to each metal. Known type I (ssDNA and camptothecin) and type II (etoposide, novobiocin and nalidixic acid) inhibitors with different mechanisms of action were used to demonstrate that MacTopoIIIα is a type IA topoisomerase. Alignment of MacTopoIIIα with characterized topoisomerases identified Y317 as the putative catalytic residue, and a Y317F mutation ablated DNA relaxation activity, demonstrating that Y317 is essential for catalysis. As the role of Domain V (C-terminal domain) is unclear, MacTopoIIIα was aligned with the canonical E. coli TopoI 67 kDa fragment in order to construct an N-terminal (1–586) and a C-terminal (587–752) fragment for analysis. Activity could neither be elicited from the fragments individually nor reconstituted from a mixture of the fragments, suggesting that native folding is impaired when the two fragments are expressed separately. Evidence that each of the split domains plays a role in Zn2+ binding of the enzyme is also provided

Defective lysosome reformation during autophagy causes skeletal muscle disease.

The regulation of autophagy-dependent lysosome homeostasis in vivo is unclear. We showed that the inositol polyphosphate 5-phosphatase INPP5K regulates autophagic lysosome reformation (ALR), a lysosome recycling pathway, in muscle. INPP5K hydrolyzes phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] to phosphatidylinositol 4-phosphate [PI(4)P], and INPP5K mutations cause muscular dystrophy by unknown mechanisms. We report that loss of INPP5K in muscle caused severe disease, autophagy inhibition, and lysosome depletion. Reduced PI(4,5)P2 turnover on autolysosomes in Inpp5k-/- muscle suppressed autophagy and lysosome repopulation via ALR inhibition. Defective ALR in Inpp5k-/- myoblasts was characterized by enlarged autolysosomes and the persistence of hyperextended reformation tubules, structures that participate in membrane recycling to form lysosomes. Reduced disengagement of the PI(4,5)P2 effector clathrin was observed on reformation tubules, which we propose interfered with ALR completion. Inhibition of PI(4,5)P2 synthesis or expression of WT INPP5K but not INPP5K disease mutants in INPP5K-depleted myoblasts restored lysosomal homeostasis. Therefore, bidirectional interconversion of PI(4)P/PI(4,5)P2 on autolysosomes was integral to lysosome replenishment and autophagy function in muscle. Activation of TFEB-dependent de novo lysosome biogenesis did not compensate for loss of ALR in Inpp5k-/- muscle, revealing a dependence on this lysosome recycling pathway. Therefore, in muscle, ALR is indispensable for lysosome homeostasis during autophagy and when defective is associated with muscular dystrophy