Neurons are highly polarized cells, the most notable feature of which are their dendrites. Within the dendrites reside many organelles, each performing their functions in a localized area away from the so-ma. Among the organelles in the dendrites, Golgi outposts (GOPs) are unique to dendrites—GOPs are rarely found in axons. Because dendrites are much thinner than the soma, many organelles such as GOPs are miniaturized and thus may have differing functional capacity to their perinuclear counter-parts. These differences, among many others, may contribute to dendritic vulnerability over other neu-ronal domains to protein toxicity. However, whether GOPs are linked to neuronal dendrite pathology in diseases has not yet been explored. Protein toxicity has been shown to be associated with dendrite de-fects, but whence protein toxicity induces those defects is difficult to understand. Protein toxicity is observed in most neurodegenerative diseases. However, most cases of neurodegenerative diseases are sporadic; the etiology is unknown and the causes are usually multiple. Therefore, a monogenic disease, such as Machado-Joseph Disease (MJD), can be a great model to understand the mechanism underlying dendrite defects via protein toxicity. Notably, MJD is known to be caused primarily by nuclear protein toxicity, making clear the pathogenic cause of the disease. During the course of my graduate work, I ex-amined the MJD toxic proteins using a Drosophila Class IV dendritic arborization (C4 da) neuronal model to understand how nuclear protein toxicity induces dendrite pathologies and how GOPs may con-tribute. I show in this Theses that polyQ protein expression in the nucleus causes terminal dendrite elon-gation and branching defects, most likely through perturbation of GOPs. PolyQ expression also led to decreased plasma membrane (PM) supply in C4 da neurons. By sequencing mRNA of fly brains express-ing polyQ proteins, I show that polyQ proteins down-regulated many genes involved in secretory path-way, including COPII genes the knockdown of which reduces the number of GOPs in C4 da neurons. Transcription factor (TF) analysis identifies CrebA as the TF disrupted by polyQ proteins and whose overexpression restores GOP loss in polyQ-expressing neurons. I demonstrate through chromatin im-munoprecipitation (ChIP)-PCR that CBP binds to CrebA promoter to regulate its transcription. Notably, I also show that CBP is an important regulator of GOP number in C4 da neurons. Next, I also show that CBP interacts with Cut to regulate the transcription of CrebA. To find out the link between polyQ pro-teins and CBP, I co-stained CBP and polyQ and found that polyQ proteins sequestered CBP. This se-questration mechanism seems to interfere with CBP function, the loss of which results in decreased CrebA expression, subsequently leading to down-regulation of COPII-related molecules and ultimately reducing the number of GOPs. I also show that Rac1-mediated terminal dendrite branching pathway may be intertwined with the CBP/Cut pathway. Taken together, I demonstrate that polyQ proteins induce dendrite pathology through the CBP/Cut-CrebA-COPII pathway. These results from my graduate study may help advance therapeutic research for diseases in which nuclear protein toxicity is the main cause. These results of mine may also be helpful in other disease fields in which dendrite pathology is present.
Table Of Contents
Abstract i List of contents ii List of tables iv List of figures v Ⅰ. Introduction 1.1 Neuronal dendrites require localized organelles for their structural growth and proper function 1 1.2 Dendrite defect is a common pathological feature of protein toxicity in various neurodegenerative diseases 3 1.3 Fly models for polyglutamine diseases are suitable for investigating dendritic pathology 4 II. Materials and Methods 7 III. Results 3.1 PolyQ toxicity in the nucleus disrupts branching and elongation of terminal dendrites in C4 da neurons 30 3.2 PolyQ toxicity in neurons induces a loss of GOP number in C4 da neurons 40 3.3 PolyQ toxicity in the nucleus leads to down-regulated cellular processes associated with GOP synTheses and PM protein supply 54 3.4 CrebA overexpression restores the down-regulation of COPII pathway and the loss of GOPs caused by polyQ toxicity in nucleus 66 3.5 Co-overexpression of CrebA and Rac1 synergistically restores polyQ-induced dendrite pathology 85 3.6 CBP directly binds to the CrebA promoter to regulate its transcription in cooperation with Cut 97 3.7 PolyQ proteins in the nucleus interfere with CBP at the upstream of CrebA in regulation of GOP synTheses 110 IV. Discussion 4.1 PolyQ proteins in the nucleus cause terminal dendrite defect through the perturbation of CBP-CrebA-COPII pathway 115 4.2 PolyQ proteins may disrupt the stoichiometric ratio of CBP to Cut for the regulation of CrebA transcription 116 4.3 CBP-CrebA pathway, among many other transcriptional pathways, contributes significantly to the loss of GOPs and terminal dendrite defects by polyQ proteins 119 4.4 PolyQ proteins in the nucleus disrupt the neuropeptide maturation process, but perinuclear Golgi are relatively spared 122 4.5 PolyQ proteins in the nucleus significantly down-regulates a substantial number of genes without causing cell death in Drosophila brain 125 4.6 MJDtr-27Q can also cause neurotoxicity 129 4.7 Conclusion 131 V. References 134 VI. Summary (국문) 140