Axonal outgrowth, neuropeptides expression and receptors tyrosine kinase phosphorylation in 3D organotypic cultures of adult dorsal root ganglia

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Abstract

Limited knowledge from mechanistic studies on adult sensory neuronal activity was generated, to some extent, in recapitulated adult in vivo 3D microenvironment. To fill this gap there is a real need to better characterize the adult dorsal root ganglia (aDRG) organotypic cultures to make these in vitro systems exploitable for different approaches, ranging from basic neurobiology to regenerative therapies, to address the sensory nervous system in adult stage. We conducted a direct head-to-head comparison of aDRG and embryonic DRG (eDRG) organotypic culture focusing on axonal growth, neuropeptides expression and receptors tyrosine kinase (RTK) activation associated with neuronal survival, proliferation and differentiation. To identify alterations related to culture conditions, these parameters were also addressed in retrieved aDRG and eDRG and compared with organotypic cultures. Under similar neurotrophic stimulation, aDRG organotypic cultures displayed lower axonal outgrowth rate supported by reduced expression of growth associated protein-43 and high levels of RhoA and glycogen synthase kinase 3 beta mRNA transcripts. In addition, differential alteration in sensory neuropeptides expression, namely calcitonin gene-related peptide and substance P, was detected and was mainly pronounced at gene expression levels. Among 39 different RTK, five receptors from three RTK families were emphasized: tropomyosin receptor kinase A (TrkA), epidermal growth factor receptors (EGFR, ErbB2 and ErbB3) and platelet-derived growth factor receptor (PDGFR). Of note, except for EGFR, the phosphorylation of these receptors was dependent on DRG developmental stage and/or culture condition. In addition, EGFR and PDGFR displayed alterations in their cellular expression pattern in cultured DRG. Overall we provided valuable information particularly important when addressing in vitro the molecular mechanisms associated with development, maturation and regeneration of the sensory nervous system.

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Most cited references 39

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Transcriptome analysis of embryonic and adult sensory axons reveals changes in mRNA repertoire localization.

Laura Gumy, Giles S H Yeo, Yi-Chun Loraine Tung … (2011)

mRNAs are transported, localized, and translated in axons of sensory neurons. However, little is known about the full repertoire of transcripts present in embryonic and adult sensory axons and how this pool of mRNAs dynamically changes during development. Here, we used a compartmentalized chamber to isolate mRNA from pure embryonic and adult sensory axons devoid of non-neuronal or cell body contamination. Genome-wide microarray analysis reveals that a previously unappreciated number of transcripts are localized in sensory axons and that this repertoire changes during development toward adulthood. Embryonic axons are enriched in transcripts encoding cytoskeletal-related proteins with a role in axonal outgrowth. Surprisingly, adult axons are enriched in mRNAs encoding immune molecules with a role in nociception. Additionally, we show Tubulin-beta3 (Tubb3) mRNA is present only in embryonic axons, with Tubb3 locally synthesized in axons of embryonic, but not adult neurons where it is transported, thus validating our experimental approach. In summary, we provide the first complete catalog of embryonic and adult sensory axonal mRNAs. In addition we show that this pool of axonal mRNAs dynamically changes during development. These data provide an important resource for studies on the role of local protein synthesis in axon regeneration and nociception during neuronal development.

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NGF-induced axon growth is mediated by localized inactivation of GSK-3beta and functions of the microtubule plus end binding protein APC.

Feng-Quan Zhou, Jiang Zhou, Shoukat Dedhar … (2004)

Little is known about how nerve growth factor (NGF) signaling controls the regulated assembly of microtubules that underlies axon growth. Here we demonstrate that a tightly regulated and localized activation of phosphatidylinositol 3-kinase (PI3K) at the growth cone is essential for rapid axon growth induced by NGF. This spatially activated PI3K signaling is conveyed downstream through a localized inactivation of glycogen synthase kinase 3beta (GSK-3beta). These two spatially coupled kinases control axon growth via regulation of a microtubule plus end binding protein, adenomatous polyposis coli (APC). Our results demonstrate that NGF signals are transduced to the axon cytoskeleton via activation of a conserved cell polarity signaling pathway. Copyright 2004 Cell Press

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Axonal protein synthesis and degradation are necessary for efficient growth cone regeneration.

E. Holt, Poonam Verma, Sabrina Chierzi … (2005)

Axonal regeneration can occur within hours of injury, the first step being the formation of a new growth cone. For sensory and retinal axons, regenerative ability in vivo correlates with the potential to form a new growth cone after axotomy in vitro. We show that this ability to regenerate a new growth cone depends on local protein synthesis and degradation within the axon. Axotomy in vitro leads to a fourfold to sixfold increase in 3H-leucine incorporation in both neurones and axons, starting within 10 min and peaking 1 h after axotomy. Application of protein synthesis inhibitors (cycloheximide and anisomycin) to cut axons, including axons whose cell bodies were removed, or proteasome inhibitors (lactacystin and N-acetyl-Nor-Leu-Leu-Al) all result in a reduction in the proportion of transected axons able to reform growth cones. Similar inhibition of growth cone formation was observed on addition of target of rapamycin (TOR), p38 MAPK (mitogen-activated protein kinase), and caspase-3 inhibitors. Comparing retinal and sensory axons of different developmental stages, levels of ribosomal protein P0 and phosphorylated translation initiation factor are high in sensory axons, lower in embryonic axons, and absent in adult retinal axons. Conditioning lesions, which increase the regenerative ability of sensory axons, lead to increases in intra-axonal protein synthetic and degradative machinery both in vitro and in vivo. Collectively, these findings suggest that local protein synthesis and degradation, controlled by various TOR-, p38 MAPK-, and caspase-dependent pathways, underlie growth cone initiation after axotomy.

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Author and article information

Contributors

Estrela Neto:

ORCID: http://orcid.org/0000-0003-2334-2292

Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: MethodologyRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing

Cecília J. Alves: Role: ConceptualizationRole: Formal analysisRole: InvestigationRole: ResourcesRole: ValidationRole: VisualizationRole: Writing – review & editing

Luís Leitão: Role: ConceptualizationRole: InvestigationRole: MethodologyRole: ResourcesRole: ValidationRole: VisualizationRole: Writing – review & editing

Daniela M. Sousa: Role: ConceptualizationRole: Funding acquisitionRole: MethodologyRole: SupervisionRole: ValidationRole: Writing – review & editing

Inês S. Alencastre: Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: MethodologyRole: ValidationRole: Writing – review & editing

Francisco Conceição: Role: ConceptualizationRole: InvestigationRole: MethodologyRole: ResourcesRole: ValidationRole: Writing – review & editing

Meriem Lamghari: Role: ConceptualizationRole: Funding acquisitionRole: Project administrationRole: ResourcesRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – review & editing

Yvette Tache: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Electronic): 1932-6203

Publication date (Electronic): 24 July 2017

Publication date Collection: 2017

Volume: 12

Issue: 7

Electronic Location Identifier: e0181612

Affiliations

[1 ] i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal

[2 ] INEB—Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal

[3 ] FMUP—Faculdade de Medicina da Universidade do Porto, Porto, Portugal

[4 ] ICBAS—Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal

University of California Los Angeles, UNITED STATES

Author notes

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: lamghari@ 123456ineb.up.pt

Author information

Estrela Neto http://orcid.org/0000-0003-2334-2292

Article

Publisher ID: PONE-D-17-08268

DOI: 10.1371/journal.pone.0181612

PMC ID: 5524368

PubMed ID: 28742111

SO-VID: 3c6c3552-7e8b-40db-b9d6-e75e317f755e

License:

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 1 March 2017

Date accepted : 5 July 2017