Thomas J. Melia, Ph.D. - US grants

DESCRIPTION (provided by applicant): Project Summary Over the last several years, macroautophagy has been implicated in a wide array of neurodegenerative disorders from the aggregation prone disorder, Huntington's disease to the lysosomal storage disorders, Neiman-Pick Type C. Despite its prevalence however, macroautophagy is still poorly understood, making it difficult to define how it contributes towards pathogenesis. Perhaps unsurprisingly, in different disorders, macroautophagy has been considered both as a potentially causative and potentially ameliorative element in disease progression. If we are to target this complex degradative pathway for therapeutics, we need to better define the autophagic process in a means we can apply it towards the brain. In this grant submission, we propose to gain new insights into macroautophagy by focusing on the key organelle involved: the autophagic vacuole (AV). Defined as an onion-like multilamellar vesicle that is positive for the marker MAP1LC3 (a mammalian homologue of ATG8), the formation and maturation of this structure is at the heart of the autophagic process and is by far the least understood. Using a novel approach which we have developed that can isolate specific populations of AV for proteomic and lipid-based analyses, we will: 1) characterize AVs from neuronal cells and brain;2) compare and contrast MAP1LC3- labeled AVs from vesicles labeled with the other four ATG8 mammalian homologues;and 3) use functional cell based assays to further define how the various ATG8- proteomes impact macroautophagy. PUBLIC HEALTH RELEVANCE: Macroautophagy is a poorly understood process that is important for allowing cells, such as neurons to get rid of proteins that no longer function. Interestingly, this process has been implicated to be at the heart of many neurodegenerative diseases such as Huntington's disease, Parkinson's disease, Alzheimer's disease, many lysosomal storage diseases and others. Here we propose to study macroautophagy as it pertains to the brain so that we can use this information to design effective treatment for these many diseases.

DESCRIPTION (provided by applicant): Macro-autophagy is the intracellular stress-response pathway by which the cell packages portions of the cytosol for delivery into the lysosome. This packaging is carried out by the de novo formation of a new organelle called the autophagosome that grows and encapsulates cytosolic material for eventual lysosomal degradation. How autophagosomes form, including especially how the membrane expands and eventually closes upon itself is an area of intense study. One factor implicated in these activitie is the ubiquitin-like protein, Atg8. During autophagy, Atg8 becomes covalently bound to phosphatidylethanolamine (PE) on the preautophagosomal membrane and remains bound through the maturation process of the autophagosome. Our preliminary results suggest that Atg8-PE is a central figure in deforming the membrane perhaps as a prelude to determining sites of membrane tethering or membrane fusion. Here we will build on these results to determine how Atg8 might control each of the membrane dynamics that underlie the late steps of autophagosome maturation.

SUMMARY Nuclear pore complexes (NPCs) are molecular sorting machines that ensure proper compartmentalization of nuclear and cytoplasmic contents in all eukaryotes. Ions, metabolites and small macromolecules pass freely across the NPC, whereas the translocation of larger (>40kD) macromolecules is impeded. Simultaneously with this barrier function, the NPC exhibits remarkable selectivity towards a group of highly mobile nuclear transport receptors (NTRs); NTRs bind signal-bearing cargo molecules and facilitate their import/export through the NPC. These selective transport events are extremely rapid and an individual NPC can transport 1000 molecules/second. How the NPC establishes this selective and efficient transport system is not completely elucidated and represents a fundamental challenge to our understanding of cellular compartmentalization. Moreover, understanding the function of the NPC will be critical for designing strategies to ameliorate a growing number of human pathologies including cancers, heart abnormalities, neurodegenerative diseases, viral infection, in addition to developmental defects that result when NPC function is perturbed. Lastly, at its core, the NPC is an efficient molecular sorting machine - defining the mechanism of transport will lead to the development of synthetic materials that mimic its properties for protein purification, biotechnology and pharmaceutical applications, including bioreactors. The molecular basis for NPC selectivity and rapid transport are interactions between NTRs and a subset of NPC proteins (nups) that are rich in phenylalanine-glycine (FG) amino acid residues. However, measured affinities between NTRs and FG-nups are too strong to support observed in vivo transport rates leading to a paradox; we (and others) suggest that this paradox reflects the limitations of examining individual FG-nups outside of their native environment, where the presence of other FG-nups with different properties, their stoichiometry, and their confinement within a cylindrical channel cumulatively contribute to a cooperative behavior that is difficult to recapitulate in vitro. By leveraging our expertise in DNA-origami we have the ability to fabricate structures termed NuPODs (Nuclear Pore Complex Organized by DNA) that mimic the dimensions of the NPC and contain defined compositions of FG-nups that are precisely spatially arranged. In Aim 1, we will use these NuPODs as binding supports to directly test how spatial-positioning and FG-density impact the cooperative behavior of FG-nups and their binding kinetics to specific NTRs. In Aim 2, we will immobilize our NuPODs on nanopores and examine how unique combinations of FG-nups establish a permeability barrier to inert macromolecules of varying sizes. These studies will open the door for the fabrication of NPC-mimics that fully recapitulate the transport properties of the NPC. Further, our understanding of the NPCs underlying design will allow us to generate NuPODs with prescribed selectivity/permeability characteristics.

Macro-autophagy is the intracellular stress-response pathway by which the cell packages portions of the cytosol for delivery into the lysosome. This ?packaging? is carried out by the de novo formation of a new organelle called the autophagosome that grows and encapsulates cytosolic material for eventual lysosomal degradation. How autophagosomes form, including especially how the membrane coordinates the capture of cytosolic toxins with its own expansion and closure is an area of intense study. One factor implicated in both cargo-capture and autophagosome dynamics is the ubiquitin-like protein, Atg8. During autophagy, Atg8 becomes covalently bound to phosphatidylethanolamine (PE) on the preautophagosomal membrane and remains bound through the maturation process of the autophagosome. Our preliminary results suggest that Atg8-PE can directly deform the membrane perhaps contributing to the unique cup-like morphology of the immature autophagosome. Further, we show that several proteins driving Atg8 recruitment are designed to recognize unique features of the autophagosome including curvature. By combining these low affinity interactions across multiple proteins in a complex, these proteins would achieve dramatic targeting selectivity for only the transient intermediate in the autophagosome growth. Once cargo-capture is complete and the autophagosome closes, curvature- sensitive components are released. Atg8-PE must also eventually be recycled and we describe how the proteases responsible for Atg8-PE release are also sensitive to the membrane structure and composition. Our discoveries are made possible by two important technological advances. First we have developed a variety of in vitro reconstitution approaches to study how Atg8-PE and other autophagy proteins influence membrane deformation and structure. In particular, we have now reconstituted Atg8-PE formation on Giant Unilamellar Vesicles that comprise both a highly tractable membrane manipulation model and also are large enough to support fluorescent-microscopy based interrogation of protein-membrane organization. Second, we can now image autophagosome intermediate structures at super resolution in three dimensions so that we can now visualize both the cup-like intermediate and its eventual resolution following fission. With this proposal, we expect to demonstrate exactly how Atg8-PE proteins coordinate the dual responsibilities of protein-protein interaction supporting cargo encapsulation with the protein- membrane complexes that shape and close the autophagosome.

Project Summary Over the last 10 years, the lysosome-mediated degradation pathway macroautophagy has gained prominence in the study of aging-related disorders and extension of lifespan. Macroautophagy is an essential cellular pathway responsible for the elimination of cytosolic proteins, lipids and organelles, and as such, the field has focused upon the role of macroautophagy in clearing protein aggregates or dysfunctional organelles (such as mitochondria) that specifically accumulate in the cytoplasm. Increasingly however, protein accumulation and organelle dysfunction are observed to occur within the nucleus, apparently shielded from cytoplasmic processes by the double-membraned nuclear envelope. Furthermore, links between aging and nuclear envelope structural defects are emerging, including nuclear envelopathies caused by mutations in the envelope scaffolding lamins. How the cell responds to these nuclear insults is not well understood, but intriguingly, lamins appear to be subject to macroautophagy-dependent turnover. Thus, it is clear that we must refocus our attention on how nuclear quality control is executed and specifically on the mechanism(s) that governs nuclear content turnover in cytoplasmic autophagosomes. Thus, in this proposal, we focus on the fundamental question of how cytoplasmic autophagy machinery and nuclear envelope remodeling are coordinated. Using S. cerevisiae, where discovery of the molecular machinery driving nuclear autophagy is the most mature, we will reveal each of the complex membrane dynamics events that occur to move nuclear envelope fragments away from the nucleus and eventually into the vacuole for degradation. We will then establish the mechanism of membrane remodeling, using fully reconstituted systems that maintain the topologic identity of each of the two nuclear envelope membranes. Importantly, within these reconstituted systems we will also introduce molecular mimics of the growing autophagosome and recapitulate the formation of the nuclear envelope-autophagosome interface that governs sequestration of nuclear envelope fragments. With the completion of this project, we will further our mechanistic understanding of a key underappreciated macroautophagic process and further our understanding of how nuclear autophagy can impact aging-impaired proteostasis.

Summary Macro-autophagy is the intracellular stress-response pathway by which the cell packages portions of the cytosol for delivery into the lysosome. This packaging is carried out by the de novo formation of a new organelle called the autophagosome that grows and encapsulates cytosolic material for eventual lysosomal degradation. How autophagosomes form, including especially from where the autophagosomes extract lipid in order to expand their membranes, has been a core problem in the field for over 50 years. Two competing models have emerged and suggest that either the autophagosome simply grows out of a pre-existing compartment (like the ER) or the autophagosome forms from the continued fusion of individual vesicles recruited from many different sites in the cell. Furthermore, lipid biogenesis pathways are intimately associated with autophagy, suggesting that one or more organelles involved in the production of lipids might also be tightly associated to the growth of the autophagosome. Temporal studies established over a decade ago that the autophagy protein ATG2, is needed during membrane expansion, but how ATG2 facilitates membrane growth has remained elusive. Our preliminary results now demonstrate that ATG2 is a member of a novel lipid-transport family of proteins and suggest a third model for membrane expansion; the bulk delivery of lipid from organelles through protein- mediated contact sites. Indeed, ATG2 binds up to 20 lipids at once, an order of magnitude more than virtually any other lipid transport protein, and thus has the capacity to move a lot of lipid during biogenesis. We show that in cells, ATG2 accumulates at an interface between autophagosomes and the ER, strongly suggesting this organelle-organelle contact site might be the location of lipid transfer. In addition, we have developed gene- edited knockouts of each of the ATG2 proteins in humans and discovered that in the absence of ATG2, not only do autophagosomes not expand, but hundreds of vesicles collect at the site of autophagosome biogenesis. This surprising observation suggests that vesicle-mediated delivery of membrane might also be essential and that the fusion of these vesicles is specifically disrupted when ATG2-mediated lipid transport is absent. With this proposal, we expect to describe how ATG2 works with proteins on both the autophagosome and the ER to drive lipid flow. Likely, this will involve proteins needed to stabilize organelle-organelle contact sites and may also involve proteins sensing or regulating lipid production in the ER. We will then establish how this lipid flow is related to the recruitment and utilization of trafficking vesicles to describe to support autophagosome growth.