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.

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.

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Within the past thirty years, the discovery and characterization of the microtubule-associated motor proteins, kinesins and cytoplasmic dynein, has radically expanded our understanding of intracellular trafficking and motile phenomena. Nevertheless, the mechanisms by which eukaryotic cells integrate motor functionality and cargo interactions over multiple subcellular domains in a spatiotemporally controlled way remain largely mysterious. During transport within the neuronal axon, dynein and the kinesins run in opposite directions along uniformly polarized microtubule tracks, so that each motor must switch between active transport and being, itself, a cargo in order to be properly positioned and carry out its function. The axon thus represents a model system in which to study the regulatory mechanisms governing intracellular transport, especially under conditions when it must be modulated in response to changing environmental cues, such as during axon outgrowth and development. Recently, the localization of certain messenger RNAs and their local translation to yield protein has emerged as a critical process for the development of axons and other neuronal compartments. I observed that transcripts encoding the dynein cofactors Lis1 and dynactin are among those localized to axons, so I hypothesized that stimulus-dependent changes in axonal transport may occur via local synthesis of dynein cofactors. In these studies, I have shown that different conditions of nerve growth factor signaling on developing axons trigger acute changes in the transport of various axonal cargoes, contemporaneous with rapid translational activation and production of Lis1 and dynactin’s main subunit, p150Glued, within the axons themselves. Differential synthesis of these cofactors in axons was confirmed to be required for the observed stimulus-dependent transport changes, which were completely prevented by axon-specific pharmacologic inhibition of protein synthesis or RNA interference targeted against Lis1 and p150Glued. In fact, Lis1 was, in an apparent paradox, locally synthesized in response to both nerve growth factor stimulation and withdrawal. I demonstrated that this is due to the fact that Lis1 is produced from a heterogeneous population of localized transcripts, differentiated chiefly by whether they interact with the RNA-binding protein APC. Preventing the binding of APC to Lis1 transcripts thus inhibited axonal synthesis of Lis1 and its resultant transport effects under conditions of nerve growth factor stimulation, while having no bearing on the similar phenomena seen during nerve growth factor withdrawal. This demonstrates that association with RNA-binding proteins can functionally distinguish sub-populations of localized messenger RNAs, which, in turn, provides a foundation for mechanistically understanding how localized protein synthesis is coupled to specific stimuli. Axonally synthesized Lis1 also was shown to have a particular role in mediating transport of a retrograde death signal originating in nerve growth factor-deprived axons, as neurons exhibited greatly reduced cell death when axonal synthesis of Lis1 was blocked. Through the application of pharmacologic agents inhibiting different steps in the propagation of this pro-apoptotic signal, I established that the signal depends upon effective endocytosis and the activity of glycogen synthase kinase 3β. It is therefore likely that the retrogradely transported signaling cargo in question is a glycogen synthase kinase 3β-containing endosome or multivesicular body—a type of large cargo consistent with Lis1’s known role in adapting the dynein motor for high-load transport. Preliminary results further indicate that axons exposed to another type of degenerative stress, in the form of toxic amyloid-β oligomers, may also employ local synthesis of Lis1 as a means of regulating transport and survival signaling. These findings establish a previously undescribed mechanism of regulating dynein activity and cargo interactions through local synthesis of its cofactors, allowing for rapid responses to environmental cues and stimuli that are especially relevant during the development of the nervous system. In addition to illustrating a regulatory principle that may be generally applicable to subcellular compartments throughout polarized cells, these studies provide new insights into intracellular transport disruptions that occur in lissencephaly, neurodegeneration, and other human disease states.

Includes bibliographical references (p. 100-114).

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Thesis (M.Sc.)--York University, 2008. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 83-89). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR38831