scholarly journals Intellectual disability-associated disruption of O-GlcNAcylation impairs neuronal development and cognitive function in Drosophila

2022 ◽  
Author(s):  
Michaela Fenckova ◽  
Villo Muha ◽  
Daniel Mariyappa ◽  
Marica Catinozzi ◽  
Ignacy Czajewski ◽  
...  
Keyword(s):  
Intellectual Disability ◽  
Cognitive Function ◽  
Neuronal Development ◽  
Critical Role ◽  
Patient Specific ◽  
Transferase Activity ◽  
Missense Mutations ◽  
Post Translational Modification ◽  
Larval Neuromuscular Junction ◽  
Glcnac Transferase

O-GlcNAcylation is a reversible co-/post-translational modification involved in a multitude of cellular processes. The addition and removal of O-GlcNAc modification is controlled by two conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Mutations in OGT have recently been discovered to cause a novel Congenital Disorder of Glycosylation (OGT-CDG) that is characterized by intellectual disability. The mechanisms by which OGT-CDG mutations affect cognition remain unclear. We manipulated O-GlcNAc transferase and O-GlcNAc hydrolase activity in Drosophila and demonstrate an important role of O-GlcNAcylation in habituation learning and synaptic development at the larval neuromuscular junction. Introduction of patient-specific missense mutations into Drosophila O-GlcNAc transferase using CRISPR/Cas9 gene editing, leads to deficits in locomotor function and habituation learning. The habituation deficit can be corrected by blocking O-GlcNAc hydrolysis, indicating that OGT-CDG mutations affect cognitive function via reduced protein O-GlcNAcylation. This study establishes a critical role for O-GlcNAc cycling and disrupted O-GlcNAc transferase activity in cognitive dysfunction. These findings suggest that blocking O-GlcNAc hydrolysis is a potential treatment strategy for OGT-CDG.

scholarly journals O-GlcNAcase contributes to cognitive function in Drosophila

2020 ◽  
Vol 295 (26) ◽  
pp. 8636-8646
Author(s):  
Villo Muha ◽  
Michaela Fenckova ◽  
Andrew T. Ferenbach ◽  
Marica Catinozzi ◽  
Ilse Eidhof ◽  
...  
Keyword(s):  
Cognitive Function ◽  
Species Loss ◽  
Post Translational Modification ◽  
Axon Terminals ◽  
Hippocampal Synaptic Plasticity ◽  
Evolutionarily Conserved ◽  
Synaptic Boutons ◽  
Larval Neuromuscular Junction ◽  
Glcnac Transferase

O-GlcNAcylation is an abundant post-translational modification in neurons. In mice, an increase in O-GlcNAcylation leads to defects in hippocampal synaptic plasticity and learning. O-GlcNAcylation is established by two opposing enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). To investigate the role of OGA in elementary learning, we generated catalytically inactive and precise knockout Oga alleles (OgaD133N and OgaKO, respectively) in Drosophila melanogaster. Adult OgaD133N and OgaKO flies lacking O-GlcNAcase activity showed locomotor phenotypes. Importantly, both Oga lines exhibited deficits in habituation, an evolutionarily conserved form of learning, highlighting that the requirement for O-GlcNAcase activity for cognitive function is preserved across species. Loss of O-GlcNAcase affected a number of synaptic boutons at the axon terminals of larval neuromuscular junction. Taken together, we report behavioral and neurodevelopmental phenotypes associated with Oga alleles and show that Oga contributes to cognition and synaptic morphology in Drosophila.

scholarly journals Distinct effects of tubulin isotype mutations on neurite growth in Caenorhabditis elegans

2017 ◽  
Author(s):  
Chaogu Zheng ◽  
Margarete Diaz-Cuadros ◽  
Ken C.Q. Nguyen ◽  
David H. Hall ◽  
Martin Chalfie
Keyword(s):  
Function Analysis ◽  
Critical Role ◽  
Neurite Growth ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Post Translational Modification ◽  
Exterior Surface ◽  
Tubulin Isotypes ◽  
C Elegans ◽  
Tubulin Isotype

AbstractTubulins, the building block of microtubules (MTs), play a critical role in both supporting and regulating neurite growth. Eukaryotic genomes contain multiple tubulin isotypes, and their missense mutations cause a range of neurodevelopmental defects. Using the C. elegans touch receptor neurons, we analyzed the effects of 67 tubulin missense mutations on neurite growth. Three types of mutations emerged: 1) loss-of-function mutations, which cause mild defects in neurite growth; 2) antimorphic mutations, which map to the GTP binding site and intradimer and interdimer interfaces, significantly reduce MT stability, and cause severe neurite growth defects; and 3) neomorphic mutations, which map to the exterior surface, increase MT stability, and cause ectopic neurite growth. Structure-function analysis reveals a causal relationship between tubulin structure and MT stability. This stability affects neuronal morphogenesis. As part of this analysis, we engineered several disease-associated human tubulin mutations into C. elegans genes and examined their impact on neuronal development at the cellular level. We also discovered an α-tubulin (TBA-7) that appears to destabilize MTs. Loss of TBA-7 led to the formation of hyperstable MTs and the generation of ectopic neurites; the lack of potential sites for polyamination and polyglutamination on TBA-7 may be responsible for this destabilization.Table of Content (TOC) Highlight SummaryDifferent tubulin isotypes perform different functions in the regulation of MT structure and neurite growth, and missense mutations of tubulin genes have three types of distinct effects on MT stability and neurite growth. One α-tubulin isotype appears to induce relative instability due to the lack of potential post-translational modification sites.

scholarly journals Distinct effects of tubulin isotype mutations on neurite growth in Caenorhabditis elegans

2017 ◽  
Vol 28 (21) ◽  
pp. 2786-2801 ◽  
Author(s):  
Chaogu Zheng ◽  
Margarete Diaz-Cuadros ◽  
Ken C. Q. Nguyen ◽  
David H. Hall ◽  
Martin Chalfie
Keyword(s):  
Caenorhabditis Elegans ◽  
Neuronal Development ◽  
Function Analysis ◽  
Critical Role ◽  
Neurite Growth ◽  
Cellular Level ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Exterior Surface ◽  
C Elegans

Tubulins, the building block of microtubules (MTs), play a critical role in both supporting and regulating neurite growth. Eukaryotic genomes contain multiple tubulin isotypes, and their missense mutations cause a range of neurodevelopmental defects. Using the Caenorhabditis elegans touch receptor neurons, we analyzed the effects of 67 tubulin missense mutations on neurite growth. Three types of mutations emerged: 1) loss-of-function mutations, which cause mild defects in neurite growth; 2) antimorphic mutations, which map to the GTP binding site and intradimer and interdimer interfaces, significantly reduce MT stability, and cause severe neurite growth defects; and 3) neomorphic mutations, which map to the exterior surface, increase MT stability, and cause ectopic neurite growth. Structure-function analysis reveals a causal relationship between tubulin structure and MT stability. This stability affects neuronal morphogenesis. As part of this analysis, we engineered several disease-associated human tubulin mutations into C. elegans genes and examined their impact on neuronal development at the cellular level. We also discovered an α-tubulin (TBA-7) that appears to destabilize MTs. Loss of TBA-7 led to the formation of hyperstable MTs and the generation of ectopic neurites; the lack of potential sites for polyamination and polyglutamination on TBA-7 may be responsible for this destabilization.

scholarly journals Nucleocytoplasmic human O-GlcNAc transferase is sufficient for O-GlcNAcylation of mitochondrial proteins

2016 ◽  
Vol 473 (12) ◽  
pp. 1693-1702 ◽  
Author(s):  
Riccardo Trapannone ◽  
Daniel Mariappa ◽  
Andrew T. Ferenbach ◽  
Daan M.F. van Aalten
Keyword(s):  
Cell Lines ◽  
Human Cell ◽  
Neuronal Development ◽  
Mitochondrial Proteins ◽  
Human Cell Lines ◽  
Post Translational Modification ◽  
Mitochondrial Proteome ◽  
Mouse Tissues ◽  
Dependent Protein ◽  
Glcnac Transferase

O-linked N-acetylglucosamine modification (O-GlcNAcylation) is a nutrient-dependent protein post-translational modification (PTM), dynamically and reversibly driven by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) that catalyse the addition and the removal of the O-GlcNAc moieties to/from serine and threonine residues of target proteins respectively. Increasing evidence suggests involvement of O-GlcNAcylation in many biological processes, including transcription, signalling, neuronal development and mitochondrial function. The presence of a mitochondrial O-GlcNAc proteome and a mitochondrial OGT (mOGT) isoform has been reported. We explored the presence of mOGT in human cell lines and mouse tissues. Surprisingly, analysis of genomic sequences indicates that this isoform cannot be expressed in most of the species analysed, except some primates. In addition, we were not able to detect endogenous mOGT in a range of human cell lines. Knockdown experiments and Western blot analysis of all the predicted OGT isoforms suggested the expression of only a single OGT isoform. In agreement with this, we demonstrate that overexpression of the nucleocytoplasmic OGT (ncOGT) isoform leads to increased O-GlcNAcylation of mitochondrial proteins, suggesting that ncOGT is necessary and sufficient for the generation of the O-GlcNAc mitochondrial proteome.

scholarly journals α-TubK40me3 is required for neuronal polarization and migration by promoting microtubule formation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xuan Xie ◽  
Shaogang Wang ◽  
Mingyi Li ◽  
Lei Diao ◽  
Xingyu Pan ◽  
...  
Keyword(s):  
Neuronal Migration ◽  
Neuronal Development ◽  
Critical Role ◽  
Post Translational Modification ◽  
Mouse Cerebral Cortex ◽  
Microtubule Formation ◽  
And Migration ◽  
Neuronal Polarization ◽  
Microtubule Function

AbstractTri-methylation on lysine 40 of α-tubulin (α-TubK40me3) is a recently identified post-translational modification involved in mitosis and cytokinesis. However, knowledge about α-TubK40me3 in microtubule function and post-mitotic cells remains largely incomplete. Here, we report that α-TubK40me3 is required for neuronal polarization and migration by promoting microtubule formation. α-TubK40me3 is enriched in mouse cerebral cortex during embryonic day (E)14 to E16. Knockdown of α-tubulin methyltransferase SETD2 at E14 leads to the defects in neuronal migration, which could be restored by overexpressing either a cytoplasm-localized SETD2 truncation or α-TubK40me3-mimicking mutant. Furthermore, α-TubK40me3 is preferably distributed on polymerized microtubules and potently promotes tubulin nucleation. Downregulation of α-TubK40me3 results in reduced microtubule abundance in neurites and disrupts neuronal polarization, which could be rescued by Taxol. Additionally, α-TubK40me3 is increased after losing α-tubulin K40 acetylation (α-TubK40ac) and largely rescues α-TubK40ac function. This study reveals a critical role of α-TubK40me3 in microtubule formation and neuronal development.

scholarly journals Catalytic deficiency of O-GlcNAc transferase leads to X-linked intellectual disability

2019 ◽  
Vol 116 (30) ◽  
pp. 14961-14970 ◽  
Author(s):  
Veronica M. Pravata ◽  
Villo Muha ◽  
Mehmet Gundogdu ◽  
Andrew T. Ferenbach ◽  
Poonam S. Kakade ◽  
...  
Keyword(s):  
Intellectual Disability ◽  
Protein Interactions ◽  
Catalytic Domain ◽  
Embryonic Stem ◽  
Global Changes ◽  
Missense Mutations ◽  
Protein Protein Interactions ◽  
Delayed Differentiation ◽  
Nucleocytoplasmic Proteins ◽  
Glcnac Transferase

O-GlcNAc transferase (OGT) is an X-linked gene product that is essential for normal development of the vertebrate embryo. It catalyses the O-GlcNAc posttranslational modification of nucleocytoplasmic proteins and proteolytic maturation of the transcriptional coregulator Host cell factor 1 (HCF1). Recent studies have suggested that conservative missense mutations distal to the OGT catalytic domain lead to X-linked intellectual disability in boys, but it is not clear if this is through changes in the O-GlcNAc proteome, loss of protein–protein interactions, or misprocessing of HCF1. Here, we report an OGT catalytic domain missense mutation in monozygotic female twins (c. X:70779215 T > A, p. N567K) with intellectual disability that allows dissection of these effects. The patients show limited IQ with developmental delay and skewed X-inactivation. Molecular analyses revealed decreased OGT stability and disruption of the substrate binding site, resulting in loss of catalytic activity. Editing this mutation into the Drosophila genome results in global changes in the O-GlcNAc proteome, while in mouse embryonic stem cells it leads to loss of O-GlcNAcase and delayed differentiation down the neuronal lineage. These data imply that catalytic deficiency of OGT could contribute to X-linked intellectual disability.

scholarly journals Generation of an Unbiased Interactome for the Tetratricopeptide Repeat Domain of O-GlcNAc Transferase Indicates a Role for the Enzyme in Intellectual Disability

2020 ◽  
Author(s):  
Hannah M Stephen ◽  
Jeremy L Praissman ◽  
Lance Wells
Keyword(s):  
Intellectual Disability ◽  
Missense Mutations ◽  
Substantial Impact ◽  
Cellular Survival ◽  
Cellular Processes ◽  
Disease States ◽  
Tetratricopeptide Repeat Domain ◽  
Nucleocytoplasmic Proteins ◽  
Glcnac Transferase ◽  
Tpr Domain

The O-GlcNActransferase (OGT) is localized to the nucleus and cytoplasm where it regulates nucleocytoplasmic proteins by modifying serine and threonine residues with a non-extended monosaccharide, b-N-Acetyl-Glucosamine (O-GlcNAc). With thousands ofknown O-GlcNAcmodifiedproteinsbut only oneOGTencoded in the mammalian genome, a prevailing question is howOGTselects its substrates. Prior work has indicated that theN-terminaltetratricopeptide repeat (TPR) domain of OGT, rather than itsC-terminalcatalytic domain, is responsible forsubcellular targeting andsubstrate selection.An additional impetus for exploring the OGT TPR domain interactome is the fact that missense mutations inOGTassociated with X-linked intellectual disability (XLID) are primarily localized to the TPR domain without substantial impact on activity or stability of the enzyme.Therefore, we adapted theBioIDlabeling method to identify interactors of a TPR-BirA* fusion protein in HeLa cells. We identified 115high confidenceinteractors representing both known and novel O-GlcNAcmodified proteins and OGT interactors. The TPR interactors are highly enriched in processes in which OGT has a known role (e.g. chromatin remodeling, cellular survival of heat stress, circadian rhythm), as well as processesin which OGT has yet to be implicated (e.g. pre-mRNA processing). Importantly,the identified TPR interactors are involved in several disease states but most notably are highly enriched in pathologies featuring intellectual disability.Theseproteinsrepresent candidateinteractors that may underlie the mechanismby which mutations in OGT lead to XLID. Furthermore, the identified interactors provide additional evidence of the importance of the TPR domain for OGT targeting and/or substrate selection.Thus, this defined interactome for the TPR domain of OGT serves as ajumping off point for future researchexploringthe role of OGT, the TPR domain, and its protein interactorsin multiple cellular processes and disease mechanisms, including intellectual disability.

Pathogenesis of Age-related Cataract: a systematic review of proteomic studies

2020 ◽  
Vol 17 ◽  
Author(s):  
Christina Karakosta ◽  
Argyrios Tzamalis ◽  
Michalis Aivaliotis ◽  
Ioannis Tsinopoulos
Keyword(s):  
Systematic Review ◽  
Oxidant Stress ◽  
Critical Role ◽  
Human Lens ◽  
Nuclear Cataract ◽  
Cataract Formation ◽  
Post Translational Modification ◽  
Ability To Act ◽  
Age Related

Background/Objective:: The aim of this systematic review is to identify all the available data on human lens proteomics with a critical role to age-related cataract formation in order to elucidate the physiopathology of the aging lens. Materials and Methods:: We searched on Medline and Cochrane databases. The search generated 328 manuscripts. We included nine original proteomic studies that investigated human cataractous lenses. Results:: Deamidation was the major age-related post-translational modification. There was a significant increase in the amount of αA-crystallin D-isoAsp58 present at all ages, while an increase in the extent of Trp oxidation was apparent in cataract lenses when compared to aged normal lenses. During aging, enzymes with oxidized cysteine at critical sites included GAPDH, glutathione synthase, aldehyde dehydrogenase, sorbitol dehydrogenase, and PARK7. Conclusion:: D-isoAsp in αA crystallin could be associated with the development of age-related cataract in human, by contributing to the denaturation of a crystallin, and decreasing its ability to act as a chaperone. Oxidation of Trp may be associated with nuclear cataract formation in human, while the role of oxidant stress in age-related cataract formation is dominant.

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