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Scott M Wilson - One of the best experts on this subject based on the ideXlab platform.
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neurotoxic mechanisms by which the USP14 inhibitor iu1 depletes ubiquitinated proteins and tau in rat cerebral cortical neurons relevance to alzheimer s disease
Biochimica et Biophysica Acta, 2017Co-Authors: Magdalena Kiprowska, Scott M Wilson, Anna Stepanova, Dustin R Todaro, Alexander Galkin, Arthur L Haas, Maria E FigueiredopereiraAbstract:Abstract In Alzheimer's disease proteasome activity is reportedly downregulated, thus increasing it could be therapeutically beneficial. The proteasome-associated deubiquitinase USP14 disassembles polyubiquitin-chains, potentially delaying proteasome-dependent protein degradation. We assessed the protective efficacy of inhibiting or downregulating USP14 in rat and mouse ( USP14 axJ ) neuronal cultures treated with prostaglandin J2 (PGJ2). IU1 concentrations ( H IU1 > 25 μM) reported by others to inhibit USP14 and be protective in non-neuronal cells, reduced PGJ2-induced Ub-protein accumulation in neurons. However, H IU1 alone or with PGJ2 is neurotoxic, induces calpain-dependent Tau cleavage, and decreases E1 ~ Ub thioester levels and 26S proteasome assembly, which are energy-dependent processes. We attribute the two latter H IU1 effects to ATP-deficits and mitochondrial Complex I inhibition, as shown herein. These H IU1 effects mimic those of mitochondrial inhibitors in general, thus supporting that ATP-depletion is a major mediator of H IU1-actions. In contrast, low IU1 concentrations ( L IU1 ≤ 25 μM) or USP14 knockdown by siRNA in rat cortical cultures or loss of USP14 in cortical cultures from ataxia ( USP14 axJ ) mice, failed to prevent PGJ2-induced Ub-protein accumulation. PGJ2 alone induces Ub-protein accumulation and decreases E1 ~ Ub thioester levels. This seemingly paradoxical result may be attributed to PGJ2 inhibiting some deubiquitinases (such as UCH-L1 but not USP14), thus triggering Ub-protein stabilization. Overall, IU1-concentrations that reduce PGJ2-induced accumulation of Ub-proteins are neurotoxic, trigger calpain-mediated Tau cleavage, lower ATP, E1 ~ Ub thioester and E1 protein levels, and reduce proteasome activity. In conclusion, pharmacologically inhibiting (with low or high IU1 concentrations) or genetically down-regulating USP14 fail to enhance proteasomal degradation of Ub-proteins or Tau in neurons.
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distinct effects of ubiquitin overexpression on nmj structure and motor performance in mice expressing catalytically inactive USP14
Frontiers in Molecular Neuroscience, 2015Co-Authors: Jada H Vaden, Pingchung Chen, Jennifer A Watson, Julie A Wilson, Alan D Howard, Scott M WilsonAbstract:Ubiquitin-specific protease 14 (USP14) is a major deubiquitinating enzyme and a key determinant of neuromuscular junction (NMJ) structure and function. We have previously reported dramatic ubiquitin depletion in the nervous systems of the USP14-deficient ataxia (axJ) mice and demonstrated that transgenic ubiquitin overexpression partially rescues the axJ neuromuscular phenotype. However, later work has shown that ubiquitin overexpression does not correct the axJ deficits in hippocampal short term plasticity, and that transgenic expression of a catalytically-inactive form of USP14 in the nervous system mimics the neuromuscular phenotype observed in the axJ mice, but causes a only a modest reduction of free ubiquitin. Instead, increased ubiquitin conjugates and aberrant activation of pJNK are observed in the nervous systems of the USP14 catalytic mutant mice. In this report, we demonstrate that restoring free ubiquitin levels in the USP14 catalytic mutant mice improved NMJ structure and reduced pJNK accumulation in motor neuron terminals, but had a negative impact on measures of NMJ function, such as motor performance and muscle development. Transgenic expression of ubiquitin had a dose-dependent effect on NMJ function in wild type mice: moderate levels of overexpression improved NMJ function while more robust ubiquitin overexpression reduced muscle development and motor coordination. Combined, these results suggest that maintenance of free ubiquitin levels by USP14 contributes to NMJ structure, but that USP14 regulates NMJ function through a separate pathway.
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ubiquitin specific protease 14 regulates c jun n terminal kinase signaling at the neuromuscular junction
Molecular Neurodegeneration, 2015Co-Authors: Jada H Vaden, Bula J Bhattacharyya, Pingchung Chen, Jennifer A Watson, Andrea G Marshall, Scott E Phillips, Julie A Wilson, Gwendalyn D King, Richard J Miller, Scott M WilsonAbstract:Ubiquitin-specific protease 14 (USP14) is one of three proteasome-associated deubiquitinating enzymes that remove ubiquitin from proteasomal substrates prior to their degradation. In vitro evidence suggests that inhibiting USP14’s catalytic activity alters the turnover of ubiquitinated proteins by the proteasome, although whether protein degradation is accelerated or delayed seems to be cell-type and substrate specific. For example, combined inhibition of USP14 and the proteasomal deubiquitinating enzyme UCH37 halts protein degradation and promotes apoptosis in multiple myeloma cells, whereas USP14 inhibition alone accelerates the degradation of aggregate-prone proteins in immortalized cell lines. These findings have prompted interest in USP14 as a therapeutic target both inside and outside of the nervous system. However, loss of USP14 in the spontaneously occurring ataxia mouse mutant leads to a dramatic neuromuscular phenotype and early perinatal lethality, suggesting that USP14 inhibition may have adverse consequences in the nervous system. We therefore expressed a catalytically inactive USP14 mutant in the mouse nervous system to determine whether USP14’s catalytic activity is required for neuromuscular junction (NMJ) structure and function. Mice expressing catalytically inactive USP14 in the nervous system exhibited motor deficits, altered NMJ structure, and synaptic transmission deficits that were similar to what is observed in the USP14-deficient ataxia mice. Acute pharmacological inhibition of USP14 in wild type mice also reduced NMJ synaptic transmission. However, there was no evidence of altered proteasome activity when USP14 was inhibited either genetically or pharmacologically. Instead, these manipulations increased the levels of non-proteasome targeting ubiquitin conjugates. Specifically, we observed enhanced proteasome-independent ubiquitination of mixed lineage kinase 3 (MLK3). Consistent with the direct activation of MLK3 by ubiquitination, we also observed increased activation of its downstrea targets MAP kinase kinase 4 (MKK4) and c-Jun N-terminal kinase (JNK). In vivo inhibition of JNK improved motor function and synapse structure in the USP14 catalytic mutant mice. USP14’s catalytic activity is required for nervous system structure and function and has an ongoing role in NMJ synaptic transmission. By regulating the ubiquitination status of protein kinases, USP14 can coordinate the activity of intracellular signaling pathways that control the development and activity of the NMJ.
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a catalytic independent function of the deubiquitinating enzyme USP14 regulates hippocampal synaptic short term plasticity and vesicle number
The Journal of Physiology, 2014Co-Authors: Brandon J Walters, Scott M Wilson, Jada J Hallengren, Christopher S Theile, Hidde L Ploegh, Lynn E DobrunzAbstract:Key points Mice carrying the ataxia (axJ) mutation have a 95% reduction in the deubiquitinating enzyme USP14, which results in a reduction in hippocampal paired pulse facilitation, a form of short-term synaptic plasticity. Hippocampal synapses in axJ mice have a 50% reduction in synaptic vesicles but no change in the initial release probability, which is a novel mechanism for regulating paired pulse facilitation. USP14 modulates hippocampal short-term plasticity and structure independent of its deubiquitinating activity, as overexpression of a catalytically inactive form of USP14 restores hippocampal paired pulse facilitation and vesicle number to the ataxia mice. Pharmacological inhibition of the proteasome also rescues the deficits in hippocampal short-term plasticity in ataxia mice, implying that the loss of USP14 causes increased protein degradation. These results suggest that USP14 plays a modulatory role in regulating protein turnover by the proteasome that is independent of its canonical role in the disassembly of polyubiquitin conjugates. Abstract The ubiquitin proteasome system is required for the rapid and precise control of protein abundance that is essential for synaptic function. USP14 is a proteasome-bound deubiquitinating enzyme that recycles ubiquitin and regulates synaptic short-term synaptic plasticity. We previously reported that loss of USP14 in axJ mice causes a deficit in paired pulse facilitation (PPF) at hippocampal synapses. Here we report that USP14 regulates synaptic function through a novel, deubiquitination-independent mechanism. Although PPF is usually inversely related to release probability, USP14 deficiency impairs PPF without altering basal release probability. Instead, the loss of USP14 causes a large reduction in the number of synaptic vesicles. Over-expression of a catalytically inactive form of USP14 rescues the PPF deficit and restores synaptic vesicle number, indicating that USP14 regulates presynaptic structure and function independently of its role in deubiquitination. Finally, the PPF deficit caused by loss of USP14 can be rescued by pharmacological inhibition of proteasome activity, suggesting that inappropriate protein degradation underlies the PPF impairment. Overall, we demonstrate a novel, deubiquitination-independent function for USP14 in influencing synaptic architecture and plasticity.
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genetic background alters the severity and onset of neuromuscular disease caused by the loss of ubiquitin specific protease 14 USP14
PLOS ONE, 2013Co-Authors: Andrea G Marshall, Jennifer A Watson, Scott E Phillips, Julie A Wilson, Brandon J Walters, Jada J Hallengren, Lynn E Dobrunz, Ludwig Francillon, Scott M WilsonAbstract:In this study, we identified and characterized an N-ethyl-N-nitrosourea (ENU) induced mutation in USP14 (nmf375) that leads to adult-onset neurological disease. The nmf375 mutation causes aberrant splicing of USP14 mRNA, resulting in a 95% reduction in USP14. We previously showed that loss of USP14 in ataxia (axJ) mice results in reduced ubiquitin levels, motor endplate disease, Purkinje cell axonal dystrophy and decreased hippocampal paired pulse facilitation (PPF) during the first 4-6 weeks of life, and early postnatal lethality by two months of age. Although the loss of USP14 is comparable between the nmf375 and axJ mice, the nmf375 mice did not exhibit these axJ developmental abnormalities. However, by 12 weeks of age the nmf375 mutants present with ubiquitin depletion and motor endplate disease, indicating a continual role for USP14-mediated regulation of ubiquitin pools and neuromuscular junction (NMJ) structure in adult mice. The observation that motor endplate disease was only seen after ubiquitin depletion suggests that the preservation of NMJ structure requires the stable maintenance of synaptic ubiquitin pools. Differences in genetic background were shown to affect ubiquitin expression and dramatically alter the phenotypes caused by USP14 deficiency.
Lynn E Dobrunz - One of the best experts on this subject based on the ideXlab platform.
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a catalytic independent function of the deubiquitinating enzyme USP14 regulates hippocampal synaptic short term plasticity and vesicle number
The Journal of Physiology, 2014Co-Authors: Brandon J Walters, Scott M Wilson, Jada J Hallengren, Christopher S Theile, Hidde L Ploegh, Lynn E DobrunzAbstract:Key points Mice carrying the ataxia (axJ) mutation have a 95% reduction in the deubiquitinating enzyme USP14, which results in a reduction in hippocampal paired pulse facilitation, a form of short-term synaptic plasticity. Hippocampal synapses in axJ mice have a 50% reduction in synaptic vesicles but no change in the initial release probability, which is a novel mechanism for regulating paired pulse facilitation. USP14 modulates hippocampal short-term plasticity and structure independent of its deubiquitinating activity, as overexpression of a catalytically inactive form of USP14 restores hippocampal paired pulse facilitation and vesicle number to the ataxia mice. Pharmacological inhibition of the proteasome also rescues the deficits in hippocampal short-term plasticity in ataxia mice, implying that the loss of USP14 causes increased protein degradation. These results suggest that USP14 plays a modulatory role in regulating protein turnover by the proteasome that is independent of its canonical role in the disassembly of polyubiquitin conjugates. Abstract The ubiquitin proteasome system is required for the rapid and precise control of protein abundance that is essential for synaptic function. USP14 is a proteasome-bound deubiquitinating enzyme that recycles ubiquitin and regulates synaptic short-term synaptic plasticity. We previously reported that loss of USP14 in axJ mice causes a deficit in paired pulse facilitation (PPF) at hippocampal synapses. Here we report that USP14 regulates synaptic function through a novel, deubiquitination-independent mechanism. Although PPF is usually inversely related to release probability, USP14 deficiency impairs PPF without altering basal release probability. Instead, the loss of USP14 causes a large reduction in the number of synaptic vesicles. Over-expression of a catalytically inactive form of USP14 rescues the PPF deficit and restores synaptic vesicle number, indicating that USP14 regulates presynaptic structure and function independently of its role in deubiquitination. Finally, the PPF deficit caused by loss of USP14 can be rescued by pharmacological inhibition of proteasome activity, suggesting that inappropriate protein degradation underlies the PPF impairment. Overall, we demonstrate a novel, deubiquitination-independent function for USP14 in influencing synaptic architecture and plasticity.
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genetic background alters the severity and onset of neuromuscular disease caused by the loss of ubiquitin specific protease 14 USP14
PLOS ONE, 2013Co-Authors: Andrea G Marshall, Jennifer A Watson, Scott E Phillips, Julie A Wilson, Brandon J Walters, Jada J Hallengren, Lynn E Dobrunz, Ludwig Francillon, Scott M WilsonAbstract:In this study, we identified and characterized an N-ethyl-N-nitrosourea (ENU) induced mutation in USP14 (nmf375) that leads to adult-onset neurological disease. The nmf375 mutation causes aberrant splicing of USP14 mRNA, resulting in a 95% reduction in USP14. We previously showed that loss of USP14 in ataxia (axJ) mice results in reduced ubiquitin levels, motor endplate disease, Purkinje cell axonal dystrophy and decreased hippocampal paired pulse facilitation (PPF) during the first 4-6 weeks of life, and early postnatal lethality by two months of age. Although the loss of USP14 is comparable between the nmf375 and axJ mice, the nmf375 mice did not exhibit these axJ developmental abnormalities. However, by 12 weeks of age the nmf375 mutants present with ubiquitin depletion and motor endplate disease, indicating a continual role for USP14-mediated regulation of ubiquitin pools and neuromuscular junction (NMJ) structure in adult mice. The observation that motor endplate disease was only seen after ubiquitin depletion suggests that the preservation of NMJ structure requires the stable maintenance of synaptic ubiquitin pools. Differences in genetic background were shown to affect ubiquitin expression and dramatically alter the phenotypes caused by USP14 deficiency.
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A catalytic independent function of the deubiquitinating enzyme USP14 regulates hippocampal synaptic short‐term plasticity and vesicle number
The Journal of Physiology, 2013Co-Authors: Brandon J Walters, Scott M Wilson, Jada J Hallengren, Christopher S Theile, Hidde L Ploegh, Lynn E DobrunzAbstract:Key points Mice carrying the ataxia (axJ) mutation have a 95% reduction in the deubiquitinating enzyme USP14, which results in a reduction in hippocampal paired pulse facilitation, a form of short-term synaptic plasticity. Hippocampal synapses in axJ mice have a 50% reduction in synaptic vesicles but no change in the initial release probability, which is a novel mechanism for regulating paired pulse facilitation. USP14 modulates hippocampal short-term plasticity and structure independent of its deubiquitinating activity, as overexpression of a catalytically inactive form of USP14 restores hippocampal paired pulse facilitation and vesicle number to the ataxia mice. Pharmacological inhibition of the proteasome also rescues the deficits in hippocampal short-term plasticity in ataxia mice, implying that the loss of USP14 causes increased protein degradation. These results suggest that USP14 plays a modulatory role in regulating protein turnover by the proteasome that is independent of its canonical role in the disassembly of polyubiquitin conjugates. Abstract The ubiquitin proteasome system is required for the rapid and precise control of protein abundance that is essential for synaptic function. USP14 is a proteasome-bound deubiquitinating enzyme that recycles ubiquitin and regulates synaptic short-term synaptic plasticity. We previously reported that loss of USP14 in axJ mice causes a deficit in paired pulse facilitation (PPF) at hippocampal synapses. Here we report that USP14 regulates synaptic function through a novel, deubiquitination-independent mechanism. Although PPF is usually inversely related to release probability, USP14 deficiency impairs PPF without altering basal release probability. Instead, the loss of USP14 causes a large reduction in the number of synaptic vesicles. Over-expression of a catalytically inactive form of USP14 rescues the PPF deficit and restores synaptic vesicle number, indicating that USP14 regulates presynaptic structure and function independently of its role in deubiquitination. Finally, the PPF deficit caused by loss of USP14 can be rescued by pharmacological inhibition of proteasome activity, suggesting that inappropriate protein degradation underlies the PPF impairment. Overall, we demonstrate a novel, deubiquitination-independent function for USP14 in influencing synaptic architecture and plasticity.
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USP14 deficiency increases tau phosphorylation without altering tau degradation or causing tau dependent deficits
PLOS ONE, 2012Co-Authors: Pingchung Chen, Jennifer A Watson, Scott E Phillips, Julie A Wilson, Brandon J Walters, Karen G Green, Robert E Schmidt, Gail V W Johnson, Erik D Roberson, Lynn E DobrunzAbstract:Regulated protein degradation by the proteasome plays an essential role in the enhancement and suppression of signaling pathways in the nervous system. Proteasome-associated factors are pivotal in ensuring appropriate protein degradation, and we have previously demonstrated that alterations in one of these factors, the proteasomal deubiquitinating enzyme ubiquitin-specific protease 14 (USP14), can lead to proteasome dysfunction and neurological disease. Recent studies in cell culture have shown that USP14 can also stabilize the expression of over-expressed, disease-associated proteins such as tau and ataxin-3. Using USP14-deficient axJ mice, we investigated if loss of USP14 results in decreased levels of endogenous tau and ataxin-3 in the nervous system of mice. Although loss of USP14 did not alter the overall neuronal levels of tau and ataxin-3, we found increased levels of phosphorylated tau that correlated with the onset of axonal varicosities in the USP14-deficient mice. These changes in tau phosphorylation were accompanied by increased levels of activated phospho-Akt, phosphorylated MAPKs, and inactivated phospho-GSK3β. However, genetic ablation of tau did not alter any of the neurological deficits in the USP14-deficient mice, demonstrating that increased levels of phosphorylated tau do not necessarily lead to neurological disease. Due to the widespread activation of intracellular signaling pathways induced by the loss of USP14, a better understanding of the cellular pathways regulated by the proteasome is required before effective proteasomal-based therapies can be used to treat chronic neurological diseases.
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differential effects of USP14 and uch l1 on the ubiquitin proteasome system and synaptic activity
Molecular and Cellular Neuroscience, 2008Co-Authors: Brandon J Walters, Pingchung Chen, Julie A Wilson, Hidde L Ploegh, Lynn E Dobrunz, Susan L Campbell, A P Taylor, D G Schroeder, K Artavanistsakonas, Scott M WilsonAbstract:The ubiquitin proteasome pathway has been implicated in the pathogenesis of many neurodegenerative diseases, and alterations in two different deubiquitinating enzymes, Uch-L1 and USP14, result in neurological phenotypes in mice. We identified a new mutation in Uch-L1 and compared the roles of Uch-L1 and USP14 in the ubiquitin proteasome system. Deficiencies in either Uch-L1 or USP14 result in decreased levels of ubiquitin, suggesting that they both regulate ubiquitin stability in the nervous system. However, the effect of ubiquitin depletion on viability and onset of symptoms is more severe in the USP14-deficient mice, and changes in hippocampal synaptic transmission were only observed in USP14-deficient mice. In addition, while USP14 appears to function at the proteasome, Uch-L1 deficiency resulted in up-regulation of lysosomal components, indicating that Uch-L1 and USP14 may differentially affect the ubiquitin proteasome system and synaptic activity by regulating different pools of ubiquitin in the cell.
Brandon J Walters - One of the best experts on this subject based on the ideXlab platform.
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a catalytic independent function of the deubiquitinating enzyme USP14 regulates hippocampal synaptic short term plasticity and vesicle number
The Journal of Physiology, 2014Co-Authors: Brandon J Walters, Scott M Wilson, Jada J Hallengren, Christopher S Theile, Hidde L Ploegh, Lynn E DobrunzAbstract:Key points Mice carrying the ataxia (axJ) mutation have a 95% reduction in the deubiquitinating enzyme USP14, which results in a reduction in hippocampal paired pulse facilitation, a form of short-term synaptic plasticity. Hippocampal synapses in axJ mice have a 50% reduction in synaptic vesicles but no change in the initial release probability, which is a novel mechanism for regulating paired pulse facilitation. USP14 modulates hippocampal short-term plasticity and structure independent of its deubiquitinating activity, as overexpression of a catalytically inactive form of USP14 restores hippocampal paired pulse facilitation and vesicle number to the ataxia mice. Pharmacological inhibition of the proteasome also rescues the deficits in hippocampal short-term plasticity in ataxia mice, implying that the loss of USP14 causes increased protein degradation. These results suggest that USP14 plays a modulatory role in regulating protein turnover by the proteasome that is independent of its canonical role in the disassembly of polyubiquitin conjugates. Abstract The ubiquitin proteasome system is required for the rapid and precise control of protein abundance that is essential for synaptic function. USP14 is a proteasome-bound deubiquitinating enzyme that recycles ubiquitin and regulates synaptic short-term synaptic plasticity. We previously reported that loss of USP14 in axJ mice causes a deficit in paired pulse facilitation (PPF) at hippocampal synapses. Here we report that USP14 regulates synaptic function through a novel, deubiquitination-independent mechanism. Although PPF is usually inversely related to release probability, USP14 deficiency impairs PPF without altering basal release probability. Instead, the loss of USP14 causes a large reduction in the number of synaptic vesicles. Over-expression of a catalytically inactive form of USP14 rescues the PPF deficit and restores synaptic vesicle number, indicating that USP14 regulates presynaptic structure and function independently of its role in deubiquitination. Finally, the PPF deficit caused by loss of USP14 can be rescued by pharmacological inhibition of proteasome activity, suggesting that inappropriate protein degradation underlies the PPF impairment. Overall, we demonstrate a novel, deubiquitination-independent function for USP14 in influencing synaptic architecture and plasticity.
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genetic background alters the severity and onset of neuromuscular disease caused by the loss of ubiquitin specific protease 14 USP14
PLOS ONE, 2013Co-Authors: Andrea G Marshall, Jennifer A Watson, Scott E Phillips, Julie A Wilson, Brandon J Walters, Jada J Hallengren, Lynn E Dobrunz, Ludwig Francillon, Scott M WilsonAbstract:In this study, we identified and characterized an N-ethyl-N-nitrosourea (ENU) induced mutation in USP14 (nmf375) that leads to adult-onset neurological disease. The nmf375 mutation causes aberrant splicing of USP14 mRNA, resulting in a 95% reduction in USP14. We previously showed that loss of USP14 in ataxia (axJ) mice results in reduced ubiquitin levels, motor endplate disease, Purkinje cell axonal dystrophy and decreased hippocampal paired pulse facilitation (PPF) during the first 4-6 weeks of life, and early postnatal lethality by two months of age. Although the loss of USP14 is comparable between the nmf375 and axJ mice, the nmf375 mice did not exhibit these axJ developmental abnormalities. However, by 12 weeks of age the nmf375 mutants present with ubiquitin depletion and motor endplate disease, indicating a continual role for USP14-mediated regulation of ubiquitin pools and neuromuscular junction (NMJ) structure in adult mice. The observation that motor endplate disease was only seen after ubiquitin depletion suggests that the preservation of NMJ structure requires the stable maintenance of synaptic ubiquitin pools. Differences in genetic background were shown to affect ubiquitin expression and dramatically alter the phenotypes caused by USP14 deficiency.
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A catalytic independent function of the deubiquitinating enzyme USP14 regulates hippocampal synaptic short‐term plasticity and vesicle number
The Journal of Physiology, 2013Co-Authors: Brandon J Walters, Scott M Wilson, Jada J Hallengren, Christopher S Theile, Hidde L Ploegh, Lynn E DobrunzAbstract:Key points Mice carrying the ataxia (axJ) mutation have a 95% reduction in the deubiquitinating enzyme USP14, which results in a reduction in hippocampal paired pulse facilitation, a form of short-term synaptic plasticity. Hippocampal synapses in axJ mice have a 50% reduction in synaptic vesicles but no change in the initial release probability, which is a novel mechanism for regulating paired pulse facilitation. USP14 modulates hippocampal short-term plasticity and structure independent of its deubiquitinating activity, as overexpression of a catalytically inactive form of USP14 restores hippocampal paired pulse facilitation and vesicle number to the ataxia mice. Pharmacological inhibition of the proteasome also rescues the deficits in hippocampal short-term plasticity in ataxia mice, implying that the loss of USP14 causes increased protein degradation. These results suggest that USP14 plays a modulatory role in regulating protein turnover by the proteasome that is independent of its canonical role in the disassembly of polyubiquitin conjugates. Abstract The ubiquitin proteasome system is required for the rapid and precise control of protein abundance that is essential for synaptic function. USP14 is a proteasome-bound deubiquitinating enzyme that recycles ubiquitin and regulates synaptic short-term synaptic plasticity. We previously reported that loss of USP14 in axJ mice causes a deficit in paired pulse facilitation (PPF) at hippocampal synapses. Here we report that USP14 regulates synaptic function through a novel, deubiquitination-independent mechanism. Although PPF is usually inversely related to release probability, USP14 deficiency impairs PPF without altering basal release probability. Instead, the loss of USP14 causes a large reduction in the number of synaptic vesicles. Over-expression of a catalytically inactive form of USP14 rescues the PPF deficit and restores synaptic vesicle number, indicating that USP14 regulates presynaptic structure and function independently of its role in deubiquitination. Finally, the PPF deficit caused by loss of USP14 can be rescued by pharmacological inhibition of proteasome activity, suggesting that inappropriate protein degradation underlies the PPF impairment. Overall, we demonstrate a novel, deubiquitination-independent function for USP14 in influencing synaptic architecture and plasticity.
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USP14 deficiency increases tau phosphorylation without altering tau degradation or causing tau dependent deficits
PLOS ONE, 2012Co-Authors: Pingchung Chen, Jennifer A Watson, Scott E Phillips, Julie A Wilson, Brandon J Walters, Karen G Green, Robert E Schmidt, Gail V W Johnson, Erik D Roberson, Lynn E DobrunzAbstract:Regulated protein degradation by the proteasome plays an essential role in the enhancement and suppression of signaling pathways in the nervous system. Proteasome-associated factors are pivotal in ensuring appropriate protein degradation, and we have previously demonstrated that alterations in one of these factors, the proteasomal deubiquitinating enzyme ubiquitin-specific protease 14 (USP14), can lead to proteasome dysfunction and neurological disease. Recent studies in cell culture have shown that USP14 can also stabilize the expression of over-expressed, disease-associated proteins such as tau and ataxin-3. Using USP14-deficient axJ mice, we investigated if loss of USP14 results in decreased levels of endogenous tau and ataxin-3 in the nervous system of mice. Although loss of USP14 did not alter the overall neuronal levels of tau and ataxin-3, we found increased levels of phosphorylated tau that correlated with the onset of axonal varicosities in the USP14-deficient mice. These changes in tau phosphorylation were accompanied by increased levels of activated phospho-Akt, phosphorylated MAPKs, and inactivated phospho-GSK3β. However, genetic ablation of tau did not alter any of the neurological deficits in the USP14-deficient mice, demonstrating that increased levels of phosphorylated tau do not necessarily lead to neurological disease. Due to the widespread activation of intracellular signaling pathways induced by the loss of USP14, a better understanding of the cellular pathways regulated by the proteasome is required before effective proteasomal-based therapies can be used to treat chronic neurological diseases.
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the proteasome associated deubiquitinating enzyme USP14 is essential for the maintenance of synaptic ubiquitin levels and the development of neuromuscular junctions
The Journal of Neuroscience, 2009Co-Authors: Pingchung Chen, Julie A Wilson, Brandon J Walters, Xiaoming Li, Scott M WilsonAbstract:Dysfunction of the ubiquitin proteasome system (UPS) has been implicated in the pathogenesis of many neurological diseases, including Alzheimer9s, spinocerebellar ataxia, and several motor neuron diseases. Recent research indicates that changes in synaptic transmission may play a critical role in the progression of neurological disease; however, the mechanisms by which the UPS regulates synaptic structure and function have not been well characterized. In this report, we show that USP14 is indispensable for synaptic development and function at neuromuscular junctions (NMJs). USP14-deficient ax J mice display a resting tremor, a reduction in muscle mass, and notable hindlimb rigidity without any detectable loss of motor neurons. Instead, loss of USP14 causes developmental defects at motor neuron endplates. Presynaptic defects include phosphorylated neurofilament accumulations, nerve terminal sprouting, and poor arborization of the motor nerve terminals, whereas postsynaptic acetylcholine receptors display immature plaque-like morphology. These structural changes in the NMJ correlated with ubiquitin loss in the spinal cord and sciatic nerve. Further studies demonstrated that the greatest loss of ubiquitin was found in synaptosomal fractions, suggesting that the endplate swellings may be caused by decreased protein turnover at the synapse. Transgenic restoration of USP14 in the nervous system corrected the levels of monomeric ubiquitin in the motor neuron circuit and the defects that were observed in the motor endplates and muscles of the ax J mice. These data define a critical role for USP14 at mammalian synapses and suggest a requirement for local ubiquitin recycling by the proteasome to control the development and function of NMJs.
Yue Ding - One of the best experts on this subject based on the ideXlab platform.
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the USP14 nlrc5 pathway inhibits titanium particle induced osteolysis in mice by suppressing nf κb and pi3k akt activities
Journal of Biological Chemistry, 2020Co-Authors: Guibin Fang, Yuan Fu, Shixun Li, Manyuan Kuang, Changchuan Li, Yue DingAbstract:: Total hip arthroplasty (THA) is a widely-used surgical intervention for treating patients with end-stage degenerative and inflammatory osteoarthropathy. However, wear particles from the artificial titanium joint can induce osteolysis, limiting the long-term survivorship of THA. Monocyte/macrophage lineage cells are the key players in the response to wear particles, and the proinflammatory NF-κB and phosphoinositide 3-kinase (PI3K)-AKT Ser/Thr kinase (AKT)-signaling pathways have been shown to be the most important contributors to wear particle-induced osteolysis. In contrast, ubiquitin-specific protease 14 (USP14) specifically removes the polyubiquitin chains from the nucleotide-binding and oligomerization domain (NOD)-like receptor family Caspase recruitment domain (CARD)-containing 5 (NLRC5) and thereby enhances the NLRC5-mediated inhibition of NF-κB signaling. In this study, we aimed to clarify the role of the USP14-NLRC5 pathway in wear particle-induced osteolysis in vitro and in vivo We found that NLRC5 or USP14 overexpression inhibits titanium particle-induced proinflammatory tumor necrosis factor α (TNFα) production and NF-κB pathway activation, and it also decreases M1 macrophage polarization and PI3K/AKT pathway activation. Of note, NLRC5 and USP14 overexpression attenuated titanium particle-induced cranial osteolysis in mice. In conclusion, the findings of our study indicate that the USP14-NLRC5 pathway inhibits titanium particle-induced osteolysis by suppressing the NF-κB and PI3K/AKT pathways both in vitro and in vivo.
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The USP14-NLRC5 pathway inhibits titanium particle-induced osteolysis in mice by suppressing NF-κB and PI3K/AKT activities.
Journal of Biological Chemistry, 2020Co-Authors: Guibin Fang, Yuan Fu, Shixun Li, Manyuan Kuang, Changchuan Li, Yue DingAbstract:: Total hip arthroplasty (THA) is a widely-used surgical intervention for treating patients with end-stage degenerative and inflammatory osteoarthropathy. However, wear particles from the artificial titanium joint can induce osteolysis, limiting the long-term survivorship of THA. Monocyte/macrophage lineage cells are the key players in the response to wear particles, and the proinflammatory NF-κB and phosphoinositide 3-kinase (PI3K)-AKT Ser/Thr kinase (AKT)-signaling pathways have been shown to be the most important contributors to wear particle-induced osteolysis. In contrast, ubiquitin-specific protease 14 (USP14) specifically removes the polyubiquitin chains from the nucleotide-binding and oligomerization domain (NOD)-like receptor family Caspase recruitment domain (CARD)-containing 5 (NLRC5) and thereby enhances the NLRC5-mediated inhibition of NF-κB signaling. In this study, we aimed to clarify the role of the USP14-NLRC5 pathway in wear particle-induced osteolysis in vitro and in vivo We found that NLRC5 or USP14 overexpression inhibits titanium particle-induced proinflammatory tumor necrosis factor α (TNFα) production and NF-κB pathway activation, and it also decreases M1 macrophage polarization and PI3K/AKT pathway activation. Of note, NLRC5 and USP14 overexpression attenuated titanium particle-induced cranial osteolysis in mice. In conclusion, the findings of our study indicate that the USP14-NLRC5 pathway inhibits titanium particle-induced osteolysis by suppressing the NF-κB and PI3K/AKT pathways both in vitro and in vivo.
Pingchung Chen - One of the best experts on this subject based on the ideXlab platform.
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distinct effects of ubiquitin overexpression on nmj structure and motor performance in mice expressing catalytically inactive USP14
Frontiers in Molecular Neuroscience, 2015Co-Authors: Jada H Vaden, Pingchung Chen, Jennifer A Watson, Julie A Wilson, Alan D Howard, Scott M WilsonAbstract:Ubiquitin-specific protease 14 (USP14) is a major deubiquitinating enzyme and a key determinant of neuromuscular junction (NMJ) structure and function. We have previously reported dramatic ubiquitin depletion in the nervous systems of the USP14-deficient ataxia (axJ) mice and demonstrated that transgenic ubiquitin overexpression partially rescues the axJ neuromuscular phenotype. However, later work has shown that ubiquitin overexpression does not correct the axJ deficits in hippocampal short term plasticity, and that transgenic expression of a catalytically-inactive form of USP14 in the nervous system mimics the neuromuscular phenotype observed in the axJ mice, but causes a only a modest reduction of free ubiquitin. Instead, increased ubiquitin conjugates and aberrant activation of pJNK are observed in the nervous systems of the USP14 catalytic mutant mice. In this report, we demonstrate that restoring free ubiquitin levels in the USP14 catalytic mutant mice improved NMJ structure and reduced pJNK accumulation in motor neuron terminals, but had a negative impact on measures of NMJ function, such as motor performance and muscle development. Transgenic expression of ubiquitin had a dose-dependent effect on NMJ function in wild type mice: moderate levels of overexpression improved NMJ function while more robust ubiquitin overexpression reduced muscle development and motor coordination. Combined, these results suggest that maintenance of free ubiquitin levels by USP14 contributes to NMJ structure, but that USP14 regulates NMJ function through a separate pathway.
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ubiquitin specific protease 14 regulates c jun n terminal kinase signaling at the neuromuscular junction
Molecular Neurodegeneration, 2015Co-Authors: Jada H Vaden, Bula J Bhattacharyya, Pingchung Chen, Jennifer A Watson, Andrea G Marshall, Scott E Phillips, Julie A Wilson, Gwendalyn D King, Richard J Miller, Scott M WilsonAbstract:Ubiquitin-specific protease 14 (USP14) is one of three proteasome-associated deubiquitinating enzymes that remove ubiquitin from proteasomal substrates prior to their degradation. In vitro evidence suggests that inhibiting USP14’s catalytic activity alters the turnover of ubiquitinated proteins by the proteasome, although whether protein degradation is accelerated or delayed seems to be cell-type and substrate specific. For example, combined inhibition of USP14 and the proteasomal deubiquitinating enzyme UCH37 halts protein degradation and promotes apoptosis in multiple myeloma cells, whereas USP14 inhibition alone accelerates the degradation of aggregate-prone proteins in immortalized cell lines. These findings have prompted interest in USP14 as a therapeutic target both inside and outside of the nervous system. However, loss of USP14 in the spontaneously occurring ataxia mouse mutant leads to a dramatic neuromuscular phenotype and early perinatal lethality, suggesting that USP14 inhibition may have adverse consequences in the nervous system. We therefore expressed a catalytically inactive USP14 mutant in the mouse nervous system to determine whether USP14’s catalytic activity is required for neuromuscular junction (NMJ) structure and function. Mice expressing catalytically inactive USP14 in the nervous system exhibited motor deficits, altered NMJ structure, and synaptic transmission deficits that were similar to what is observed in the USP14-deficient ataxia mice. Acute pharmacological inhibition of USP14 in wild type mice also reduced NMJ synaptic transmission. However, there was no evidence of altered proteasome activity when USP14 was inhibited either genetically or pharmacologically. Instead, these manipulations increased the levels of non-proteasome targeting ubiquitin conjugates. Specifically, we observed enhanced proteasome-independent ubiquitination of mixed lineage kinase 3 (MLK3). Consistent with the direct activation of MLK3 by ubiquitination, we also observed increased activation of its downstrea targets MAP kinase kinase 4 (MKK4) and c-Jun N-terminal kinase (JNK). In vivo inhibition of JNK improved motor function and synapse structure in the USP14 catalytic mutant mice. USP14’s catalytic activity is required for nervous system structure and function and has an ongoing role in NMJ synaptic transmission. By regulating the ubiquitination status of protein kinases, USP14 can coordinate the activity of intracellular signaling pathways that control the development and activity of the NMJ.
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USP14 deficiency increases tau phosphorylation without altering tau degradation or causing tau dependent deficits
PLOS ONE, 2012Co-Authors: Pingchung Chen, Jennifer A Watson, Scott E Phillips, Julie A Wilson, Brandon J Walters, Karen G Green, Robert E Schmidt, Gail V W Johnson, Erik D Roberson, Lynn E DobrunzAbstract:Regulated protein degradation by the proteasome plays an essential role in the enhancement and suppression of signaling pathways in the nervous system. Proteasome-associated factors are pivotal in ensuring appropriate protein degradation, and we have previously demonstrated that alterations in one of these factors, the proteasomal deubiquitinating enzyme ubiquitin-specific protease 14 (USP14), can lead to proteasome dysfunction and neurological disease. Recent studies in cell culture have shown that USP14 can also stabilize the expression of over-expressed, disease-associated proteins such as tau and ataxin-3. Using USP14-deficient axJ mice, we investigated if loss of USP14 results in decreased levels of endogenous tau and ataxin-3 in the nervous system of mice. Although loss of USP14 did not alter the overall neuronal levels of tau and ataxin-3, we found increased levels of phosphorylated tau that correlated with the onset of axonal varicosities in the USP14-deficient mice. These changes in tau phosphorylation were accompanied by increased levels of activated phospho-Akt, phosphorylated MAPKs, and inactivated phospho-GSK3β. However, genetic ablation of tau did not alter any of the neurological deficits in the USP14-deficient mice, demonstrating that increased levels of phosphorylated tau do not necessarily lead to neurological disease. Due to the widespread activation of intracellular signaling pathways induced by the loss of USP14, a better understanding of the cellular pathways regulated by the proteasome is required before effective proteasomal-based therapies can be used to treat chronic neurological diseases.
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enhancement of proteasome activity by a small molecule inhibitor of USP14
Nature, 2010Co-Authors: Soyeon Park, Pingchung Chen, Suzanne Elsasser, Dong Chan Oh, Carlos A Gartner, Nevena Dimova, John HannaAbstract:Proteasomes, the primary mediators of ubiquitin–protein conjugate degradation, are regulated through complex and poorly understood mechanisms. Here we show that USP14, a proteasome-associated deubiquitinating enzyme, can inhibit the degradation of ubiquitin–protein conjugates both in vitro and in cells. A catalytically inactive variant of USP14 has reduced inhibitory activity, indicating that inhibition is mediated by trimming of the ubiquitin chain on the substrate. A high-throughput screen identified a selective small-molecule inhibitor of the deubiquitinating activity of human USP14. Treatment of cultured cells with this compound enhanced degradation of several proteasome substrates that have been implicated in neurodegenerative disease. USP14 inhibition accelerated the degradation of oxidized proteins and enhanced resistance to oxidative stress. Enhancement of proteasome activity through inhibition of USP14 may offer a strategy to reduce the levels of aberrant proteins in cells under proteotoxic stress. In the ubiquitin–proteasome system, which serves an important role in the eukaryotic cell by degrading proteins that are damaged or surplus to requirements, substrates destined for destruction are covalently modified with ubiquitin chains and subsequently degraded by the proteasome. A novel regulatory mechanism in which proteasomal activity is modulated by the length of ubiquitin chains has now been identified in human cells. The deubiquitinating enzyme USP14 can inhibit the degradation of ubiquitin-conjugated substrates by trimming ubiquitin chains. Furthermore, a chemical screen identified a small-molecule inhibitor of USP14, and treatment of mammalian cells with this compound resulted in increased clearance of a variety of substrates, including oxidized proteins and disease-causing toxic proteins. Thus, stimulation of proteasome activity may offer a strategy to reduce the levels of toxic proteins in cells. In the ubiquitin–proteasome system, substrates destined for destruction are modified with ubiquitin chains and then degraded by the proteasome. These authors reveal a regulatory mechanism in which proteasomal activity is modulated by the length of ubiquitin chains in human cells. They find that deubiquitinating enzyme USP14 can inhibit the degradation of ubiquitin-conjugated substrates by trimming ubiquitin chains, and that stimulation of proteasome activity may be used to reduce the levels of toxic proteins in cells.
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the proteasome associated deubiquitinating enzyme USP14 is essential for the maintenance of synaptic ubiquitin levels and the development of neuromuscular junctions
The Journal of Neuroscience, 2009Co-Authors: Pingchung Chen, Julie A Wilson, Brandon J Walters, Xiaoming Li, Scott M WilsonAbstract:Dysfunction of the ubiquitin proteasome system (UPS) has been implicated in the pathogenesis of many neurological diseases, including Alzheimer9s, spinocerebellar ataxia, and several motor neuron diseases. Recent research indicates that changes in synaptic transmission may play a critical role in the progression of neurological disease; however, the mechanisms by which the UPS regulates synaptic structure and function have not been well characterized. In this report, we show that USP14 is indispensable for synaptic development and function at neuromuscular junctions (NMJs). USP14-deficient ax J mice display a resting tremor, a reduction in muscle mass, and notable hindlimb rigidity without any detectable loss of motor neurons. Instead, loss of USP14 causes developmental defects at motor neuron endplates. Presynaptic defects include phosphorylated neurofilament accumulations, nerve terminal sprouting, and poor arborization of the motor nerve terminals, whereas postsynaptic acetylcholine receptors display immature plaque-like morphology. These structural changes in the NMJ correlated with ubiquitin loss in the spinal cord and sciatic nerve. Further studies demonstrated that the greatest loss of ubiquitin was found in synaptosomal fractions, suggesting that the endplate swellings may be caused by decreased protein turnover at the synapse. Transgenic restoration of USP14 in the nervous system corrected the levels of monomeric ubiquitin in the motor neuron circuit and the defects that were observed in the motor endplates and muscles of the ax J mice. These data define a critical role for USP14 at mammalian synapses and suggest a requirement for local ubiquitin recycling by the proteasome to control the development and function of NMJs.
