Defects in the biogenesis and function of lysosomes result in lysosomal storage disorders (LSDs). The underlying mechanisms that lead to disease pathology are poorly understood, representing a barrier in identifying therapeutic targets. In the lysosomal disease mucolipidosis II (ML-II), the enzyme (GlcNAc-1- phosphotransferase) that synthesizes the carbohydrate-based tag (mannose 6-phosphate) needed for receptor-mediated lysosomal targeting is missing. This causes hypersecretion of newly made cathepsins into the extracellular space. Cathepsin mistargeting places these enzymes in contact with the matrix-deposited growth factors that control the timing and fidelity of chondrocyte maturation. The primary objective of this proposal is to address how secreted lysosomal cathepsins modulate key growth factor pathways in chondrocytes and cause ML-II cartilage pathology. Most work on ML-II (and other LSDs) focuses on the consequences of storage on cellular function. Our approach is novel in that it focuses instead on the consequences of mistargeted lysosomal hydrolases, a unique feature of ML-II. Using a morpholino- knockdown strategy, we generated the first zebrafish model for ML-II to study the impact of impaired M6P- dependent lysosomal targeting during development. We demonstrated chondrogenesis defects accompanied by sustained expression of the transforming growth factor beta (TGFss)-regulated matrix protein, type II collagen, and sustained and elevated activity of the cathepsin K. Lowering cathepsin K activity in the ML-II background reduced this sustained expression of type II collagen and alleviated the cartilage phenotypes. The finding that inhibition of cathepsin K reduces the abundance of type II collagen was unexpected since decreased activity of this potent collagenase was predicted instead to lead to collagen accumulation. This observation supports a broader function for this protease - beyond collagen turnover - during pathogenic cartilage development. Our collective data support a model whereby ML-II chondrocytes are unable to proceed beyond early phases of chondrogenesis. Since progression through the normal chondrogenic program requires a transition from TGFss to bone morphogenetic protein (BMP) signaling, we hypothesize that hypersecretion and extracellular activity of cathepsin K disrupts the balance of these growth factor pathways, in turn causing abnormal chondrocyte differentiation. ML-II is untreatable. By addressing the molecular mechanisms that underlie abnormal chondrogenesis in ML-II, we hope to identify new downstream targets for therapy and develop a powerful system to investigate the physiological relevance of secreted cathepsins during normal cartilage formation. The proposed specific aims will 1) address how cathepsin K hypersecretion alters the balance of TGFss and BMP signaling during chondrocyte differentiation, 2) define cathepsin K regulation and activity within the stages of normal cartilage development, and 3) uncover the structural elements within the GlcNAc-1-phosphotransferase enzyme that mediate specific recognition of cathepsins.

Public Health Relevance

The overall goal of this proposal is to investigate how lysosomal cathepsin proteases modulate the signaling pathways that drive normal and pathological cartilage development. This work leverages a zebrafish model for mucolipidosis II, a lysosomal disease in which cathepsins are mistargeted outside the cell and placed in contact with key growth factors. We seek to translate the mechanistic insight gained into new therapies for lysosomal disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM086524-06
Application #
8630244
Study Section
Intercellular Interactions (ICI)
Program Officer
Marino, Pamela
Project Start
2009-01-01
Project End
2017-11-30
Budget Start
2014-01-15
Budget End
2014-11-30
Support Year
6
Fiscal Year
2014
Total Cost
$333,760
Indirect Cost
$109,760
Name
University of Georgia
Department
None
Type
Organized Research Units
DUNS #
004315578
City
Athens
State
GA
Country
United States
Zip Code
30602
Flanagan-Steet, Heather; Matheny, Courtney; Petrey, Aaron et al. (2016) Enzyme-specific differences in mannose phosphorylation between GlcNAc-1-phosphotransferase αβ and γ subunit deficient zebrafish support cathepsin proteases as early mediators of mucolipidosis pathology. Biochim Biophys Acta 1860:1845-53
Flanagan-Steet, Heather; Aarnio, Megan; Kwan, Brian et al. (2016) Cathepsin-Mediated Alterations in TGFß-Related Signaling Underlie Disrupted Cartilage and Bone Maturation Associated With Impaired Lysosomal Targeting. J Bone Miner Res 31:535-48
Gurda, Brittney L; De Guilhem De Lataillade, Adrien; Bell, Peter et al. (2016) Evaluation of AAV-mediated Gene Therapy for Central Nervous System Disease in Canine Mucopolysaccharidosis VII. Mol Ther 24:206-16
Han, Sang-Oh; Pope, Rand; Li, Songtao et al. (2016) A beta-blocker, propranolol, decreases the efficacy from enzyme replacement therapy in Pompe disease. Mol Genet Metab 117:114-9
Bammens, Riet; Mehta, Nickita; Race, Valérie et al. (2015) Abnormal cartilage development and altered N-glycosylation in Tmem165-deficient zebrafish mirrors the phenotypes associated with TMEM165-CDG. Glycobiology 25:669-82
Qian, Yi; van Meel, Eline; Flanagan-Steet, Heather et al. (2015) Analysis of mucolipidosis II/III GNPTAB missense mutations identifies domains of UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase involved in catalytic function and lysosomal enzyme recognition. J Biol Chem 290:3045-56
Leroy, Jules G; Sillence, David; Wood, Tim et al. (2014) A novel intermediate mucolipidosis II/III** caused by GNPTAB mutation in the cytosolic N-terminal domain. Eur J Hum Genet 22:594-601
Boccuto, Luigi; Aoki, Kazuhiro; Flanagan-Steet, Heather et al. (2014) A mutation in a ganglioside biosynthetic enzyme, ST3GAL5, results in salt & pepper syndrome, a neurocutaneous disorder with altered glycolipid and glycoprotein glycosylation. Hum Mol Genet 23:418-33
Chu, Jaime; Mir, Alexander; Gao, Ningguo et al. (2013) A zebrafish model of congenital disorders of glycosylation with phosphomannose isomerase deficiency reveals an early opportunity for corrective mannose supplementation. Dis Model Mech 6:95-105
Mbua, Ngalle Eric; Flanagan-Steet, Heather; Johnson, Steven et al. (2013) Abnormal accumulation and recycling of glycoproteins visualized in Niemann-Pick type C cells using the chemical reporter strategy. Proc Natl Acad Sci U S A 110:10207-12

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