Defects in the protein machinery responsible for the degradation and recycling of lysosomal substrates result in a group of human diseases collectively known as lysosomal storage disorders (LSDs). Despite advances in defining the genetic basis for LSDs, surprisingly little is known about the underlying mechanisms that lead to disease pathology. Identifying the sensitive biochemical pathways responsible for the developmental and progressive features of LSDs is essential since it will provide the underpinnings for new therapeutic strategies. The long-term goal of the proposed research is to investigate the molecular pathogenesis of mucolipidosis II (ML-II), the devastating LSD caused by defects in mannose 6-phosphate (M6P) biosynthesis. In an effort to link the cartilage defects associated with this disease to specific biochemical pathways, we have established a zebrafish model for ML-II that shares many of the pathologies noted in human patients, such as abnormalities in craniofacial cartilage development. The cartilage morphogenesis defects in these zebrafish are accompanied by alterations in i) chondrocyte differentiation, ii) deposition of type II collagen and iii) the expression of ECM modifying enzymes. Furthermore, we have noted changes in the secretion and storage of latent TGF-ss1 and integrity of fibronectin in ML-II patient fibroblasts. A specific role for the TGF-ss pathway in ML-II cartilage pathogenesis is further suggested by the fact i) TGF-ss growth factors are central to multiple aspects of cartilage development and homeostasis, ii) latent TGF-ss is M6P-modified and iii) the activation of latent TGF-ss complexes involves M6P receptor-mediated events. Using complementary zebrafish and cell- based approaches, we will test the hypothesis that M6P-dependent TGF-ss dysregulation and impaired ECM maintenance cause the abnormal cartilage development and homeostasis associated with ML-II. In the first aim, we will further elucidate the cellular mechanisms that underlie abnormal chondrogenesis in ML-II zebrafish. With the aid of in vivo confocal microscopy, we will investigate how the behavior of chondrocyte precursors is altered during craniofacial development in ML-II zebrafish and how changes in the expression of ECM proteins and regulators of ECM synthesis and turnover impact specific properties of chondrocytes within developing cartilage. In the second aim, we will take advantage of multiple cell culture systems to investigate the molecular mechanisms responsible for M6P-dependent TGF-ss dysregulation and fibronectin fragmentation, and how such events lead to the abnormal development and progressive destruction of cartilage noted in ML-II. In the third aim, we will assess the contribution of the cation-independent M6P receptor (CI-MPR) on TGF-ss regulation and cartilage development. A unique set of zebrafish CI-MPR mutants, restricted in their ability to bind specific ligands or properly traffic and internalize these ligands, will be introduced into CI-MPR null zebrafish and the resulting embryos analyzed on a phenotypic and cellular level with regard to cartilage development.
Despite advances in uncovering the genetic basis for many lysosomal storage disorders, surprisingly little is known about the underlying mechanisms that lead to disease pathology. Using the zebrafish model system, the broad goal of this proposal is to link the developmental and progressive bone and cartilage defects associated with these disorders with specific biochemical pathways. By doing so, we hope to identify novel therapeutic targets.
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