Cathepsin-Mediated Alterations in TGFß-Related Signaling Underlie Disrupted Cartilage and Bone Maturation Associated With Impaired Lysosomal Targeting

Flanagan-Steet, Heather; Aarnio, Megan; Kwan, Brian; Guihard, Pierre; Petrey, Aaron C.; Haskins, Mark E.; Blanchard, Frédéric; Steet, Richard

doi:10.1002/jbmr.2722

Cited by 20 publications

(30 citation statements)

References 62 publications

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“…In culture Kif5B contributes to mitochondrial transport [ 24 ] but no obvious cartilage defects were reported, and mitochondria transport defects have not yet been linked to chondrocyte maintenance. Cathepsins are known lysosomal effectors of cell death [ 64 ] and function in chondrogenesis [ 65 , 66 ]. In zebrafish and mammals, interference with lysosomal trafficking disrupts chondrocyte maturation via TGFβ signaling and Cathepsin K, rather than maintenance [ 65 , 67 ].…”

Section: Discussionmentioning

confidence: 99%

“…Cathepsins are known lysosomal effectors of cell death [ 64 ] and function in chondrogenesis [ 65 , 66 ]. In zebrafish and mammals, interference with lysosomal trafficking disrupts chondrocyte maturation via TGFβ signaling and Cathepsin K, rather than maintenance [ 65 , 67 ]. Cathepsin D mediates early stages of perichondral ossification [ 68 ], and although myopathy was reported in zebrafish morphants no cartilage phenotypes were noted [ 69 ].…”

Section: Discussionmentioning

confidence: 99%

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Kinesin-1 promotes chondrocyte maintenance during skeletal morphogenesis

et al. 2017

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During skeletal morphogenesis diverse mechanisms are used to support bone formation. This can be seen in the bones that require a cartilage template for their development. In mammals the cartilage template is removed, but in zebrafish the cartilage template persists and the bone mineralizes around the cartilage scaffold. Remodeling of unmineralized cartilage occurs via planar cell polarity (PCP) mediated cell rearrangements that contribute to lengthening of elements; however, the mechanisms that maintain the chondrocyte template that supports perichondral ossification remain unclear. We report double mutants disrupting two zebrafish kinesin-I genes (hereafter kif5Blof) that we generated using CRISPR/Cas9 mutagenesis. We show that zygotic Kif5Bs have a conserved function in maintaining muscle integrity, and are required for cartilage remodeling and maintenance during craniofacial morphogenesis by a PCP-distinct mechanism. Further, kif5Blof does not activate ER stress response genes, but instead disrupts lysosomal function, matrix secretion, and causes deregulated autophagic markers and eventual chondrocyte apoptosis. Ultrastructural and transplantation analysis reveal neighboring cells engulfing extruded kif5Blof chondrocytes. Initial cartilage specification is intact; however, during remodeling, kif5Blof chondrocytes die and the cartilage matrix devoid of hypertrophic chondrocytes remains and impedes normal ossification. Chimeric and mosaic analyses indicate that Kif5B functions cell-autonomously in secretion, nuclear position, cell elongation and maintenance of hypertrophic chondrocytes. Interestingly, large groups of wild-type cells can support elongation of neighboring mutant cells. Finally, mosaic expression of kif5Ba, but not kif5Aa in cartilage rescues the chondrocyte phenotype, further supporting a specific requirement for Kif5B. Cumulatively, we show essential Kif5B functions in promoting cartilage remodeling and chondrocyte maintenance during zebrafish craniofacial morphogenesis.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Kinesin-1 promotes chondrocyte maintenance during skeletal morphogenesis

et al. 2017

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“…The lack of cathepsin hypersecretion is further supported by confocal analyses of Ctsk localization. We previously demonstrated Ctsk’s presence in the extracellular space outside chondrocytes lacking the αβ subunit (22). Analyses of GFP-labeled gnptg −/− chondrocytes, however, showed only intracellular Ctsk (Figure 2F).…”

Section: Resultsmentioning

confidence: 99%

“…This is consistent with their lack of overt phenotypes, which were previously linked in gnptab -deficient animals to reduced mannose phosphorylation of Ctsk. Loss of M6P on Ctsk was associated with its increased extracellular activity and phenotypic onset (22,23). The fact that both parameters are unaltered in gnptg −/− animals further supports the idea that cathepsin proteases play a central and specific role in MLII disease pathology.…”

Section: Discussionmentioning

confidence: 99%

“…Despite lacking intralysosomal storage, gnptab -deficient zebrafish also recapitulate many of the molecular phenotypes associated with MLII (21). Although both glycosidase and cathepsin protease mannose phosphorylation are impaired, the cartilage phenotypes in gnptab -deficient zebrafish embryos have been specifically linked to increased extracellular activity of certain cathepsin proteases (22,23). Furthermore, analysis of specific patient mutations in cells and in zebrafish suggests that the phenotypic variability noted in human patients may lie in the differential impact of these mutations on the mannose phosphorylation of specific lysosomal substrates (5,24,25).…”

Section: Introductionmentioning

confidence: 99%

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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

Flanagan-Steet

Matheny

Petrey

et al. 2016

Biochimica et Biophysica Acta (BBA) - General Subjects

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Targeting soluble acid hydrolases to lysosomes requires the addition of mannose 6-phosphate residues on their N-glycans. This process is initiated by GlcNAc-1-phosphotransferase, a multi-subunit enzyme encoded by the GNPTAB and GNPTG genes. The GNPTAB gene products (the α and β subunits) are responsible for recognition and catalysis of hydrolases whereas the GNPTG gene product (the γ subunit) enhances mannose phosphorylation of a subset of hydrolases. Here we identify and characterize a zebrafish gnptg insertional mutant and show that loss of the gamma subunit reduces mannose phosphorylation on a subset glycosidases but does not affect modification of several cathepsin proteases. We further show that glycosidases, but not cathepsins, are hypersecreted from gnptg−/− embryonic cells, as evidenced by reduced intracellular activity and increased circulating serum activity. The gnptg−/− embryos lack the gross morphological or craniofacial phenotypes shown in gnptab-deficient morphant embryos to result from altered cathepsin activity. Despite the lack of overt phenotypes, decreased fertilization and embryo survival were noted in mutants, suggesting that gnptg associated deposition of mannose 6-phosphate modified hydrolases into oocytes is important for early embryonic development. Collectively, these findings demonstrate that loss of the zebrafish GlcNAc-1-phosphotransferase γ subunit causes enzyme-specific effects on mannose phosphorylation. The finding that cathepsins are normally modified in gnptg−/− embryos is consistent with data from gnptab-deficient zebrafish suggesting these proteases are the key mediators of acute pathogenesis. This work also establishes a valuable new model that can be used to probe the functional relevance of GNPTG mutations in the context of a whole animal.

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A tapt1 knock-out zebrafish line with aberrant lens development and impaired vision models human early-onset cataract

et al. 2023

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Bi-allelic mutations in the gene coding for human trans-membrane anterior-posterior transformation protein 1 (TAPT1) result in a broad phenotypic spectrum, ranging from syndromic disease with severe skeletal and congenital abnormalities to isolated early-onset cataract. We present here the first patient with a frameshift mutation in the TAPT1 gene, resulting in both bilateral early-onset cataract and skeletal abnormalities, in addition to several dysmorphic features, in this way further expanding the phenotypic spectrum associated with TAPT1 mutations. A tapt1a/tapt1b double knock-out (KO) zebrafish model generated by CRISPR/Cas9 gene editing revealed an early larval phenotype with eye malformations, loss of vision, increased photokinetics and hyperpigmentation, without visible skeletal involvement. Ultrastructural analysis of the eyes showed a smaller condensed lens, loss of integrity of the lens capsule with formation of a secondary lens and hyperplasia of the cells in the ganglion and inner plexiform layers of the retina. Transcriptomic analysis pointed to an impaired lens development with aberrant expression of many of the crystallin and other lens-specific genes. Furthermore, the phototransduction and visual perception pathways were found to be significantly disturbed. Differences in light perception are likely the cause of the increased dark photokinetics and generalized hyperpigmentation observed in this zebrafish model. In conclusion, this study validates TAPT1 as a new gene for early-onset cataract and sheds light on its ultrastructural and molecular characteristics.

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Cathepsin-Mediated Alterations in TGFß-Related Signaling Underlie Disrupted Cartilage and Bone Maturation Associated With Impaired Lysosomal Targeting

Cited by 20 publications

References 62 publications

Kinesin-1 promotes chondrocyte maintenance during skeletal morphogenesis

Kinesin-1 promotes chondrocyte maintenance during skeletal morphogenesis

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

A tapt1 knock-out zebrafish line with aberrant lens development and impaired vision models human early-onset cataract

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