A splicing mutation in the novel mitochondrial protein DNAJC11 causes motor neuron pathology associated with cristae disorganization, and lymphoid abnormalities in mice.

Mitochondrial structure and function is emerging as a major contributor to neuromuscular disease, highlighting the need for the complete elucidation of the underlying molecular and pathophysiological mechanisms. Following a forward genetics approach with N-ethyl-N-nitrosourea (ENU)-mediated random mutagenesis, we identified a novel mouse model of autosomal recessive neuromuscular disease caused by a splice-site hypomorphic mutation in a novel gene of unknown function, DnaJC11. Recent findings have demonstrated that DNAJC11 protein co-immunoprecipitates with proteins of the mitochondrial contact site (MICOS) complex involved in the formation of mitochondrial cristae and cristae junctions. Homozygous mutant mice developed locomotion defects, muscle weakness, spasticity, limb tremor, leucopenia, thymic and splenic hypoplasia, general wasting and early lethality. Neuropathological analysis showed severe vacuolation of the motor neurons in the spinal cord, originating from dilatations of the endoplasmic reticulum and notably from mitochondria that had lost their proper inner membrane organization. The causal role of the identified mutation in DnaJC11 was verified in rescue experiments by overexpressing the human ortholog. The full length 63 kDa isoform of human DNAJC11 was shown to localize in the periphery of the mitochondrial outer membrane whereas putative additional isoforms displayed differential submitochondrial localization. Moreover, we showed that DNAJC11 is assembled in a high molecular weight complex, similarly to mitofilin and that downregulation of mitofilin or SAM50 affected the levels of DNAJC11 in HeLa cells. Our findings provide the first mouse mutant for a putative MICOS protein and establish a link between DNAJC11 and neuromuscular diseases.

A RANKL G278R mutation causing osteopetrosis identifies a functional amino acid essential for trimer assembly in RANKL and TNF.

Receptor activator of nuclear factor-κB ligand (RANKL), a trimeric tumor necrosis factor (TNF) superfamily member, is the central mediator of osteoclast formation and bone resorption. Functional mutations in RANKL lead to human autosomal recessive osteopetrosis (ARO), whereas RANKL overexpression has been implicated in the pathogenesis of bone degenerative diseases such as osteoporosis. Following a forward genetics approach using N-ethyl-N-nitrosourea (ENU)-mediated random mutagenesis, we generated a novel mouse model of ARO caused by a new loss-of-function allele of Rankl with a glycine-to-arginine mutation at codon 278 (G278R) at the extracellular inner hydrophobic F ß-strand of RANKL. Mutant mice develop severe osteopetrosis similar to Rankl-deficient mice, whereas exogenous administration of recombinant RANKL restores osteoclast formation in vivo. We show that RANKL(G278R) monomers fail to assemble into homotrimers, are unable to bind and activate the RANK receptor and interact with wild-type RANKL exerting a dominant-negative effect on its trimerization and function in vitro. Since G278 is highly conserved within the TNF superfamily, we identified that a similar substitution in TNF, G122R, also abrogated trimerization, binding to TNF receptor and consequently impaired TNF biological activity. Notably, SPD304, a potent small-molecule inhibitor of TNF trimerization that interacts with G122, also inhibited RANKL activity, suggesting analogous inhibitory mechanisms. Our results provide a new disease model for ARO and identify a functional amino acid in the TNF-like core domain essential for trimer formation both in RANKL and in TNF that could be considered a novel potential target for inhibiting their biological activities.

The human desmin locus: gene organization and LCR-mediated transcriptional control.

Locus control regions (LCRs) are defined by their ability to confer reproducible physiological levels of transgene expression in mice and therefore thought to possess the ability to generate dominantly a transcriptionally active chromatin structure. We report the first characterization of a muscle-cell-specific LCR, which is linked to the human desmin gene (DES). The DES LCR consists of five regions of muscle-specific DNase I hypersensitivity (HS) localized between -9 and -18 kb 5' of DES and reproducibly drives full physiological levels of expression in all muscle cell types. The DES LCR DNase I HS regions are highly conserved between humans and other mammals and can potentially bind a broad range of muscle-specific and ubiquitous transcription factors. Bioinformatics and direct molecular analysis show that the DES locus consists of three muscle-specific (DES) or muscle preferentially expressed genes (APEG1 and SPEG, the human orthologue of murine striated-muscle-specific serine/threonine protein kinase, Speg). The DES LCR may therefore regulate expression of SPEG and APEG1 as well as DES.

Genomic characterization of the human prion protein (PrP) gene locus.

Prion protein (PrP) is intimately linked with a class of neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). Employing bioinformatics and direct molecular analysis, we demonstrated that the human PrP gene (PRNP) locus, which is situated at Chromosome (Chr) position 20p12-ter, contains three genes within a 55-kb interval: PRNP; DOPPEL or PRND, located 20 kb 3' of PRNP; and a novel gene, designated PRNT, that maps 3 kb 3' to PRND and is transcribed to generate at least three alternatively spliced mRNAs. All three genes of this locus demonstrate low sequence homology, implying that, although they may be evolutionarily related, they are functionally distinct. Analysis of both adult and fetal human tissues confirmed the ubiquitous but variable expression profile of PRNP, with the highest levels observed in the CNS and testis. Contrastingly, although PRND shows a wide tissue expression pattern in fetal tissues, it is expressed exclusively in adult testis, whereas all three PRNT isoforms were detected only in adult testis, implying that PRND is developmentally regulated. An investigation of the regulatory mechanisms underlying this complex gene expression pattern from the PRNP locus should provide insight into the function of these genes and the possible involvement of the non-PrP proteins in the development of TSEs.