Cytoplasmic dynein is a highly conserved multi-subunit motor protein that transports a variety of cellular cargoes, including proteins and organelles, towards minus ends of microtubules. Dynein is recruited to specific subclasses of cellular organelles via a specialized class of adaptor proteins, that serve as physical scaffolds for dynein recruitment to cargoes. Recent work shows that these adaptor proteins are also capable of altering biophysical properties of dynein in vitro and in vivo. This work now finds that a dynein adaptor protein, RILP, through multiple interactors, coordinates the progression of a complex biological pathway. Autophagy is a multi-step, highly conserved pathway that involves de novo formation of a double-membraned autophagosome around ubiquitinated cellular cargoes including long-lived proteins and damaged organelles for subsequent degradation by the lysosome. My work finds a dynein adaptor protein, RILP, to control not only retrograde microtubule-based autophagosome transport but their formation as well. RILP achieves these functions by sequentially interacting with the isolation membrane protein, ATG5, and the autophagosome membrane protein, LC3. During autophagosome formation, ATG5 competes with dynein to bind to a common site within the RILP N-terminus to prevent premature initiation of autophagosome motility. Depletion or LC3-interacting site mutations in RILP prevent formation of autophagosomes as well as impede their retrograde transport. This in turn results in an accumulation of ubiquitinated cargoes, including p62/ Sequestosome-1 in cells, showing that RILP is essential for autophagic clearance in cells, a finding that has broad implications for aggregate-prone neurodegenerative diseases. Finally, this work characterizes the molecular composition of the RILP-dynein supercomplex, and identifies Lis1 (implicated in lissencephaly) as an obligate component of the RILP supercomplex. Interestingly, another dynein regulator, NudE (implicated in microcephaly) is absent. Lis1 depletion results in RILP vesicle dispersion, suggesting that it is needed for RILP-mediated dynein driven transport. Altogether, these findings show for the first time that dynein adaptor RILP controls a complex multi-step biological pathway. The unique composition of RILP supercomplex holds new possibilities for dynein regulation in vivo.

Macroautophagy is a degradative system that cells employ to degrade proteins, lipids, pathogens or whole organelles. Dysfunctional autophagy has been implicated in diseases ranging from cancer to neurodegeneration. Animal models lacking macroautophagy fail to preserve a functional liver or central nervous system, supporting the importance of autophagy in maintaining the health of these tissues. However, it is unclear why this degradative pathway is critical in maintaining homeostasis. All macroautophagic cargo are sequestered by the multilamellar organelle called the autophagosome. The formation of the autophagosome depends on the lipidation of a cytosolic protein LC3, so that it associates with the autophagosomal membrane throughout the autophagic process. Using a mouse model expressing GFP-LC3, we have developed an approach to immunopurify autophagosomes from different tissue, then identified their autophagosomal content using tandem-mass-tag (TMT) quantitative proteomics. We have found that the tissues rely on autophagy differently based on the turnover of their organelles as liver depended more on autophagy for ER turnover and brain relied on autophagy more for mitochondrial turnover and its synaptic vesicle homeostasis. Starvation can activate macroautophagy, and is the most studied means through which this pathway has been studied. The importance of autophagy activation in the liver during starvation has been well characterized whereas its importance in the brain has been debated. In this study, we have found that both the liver and brain rely on autophagic degradation of mitochondria differently during starvation. As expected, liver increases its autophagic response upon 24 hr nutrient deprivation, but surprisingly cargo capture transitions from whole mitochondrial turnover to piecemeal mitochondrial turnover. In contrast, in brain, mitochondria-turnover remains largely unchanged. Moreover, although neuronal cargo proteins also remained largely unchanged in response to nutrient deprivation, there was a robust response driven by the non-neuronal cells of the CNS including glial cells and brain endothelial cells, indicating how the discrete cell types of the CNS respond to this physiologic stressor differently. Taken together, this work reveals the tissue-specificity and cell-specificity in the physiological role of autophagy, providing insight in how vertebrates use autophagy to maintain health and react to stress.

No description available.

p38 mitogen-activated protein kinases (MAPKs) control the endocytic trafficking of various growth-related cell surface receptors and transporters. Herein, I demonstrate that p38 MAPKs also regulate autophagy, or the process of self-cannibalism. In my studies, inhibition of p38 MAPKs triggered rapid formation of autophagosomes in prostate cancer cells, even under nutrient-rich conditions, and remarkably, the autophagosomal membranes emanated from endoplasmic reticulum exit sites via the concerted actions of the small GTPases, ARF1 and SAR1. Once formed, the autophagosomes fused with late endosomes and/or lysosomes, in a Rab7-dependent manner, to form “hybrid organelles” that were co-labeled with ER, autophagic, late endosomal, and lysosomal markers. Unlike other inducers of autophagy, however, inhibition of p38 MAPKs suppressed the fission of hybrid organelles, resulting in a profound but reversible accumulation of large cytoplasmic vacuoles. Thus, in addition to their previously reported roles in endocytosis, p38 MAPKs appear to coordinately regulate autophagy and the downstream biogenesis and fission of hybrid organelles. / text

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 135-155). Also available in print.

The aberrant accumulation of aggregated proteins is a pathologic hallmark across adult-onset neurodegenerative diseases, the majority of which have no effective treatment. Although the relative importance of these structures to pathogenesis has been proposed in several diseases, there is little understanding of how we might accelerate the turnover of aggregated proteins, and in turn, a lack of consensus about whether targeting them would provide any therapeutic benefit. The overarching goal of my dissertation is to address both of these questions by focusing on how the pathway macroautophagy might handle protein aggregates in the adult brain. Aggregation-prone proteins are preferentially degraded through the lysosome-mediated degradation pathway macroautophagy (referred to hereafter as autophagy) (Ravikumar 2002; Iwata 2005; Yamamoto 2006). Although studies suggest that aggregates are degraded in bulk by autophagy (Ravikumar 2002; Iwata 2005), studies show that they are more likely degraded in an adaptor-protein dependent manner (Lemasters, 2005; Kraft, 2008; Hanna, 2012; Isakon, 2012; Filimonenko, 2010). In the Yamamoto lab, we have found that the adaptor, the Autophagy-linked FYVE protein (Alfy/WDFY3), is required for the degradation of detergent-insoluble aggregated proteins through selective autophagy in cell-based systems and the adult brain (Simonsen, 2004; Eenjes, 2016; Filimonenko, 2010; Fox, 2020). Through immunohistochemical and loss-of-function studies, Alfy has been implicated in the turnover of disease-relevant protein aggregates including mHtt, α-synuclein, SOD1, and TDP-43, as well as protein complexes such as the midbody ring (Filimonenko, 2010; Clausen, 2010; Han, 2014; Hocking, 2010; Isakson, 2013; Kadir, 2016). Here, I present a potential strategy to suppress disease progression across neurodegenerative disorders by increasing the levels, and thereby the function, of Alfy. I hypothesized that genetically augmenting Alfy levels in the brain will be sufficient to alleviate aggregate burden and delay the onset of proteotoxic stress in different mouse models of neurodegeneration. Using biochemical and genetic approaches, I conducted an extensive in vivo study, demonstrating that augmenting Alfy expression levels in mice can be neuroprotective, and that Alfy may be an influential genetic modifier of neurodegenerative disease. Using two independent genetic approaches that upregulate Alfy expression, I found that they both dramatically delay the onset of disease phenotypes in mouse models of Huntington’s disease, synucleinopathy and TDP-43 proteinopathy. First, I found that ectopic overexpression of Alfy has a pronounced, neuroprotective effect on reducing aggregation, improving motor function, and extending survival in disease models. In parallel, I used mouse genetics to verify the potency of a rare Alfy variant identified in a large Venezuelan cohort of Huntington’s disease that correlated with delayed onset in Huntington’s disease by 10-20 years. Excitingly, in support of our hypothesis, I found that the presence of this single nucleic acid polymorphism led to an increase in steady state levels of Alfy in both humans and in mice, and it was sufficient to recapitulate the benefits of ectopic Alfy overexpression. Taken together, these studies demonstrate that increasing Alfy levels in the brain are sufficient to augment the turnover of aggregated proteins, and may be an effective therapeutic strategy that can be beneficial across neurodegenerative diseases.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Fan, Dahua. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 117-155). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes numerous human pathologies. Biochemical evidence suggests that soluble misfolded proteins are retrotranslocated out of the ER, via the endoplasmic reticulum associated degradation (ERAD) pathway, for proteosome-mediated cytoplasmic degradation. Excess, misfolded- or insoluble proteins, are suggested to cause induction of “overflow” degradation pathways. For soluble proteins, overflow to vacuole-mediated destruction is suggested to occur via two Golgi-to-vacuole (Gvt) routes, the alkaline phosphatase (ALP), direct route, or, a carboxypeptidase Y- (CPY-), prevacuolar compartmentvacuole, indirect route, though only the CPY route is thought to degrade soluble proteins. Insoluble aggregate-containing structures are suggested to be degraded by engulfment by membranes of unknown origin and trafficking to the vacuole for destruction, via an autophagic pathway. To confirm biochemical evidence, wild-type (BY4742), autophagosome- (W303/ATG14), CPY- and autophagy pathway- (W303/VPS30), and proteosome (WCG/2) mutants of S. cerevisiae yeasts were transformed with a high expression pYES plasmid and mutant (Z) human alpha-1- proteinase inhibitor (A1PiZ), giving rise to the derivatives cells BY4742/Z, W303/ATG14/Z, W303/VPS30/Z and WCG/2/Z, respectively. Electron microscopy using gold labeling for A1PiZ, markers for the ER, the ERAD ER channel protein, Sec61, or the chaperone, binding protein (BiP), ALP for the ALP pathway, and CPY for the CPY pathway, was used. Overexpression of A1PiZ seems to result in targeting to the vacuole via a prevacuolar, CPY-like compartment (PVC, 200-500 nm), though CPY and A1PiZ appears not to colocalise, unconvincingly confirming collaborative biochemical data. Large amounts of A1PiZ localise in the cytosol, possibly indicating a largely proteasome-mediated degradation. ER-resident A1PiZ targeting to the vacuole seems also to occur by the budding of the ER and peripheral plasma membrane or ER membrane only. This occurs in all cells, but especially in ATG14 gene (ΔATG14) mutants, possibly indicating autophagosome-mediated degradation independence, in the latter mutants. The ATG14 mutation gave rise to crescent-shaped, initiating membranelike (IM-like) structures of approximately Cvt vesicle-diameter, possibly indicating that ΔATG14 blocks autophagosome- (500-1000 nm) and Cvt vesicle (100-200 nm) enclosure, after core IM formation. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.