Author: Linyan Wu
Wu, Linyan, 2007 BRAIN DERIVED NEUROTROPHIC FACTOR TRANSPORT AND PHYSIOLOGICAL SIGNIFICANCE, Flinders University, School of Medicine
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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.
Keywords: BDNF,HAP1,axonal transport
Subject: Psychology thesis
Thesis type: Doctor of Philosophy
School: School of Medicine
Supervisor: Xin-Fu Zhou