Impaired Neurite Contact Guidance in Ubiquitin Ligase E3a (Ube3a)-Deficient Hippocampal Neurons on Nanostructured Substrates

2016 ◽  
Vol 5 (7) ◽  
pp. 850-862 ◽  
Author(s):  
I. Tonazzini ◽  
S. Meucci ◽  
G. M. Van Woerden ◽  
Y. Elgersma ◽  
M. Cecchini
Keyword(s):  
Ubiquitin Ligase ◽  
Hippocampal Neurons ◽  
Contact Guidance ◽  
Nanostructured Substrates

scholarly journals The X-linked intellectual disability gene product and E3 ubiquitin ligase KLHL15 degrades  doublecortin proteins to constrain neuronal dendritogenesis

2020 ◽  
pp. jbc.RA120.016210
Author(s):  
Jianing Song ◽  
Ronald A. Merrill ◽  
Andrew Y. Usachev ◽  
Stefan Strack
Keyword(s):  
Intellectual Disability ◽  
Brain Development ◽  
Ubiquitin Ligase ◽  
Hippocampal Neurons ◽  
E3 Ubiquitin Ligase ◽  
Gene Mutations ◽  
Disease Protein ◽  
Associated Proteins ◽  
Interaction Regions ◽  
And Function

Proper brain development and function requires finely controlled mechanisms for protein turnover and disruption of genes involved in proteostasis is a common cause of neurodevelopmental disorders. Kelch-like 15 (KLHL15) is a substrate adaptor for cullin3 (Cul3)-containing E3 ubiquitin ligases and KLHL15 gene mutations were recently described as a cause of severe X-linked intellectual disability (XLID). Here, we used a bioinformatics approach to identify a family of neuronal microtubule-associated proteins (MAPs) as KLHL15 substrates, which are themselves critical for early brain development. We biochemically validated doublecortin (DCX), also an X-linked disease protein, and doublecortin-like kinase 1 and 2 (DCLK1/2) as bona fide KLHL15 interactors and mapped KLHL15 interaction regions to their tandem DCX domains. Shared with two previously identified KLHL15 substrates, a FRY tripeptide at the C-terminal edge of the second DCX domain is necessary for KLHL15-mediated ubiquitination of DCX and DCLK1/2 and subsequent proteasomal degradation. Conversely, silencing endogenous KLHL15 markedly stabilizes these DCX domain-containing proteins and prolongs their half-life. Functionally, overexpression of KLHL15 in the presence of wild-type DCX reduces dendritic complexity of cultured hippocampal neurons, whereas neurons expressing FRY-mutant DCX are resistant to KLHL15. Collectively, our findings highlight the critical importance of the E3 ubiquitin ligase adaptor KLHL15 in proteostasis of neuronal MAPs and identify a regulatory network important for development of the mammalian nervous system.

scholarly journals Negative Regulation of Autophagy by UBA6-BIRC6–Mediated Ubiquitination of LC3

2019 ◽  
Author(s):  
Rui Jia ◽  
Juan S. Bonifacino
Keyword(s):  
Ubiquitin Ligase ◽  
Neurodegenerative Disorders ◽  
Hippocampal Neurons ◽  
Neuroblastoma Cells ◽  
Autophagic Flux ◽  
Protein Synthesis Inhibition ◽  
Human Neuroblastoma Cells ◽  
Human Neuroblastoma ◽  
Ubiquitin Ligase E3 ◽  
Autophagy Inhibition

AbstractAlthough the process of autophagy has been extensively studied, the mechanisms that regulate it remain insufficiently understood. The ability to manipulate autophagy is important not only for addressing fundamental biological questions, but also for its possible application to the treatment of various human diseases. To identify novel regulators of autophagy, we performed a whole-genome CRISPR/Cas9 knockout screen in H4 human neuroblastoma cells gene-edited to express the endogenous autophagy effector LC3B fused to a tandem of GFP and mCherry. Using this methodology, we identified the ubiquitin-activating (E1) enzyme UBA6 and the hybrid ubiquitin-conjugating (E2)/ubiquitin-ligase (E3) enzyme BIRC6 as important autophagy regulators. We found that these two enzymes cooperate to monoubiquitinate LC3B on lysine-51, targeting it for degradation by the proteasome. Knockout of UBA6 or BIRC6 increased the levels of LC3B as well as autophagic flux under conditions of nutrient deprivation or protein synthesis inhibition. Moreover, depletion of UBA6 or BIRC6 KO decreased the formation of aggresome-like induced structures in H4 cells, and aggregates of an α-synuclein mutant in the axon of rat hippocampal neurons. These findings demonstrate that UBA6 and BIRC6 negatively regulate autophagy by limiting the availability of LC3B, possibly to prevent the deleterious effects of excessive autophagy. Inhibition of UBA6 or BIRC6, on the other hand, could be used to enhance autophagic clearance of protein aggregates in neurodegenerative disorders.

Contact guidance of CNS neurites on grooved quartz: influence of groove dimensions, neuronal age and cell type

1997 ◽  
Vol 110 (23) ◽  
pp. 2905-2913 ◽  
Author(s):  
A. Rajnicek ◽  
S. Britland ◽  
C. McCaig
Keyword(s):  
Hippocampal Neurons ◽  
Growth Cones ◽  
Companion Paper ◽  
Contact Guidance ◽  
Neuronal Polarity ◽  
Cell Type ◽  
Spinal Cord Neurons ◽  
In Vitro System ◽  
Guidance Information

We used an in vitro system that eliminates competing guidance cues found in embryos to determine whether substratum topography alone provides important neurite guidance information. Dissociated embryonic Xenopus spinal cord neurons and rat hippocampal neurons were grown on quartz etched with a series of parallel grooves. Xenopus neurites grew parallel to grooves as shallow as 14 nm and as narrow as 1 microm. Hippocampal neurites grew parallel to deep, wide grooves but perpendicular to shallow, narrow ones. Grooved substrata determined the sites at which neurites emerged from somas: Xenopus neurites sprouted from regions parallel to grooves but presumptive axons on rat hippocampal neurons emerged perpendicular to grooves and presumptive dendrites emerged parallel to them. Neurites grew faster in the favored direction of orientation and turned through large angles to align on grooves. The frequency of perpendicular alignment of hippocampal neurites depended on the age of the embryos from which neurons were isolated, suggesting that contact guidance is regulated in development. Collectively, the data indicate that substratum topography is a potent morphogenetic factor for developing CNS neurons and suggest that in addition to a role in pathfinding the geometry of the embryo assists in establishing neuronal polarity. In the companion paper (A. M. Rajnicek and C. D. McCaig (1997) J. Cell Sci. 110, 2915–2924) we explore the cellular mechanism for contact guidance of growth cones.

scholarly journals The X-linked intellectual disability gene product and E3 ubiquitin ligase KLHL15 degrades doublecortin proteins to constrain neuronal dendritogenesis

2020 ◽  
Author(s):  
Jianing Song ◽  
Ronald A. Merrill ◽  
Andrew Y. Usachev ◽  
Stefan Strack
Keyword(s):  
Intellectual Disability ◽  
Brain Development ◽  
Ubiquitin Ligase ◽  
Hippocampal Neurons ◽  
E3 Ubiquitin Ligase ◽  
Gene Mutations ◽  
Associated Proteins ◽  
Bona Fide ◽  
Interaction Regions ◽  
And Function

ABSTRACTProper brain development and function requires finely controlled mechanisms for protein turnover and disruption of genes involved in proteostasis is a common cause of neurodevelopmental disorders. Kelch-like 15 (KLHL15) is a substrate adaptor for cullin3 (Cul3)-containing E3 ubiquitin ligases and KLHL15 gene mutations were recently described as a cause of severe X-linked intellectual disability. Here, we used a bioinformatics approach to identify a family of neuronal microtubule-associated proteins (MAPs) as KLHL15 substrates, which are themselves critical for early brain development. We biochemically validated doublecortin (DCX), also an X-linked disease gene, and doublecortin-like kinases 1 and 2 (DCLK1/2) as bona fide KLHL15 interactors and mapped KLHL15 interaction regions to their tandem DCX domains. Shared with two previously identified KLHL15 substrates, a FRY tripeptide at the C-terminal edge of the second DCX domain is necessary for KLHL15-mediated ubiquitination of DCX and DCLK1/2 and subsequent proteasomal degradation. Conversely, silencing endogenous KLHL15 markedly stabilizes these DCX domain-containing proteins and prolongs their half-life. Functionally, overexpression of KLHL15 in the presence of wild-type DCX reduces dendritic complexity of cultured hippocampal neurons, whereas neurons expressing FRY-mutant DCX are resistant to KLHL15. Collectively, our findings highlight the critical importance of the E3 ubiquitin ligase adaptor KLHL15 in proteostasis of neuronal MAPs and identify a regulatory network important for development of the mammalian nervous system.

scholarly journals The role of ubiquitin ligase E3A in polarized contact guidance and rescue strategies in UBE3A-deficient hippocampal neurons

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Ilaria Tonazzini ◽  
Geeske M. Van Woerden ◽  
Cecilia Masciullo ◽  
Edwin J. Mientjes ◽  
Ype Elgersma ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Hippocampal Neurons ◽  
Treatment Strategies ◽  
Contact Guidance ◽  
Cell Contractility ◽  
Actin Fiber ◽  
Mediated Contact ◽  
Focal Adhesion Pathway

Abstract Background Although neuronal extracellular sensing is emerging as crucial for brain wiring and therefore plasticity, little is known about these processes in neurodevelopmental disorders. Ubiquitin protein ligase E3A (UBE3A) plays a key role in neurodevelopment. Lack of UBE3A leads to Angelman syndrome (AS), while its increase is among the most prevalent genetic causes of autism (e.g., Dup15q syndrome). By using microstructured substrates that can induce specific directional stimuli in cells, we previously found deficient topographical contact guidance in AS neurons, which was linked to a dysregulated activation of the focal adhesion pathway. Methods Here, we study axon and dendrite contact guidance and neuronal morphological features of wild-type, AS, and UBE3A-overexpressing neurons (Dup15q autism model) on micrograting substrates, with the aim to clarify the role of UBE3A in neuronal guidance. Results We found that loss of axonal contact guidance is specific for AS neurons while UBE3A overexpression does not affect neuronal directional polarization along microgratings. Deficits at the level of axonal branching, growth cone orientation and actin fiber content, focal adhesion (FA) effectors, and actin fiber–binding proteins were observed in AS neurons. We tested different rescue strategies for restoring correct topographical guidance in AS neurons on microgratings, by either UBE3A protein re-expression or by pharmacological treatments acting on cytoskeleton contractility. Nocodazole, a drug that depolymerizes microtubules and increases cell contractility, rescued AS axonal alignment to the gratings by partially restoring focal adhesion pathway activation. Surprisingly, UBE3A re-expression only resulted in partial rescue of the phenotype. Conclusions We identified a specific in vitro deficit in axonal topographical guidance due selectively to the loss of UBE3A, and we further demonstrate that this defective guidance can be rescued to a certain extent by pharmacological or genetic treatment strategies. Overall, cytoskeleton dynamics emerge as important partners in UBE3A-mediated contact guidance responses. These results support the view that UBE3A-related deficits in early neuronal morphogenesis may lead to defective neuronal connectivity and plasticity.

scholarly journals Polarization of hippocampal neurons with competitive surface stimuli: contact guidance cues are preferred over chemical ligands

2006 ◽  
Vol 4 (13) ◽  
pp. 223-233 ◽  
Author(s):  
Natalia Gomez ◽  
Shaochen Chen ◽  
Christine E Schmidt
Keyword(s):  
Hippocampal Neurons ◽  
Artificial Substrates ◽  
Contact Guidance ◽  
Neural Engineering ◽  
Environmental Signals ◽  
Guidance Cues ◽  
Unique System ◽  
Extracellular Stimuli ◽  
Regeneration Processes ◽  
Microfabrication Techniques

Neuronal behaviour is profoundly influenced by extracellular stimuli in many developmental and regeneration processes. Understanding neuron responses and integration of environmental signals could impact the design of successful therapies for neurodegenerative diseases and nerve injuries. Here, we have investigated the influence of localized extracellular cues on polarization (i.e. axon formation) of hippocampal neurons. Electron-beam lithography, microfabrication techniques and protein immobilization were used to create a unique system that provided simultaneous and independent chemical and physical cues to individual neurons. In particular, we analysed competitive responses between simultaneous stimulation with chemical ligands, including immobilized nerve growth factor and laminin, and contact guidance cues mediated by surface topography (i.e. microchannels). Contact guidance cues were preferred 70% of the time over chemical ligands by neurons extending axons, which suggests a stronger stimulation mechanism triggered by topography. This investigation contributes to the understanding of neuronal behaviour on artificial substrates, which is applicable to the creation of artificial environments for neural engineering applications.

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