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  1. Article ; Online: Optimized protocol for in vivo affinity purification proteomics and biochemistry using C. elegans

    Muriel Desbois / Joseph S. Pak / Karla J. Opperman / Andrew C. Giles / Brock Grill

    STAR Protocols, Vol 4, Iss 2, Pp 102262- (2023)

    2023  

    Abstract: Summary: We present an optimized protocol for in vivo affinity purification proteomics and biochemistry using the model organism C. elegans. We describe steps for target tagging, large-scale culture, affinity purification using a cryomill, mass ... ...

    Abstract Summary: We present an optimized protocol for in vivo affinity purification proteomics and biochemistry using the model organism C. elegans. We describe steps for target tagging, large-scale culture, affinity purification using a cryomill, mass spectrometry and validation of candidate binding proteins. Our approach has proven successful for identifying protein-protein interactions and signaling networks with verified functional relevance. Our protocol is also suitable for biochemical evaluation of protein-protein interactions in vivo.For complete details on the use and execution of this protocol, please refer to Crawley et al.,1 Giles et al.,2 and Desbois et al.3 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
    Keywords Model Organisms ; Neuroscience ; Protein Biochemistry ; Proteomics ; Protein expression and purification ; Mass Spectrometry ; Science (General) ; Q1-390
    Subject code 570
    Language English
    Publishing date 2023-06-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: O-GlcNAc transferase OGT-1 and the ubiquitin ligase EEL-1 modulate seizure susceptibility in C. elegans.

    Nirthieca Suthakaran / Jonathan Wiggins / Andrew Giles / Karla J Opperman / Brock Grill / Ken Dawson-Scully

    PLoS ONE, Vol 16, Iss 11, p e

    2021  Volume 0260072

    Abstract: Neurodevelopmental disorders such as epilepsy and autism have been linked to an imbalance of excitation and inhibition (E/I) in the central nervous system. The simplicity and tractability of C. elegans allows our electroconvulsive seizure (ES) assay to ... ...

    Abstract Neurodevelopmental disorders such as epilepsy and autism have been linked to an imbalance of excitation and inhibition (E/I) in the central nervous system. The simplicity and tractability of C. elegans allows our electroconvulsive seizure (ES) assay to be used as a behavioral readout of the locomotor circuit and neuronal function. C. elegans possess conserved nervous system features such as gamma-aminobutyric acid (GABA) and GABA receptors in inhibitory neurotransmission, and acetylcholine (Ach) and acetylcholine receptors in excitatory neurotransmission. Our previously published data has shown that decreasing inhibition in the motor circuit, via GABAergic manipulation, will extend the time of locomotor recovery following electroshock. Similarly, mutations in a HECT E3 ubiquitin ligase called EEL-1 leads to impaired GABAergic transmission, E/I imbalance and altered sensitivity to electroshock. Mutations in the human ortholog of EEL-1, called HUWE1, are associated with both syndromic and non-syndromic intellectual disability. Both EEL-1 and its previously established binding protein, OGT-1, are expressed in GABAergic motor neurons, localize to GABAergic presynaptic terminals, and function in parallel to regulate GABA neuron function. In this study, we tested behavioral responses to electroshock in wildtype, ogt-1, eel-1 and ogt-1; eel-1 double mutants. Both ogt-1 and eel-1 null mutants have decreased inhibitory GABAergic neuron function and increased electroshock sensitivity. Consistent with EEL-1 and OGT-1 functioning in parallel pathways, ogt-1; eel-1 double mutants showed enhanced electroshock susceptibility. Expression of OGT-1 in the C. elegans nervous system rescued enhanced electroshock defects in ogt-1; eel-1 double mutants. Application of a GABA agonist, Baclofen, decreased electroshock susceptibility in all animals. Our C. elegans electroconvulsive seizure assay was the first to model a human X-linked Intellectual Disability (XLID) associated with epilepsy and suggests a potential novel role for the OGT-1/EEL-1 complex in seizure susceptibility.
    Keywords Medicine ; R ; Science ; Q
    Subject code 571
    Language English
    Publishing date 2021-01-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: O-GlcNAc transferase OGT-1 and the ubiquitin ligase EEL-1 modulate seizure susceptibility in C. elegans

    Nirthieca Suthakaran / Jonathan Wiggins / Andrew Giles / Karla J. Opperman / Brock Grill / Ken Dawson-Scully

    PLoS ONE, Vol 16, Iss

    2021  Volume 11

    Abstract: Neurodevelopmental disorders such as epilepsy and autism have been linked to an imbalance of excitation and inhibition (E/I) in the central nervous system. The simplicity and tractability of C. elegans allows our electroconvulsive seizure (ES) assay to ... ...

    Abstract Neurodevelopmental disorders such as epilepsy and autism have been linked to an imbalance of excitation and inhibition (E/I) in the central nervous system. The simplicity and tractability of C. elegans allows our electroconvulsive seizure (ES) assay to be used as a behavioral readout of the locomotor circuit and neuronal function. C. elegans possess conserved nervous system features such as gamma-aminobutyric acid (GABA) and GABA receptors in inhibitory neurotransmission, and acetylcholine (Ach) and acetylcholine receptors in excitatory neurotransmission. Our previously published data has shown that decreasing inhibition in the motor circuit, via GABAergic manipulation, will extend the time of locomotor recovery following electroshock. Similarly, mutations in a HECT E3 ubiquitin ligase called EEL-1 leads to impaired GABAergic transmission, E/I imbalance and altered sensitivity to electroshock. Mutations in the human ortholog of EEL-1, called HUWE1, are associated with both syndromic and non-syndromic intellectual disability. Both EEL-1 and its previously established binding protein, OGT-1, are expressed in GABAergic motor neurons, localize to GABAergic presynaptic terminals, and function in parallel to regulate GABA neuron function. In this study, we tested behavioral responses to electroshock in wildtype, ogt-1, eel-1 and ogt-1; eel-1 double mutants. Both ogt-1 and eel-1 null mutants have decreased inhibitory GABAergic neuron function and increased electroshock sensitivity. Consistent with EEL-1 and OGT-1 functioning in parallel pathways, ogt-1; eel-1 double mutants showed enhanced electroshock susceptibility. Expression of OGT-1 in the C. elegans nervous system rescued enhanced electroshock defects in ogt-1; eel-1 double mutants. Application of a GABA agonist, Baclofen, decreased electroshock susceptibility in all animals. Our C. elegans electroconvulsive seizure assay was the first to model a human X-linked Intellectual Disability (XLID) associated with epilepsy and suggests a potential novel role for the ...
    Keywords Medicine ; R ; Science ; Q
    Subject code 571
    Language English
    Publishing date 2021-01-01T00:00:00Z
    Publisher Public Library of Science (PLoS)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article ; Online: Autophagy is inhibited by ubiquitin ligase activity in the nervous system

    Oliver Crawley / Karla J. Opperman / Muriel Desbois / Isabel Adrados / Melissa A. Borgen / Andrew C. Giles / Derek R. Duckett / Brock Grill

    Nature Communications, Vol 10, Iss 1, Pp 1-

    2019  Volume 17

    Abstract: Despite growing interest in the role of autophagy in neurons, it remains unclear how this process is regulated, in particular, how autophagy is spatially restricted in subcellular compartments in neurons. In this study, the authors use an unbiased ... ...

    Abstract Despite growing interest in the role of autophagy in neurons, it remains unclear how this process is regulated, in particular, how autophagy is spatially restricted in subcellular compartments in neurons. In this study, the authors use an unbiased proteomic approach to show that the autophagy initiating kinase UNC-51/ULK and autophagosome formation are inhibited by the ubiquitin ligase RPM-1, and demonstrate that this interaction is within specific axonal compartments.
    Keywords Science ; Q
    Language English
    Publishing date 2019-11-01T00:00:00Z
    Publisher Nature Portfolio
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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    Inter-library loan at ZB MED

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  5. Article ; Online: Autophagy is inhibited by ubiquitin ligase activity in the nervous system

    Oliver Crawley / Karla J. Opperman / Muriel Desbois / Isabel Adrados / Melissa A. Borgen / Andrew C. Giles / Derek R. Duckett / Brock Grill

    Nature Communications, Vol 10, Iss 1, Pp 1-

    2019  Volume 17

    Abstract: Despite growing interest in the role of autophagy in neurons, it remains unclear how this process is regulated, in particular, how autophagy is spatially restricted in subcellular compartments in neurons. In this study, the authors use an unbiased ... ...

    Abstract Despite growing interest in the role of autophagy in neurons, it remains unclear how this process is regulated, in particular, how autophagy is spatially restricted in subcellular compartments in neurons. In this study, the authors use an unbiased proteomic approach to show that the autophagy initiating kinase UNC-51/ULK and autophagosome formation are inhibited by the ubiquitin ligase RPM-1, and demonstrate that this interaction is within specific axonal compartments.
    Keywords Science ; Q
    Language English
    Publishing date 2019-11-01T00:00:00Z
    Publisher Nature Publishing Group
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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    Inter-library loan at ZB MED

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  6. Article ; Online: The HECT Family Ubiquitin Ligase EEL-1 Regulates Neuronal Function and Development

    Karla J. Opperman / Ben Mulcahy / Andrew C. Giles / Monica G. Risley / Rayna L. Birnbaum / Erik D. Tulgren / Ken Dawson-Scully / Mei Zhen / Brock Grill

    Cell Reports, Vol 19, Iss 4, Pp 822-

    2017  Volume 835

    Abstract: Genetic changes in the HECT ubiquitin ligase HUWE1 are associated with intellectual disability, but it remains unknown whether HUWE1 functions in post-mitotic neurons to affect circuit function. Using genetics, pharmacology, and electrophysiology, we ... ...

    Abstract Genetic changes in the HECT ubiquitin ligase HUWE1 are associated with intellectual disability, but it remains unknown whether HUWE1 functions in post-mitotic neurons to affect circuit function. Using genetics, pharmacology, and electrophysiology, we show that EEL-1, the HUWE1 ortholog in C. elegans, preferentially regulates GABAergic presynaptic transmission. Decreasing or increasing EEL-1 function alters GABAergic transmission and the excitatory/inhibitory (E/I) balance in the worm motor circuit, which leads to impaired locomotion and increased sensitivity to electroshock. Furthermore, multiple mutations associated with intellectual disability impair EEL-1 function. Although synaptic transmission defects did not result from abnormal synapse formation, sensitizing genetic backgrounds revealed that EEL-1 functions in the same pathway as the RING family ubiquitin ligase RPM-1 to regulate synapse formation and axon termination. These findings from a simple model circuit provide insight into the molecular mechanisms required to obtain E/I balance and could have implications for the link between HUWE1 and intellectual disability.
    Keywords EEL-1 ; HUWE1 ; intellectual disability ; motor neuron ; C. elegans ; RPM-1 ; GABA ; synaptic transmission ; acetylcholine ; seizure ; Biology (General) ; QH301-705.5
    Subject code 571
    Language English
    Publishing date 2017-04-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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    Inter-library loan at ZB MED

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