Show simple item record

dc.contributor.authorNieuwenhuis, Bart
dc.date.accessioned2019-06-24T16:10:30Z
dc.date.available2019-06-24T16:10:30Z
dc.date.issued2019-10-26
dc.date.submitted2019-03-06
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/293896
dc.description.abstractInjury to the brain and spinal cord has devastating consequences because adult neurons in the central nervous system (CNS) do not regenerate. Traumatic CNS injuries are a significant clinical problem and have life-changing consequences for patients. There are 130.000 new cases of spinal cord injury (SCI) per year worldwide and currently affects more than 2.5 million people (International Spinal Research Trust). Spinal cord injury often results in loss of essential bodily functions including sexual function, bladder control, and motor and sensory function. There are many factors that contribute to the failure of axon regeneration in the CNS. Generally speaking, the problem of axon regeneration can be subdivided in at least two aspects: 1) the growth-repulsive environment surrounding the injured axon and 2) the low intrinsic regeneration potential of adult CNS neurons. In this dissertation, the focus is on the biology inside neurons and genetic approaches to enhance the axonal regeneration capacity of cortical neurons. Gene therapy is a promising strategy to promote axon regeneration. Gene therapy could be described as the delivery of a foreign gene into cells to treat a genetic disease or acquired condition. In this dissertation, adeno-associated viral (AAV) vectors were used to deliver transgenes into cortical neurons and the corticospinal tract, because regeneration of this neuronal pathway is a key event to restore motor function. One of the advantages of gene therapy is the local and long-term expression of transgenes following direct injection. To achieve a successful gene therapy for axonal regeneration, it is important to have good transduction efficiency in neurons and to deliver a transgene (or a combination of transgenes) that promotes axon regeneration over long distances and past the site of injury. The overall objective of this dissertation was to optimize AAV-mediated transduction of the corticospinal tract, and to enhance axon regeneration in cortical neurons by using vectors containing CRISPR-Cas9 and phosphoinositide 3-kinases. Chapter I is a literature review about cell surface receptors called integrins and their role in axonal regeneration. Integrins contribute to the spontaneous axon regeneration in the peripheral nervous system by interacting with ligands in the extracellular matrix. Integrin biology is different in the CNS. These receptors are transported into the axon of developing neurons, but selective axonal transport limits the regenerative response in adult CNS neurons. Targeting integrins and related molecules that control their transport and activation state is therefore a promising tool to promote robust regeneration in the CNS. The platforms and experimental therapies used in integrin research are also relevant for other mechanisms limiting axon regeneration. Chapter II is based on the hypothesis that the axon initial segment is a barrier for the axonal transport of integrins and other growth-promoting receptors. The aim was to dismantle the axon initial segment by knockout of the cytoskeletal scaffolding protein Ankyrin-G. Two genetic tools were made and tested to knockout Ankyrin-G: (1) a short hairpin RNA, and (2) a dual promoter AAV viral vector that drives the expression of a CRISPR-associated endonuclease 9 from Staphylococcus aures and one guide RNA targeting Ankyrin-G. The preliminary data shows that the vector-mediated RNA interference results in cellular toxicity in cultured cortical neurons, and that knockout of the axon initial segment was achieved in a proportion of neurons following CRISPR interference. Chapter III is based on the hypothesis that low axonal PtdIns-3,4,5-P3 (PIP3) signalling, due to the absence of growth-promoting receptors in the axon, contributes to regenerative failure in the CNS. This chapter investigates whether overexpression of phosphoinositide 3-kinases (PI3Ks), which generates PIP3, can promote axon regeneration in cultured cortical neurons. The first immunocytochemistry experiment confirmed our hypothesis that there is a decline of PIP3 levels in the axon in line with maturation. Expression of activated PI3Ks in developing neurons resulted in increased axonal growth, and expression in maturing neurons enlarged the soma size and resulted in more complex dendritic morphology in vitro. Consistently, the expression of constitutively activated PI3K, but not wild type PI3K, enhanced the PIP3 signaling pathway in these neurons. Importantly, overexpression of PI3Ks increased the success rate of axon regeneration in cortical neurons that underwent in vitro laser axotomy. Chapter IV aimed to identify the best method to deliver transgenes into the corticospinal tract by using AAVs. The choice of serotype and promoter has a crucial impact on gene therapy as it can ultimately depict whether it will be successful or unsuccessful. This study consisted of a direct comparison between the AAV1 and AAV5 viral vector serotypes and four promoters (CMV, mPGK, sCAG, synapsin) in their efficiency to express eGFP following injection at the sensory-motor cortex in mice and rats. The data suggests that AAV1 is the superior serotype to target layer V cortical neurons. Furthermore, the mPGK and synapsin promoters are superior over the other promoters to transduce the corticospinal tract. Chapter V is a summary and general discussion of the obtained results in dissertation. It highlights future perspectives of the current work and how the research contributes to advance the field of gene therapy and axon regeneration.
dc.description.sponsorshipThis work was funded by a Nathalie Rose Barr award (NRB110) from the International Spinal Research Trust, a grant from the Medical Research Council (G1000864), an ERA-NET NEURON grant AxonRepair (013-16-002), and support from the laboratory for regeneration of sensorimotor systems at the Netherlands Institute for Neuroscience.
dc.language.isoen
dc.rightsAttribution 4.0 International (CC BY 4.0)
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.subjectAxon regeneration
dc.subjectIntegrins
dc.subjectCRISPR-Cas9
dc.subjectAxon initial segment
dc.subjectPhosphoinositide 3-kinases
dc.subjectAdeno-associated viral vectors
dc.subjectCorticospinal tract
dc.titleGene therapy for axon regeneration
dc.typeThesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnameDoctor of Philosophy (PhD)
dc.publisher.institutionUniversity of Cambridge
dc.publisher.departmentClinical Neurosciences
dc.date.updated2019-06-24T13:47:18Z
dc.identifier.doi10.17863/CAM.41004
dc.contributor.orcidNieuwenhuis, Bart [0000-0002-2065-2271]
dc.publisher.collegeKing's
dc.type.qualificationtitlePhD in Clinical Neurosciences
cam.supervisorFawcett, James
cam.supervisorVerhaagen, Joost
cam.supervisor.orcidFawcett, James [0000-0002-7990-4568]
cam.supervisor.orcidVerhaagen, Joost [0000-0002-8341-1096]
cam.thesis.fundingfalse
rioxxterms.freetoread.startdate2020-06-24


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record

Attribution 4.0 International (CC BY 4.0)
Except where otherwise noted, this item's licence is described as Attribution 4.0 International (CC BY 4.0)