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No description available.

Axons possess a continuous network of smooth tubular endoplasmic reticulum (ER), extending from the nuclear envelope throughout the neuron to synapses. Mutations affecting proteins with intramembrane hairpin domains that model tubular ER membrane can lead to the axon degenerative disease, hereditary spastic paraplegia (HSP). However, the extent and mechanisms by which HSP proteins contribute to axonal ER organization and dynamics are unclear. To understand these mechanisms, there is a need to visualize axonal ER in wild-type and mutant live axons. I have therefore aimed to develop these tools in Drosophila larvae and adults, and use them to visualize mutant phenotypes. Firstly, I developed a system to visualize fluorescently marked ER in individual axons in adult fly legs, and tested how this can be used to investigate the effects of loss of intramembrane hairpin HSP proteins on ER in adult legs. Secondly, known mutations affecting HSP hairpin proteins reduce the axonal ER network but not severely; I hypothesized that additional HSP ER membrane proteins might contribute to residual tubule formation; these include Arl6IP, also reported to promote ER tubule formation. I generated transgenic flies to overexpress a fluorescently tagged eGFP::Arl6IP1, and found that this fusion protein localizes within axonal ER. To study whether loss of Arl6IP1 function affects axonal ER, I tested the effects of knockdown on this compartment, but found no consistent effects. To achieve stronger loss of function, I also generated a mutant stock that lacked one of the transmembrane domains and showed a slight developmental delay in homozygous Drosophila larvae. Like mutations in a number of other HSP hairpin proteins, this lesion is homozygous viable, and further characterization of its phenotype will help elucidate how Arl6IP1 contributes to modeling the axonal ER network. In conclusion, my work shows the utility of GFP markers of axonal ER, it can facilitate faster screening for other genes that potentially regulate ER structure and for ageing phenotypes that are not apparent in larval stages, and suggests Arl6IP1 as another HSP protein with a role in axonal ER organization.

Neurotrophins are important signaling molecules in neuronal survival and differentiation. The precursor forms of neurotrophins (proneurotrophins) are the dominant form of gene products in animals, which are cleaved to generate prodomain and mature neurotrophins, and are sorted to constitutive or regulated secretory pathway and released. Brain-derived neurotrophic factor (BDNF) plays a pivotal role in the brain development and in the pathogenesis of neurological diseases. In Huntington’s disease, the defective transport of BDNF in cortical and striatal neurons and the highly expressed polyQ mutant huntingtin (Htt) result in the degeneration of striatal neurons. The underlying mechanism of BDNF transport and release is remains to be investigated. Current studies were conducted to identify the mechanisms of how BDNF is transported in axons post Golgi trafficking. By using affinity purification and 2D-DIGE assay, we show Huntingtin-associated protein 1 (HAP1) interacts with the prodomain and mature BDNF. The GST pull-down assays have addressed that HAP1 directly binds to the prodomain but not to mature BDNF and this binding is decreased by PolyQ Htt. HAP1 immunoprecipitation shows that less proBDNF is associated with HAP1 in the brain homogenate of Huntington’s disease compared to the control. Co-transfections of HAP1 and BDNF plasmids in PC12 cells show HAP1 is colocalized with proBDNF and the prodomain, but not mature BDNF. ProBDNF was accumulated in the proximal and distal segments of crushed sciatic nerve in wild type mice but not in HAP1-/- mice. The activity-dependent release of the prodomain of BDNF is abolished in HAP1-/- mice. We conclude that HAP1 is the cargo-carrying molecule for proBDNF-containing vesicles and plays an essential role in the transport and release of BDNF in neuronal cells. 20-30% of people have a valine to methionine mutation at codon 66 (Val66Met) in the prodomain BDNF, which results in the retardation of transport and release of BDNF, but the mechanism is not known. Here, GST-pull down assays demonstrate that HAP1 binds Val66Met prodomain with less efficiency than the wild type and PolyQ Htt further reduced the binding, but the PC12 cells colocalization rate is almost the same between wt prodomain/HAP1 and Val66Met prodomain/HAP1, suggesting that the mutation in the prodomain may reduce the release by impairing the cargo-carrying efficiency of HAP1, but the mutation does not disrupt the sorting process. Recent studies have shown that proneurotrophins bind p75NTR and sortilin with high affinity, and trigger apoptosis of neurons in vitro. Here, we show that proBDNF plays a role in the death of axotomized sensory neurons. ProBDNF, p75NTR and sortilin are highly expressed in DRG neurons. The recombinant proBDNF induces the dose-dependent death of PC12 cells and the death activity is completely abolished in the presence of antibodies against the prodomain of BDNF. The exogenous proBDNF enhances the death of axotomized sensory neurons and the antibodies to the prodomain or exogenous sortilin-extracellular domain-Fc fusion molecule reduces the death of axotomized sensory neurons. We conclude that proBDNF induces the death of sensory neurons in neonatal rats and the suppression of endogenous proBDNF rescued the death of axotomized sensory neurons.

The ability to repair damaged mammalian axons to re-establish functional connections continues to be a goal for neuroscientists. Following axonal severance, proximal segments of mammalian axons seal themselves rapidly at the lesion site. Distal segments of severed mammalian axons undergo Wallerian degeneration within 24-72 hours. Prior to the onset of degeneration, distal axonal segments remain electrically excitable. The work described in this dissertation demonstrates that polyethylene glycol (PEG), a hydrophilic polymer, can rapidly repair severed axons by fusing the plasmalemmas of two closely apposed distal and proximal axonal segments. This plasmalemmal fusion restores morphological integrity of severed axons and their ability to conduct action potentials across the injury site. The ability to fuse proximal and distal severed axonal segments using PEG is improved when the axonal segments are exposed to antioxidants, such as melatonin and methylene blue, and also when microsutures provide additional support in transected sciatic nerves. The restoration of axonal continuity by PEG-fusion restores function, improving behavioral recovery in rats with crush-injured sciatic nerves, as well as those in which the sciatic is complete transected. / text

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Isolated and identified crab axons have been used to study the forms of the repetitive responses to direct current. Using techniques which enable the responses of isolated axons to be studied at the site of imposed electrical currents, the responses can be classified into five major groups with two subdivisions:- Group 1. Axons showing no marked supernormality during the recovery cycle, that repeat over a wide range of frequencies when stimulated by direct current, with frequency increasing smoothly with the strength of applied current, Group ia. To direct current these axons yield a train of impulses, the intervals between which progressively lengthen. Group ib. To direct current these axons yield a train of impulses the Intervals between which, for some time at least, progressively shorten. Group ll. Axons showing a pronounced supemomality during the recovery cycle, that repeat over only a limited frequency range. Group lla. Axons capable of long latencies, with oscillatory subthreshold potentials before and after the repetitive response. Group llb. Axons showing only short latencies, and lacking subthreshold oscillations before the repetitive response, but nevertheless with oscillations following the response. Group lll. Axons with a prolonged long-lived supemormality during the recovery cycle, which can be correlated with a prolonged action potential. They can repeat over a wide range of frequencies stimulated by direct current, but lack true local potentials for all action potentials except the first. Group lV. Axons with a relatively prolonged subnormality during the recovery cycle. They show short trains of action potentials, the amplitude of which progressively decreases even to near threshold currents, and the interspike intervals show a smooth increase. Group V. Axons unable to repeat to direct current, having a low safety factor and high threshold. They are capable of only short latencies. The single action potential shows a considerable variation in amplitude. A wide varied of experiments have been carried out, which have shown that several factors influence the form of the repetitive response in crab axons, and that the inadequacy of previous theories stems from their oversimplification. The factors show to operate in determining the form of these responses are:- 1. Changes in the resistance of the axon membrane, so that a constant current pulse will not cause the sane potential displacement while it acts. These changes can occur as the result of ionic accumulation outside the axon, or from the active process of delayed rectification. 2. The duration and from of the recovery cycle limits the upper frequency of the repetitive response, as well as influencing it at other times. 3. Sustained depolarisation depresses excitability, by lengthening the repolarisation time of an action potential and the period of recovery following it, as can be seen when the threshold potential for the spike rises throughout a repetitive response. 4. Changes in the membrane potential that result from the accumulation of ions in the near vicinity of the axon membrane. These changes, although they show some interdependence, are often difficult to completely eliminate any particular one by experiment. Although these factors have not been measured quantitatively, on account of technical difficulties inherent in the use of crab axons, they are sufficient to provide a coherent interpretation of repetition.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20244号 / 医博第4203号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙橋 良輔, 教授 伊佐 正, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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