Osteoclasts are cells that degrade bone, by forming a ruffled border (RB) membrane, contained within an actin-rich attachment site (the sealing zone; SZ). Lysosomal vesicles fuse to the RB, and release their contents into the extracellular space to degrade bone matrix. LC3, a marker of autophagosomes, localises to the RB, implying that either canonical autophagy (i.e. autophagosomes) or non-canonical autophagy (a process where LC3 localises to non-autophagic membrane) is involved in the resorptive function of osteoclasts. To examine this in detail, this study used a model with reduced canonical autophagy (FIP200 conditional knockout mouse), and two non-canonical autophagy deficient models (Rubicon knockdown in RAW 264.7-cell derived osteoclasts and an Atg16L1 WD40 domain knockout mouse). Using advanced imaging and molecular techniques I examined whether impairing either process affected LC3 RB localisation and resorption. Reducing canonical autophagy through FIP200 deficiency did not significantly affect GFP-LC3 RB localisation or bone homeostasis. However, impairing non-canonical autophagy resulted in a trend towards increased resorption in vitro. In Atg16L1 WD40 domain-deficient osteoclasts, this may be due to the significantly larger SZs formed in the mutants, which were often stable and contained LysoTracker-positive acidic vesicles within them, putatively signalling increased resorption. As LC3 was frequently observed at the RB, I then examined the LC3-interacting lysosomal adaptor protein, PLEKHM1. I showed that in the PLEKHM1 functional knockout mouse model (R714 STOP), osteoclasts still form RBs but have impaired resorption in vitro. Detailed analysis of multiple aspects of resorption in PLEKHM1 deficient osteoclasts, was required to uncover these defects which may underlie the osteopetrotic phenotype observed in PLEKHM1 deficient mice. Overall this work reveals the potential role of non-canonical autophagy in osteoclast function. Additional dissection of this pathway in osteoclasts may uncover further new insights regarding the regulation of bone resorption and defects underlying bone disorders.

No description available.

No description available.

在真核细胞中，细胞自噬是一个将细胞物质吞噬到自噬体降解的保守的代谢过程。自噬体起始于自噬前体结构(PAS) 并由其逐渐扩展和延伸形成其最后的双膜结构，最后和溶酶体（lysosome）或液泡（vacuole）融合得以降解。在酵母和动物细胞中，研究已发现一系列自噬体相关基因（ATG）蛋白参与调控自噬体的形成。自噬体形成的相关研究存在两个主要的未解决的问题，它们包括自噬体的膜来源和膜变形机制。而在植物中，相当一部分关键的自噬体同源蛋白的缺失导致其分子机制研究仍处于初步阶段。在本研究中，我主要通过利用SH3P2，一个N 端含有BAR (Bin-Amphiphysin-Rvs) 结构域及C 端含有SH3（Src homology 3）结构域的蛋白作为探针, 在拟南芥中研究自噬体的形成. 在进一步的研究中，借助了免疫细胞化学技术（抗体），分子技术（萤光蛋白标记），基因技术（RNAi 干扰）以及蛋白作用（酵母双杂交和免疫共沉淀）等不同的生化以及细胞生物学手段，我发现在植物细胞中也存在保守的自噬体的形成模式，而在其过程中, SH3P2 起着重要的调控作用。通过研究我发现：1）在拟南芥植物中，绿色荧光蛋白标记的SH3P2-GFP 蛋白具有对自噬诱导的应答反应；；2）在拟南芥转基因植物和PSBD 悬浮细胞中，SH3P2-GFP 蛋白与自噬体标记蛋白共定位； 3） 在自噬途径中，SH3P2-GFP 活跃地参与在自噬体膜变形过程中并且定位在自噬前体结构包括其扩展结构的膜上； 4）基因敲低SH3P2 在拟南芥植物中是致死的并且抑制自噬体的形成过程；5) SH3P2 能通过它的BAR 结构域互相聚合; 6）SH3P2 可以结合磷脂酰肌醇-3-磷酸（PI3P）并且与磷脂酰肌醇-3-激酶复合体存在联系；7）SH3P2 通过它的SH3 结构域直接与ATG8 结合。综上所述，此项研究发掘了一个新型的膜相关蛋白SH3P2 参与在拟南芥植物自噬途径中，而其与ATG8 的直接相互结合同时也揭示了一个新的自噬形成调控机制。 / In eukaryotic cells, autophagy is a conserved catabolic mechanism by engulfing the cytoplasmic cargoes into a structure termed autophagosome. In general, autophagosome is initiated from a site named PAS (phagophore assembly site preautophagosome structure), which then expands and elongates to form a double membrane structure. Ultimately, the outer membrane of autophagosome will fuse with the lysosome or vacuole membrane and deliver the cargoes for degradation or recycling. In yeast and animal cells, a number of ATGs (autophagy related genes) have been identified to regulate the autophagosome formation. Studies of the autophagosome formation involve two main unsolved questions: the membrane origin and the membrane deformation mechanism. In plants, several key players responsible for autophagosome biogenesis are missing and the molecular mechanisms for the autophagosome formation remain elusive. In this study, I have used SH3P2, which contains a N-terminus BAR (Bin-Amphiphysin-Rvs) domain and C-terminus SH3 (Src homology 3) domain, as a probe, to study the autophagosome formation in plants. Using a combination of immunocytochemical (antibodies), molecular (GFP fusions), genetic (RNAi) and interaction (Yeast two-hybrid and Co-IP) approaches, I have shown that a conserved autophagosome formation model exists in plant cells and SH3P2 plays an essential role in the autophagy pathway in Arabidopsis thaliana. I have found that 1) SH3P2-GFP fusion proteins response to autophagic induction in transgenic Arabidopsis plants; 2) SH3P2-GFP colocalize with the known autophagosome markers in both transgenic Arabidopsis plant and PSBD cells; 3) SH3P2-GFP localizes on the PAS membrane and actively participates in membrane deformation events during autophagosome formation throughout its expansion process via the dynamic and ultra structural analysis; 4) Knock-down of SH3P2 is developmental lethal and suppresses the autophagosome formation and autophagic flux; 5) SH3P2 has a self-interaction via its BAR domain; 6) SH3P2 binds to PI3P (Phosphatidylinositol-3-Phosphate) and associates with the PI3K (Phosphatidylinositol-3-Phosphate Kinase) complex; 7) SH3P2 directly interacts with ATG8 via its SH3 domain. Taken together, this thesis research has identified a novel membrane-associated protein and demonstrated its essential role in autophagy in plant. The demonstration for the direct association between SH3P2 and the ATG8 complex may provide an insightful mechanism for autophagosome regulation in Arabidopsis thaliana. / Detailed summary in vernacular field only. / Zhuang, Xiaohong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 97-104). / Abstracts also in Chinese. / Statement --- p.I / Abstract --- p.II / 摘要 --- p.IV / Acknowledgements --- p.VI / Table of Contents --- p.VIII / List of Tables --- p.X / List of Figures --- p.XI / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Introduction of Autophagy --- p.2 / Chapter 1.2 --- Molecular Machinery for Autophagy --- p.5 / Chapter 1.3 --- Membrane Origins of Autophagosome --- p.8 / Chapter 1.4 --- Membrane Sensors for Autophagosome Formation --- p.10 / Chapter 1.4.1 --- ATG14 --- p.10 / Chapter 1.4.2 --- Bif1 (Bax-interacting factor 1) --- p.11 / Chapter 1.5 --- Autophagy in Plants --- p.12 / Chapter 1.6 --- Research Objectives --- p.13 / Chapter Chapter 2 --- SH3P2 Defines a Conserved Autohagosome Formation Process in Arabidopsis --- p.15 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.2 --- Materials and Methods --- p.19 / Chapter 2.2.1 --- Plasmid Construction --- p.19 / Chapter 2.2.2 --- Plant Materials, Growth and Treatment Conditions --- p.24 / Chapter 2.2.3 --- Transient Expression in Protoplasts and Confocal Imaging --- p.24 / Chapter 2.2.4 --- Antibody Generation, Protein Extraction and Western Blot Analysis --- p.25 / Chapter 2.2.5 --- Immunofluorescence Confocal Study --- p.26 / Chapter 2.2.6 --- Electron Microscopy (EM) Study --- p.26 / Chapter 2.2.7 --- Accession Numbers --- p.27 / Chapter 2.3. --- Results --- p.28 / Chapter 2.3.1. --- SH3P2-GFP Fusion Proteins Response to Autophagic Induction in Arabidopsis --- p.28 / Chapter 2.3.2 --- The SH3P2-GFP Positive Compartments are Overlapped with Autophagosome Markers --- p.36 / Chapter 2.3.3 --- Dynamic Analysis of SH3P2-GFP Positive Compartments in Arabidopsis Transgenic Plants upon Autophagic Induction --- p.42 / Chapter 2.3.4 --- EM Analysis of the subcellular localization of SH3P2 after autophagic induction --- p.44 / Chapter 2.4 --- Discussion --- p.52 / Chapter Chapter 3 --- SH3P2 is Essential for Plant Development and Autophagic Pathway in Arabidopsis --- p.54 / Chapter 3.1 --- Introduction. --- p.55 / Chapter 3.2.1 --- Plasmid Construction --- p.57 / Chapter 3.2.2 --- Plant Materials, Growth and Treatment Conditions --- p.57 / Chapter 3.2.3 --- Transient Expression in Protoplasts and Confocal Imaging --- p.58 / Chapter 3.2.4 --- Protein Extraction and Immunoblot Analysis --- p.58 / Chapter 3.2.5 --- RT-PCR --- p.59 / Chapter 3.3 --- Results --- p.60 / Chapter 3.3.1 --- RNAi Knockdown of SH3P2 is Developmental Lethal --- p.60 / Chapter 3.3.2 --- RNAi Knockdown of SH3P2 Suppresses the Autophagosome Formation and Autophagic Flux --- p.63 / Chapter 3.4 --- Discussion --- p.71 / Chapter Chapter 4 --- SH3P2 is Associated with the ATG Machinery --- p.73 / Chapter 4.1 --- Introduction --- p.74 / Chapter 4.2 --- Materials and Methods --- p.76 / Chapter 4.2.1 --- Plasmid Construction --- p.76 / Chapter 4.2.2 --- Plant Materials, Growth and Treatment Conditions --- p.76 / Chapter 4.2.3 --- Recombinant Protein Expression --- p.77 / Chapter 4.2.4 --- In Vitro Lipid Binding Assay --- p.77 / Chapter 4.2.5 --- Yeast-two Hybrid Analysis --- p.78 / Chapter 4.2.5 --- Immunoprecipitation Analysis --- p.78 / Chapter 4.3 --- Results --- p.80 / Chapter 4.3.1 --- SH3P2 Binds to PI3P --- p.80 / Chapter 4.3.2 --- SH3P2 Has a Strong Self-interaction via the BAR Domain --- p.82 / Chapter 4.3.3 --- SH3P2 is Associated with the PI3K Complex and Interacts with ATG8 --- p.84 / Chapter 4.4 --- Discussion --- p.86 / Chapter Chapter 5 --- Discussions and Perspectives --- p.87 / Chapter 5.1 --- Discussions --- p.88 / Chapter 5.1.1 --- Autophagosome Formation is Conserved in Arabidopsis thaliana --- p.88 / Chapter 5.1.2 --- SH3P2 Interacts with the ATG8 Complex and is Required for the Autophagic Pathway in Arabidopsis thaliana --- p.90 / Chapter 5.1.3 --- A Novel Membrane-associated Regulator for Autophagosome formaiton in Arabidopsis thaliana --- p.92 / Chapter 5.2 --- Working Model of SH3P2 during Autophagosome Formation in Arabidopsis --- p.93 / Chapter 5.3 --- Future Perspectives --- p.96 / References --- p.97 / List of Publications --- p.104

Paget's disease of bone (PDB) is characterised by focal lesions of increased bone turnover driven by overactive osteoclasts, which often contain nuclear and cytoplasmic inclusion bodies. Mutations affecting the sequestosome-1 (SQSTM1) ubiquitin-associated (UBA) domain have been identified in individuals with PDB. SQSTM1, also known as p62, is a ubiquitously expressed multidomain scaffold protein of 62 kDa that functions in multiple signalling pathways important for cell survival and osteoclast activity. The mechanisms by which SQSTM1 mutations cause PDB remain unclear. Using immunohistochemistry, I showed evidence that protein degradation pathway components, both from the UPS and the autophagy pathway, are elevated in osteoclasts in patients with PDB compared with control osteoclasts from patients without PDB. Using molecular and microscopical methods to examine Pagetic bone biopsies, osteoclast cultures and various cell lines, I have identified two isoforms of SQSTM1. In all cell types examined, four SQSTM1 transcripts were detected, differing in their 5′-untranslated region; one transcript encodes p62, while the other three encode a 55 kDa isoform of SQSTM1. The newly identified isoform also contains the UBA domain mutated in PDB. Using biochemical and microscopical methods, I found that both SQSTM1 isoforms are degraded by autophagy. The isoforms interact with each other and form aggregates upon autophagy inhibition. SQSTM1-55 is ~21× more abundant in osteoclasts than SQSTM1/p62. Biochemical and microscopical methods showed that PDB-causing mutations in SQSTM1/p62 impair its autophagic degradation. Cell lines expressing SQSTM1/p62 mutations form paracrystalline inclusion bodies that by immuno-transmission electron microscopy (TEM) were found to contain SQSTM1 and ubiquitin and were ultrastructurally identical to those found in PDB. As observed by TEM, these inclusions can be degraded by autophagy. The effects of mutations in SQSTM1-55 have yet to be characterised. Abstract Taken together, these data show that mutations in SQSTM1 isoforms impair protein degradation and can lead to inclusion body formation suggesting that PDB results from dysregulated protein degradation in osteoclasts.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2012 / Cell-extracellular matrix (ECM) detachment triggers a cell survival mechanism known as autophagy. A link between attachment and autophagy suggests a form of adhesion-based regulation, involving mechanotransduction of extracellular-originating signals to the cellular machinery controlling autophagy induction. This implies a role for integrin-linked kinase (ILK), which transmits mechanical stimuli to the mammalian target of rapamycin (mTOR) signalling pathway. Cells with a propensity for metastasis may negate these adhesive signals, inducing autophagy inappropriately. Metastasis is a hallmark of transformation frequently associated with human oesophageal squamous cell carcinoma (HOSCC). Additionally, hyperactive mTOR/mTORC1 signalling correlates increasingly with HOSCC. Therefore, the protein expression of significant signal transduction pathway intermediates was investigated in response to both soluble and ECM-originating stimuli. Measurements by SDS-PAGE and western-blotting coupled to semi-quantitative densitometry, during standard tissue culture conditions, revealed that HOSCC’s expressed moderate-to-high levels of mTOR, p-RPS6(Ser 235/236) and mATG-13; indicating elevated levels of autophagy induction despite aberrant signalling through mTOR/mTORC1. Additionally, an 80 kDa mTORβ isoform was identified in HOSCC cells with lower mTOR abundance, presumably to maintain aberrant mTORC1 signalling. A canonical role for the PI3K/PKB pathway was also identified; where autophagy induction accompanied diminished mTORC1 signalling in response to specific PI3K inhibition with LY294002 and serum withdrawal. However, autophagy induction varied in response to a dose-dependent decrease in mTORC1 signalling after exposure of HOSCC cells to rapamycin. Moreover, specific inhibition of p90RSK with BI-D1870, suggests that mTORC1 phosphorylates RPS6(Ser 235/236) in the absence of MAPK signals. Furthermore, ectopic ILK expression indicated an enhanced potential for adhesion-based signalling. Correspondingly, HOSCC cells commonly increased mTOR and p-RPS6(Ser 235/236) expression following growth on fibronectin or collagen. However, co-immunoprecipitation analysis revealed that signals transduction to mTOR precludes a direct interaction with ILK or FAK. Rather, ECM-modulation of mTOR occurs in a integrin-triggered, but PI3K-depedant manner; since specific inhibition of PI3K negated fibronectin-induced increases of mTOR concentration and RPS6(Ser 235/236) phosphorylation. Thus, these data strongly suggest mTOR is a target for adhesion-based signal transduction, where the ECM influences cell survival through mTORC1. Moreover, exploitation of autophagy induction post cell-ECM detachment in HOSCC may promote the survival of metastases during dissemination.

Autophagy requires the biogenesis of autophagosomes (APs), which are large multilamellar vesicles that sequester cytoplasmic substrates and undergo a maturation process that ultimately leads to their fusion with lysosomes. Previous studies have suggested that local production of phosphatidylinositol-3-phosphate (PI3P) by class III phosphatidylinositol 3-kinase (PI3K) (i.e., Vps34) is required for AP biogenesis at specialized sites of the endoplasmic reticulum called "omegasomes". Although Vps34 is the sole source of PI3P in budding yeast, mammalian cells can produce PI3P through alternate pathways, including direct synthesis by the class II PI3Ks; however, the physiological relevance of these alternate pathways in the context of autophagy is unknown. To address this question, we generated Vps34 knock-out mouse embryonic fibroblasts (MEFs) and analyzed the impact of Vps34 deletion on autophagy in mammalian cells. Using a novel higher affinity 4x-FYVE finger PI3P-binding probe, we found a Vps34-independent pool of PI3P accounting for ~35% of the total amount of this lipid species by biochemical analysis. Importantly, WIPI-1, an autophagy-relevant PI3P probe, still formed some puncta upon starvation-induced autophagy in the Vps34 knock-out MEFs. Additional characterization of autophagy by electron microscopy as well as protein degradation assays showed that while Vps34 is important for starvation-induced autophagy there is a significant component of functional autophagy occurring in the absence of Vps34. Given these findings, class II PI3Ks (α and β isoforms) were examined as potential positive regulators of autophagy. Depletion of class II PI3Ks reduced recruitment of WIPI-1 and LC3 to AP nucleation sites and caused an accumulation of the autophagy substrate, p62, which was exacerbated upon the concomitant ablation of Vps34. Our studies indicate that while Vps34 is the main PI3P source during autophagy, class II PI3Ks also significantly contribute to PI3P generation and regulate AP biogenesis. In addition, we used a lipidomic approach to capture the lipid profile of cells in the presence and absence of Vps34 under steady-state and during starvation-induced autophagy. Lipidomics is an emerging powerful tool with the potential to identify new interconnected metabolic lipid networks as well as generate new hypotheses. Here, we identified a new relationship between Vps34 and cholesterol homeostasis. Additionally, we identified specific changes in lysolipids during autophagy. Lastly, we investigated whether the retromer complex plays a role in autophagy. Retromer is a protein complex that binds PI3P on the endosomal membrane and mediates retrograde trafficking of transmembrane proteins from the endosome to the trans-Golgi network. Recent studies have shown a downregulation of this complex associated with sporadic Alzheimer's disease (AD) and have demonstrated aberrant trafficking and processing of APP, a pathological feature of AD, as a result of retromer deficiency. Because retromer is important for maintaining endo-lysosomal system function, we hypothesized that it promote efficient autophagy and may contribute to the dysfunctional autophagy observed in AD when impaired. Using standard autophagy assays, such as assessing LC3 conjugation and puncta formation, our preliminary studies suggest a negative regulatory role for retromer in autophagy. Additionally, we observed a strong association of retromer with Atg9, an autophagy-related gene transmembrane protein that is believe to traffic lipids to the growing autophagosome membrane and recycle autophagy proteins from this compartment.

No description available.

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

Background and objectives: Growing bodies of evidence demonstrate that adipocyte fatty acid binding protein (A-FABP) mediates the pathogenesis of atherosclerosis through its direct impacts on macrophages. Loss-of-function study was conducted by utilizing peritoneal macrophages derived from A-FABP knockout (KO) mice, to investigate the role of A-FABP in autophagy and macrophage foam cell formation. Key findings: 1. No morphological changes between the peritoneal macrophages derived from A-FABP knockout (KO) or their wild-type (WT) littermates. 2. Foam cell formation was successfully induced by the treatment of acetylated low-density lipoproteins (LDL) in peritoneal macrophages derived from A-FABP WT and KO mice. 3. LDL treatment induces autophagy in peritoneal macrophages from both A-FABP WT and KO mice. 4. The extent of LDL-induced autophagy is reduced in peritoneal macrophages of WT mice and is accompanied by increased lipid droplet accumulation when compared with A-FABP KO mice. Conclusions: A-FABP is a suppressor of autophagy and contributes to the attenuation of cholesterol efflux, subsequently resulting in enhancement of lipid droplets accumulation in peritoneal macrophages. A-FABP mediates the formation of macrophage foam cell via the suppression of autophagy. The results suggest that A-FABP is a potential therapeutic target to suspend the progression of atherosclerosis and remit the atherosclerotic lesion. / published_or_final_version / Medicine / Master / Master of Medical Sciences