NGLY1 Deficiency Zebrafish Model Manifests Abnormalities of the Nervous and Musculoskeletal Systems

Mesika, Aviv; Nadav, Golan; Shochat, Chen; Kalfon, Limor; Jackson, Karen; Khalaileh, Ayat; Karasik, David; Falik‐Zaccai, Tzipora C.

doi:10.3389/fcell.2022.902969

Cited by 6 publications

(3 citation statements)

References 33 publications

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“… 68 , 69 ECM proteins are involved in the development of skeletal muscles in different ages. 69 The deficiency of NGLY1 leads to deformities in skeletal muscle and develops abnormalities in central and peripheral nervous system, 70 , 71 additionally cells undergo abnormal glycosylation. Therefore, in the absence of NGLY1, the changes in ECM protein glycosylation may modulate phenotypic characteristics in the cells.…”

Section: Discussionmentioning

confidence: 99%

N‐glycoproteomics reveals distinct glycosylation alterations in NGLY1‐deficient patient‐derived dermal fibroblasts

Budhraja

Saraswat

Graef

et al. 2022

J of Inher Metab Disea

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Congenital disorders of glycosylation are genetic disorders that occur due to defects in protein and lipid glycosylation pathways. A deficiency of N-glycanase 1, encoded by the NGLY1 gene, results in a congenital disorder of deglycosylation. The NGLY1 enzyme is mainly involved in cleaving N-glycans from misfolded, retrotranslocated glycoproteins in the cytosol from the endoplasmic reticulum before their proteasomal degradation or activation. Despite the essential role of NGLY1 in deglycosylation pathways, the exact consequences of NGLY1 deficiency on global cellular protein glycosylation have not yet been investigated. We undertook a multiplexed tandem mass tags-labeling-based quantitative glycoproteomics and proteomics analysis of fibroblasts from NGLY1-deficient individuals carrying different biallelic pathogenic variants in NGLY1. This quantitative mass spectrometric analysis detected 8041 proteins and defined a proteomic signature of differential expression across affected individuals and controls. Proteins that showed significant differential expression included phospholipid phosphatase 3, stromal cellderived factor 1, collagen alpha-1 (IV) chain, hyaluronan and proteoglycan link protein 1, and thrombospondin-1. We further detected a total of 3255 Nglycopeptides derived from 550 glycosylation sites of 407 glycoproteins by multiplexed N-glycoproteomics. Several extracellular matrix glycoproteins and adhesion molecules showed altered abundance of N-glycopeptides. Overall, we observed distinct alterations in specific glycoproteins, but our data revealed no global accumulation of glycopeptides in the patient-derived fibroblasts, despite the genetic defect in NGLY1. Our findings highlight new molecular and system-level insights for understanding NGLY1-CDDG.

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Section: Discussionmentioning

confidence: 99%

N‐glycoproteomics reveals distinct glycosylation alterations in NGLY1‐deficient patient‐derived dermal fibroblasts

Budhraja

Saraswat

Graef

et al. 2022

J of Inher Metab Disea

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“…The strategy for establishing a stable zebrafish knockout followed a published CRISPR/Cas9 protocol 43 . In brief, the guide RNA (gRNA) was designed to target sequence 5'-AGTTCTGTGACGTCTGCGCACGG-3' in exon 4 of the stac3 gene.…”

Section: Generation Of Stac3 -/By Crispr/cas9 Systemmentioning

confidence: 99%

Early life lipid overload in Native American myopathy is phenocopied by stac3 knock out in zebrafish

Donaka

Zheng

Karasik

2023

Preprint

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Understanding the early stages of human congenital myopathies is critical for proposing strategies for improving skeletal muscle performance by restoring the functional integrity of cytoskeleton. SH3 and cysteine-rich domain 3 (Stac3) is a protein involved in nutrient sensing, and is an essential component of the excitation-contraction (EC) coupling machinery for Ca2+ releasing. A mutation in STAC3 causes debilitating Native American myopathy (NAM) in humans, and loss of this gene in mice and zebrafish resulted in death in early life. Previously, NAM patients demonstrated increased lipids in skeletal muscle biopsy. However, whether elevated neutral lipids could alter muscle function in NAM disease is now well understood. Here, using a CRISPR/Cas9 induced stac3 knockout (KO) zebrafish model, we determined that loss of stac3 led to muscle weakness, as evidenced by delayed larval hatching. We observed decreased whole-body Ca2+ level at 5 days post-fertilization (dpf) and defects in the skeletal muscle cytoskeleton, i.e., F-actin and slow muscle fibers at 5 and 7 dpf. To this end, myogenesis regulators such as myoD and myf5, were significantly altered in stac3−/− larvae at 5 dpf. Homozygous larvae exhibited elevated neutral lipid levels at 5 dpf, which persisted beyond 7 dpf, followed by a progressive demise of the KO larva by 11 dpf. In summary, the presented findings suggest that stac3−/− can serve as a non-mammalian model to identify lipid-lowering molecules for refining muscle function in NAM patients.

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“…The strategy for establishing a stable zebrafish knockout followed a published CRISPR/Cas9 protocol 26 . In brief, the guide RNA (gRNA) was designed to target sequence 5'-AGTTCTGTGACGTCTGCGCACGG-3' in exon 4 of the stac3 gene.…”

Section: Generation Of Stac3 -/By Crispr/cas9 Systemmentioning

confidence: 99%

Early life lipid overload in Native American myopathy is phenocopied bystac3knock out in zebrafish

Donaka¹,

Zheng

Karasik³

2023

Preprint

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Understanding the early stages of human congenital myopathies is critical for proposing strategies for improving skeletal muscle performance by the functional integrity of cytoskeleton. SH3 and cysteine-rich domain 3 (Stac3) is a protein involved in nutrient sensing, and is an essential component of the excitation-contraction (EC) coupling machinery for Ca2+ releasing. A mutation in STAC3 causes debilitating Native American myopathy (NAM) in humans, and loss of this gene in mice and zebrafish resulted in death in early life. Previously, NAM patients demonstrated increased lipids in skeletal muscle biopsy. However, elevated neutral lipids could alter muscle function in NAM disease via EC coupling apparatus is yet undiscovered in early development. Here, using a CRISPR/Cas9 induced stac3 knockout (KO) zebrafish model, we determined that loss of stac3 led to muscle weakness, as evidenced by delayed larval hatching. We observed decreased whole-body Ca2+ level at 5 days post-fertilization (dpf) and defects in the skeletal 35 muscle cytoskeleton, i.e., F-actin and slow muscle fibers at 5 and 7 dpf. Homozygous larvae 36 exhibited elevated neutral lipid levels at 5 dpf, which persisted beyond 7 dpf. Myogenesis regulators such as myoD and myf5, were significantly altered in stac3-/- larvae at 5 dpf, thus a progressive death of the KO larva by 11 dpf. In summary, the presented findings suggest that stac3-/- can serve as a non-mammalian model to identify lipid-lowering molecules for refining muscle function in NAM patients.

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NGLY1 Deficiency Zebrafish Model Manifests Abnormalities of the Nervous and Musculoskeletal Systems

Cited by 6 publications

References 33 publications

N‐glycoproteomics reveals distinct glycosylation alterations in NGLY1‐deficient patient‐derived dermal fibroblasts

N‐glycoproteomics reveals distinct glycosylation alterations in NGLY1‐deficient patient‐derived dermal fibroblasts

Early life lipid overload in Native American myopathy is phenocopied by stac3 knock out in zebrafish

Early life lipid overload in Native American myopathy is phenocopied bystac3knock out in zebrafish

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