京都大学 / 新制・課程博士 / 博士(医学) / 甲第24500号 / 医博第4942号 / 新制||医||1064(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 生田 宏一, 教授 萩原 正敏, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

La fragmentation de l'appareil de Golgi représente un des changements les plus précoces et les plus répandus dans les maladies neurodégénératives. Afin de comprendre les mécanismes moléculaires de ces changements, j'ai étudié deux modèles expérimentaux de maladie du neurone moteur. 1. Les souris pmn (progressive motor neuronopathy) : Celles-ci sont atteintes d'une forme très grave de dégénérescence des neurones moteurs et des défauts moléculaires sont liés à une mutation faux-sens d'une protéine localisée au niveau du Golgi, la chaperonne des tubulines TBCE, identifiée par (Martin, Jaubert et al. 2002; Schaefer, Schmalbruch et al. 2007). Au cours de ma thèse, nous avons identifié des anomalies importantes du Golgi dans les neurones moteurs lombaires de souris pmn et déterminé leur relevance fonctionnelle ainsi que les mécanismes moléculaires. D'après les immunomarquages et la modélisation 3D des membranes, la fragmentation et l'atrophie du Golgi dans les neurones lombaires moteurs pmn ressemblent à celles rapportées dans la SLA et se produit dans des cinétiques similaires. Les analyses en microcopie électronique montrent que l'empilement des citernes golgiennes est progressivement remplacé par des petites vésicules. Les analyses biochimiques révèlent : 1/ une redistribution cytosolique des protéines d'arrimage tel que GM130, 2/ une diminution des protéines β-COP et 3/ une augmentation considérable des protéines golgiennes d'amarrage v-SNARE GS15 et GS28 contrôlant la fusion des vésicules. / Fragmentation of the Golgi apparatus represents one of the earliest and most constant pathological changes in neurodegenerative diseases. To understand the molecular mechanisms of these changes I investigated two experimental models of motor neuron diseases. 1. pmn mice with progressive motor neuronopathy. The pmn mice were chosen since they suffer from a very aggressive form of motor neuron degeneration and since their molecular defects represents a missense mutation in a Golgi-localized tubulin chaperone TBCE, as shown by previous (Martin et al 2002, Schäfer et al 2007). In the last years, we identified severe Golgi abnormalities in motor neurons of pmn mice and dissected out their functional relevance and molecular mechanisms. According to immunolabelings and 3D membrane modelings, Golgi fragmentation and atrophy in lumbar pmn motor neurons resembled those reported in human ALS and proceeded with similar kinetics. Electron microscopy illustrated that Golgi cisternae were progressively transformed into small vesicles. Biochemical analyses revealed : 1/ a cytosolic redistribution of tethering factor such as GM130, 2/ a decrease in β-COP protein level and 3/ a massive increase in the Golgi v-SNARE proteins GS15 and GS28 controlling vesicle fusion. These pathological changes were due to loss of TBCE expression since they could be rescued by transgenic expression of wildtype TBCE but not mimicked by sciatic nerve axotomy. They involved defective dynamics of Golgi-derived microtubules rather than accumulation of misfolded tubulins as shown by the differential effects of TBCE-depletion, Nocodazole and a folding-incompetent tubulin mutant.

Sialyltransferases, CMP-sialic acid synthetases and CMP-sialic acid transport proteins play a crucial role in the construction of cell surface glycoconjugates. These proteins also have a pivotal role to play in a number of diseases, including cancer. The sialyltransferase enzymes are responsible for transfering sialic acids from the donor substrate (CMP-sialic acid) to growing cell surface glycoconjugate chains within the Golgi apparatus. The CMP-sialic acid synthetase enzymes are responsible for the synthesis of the CMP-sialic acid, the donor substrate of the sialyltransferases in the nucleus, while the CMP-sialic acid transport proteins are responsible for transporting CMP-sialic acid from the Cytosol to the Golgi apparatus. When these proteins function in an abnormal way, hypersialylation results, leading to an increased level of sialylation on the cell surface. This increased level of sialylation aids in the detachment of primary tumour cells due to an increase in the level of overall negative charge, causing repulsion between the cancer cells. Therefore, the sialyltransferase enzymes, CMP-sialic acid synthetases and CMP-sialic acid transport proteins are intimately involved in the metastatic cascade associated with cancer. Chapter 1 provides a general introduction of cancer metastasis, discussing the roles of three target proteins (CMP-sialic acid synthetases, CMP-sialic acid transport proteins and sialyltransferases), as well as discussing their substrate specificities, with an emphasis on their involvements in cancer metastasis. The Chapter concludes with an overview of the types of compounds intended to be utilised as probes or inhibitors of these proteins. Chapter 2 describes the general approach towards the synthesis of CMP-Neu5Ac mimetics with a sulfur linkage in the presence of a phosphate group in the general structure 38. The precursor phosphoramidite derivative 45 was prepared and isolated in a good yield using Py.TFA. Unfortunately, the target compound 38 could not be prepared. Chapter 3 describes an alternative strategy wherein S-linked sialylnucleoside mimetics, of the general structure 39, with a sulfur linkage, but no phosphate group, between the sialylmimetic and the ribose moiety in the base is targeted. A series of these S-linked sialylnucleoside mimetics were successfully prepared. Cytidine, uridine, adenosine and 5-fluorouridine nucleosides were used to create a library of different nucleosides and with structural variability also present in the sialylmimetic portion. This small 'library' of 15 compounds was designed to shed light on the interaction of these compounds with the binding sites of the sialyltranferase, CMP-sialic acid synthetase and/or CM-sialic acid transport protein. Approaches towards the synthesis of O-linked sialylnucleoside mimetics of the general structure 40 are described in Chapter 4. Several methodologies are reported, as well as protecting group manipulations, for successful preparation of these sialylnucleoside mimetics. Cytidine and uridine were employed as the nucleosides, thus allowing a direct comparison between the O- and S-linked sialylnucleoside mimetics in biological evaluation. It appears from these synthetic investigations that gaining access into the O-linked series is not as straightforward as for the S-linked series, with alternative protecting group strategies required for the different nucleosides. The biological evaluation of some of the compounds reported in Chapters 3 and 4 is detailed in Chapter 5. The sialylnucleoside mimetics were evaluated, by 1H NMR spectroscopy, for their ability to inhibit CMP-KDN synthetase. In addition, an initial 1H NMR spectroscopic-based assay was investigated for inhibition studies of α(2,6)sialyltranferase in the absence of potential inhibitors. The final chapter (Chapter 6) brings together full experimental details in support of the compounds described in the preceding Chapters.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 177-189). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 177-189). Also available on the Internet.

Thesis (Ph.D.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2009/06/09). Includes bibliographical references.

ABSTRACT The Golgi apparatus has always been an interesting organelle of study because of its unique morphology as well as the critical roles it plays in cell biology. It is situated next to the endoplasmic reticulum and secreted proteins must pass through the Golgi vesicular pathway for modifications and targeting. In addition, the Golgi apparatus plays an essential role in establishing cellular polarity. Cell polarity refers to difference in orientation of cell structures spatially, and is involved in establishing functionality. The Golgi apparatus establishes cell polarity in various ways including orienting itself spatially, biasing vesicular trafficking within the cell, and most importantly through its role as a microtubule organizing center. The cytoskeleton provides the structural framework for cells. Microtubules nucleated from the Golgi-dependent microtubule organizing center result in an asymmetric cytoskeleton. An asymmetric cytoskeleton is essential to establishing cell polarity. Neurons require cell polarity to establish the essential structures such as the axon and dendrites. The Golgi apparatus establishes neuronal polarity through its extensive network of associated proteins. In this review, we will discuss the growing evidence supporting the role of the Golgi apparatus in establishing neuronal polarity.

Formins are proteins involved in regulation and construction of actin filaments of eucaryotic organism. They parcipitate in regulating cytokinesis, polar tip growth, and thus participate in development of whole organisms. There are 2 classes of formins in Arabidopsis thaliana. Both classes include FH1 and FH2 domains (formin homology 1 a 2). Class I formins have N-terminal transmembrane domain, unlike class II formins. Some formins of class II have a N-terminal PTEN domain (Phosphatase and Tensin Homolog). Sequence analyses predicted membrane binding via phosphatase or C2 subdomain of PTEN. This thesis was focused on the formin AtFH14, specifically its PTEN domain. Based on predicted sequence, a DNA fragment encoding the PTEN domain was amplified, sequenced and cloned to destination vectors for YFP and EOS phusions. Marked protein was visualized by transient expression in Nicotiana benthamiana. Stably transformed Arabidopsis lines were prepared for stably expression of protein. The tagged protein was localized in cortical cytoplasm, cytoplasmatical strands, probably in nuclear membrane or perinuclear cytoplasm, as well as in peculiar "folicle-like" structures that might be due to binding of PTEN at the periphery of some membrane organelles. Also were seen filament structures, maybe caused by PTEN binding...

The use of peptides in drug development has been hampered by their poor pharmaceutical properties, most notably their inability to reliably permeate biological membranes and lack of targeting. To overcome these disadvantages, the AMino acid Intracellular Delivery SysTem (AMIDST) was developed. This modular peptide-based delivery system confers cellular permeability and organelle-specific targeting for therapeutic peptides. As demonstrated in this study, the delivery of a HER2-binding peptide to the secretory organelles of breast cancer cells resulted in intracellular sequestration, a reduction in downstream signalling, and reduced viability compared to the delivery of a control peptide. Given its modular design and ease of production, AMIDST has the potential to enhance the use of peptides as therapeutic agents.

Transport of different organelles along the Microtubule cytoskeleton is carried out mainly by motor proteins Dynein and Kinesin. The tubulin monomers in Microtubules are organized in such a way that the generate polarity (a minus and a plus end) that is recognized by Motor proteins. Dynein usually acts with a binding partner, Dynactin, and is in charge of moving cargoes to the minus end of microtubules (mainly towards the center of the cell). There are different kinesins, the most studied is Kinesin-1, which moves cargoes towards the plus end of microtubules. In order to fulfil their function Motors usually bind to their cargoes indirectly through adaptor proteins. Chapter 1 explains the general concepts related to a group of Adaptors that recognize the small GTP-ases, Rabs, in cargoes that need to be transported under certain physiological circumstances and help recruiting the Dynein/Dynactin complexes to them so they can move in the minus end direction. This family of Adaptors is called Rab-Dynein-Dynactin (RDD) adaptors and in this project I focused on two of them: BicD2 and RILP. In chapter 2, I will focus on BicD2 and its role in Golgi morphology. BicD2 is an RDD adaptor that mediates binding of Dynein/Dynactin to Rab6-positive vesicles. Some mutations in BicD2 have been associated to Golgi apparatus morphology disruption, but the mechanism is unclear. It has been suggested that mutated BicD2 abnormally binds Dynein/Dynactin, sequestering this motor complex, producing Golgi disruption indirectly since this organelle depends heavily on minus-directed transport to maintain its localization and structure. I test this hypothesis and conclude that even when most pathological mutations disrupt the Golgi, a Dynein/Dynactin-mediated mechanisms is probably true only to some of them, proposing alternatives mechanisms such as Rab6 abnormal accumulation and non-Golgi related mechanisms of pathogenesis. In chapter 3, I will focus on RILP and its role in autophagosome movement. RILP is an RDD adaptor that mediates binding of Dynein/Dynactin to Rab7-positive vesicles such as Lysosomes. During autophagy, autophagosomes (which are LC3-positive) are formed mainly in the ER and mature to finally fuse with the Late Endosomes or Lysosomes (both acidic) in the center of the cell. It has been described by our lab that RILP can transport LC3-vesicles in axons. Nevertheless, these vesicles are acidic, which suggest these LC3-vesicles are already fused with either Lysosomes or Late endosomes. I will work under the Hypothesis that RILP can move autophagosomes in early stages (before fusion with Lysosomes or Late endosomes) in non-neuronal cells. I show that RILP can move autophagosomes to the center and FYCO1 (a Kinesin-1 adaptor) can move them to the periphery. RILP-mediated movement of autophagosomes depends on Rab7 activation status and seems to be controlled by PKA. I proposed a phosphorylation in Rab7 as a control mechanism. Finally, the discovery of 3 LC3 interacting regions (LIRs) in the RILP molecule is discussed and their contribution to autophagosome movement is analyzed. My results highlight the relevance of RDD proteins in physiological and pathological context.