No description available.

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.

Amyotrophic Lateral Sclerosis (ALS) is a neurological disease characterized by the degeneration of upper and lower motor neurons. Genetic studies have revealed that many ALS-associated genes are involved in autophagy, but the role of this pathway in motor neurons remains poorly understood. Here, we use the SOD1G93A mouse model to investigate the role of autophagy in ALS. We find neuronal subtype-specific regulation of autophagy over the course of disease progression. Vulnerable motor neurons form large GABARAPL1-positive autophagosomes that engulf ubiquitinated cargo recognized by the selective autophagy receptor p62. Other motor neurons and interneurons do not engulf cargo within GABARAPL1-positive autophagosomes and instead accumulate somatodendritic aggregates. To investigate whether motor neuron autophagy is protective or detrimental, we generated mice in which the critical autophagy gene Atg7 is specifically disrupted in motor neurons. Phenotypic analysis of these mice revealed that autophagy is dispensable for motor neuron survival but plays a key role in regulating presynaptic structure and function. By crossing these mice to the SOD1G93A mouse model, we find that autophagy inhibition accelerates early neuromuscular denervation and neurological dysfunction. However, loss of autophagy in motor neurons eventually leads to an extension of lifespan, and this is associated with reduced pathology in interneurons and glial cells. These data suggest that vulnerable motor neurons rely on autophagy to maintain neuromuscular innervation early in disease. However, autophagy eventually acts in a non-cell autonomous manner to promote disease spread and neuroinflammation. Our results reveal counteracting roles for motor neuron autophagy early and late in ALS disease progression.

No description available.

Autophagy is the process by which cells eliminate its unwanted or dysfunctional components. A major step in autophagy is the formation of autophagosome, the double membrane that engulfs the unwanted cellular components. Dysregulation of autophagy affects neurodegenerative disorders, infectious diseases, cancer, and aging. In yeast, Atg8 protein is considered to play a crucial role in autophagosome maturation. Studies have shown that yeast lacking Atg8 protein form extremely small autophagosomes. Similarly, mammalian cells lacking Atg8 homologues produced “open” autophagosomes. Microtubule-associated protein (MAP) light chain3 (LC3), a human homologue of Atg8 protein is considered to play a major role in autophagosome maturation. However the exact mechanism by which Atg8/LC3 affects the autophagosome maturation is not completely known. A possible mechanism evolving from various studies is the following: Upon binding to the autophagosome, Atg8 family undergoes a conformational transition, which allows it to associate with another membrane-bound Atg8 in a trans-fashion. The proposed goals of this research include testing this hypothesis, identifying the stable conformations of LC3 and LC3-II (membrane bound LC3) and getting insights into the molecular mechanism by which LC3 influence autophagosome maturation. To accomplish this, we are performing Hamiltonian replica exchange molecular dynamics (HREMD) simulations on LC3 and on LC3-II. The most stable conformations of LC3, and LC3-II are identified via clustering analysis. As autophagy modulation is considered as a potential therapeutic target for various diseases, understanding the molecular mechanisms of different stages of autophagy is very important.

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.