Alzheimer’s disease (AD) is an age-related neurodegenerative disorder, characterized by global cognitive decline and progressive memory loss. As many other neurological disorders characterized by “proteinopathy”, pathology of AD includes beta-amyloid plaques and tau neurofibrillary tangles, which imply a crucial role of the cellular degradation systems in maintaining homeostasis of protein turnover. This is especially important for post-mitotic neuronal cells since aggravating protein crisis cannot be alleviated by cell division. Autophagy is a cellular degradation process that removes or recycles long-lived proteins and damaged organelles, with its enhancement being remarkably implicated during the progression of Alzheimer’s disease (AD). The majority of studies have hitherto focused on the mechanism of how oligomeric Ah, as one of the potent toxic species in AD, activates autophagy. However, how autophagy is activated remains to be elucidated. The goal of this study is to reveal the underlying mechanisms of autophagy and the subsequent events. Using imaging and biochemical analysis in primary cultures of rat hippocampal neurons, I found that oligomeric An-induced autophagy was initiated by aggregation of the endoplasmic reticulum (ER), in an mTOR-independent pathway. Ao-triggered autophagosomes were derived from omegasomes, starting from the ER aggregation sites. Aggregation of the ER facilitated the clustering of Atg14L to propel the recruitment of Beclin1 and Vps34, which contributes to generation of omegasomes. I further found that p62 targeted to ER aggregates possibly through the enhanced ubiquitinated ER chaperones trapped at ER aggregation sites, implicating the underlying mechanism for how p62 are recruited to autophagosome formation sites (omegasomes). Herein, I report key steps for activation of AH-triggered autophagy, whereby a mechanistic link between ER aggregation, autophagic activation and recruitment of p62 to autophagosome formation sites is revealed. First, Ao-induced ER aggregation triggers autophagy, via the recruitment of Beclin 1 and Vps34 to Atg14L clusters, which is a promoting factor for omegasome formation at the ER aggregation site. Second, the recruitment of p62 to omegasomes is likely mediated by the attraction of the underlying accumulation of ubiquitinated ER chaperones at the ER aggregation site. Up-regulation of autophagy is an early sign of AD. The activation of autophagy without tightly manipulation may contribute to neuronal damage in AD. In addition, how the autophagic substrates can be efficiently incorporated into the autophagic pathway is important for understanding the sustainability of autophagy. Therefore, my study on elucidating how ER aggregation initiates autophagy and the autophagic substrate/cargo receptor p62 are loaded onto autophagosome formation sites may help us to identify a potential therapeutic strategy or target for AD patients. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Inhibitory synaptic transmission within the vestibular circuits plays an essential regulatory role in coordinating vestibular functions. The maturation profile of γ- aminobutyric acid (GABA) synapses in the vestibular system remains unknown. To address this, we first used double immunohistochemistry to document the postnatal expression profile of GABAA receptors in canal-related and saccule-related vestibular nuclear neurons of rats. The proportion of Fos / GABAA receptors α1 subunit doublelabeled neurons progressively increased with age. Whole-cell patch-clamp experiments on brainstem slice preparations were also employed to characterize the developmental properties of these synapses within the medial vestibular nucleus. The frequency of GABAA receptor-mediated miniature inhibitory postsynaptic currents (IPSC) progressively increased during the first two postnatal weeks and reached a plateau thereafter. This is in agreement with an increase in sensitivity to GABAA receptor α1 subunit agonist zolpidem during the same period. The rise time and decay time however decreased by 2-fold. These results suggest that change in the composition of GABAA receptor occurs during the functional maturation of medial vestibular neurons. To further investigate whether GABA receptors contribute to synaptic plasticity in the developing vestibular nucleus, two stimulus protocols were used. Repetitive depolarizing pulses induced long-lasting decrease in the frequency of GABAA receptormediated spontaneous IPSCs between P3 and P7. The probability of inducing such frequency decline of sIPSCs decreased after the first postnatal week. High frequency stimulation on the other hand, induced long-term depression (LTD) of GABAA receptormediated evoked IPSCs between P3 and P5. The probability of inducing LTD decreased after P14. These results indicate that LTD at GABAergic synapses could be easily induced in developing medial vestibular neurons before maturation of GABAergic synaptic transmission. To examine if GABAergic transmission within the vestibular nucleus is crucial for establishment of gravity-related spatial organization, an intervention approach was adopted to perturb GABAergic transmission within the postnatal vestibular nucleus. A slice of Elvax loaded with either GABAA receptor agonist muscimol or antagonist bicuculline was inserted into the fourth ventricle and covered the bilateral vestibular nuclei at different ages. Expression of Fos protein in functionally activated neurons was used to demarcate the topographic spatial map in the inferior olive. The spatial map in subnuclei IOβ and DMCC was disturbed in each adult rat that was implanted with bicuculline- or muscimol-loaded Elvax at P1. However, no change was observed in adult rats that were pretreated with bicuculline or muscimol at P14 or P21. Vestibularrelated behavior tests were also performed. The acquisition of negative geotaxis, an otolith-related orientation reflex, was delayed in postnatal rats pretreated with bicuculline but was advanced in those rats pretreated with muscimol. Furthermore, the acquisition of motor learning, evaluated by rotarod test, was impaired in adult rats treated with bicuculline or muscimol. Taken together, our results indicated that maturation of GABAergic transmission within the vestibular nucleus play important roles in development of spatial recognition and vestibular-related behavior. / published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

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published_or_final_version / abstract / toc / Molecular Biology / Master / Master of Philosophy

published_or_final_version / abstract / toc / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Physics / Doctoral / Doctor of Philosophy