Studying RANKL-mediated pathogenic mechanisms

Receptor Activator of Nuclear Factor-κB Ligand (RANKL) is the primary mediator of bone loss through osteoclast-induced bone resorption. RANKL binds as a trimer to its receptor RANK that is expressed on the surface of osteoclast precursors initiating a signaling cascade that results in osteoclast differentiation which leads to bone resorption. The importance of RANKL in physiology has emerged from studies in RANKL knockout mice and human patients with mutations in the RANKL gene that develop severe recessive osteopetrosis due to complete osteoclast absence. RANKL is also involved in bone pathologies associated with excessive osteoclast activity and increased bone resorption such as osteoporosis, rheumatoid arthritis or bone metastasis. Therefore, its inhibition is considered one of the most promising therapeutic targets in such pathologies. Indeed, a fully-human monoclonal antibody against RANKL was approved by FDA for use in postmenopausal women with risk of osteoporosis in June 2010. Our lab has generated unique models of RANKL-mediated osteopetrosis and osteoporosis to study the molecular mechanisms involved in RANKL-mediated procedures and pathologies and to provide such models for the evaluation of novel therapeutic approaches in bone diseases.

RANKL-mediated osteopetrosis: We have recently isolated an ENU-induced mouse mutant of osteopetrosis, which is characterized by complete absence of osteoclasts. Our genetic analysis identified a recessive point mutation in the Rankl gene that causes a single aminoacid substitution at the extracellular domain of RANKL that inhibits trimer assembly exerting also a dominant negative effect. This knowledge creates new possibilities for designing highly effective drugs to inhibit RANKL function by targeting its trimerization. Moreover, our novel osteopetrotic model of human autosomal recessive osteopetrosis (ARO), offers an exceptional tool for applying new therapeutic approaches.

RANKL-mediated osteoporosis: We have recently generated transgenic mice overexpressing human RANKL (TghuRANKL) in order to model human RANKL-mediated pathologies. TghuRANKL mice develop increased osteoclastogenesis, excessive trabecular bone loss and cortical bone porosity which are the main characteristics of osteoporosis. Our RANKL transgenic mice represent a unique tool for understanding the pathogenic mechanisms that cause bone resorption and for the evaluation of novel therapeutic approaches targeting RANKL-mediated pathologies such as osteoporosis.

A novel SLC25 member causes autosomal recessive ataxia

We have recently generated by random ENU mutagenesis, a novel mouse model of severe autosomal recessive neurological disease characterized by ataxia, unsteady locomotion, episodic crises, lymphoid abnormalities, growth retardation and premature death. Using genome-wide linkage analysis and sequencing of the candidate genes we identified a nonsense point mutation in the coding region of a novel gene member of the Solute Carrier Family 25 (SLC25) that introduces a premature stop codon and results in a loss-of-function protein. All SLC25 members are nuclear-coded proteins that are imported into the inner mitochondrial membrane where they shuttle a variety of metabolites across it. Until now, almost 50 SLC25 members have been identified whereas most of them remain uncharacterized. Mutations in SLC25 genes impair mitochondria functions and result in at least 10 various human diseases, by affecting either the synthesis of ATP through oxidative phosphorylation, or the selective transport of solutes in and out of the mitochondrial matrix. This novel SLC25 member is highly conserved among various species, but its function remains completely unknown. Our ongoing studies are focused on a) the identification of the primary site of lesion in order to align the brain pathology of the mouse model with that of similar human neurodegenerative diseases, b) the identification of mitochondrial dysfunctions, and c) the analysis of the expression profile and the function of this novel SLC25 protein which constitutes a novel pathogenic target in neurological diseases such as ataxia.

A novel DnaJC family member causes neuromuscular disease

Neuromuscular diseases encompass a wide range of clinical conditions remaining incurable while the genetic and molecular basis of most of these conditions remains unknown. Using mouse ENU mutagenesis we have identified a novel autosomal recessive neuromuscular phenotype characterized by hind limb weakness, progressing with muscle atrophy, reduced body weight, atrophy of lymphoid organs, generalized paralysis and death within 30 days after birth. Genetic linkage analysis and sequencing revealed an intronic point mutation in a member of the DnaJ homolog, subfamily C (DnaJC) of heat shock proteins (Hsp40s). This is a novel gene with completely unknown function, whose protein is suggested to be located in the mitochondrion. The intronic mutation generates a novel splicing acceptor site resulting in the insertion of a small intronic region in the mature transcript, causing a replacement of the C terminus of the protein by a new sequence. This DnaJC family member shares almost 100% identity with its human ortholog, highlighting the functional importance of the protein. Our current studies focus on the histological and immunohistochemical characterization of the CNS in our mutant mice, and analysis of the expression pattern and the function of the normal and the mutated protein. The identification of a novel DnaJC protein involved in neuromuscular disease and future functional characterization of this protein can shed light into new pathogenetic pathways involved in neuromuscular diseases.

Collaborative research

In collaboration with Dr Kollias’ lab we have established a sensitized ENU mutagenesis screen in the TNF^ΔARE model of arthritis and IBD for the identification of genes that are involved in the pathogenesis of such diseases. Once identified these novel gene functions may constitute novel pharmaceutical targets for disease neutralization.