Abstract: Polarized cargo transport is essential for neuronal function. However, the minimal basic components required for selective cargo sorting and distribution in neurons remain elusive. We found that in sensory neurons the axon initial segment is largely absent and that microtubule-associated protein 2 (MAP2) defines the cargo-filtering zone in the proximal axon. Here, MAP2 directs axonal cargo entry by coordinating the activities of molecular motors. We show that distinct kinesins differentially regulate cargo velocity: kinesin-3 drives fast axonal cargo trafficking, while kinesin-1 slows down axonal cargo transport. MAP2 inhibits “slow” kinesin-1 motor activity and allows kinesin-3 to drive robust cargo transport from the soma into the axon. In the distal axon, the inhibitory action of MAP2 decreases, leading to regained kinesin-1 activity and vesicle distribution. We propose that selective axonal cargo trafficking requires the MAP2-defined pre-axonal filtering zone and the ability of cargos to switch between distinct kinesin motor activities.
Abstract: Microtubules and their associated proteins (MAPs) underpin the polarity of specialised cells. Adenomatous polyposis coli (APC) is one such MAP with a multifunctional agenda that requires precise intracellular localisations. Although APC has been found to associate with kinesin-2 subfamily members, the exact mechanism for the peripheral localization of APC remains unclear. Here we show that the heavy chain of kinesin-1 directly interacts with the APC C-terminus, contributing to the peripheral localisation of APC in fibroblasts. In rat hippocampal neurons the kinesin-1 binding domain of APC is required for its axon tip enrichment. Moreover, we demonstrate that APC requires interactions with both kinesin-2 and kinesin-1 for this localisation. Underlining the importance of the kinesin-1 association, neurons expressing APC lacking kinesin-1-binding domain have shorter axons. The identification of this novel kinesin-1-APC interaction highlights the complexity and significance of APC localisation in neurons.
Abstract: In recent years, it has been demonstrated that mRNAs localize to axons of young and mature central and peripheral nervous system neurons in culture and in vivo. Increasing evidence is supporting a fundamental role for the local translation of these mRNAs in neuronal function by regulating axon growth, maintenance and regeneration after injury. Although most mRNAs found in axons are abundant transcripts and not restricted to the axonal compartment, they are sequestered into transport ribonucleoprotein particles and their axonal localization is likely the result of specific targeting rather than passive diffusion. It has been reported that long-distance mRNA transport requires microtubule-dependent motors, but the molecular mechanisms underlying the sorting and trafficking of mRNAs into axons have remained elusive. This review places particular emphasis on motor-dependent transport of mRNAs and presents a mathematical model that describes how microtubule-dependent motors can achieve targeted trafficking in axons. A future challenge will be to systematically explore how the numerous axonal mRNAs and RNA-binding proteins regulate different aspects of specific axonal mRNA trafficking during development and after regeneration.
Abstract: Axon regeneration after injury requires the extensive reconstruction, reorganization, and stabilization of the microtubule cytoskeleton in the growth cones. Here, we identify KIF3C as a key regulator of axonal growth and regeneration by controlling microtubule dynamics and organization in the growth cone. KIF3C is developmentally regulated. Rat embryonic sensory axons and growth cones contain undetectable levels of KIF3C protein that is locally translated immediately after injury. In adult neurons, KIF3C is axonally transported from the cell body and is enriched at the growth cone where it preferentially binds to tyrosinated microtubules. Functionally, the interaction of KIF3C with EB3 is necessary for its localization at the microtubule plus-ends in the growth cone. Depletion of KIF3C in adult neurons leads to an increase in stable, overgrown and looped microtubules because of a strong decrease in the microtubule frequency of catastrophes, suggesting that KIF3C functions as a microtubule-destabilizing factor. Adult axons lacking KIF3C, by RNA interference or KIF3C gene knock-out, display an impaired axonal outgrowth in vitro and a delayed regeneration after injury both in vitro and in vivo. Murine KIF3C knock-out embryonic axons grow normally but do not regenerate after injury because they are unable to locally translate KIF3C. These data show that KIF3C is an injury-specific kinesin that contributes to axon growth and regeneration by regulating and organizing the microtubule cytoskeleton in the growth cone.
Abstract: 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.
Abstract: Poorly-controlled hyperglycaemia reduces peripheral nerve regeneration in diabetes through ill-understood mechanisms. Apoptosis is one proposed primary response. We examined how hyperglycaemia affects regeneration of axons and Schwann cells (SC) from cultured adult mouse Dorsal Root Ganglia (DRG) to separate cell-autonomous responses from systemic influences. Hyperglycaemia reduced neurite growth rate by 20-30% without altering growth cone density, indicating neuronal apoptosis was negligible. Moderate hyperglycaemia also profoundly retarded SC migration from DRG explants. This effect was independent of neuritogenesis and was reversible, indicating that SC had not died. In purified SC, even mild hyperglycaemia inhibited neuregulin-beta1-induced bromodeoxyuridine-incorporation and phosphorylation of retinoblastoma protein, indicating a block at the G1-S boundary. Moreover, migration of purified SC was inhibited by >90%. Thus, SC proliferation and migration, and axon regeneration from DRG neurons, are impaired by hyperglycaemia cell autonomously, while apoptosis is negligible. Impairment of these functions over time may exacerbate nerve injury-related diabetic neuropathy.
PhD in Natural Sciences, Department of Biochemistry, University of Cambridge, UK.
Thesis Title: Investigations into Schwann cell dysfunction under hyperglycaemic conditions.
Supervisor: Dr. Aviva Tolkovsky.
MSc in Molecular Genetics (summa cum laude), University of Salamanca, Spain.
Thesis Title: Characterisation of Patched gene in Gorlin Syndrome and in basal cell carcinomas.
Supervisor: Prof. Rogelio Gonzalez-Sarmiento.
BSc with Honours in Biochemistry (First Class), University of Salamanca, Spain.
Research Fellow Academic Lead Microscopy at Department of Anatomy, School of Biomedical Sciences, University of Otage, New Zealand.
Division head: Prof. Neil Gemmell.
Research: Ultrastructural characterisation of the peripheral and central axons of adult sensory neurons.
Postdoctoral researcher in the Cell Biology Division at the University of Utrecht, the Netherlands.
Division heads: Prof. Casper Hoogenraad and Prof. Anna Akhmanova.
Research: Role of microtubule dynamics and protein trafficking in axon regeneration.
Techniques: Live-neuronal imaging using spinning disc confocal microscopy and TIRF microscopy, live laser-photoablation, FRAP, image analysis, cell biology, biochemistry, molecular cloning.
Funding: Marie Curie Intra-European fellowship and a research grant from the Netherlands Organisation for Scientific Research (NWO).
Postdoctoral researcher in the Centre for Brain Repair at the University of Cambridge, UK.
Laboratory head: Prof. James Fawcett.
Research: Intrinsic factors in axonal growth and regeneration.
Techniques: Microarray, Quantitative RT-PCR, primary neuronal cultures, immunocytochemistry, confocal microscopy, biochemistry.
Funding: Medical Research Council (MRC) Career Development Fellowship.
Immunocytochemistry, In situ Hybridisation and Live Imaging course, Cold Spring Harbor Laboratories, NY, USA.
Instructors: Prof. Viki Allan, Dr. Ke Hu and Dr. Sui Huang.
Competitive course to learn advanced live cell-imaging techniques.
Funding: CSHL travel award.
Research assistant in the Department of Biochemistry, University of Cambridge, UK.
Supervisor: Dr. Aviva Tolkovsky.
Research: Effect of hyperglycaemia on Schwann cell proliferation, migration and myelination.
Funding: Diabetes Wellness Foundation research grant.
Advanced Techniques in Molecular Neuroscience Course, Cold Spring Harbor Laboratories, NY, USA.
Instructors: Dr. Cary Lai, Dr. Dan Lavery and Dr. Jim Boulter.
Competitive course to learn advanced molecular biology techniques applied to neurobiology.
Funding: Boehringer Ingelheim Fonds Travel Allowance.
PhD student in the Department of Biochemistry, University of Cambridge, UK.
Supervisor: Dr. Aviva Tolkovsky.
Techniques: primary Schwann cell and Dorsal Root Ganglion neuron cultures, cell line culture, cell transfection, proliferation, migration and myelination assays, immunocytochemistry, immunohistochemistry, biochemistry.
MSc student at the University of Salamanca, Spain.
Supervisor: Prof. Rogelio Gonzalez-Sarmiento.
Techniques: PCR, Single Strand Conformational Polymorphism Analysis (SSCP), molecular cloning.
Funding: “Fundacion Mapfre-Medicina” scholarship.
Internship student at St. Bart’s Hospital, University of London, UK.
Supervisor: Prof. Paolo Pozzilli.
Research: Examination of gut tissue from pre-diabetic non-obese diabetic (NOD) mice.
Funding: Leonardo Da Vinci European fellowship.
Currently I'm Research Fellow at the Department of Anatomy of the University of Otago, New Zealand.
My main research interests are the roles of cytoskeletal dynamics and cargo trafficking during axon regeneration. To study this fascinating topic I use a set of different techniques: live-cell imaging, immunostaining, biochemistry and theoretical modelling.