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

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          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.

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          Most cited references65

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          Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic p53 and deficits of contextual learning and long-term potentiation.

          The E6-AP ubiquitin ligase (human/mouse gene UBE3A/Ube3a) promotes the degradation of p53 in association with papilloma E6 protein, and maternal deficiency causes human Angelman syndrome (AS). Ube3a is imprinted with silencing of the paternal allele in hippocampus and cerebellum in mice. We found that the phenotype of mice with maternal deficiency (m-/p+) for Ube3a resembles human AS with motor dysfunction, inducible seizures, and a context-dependent learning deficit. Long-term potentiation (LTP) was severely impaired in m-/p+ mice despite normal baseline synaptic transmission and neuroanatomy, indicating that ubiquitination may play a role in mammalian LTP and that LTP may be abnormal in AS. The cytoplasmic abundance of p53 was increased in postmitotic neurons in m-/p+ mice and in AS, providing a potential biochemical basis for the phenotype through failure to ubiquitinate and degrade various effectors.
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            Microtubule stabilization specifies initial neuronal polarization

            Axon formation is the initial step in establishing neuronal polarity. We examine here the role of microtubule dynamics in neuronal polarization using hippocampal neurons in culture. We see increased microtubule stability along the shaft in a single neurite before axon formation and in the axon of morphologically polarized cells. Loss of polarity or formation of multiple axons after manipulation of neuronal polarity regulators, synapses of amphids defective (SAD) kinases, and glycogen synthase kinase-3β correlates with characteristic changes in microtubule turnover. Consistently, changing the microtubule dynamics is sufficient to alter neuronal polarization. Application of low doses of the microtubule-destabilizing drug nocodazole selectively reduces the formation of future dendrites. Conversely, low doses of the microtubule-stabilizing drug taxol shift polymerizing microtubules from neurite shafts to process tips and lead to the formation of multiple axons. Finally, local stabilization of microtubules using a photoactivatable analogue of taxol induces axon formation from the activated area. Thus, local microtubule stabilization in one neurite is a physiological signal specifying neuronal polarization.
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              Neuronal polarity: from extracellular signals to intracellular mechanisms.

              After they are born and differentiate, neurons break their previous symmetry, dramatically change their shape, and establish two structurally and functionally distinct compartments - axons and dendrites - within one cell. How do neurons develop their morphologically and molecularly distinct compartments? Recent studies have implicated several signalling pathways evoked by extracellular signals as having essential roles in a number of aspects of neuronal polarization.
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                Author and article information

                Contributors
                ilaria.tonazzini@sns.it
                g.vanwoerden@erasmusmc.nl
                cecilia.masciullo@sns.it
                e.mientjes@erasmusmc.nl
                y.elgersma@erasmusmc.nl
                marco.cecchini@nano.cnr.it
                Journal
                Mol Autism
                Mol Autism
                Molecular Autism
                BioMed Central (London )
                2040-2392
                29 November 2019
                29 November 2019
                2019
                : 10
                : 41
                Affiliations
                [1 ]Istituto Nanoscienze- Consiglio Nazionale delle Ricerche (CNR) & Scuola Normale Superiore, NEST, Piazza San Silvestro 12, 56127 Pisa, Italy
                [2 ]ISNI 000000040459992X, GRID grid.5645.2, Department of Neuroscience, ENCORE Expertise Center for Neurodevelopmental Disorders, , Erasmus MC, ; Wytemaweg 80, 3000 CA Rotterdam, the Netherlands
                Author information
                http://orcid.org/0000-0002-7323-2174
                Article
                293
                10.1186/s13229-019-0293-1
                6884852
                5ddbae61-d89c-45d5-8217-a8426f683e6f
                © The Author(s). 2019

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 3 May 2019
                : 17 October 2019
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100003246, Nederlandse Organisatie voor Wetenschappelijk Onderzoek;
                Award ID: Netherlands Organ on a Chip Initiative (NOCI)
                Award Recipient :
                Funded by: MSCA IF 2017
                Award ID: 795948
                Award Recipient :
                Funded by: Fondazione Umberto Veronesi
                Award ID: RE-guide
                Award ID: pUbe3azzle
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2019

                Neurosciences
                ubiquitin ligase e3a (ube3a),contact guidance,angelman syndrome,axonal guidance,15q duplication autism,nocodazole,microgratings,cytoskeleton

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