Autophagy as a Therapeutic Target in Multiple Diseases: From Molecular Mechanisms to Drug Discovery

Agenda

* Presentation times are subject to change.

Abstracts

The Regulation of Macroautophagy
Daniel J. Klionsky, University of Michigan

Over thirty genes have been identified in yeast that are specific to macroautophagy, and homologs have been identified for many of these genes in higher eukaryotes. Although some of the detailed mechanism of macroautophagy has been elucidated, there are still many questions concerning the molecular basis of the pathway that remain unanswered. For example, how is the initial sequestering compartment, the phagophore, nucleated? What is the origin of the membrane used for expansion of the phagophore to form the autophagosome? What are the roles of the various autophagy-related proteins in the process of autophagosome biogenesis?
 
Macroautophagy occurs at basal levels, but can be induced in response to different types of stress. Recent work connects macroautophagy to a range of diseases in humans, and there is a potential for modulating macroautophagy for therapeutic purposes; however, such use will require careful control of macroautophagic induction because excessive macroautophagy may lead to cell death. Thus, either too little or too much of this process can have detrimental consequences. For many years it has been known that macroautophagy is regulated by the Tor kinase; however, surprisingly few details of the regulatory mechanism have been elucidated. In addition, macroautophagy responds to a range of stimuli, and the network that connects these regulatory pathways is complex.
 
We have been analyzing the regulation of macroautophagy in Saccharomyces cerevisiae. One of the central autophagy-related (Atg) proteins is Atg8, a ubiquitin-like protein that is conjugated to phosphatidylethanolamine. The amount of Atg8 determines the size of autophagosomes; however, the factors controlling the expression of ATG8 have not been determined. Recently, we have been analyzing the regulation of ATG8 transcription. Because of the conserved nature of this process, the insight we gain from understanding regulation in the genetically tractable yeast system will provide additional direction for analyses in higher eukaryotes.
 
Supported by NIH Public Health Service grant GM53396

Autophagy in Nervous System and Neurodegeneration
Zhenyu Yue, Mount Sinai School of Medicine

Autophagy is a cell self-eating pathway involving delivery and digestion of cellular content in lysosomes. While autophagy is responsible for the turnover of aggregate-prone proteins, macromolecular complexes and damaged cellular organelles (e.g. mitophagy), the precise mechanism of autophagy process and regulation in the nervous system and its role in the neurodegenerative disease remain largely elusive. We have investigated in vivo function of key autophagy genes in the CNS neurons. Our studies demonstrate the neuroprotective role of autophagy in the clearance of ubiquitinated proteins aggregates and prevention of axonopathies and neurodegeneration. We find that different neurons differ in their response to the disruption of autophagy and the study suggests different vulnerability of neurons to autophagy related pathogenesis. Moreover, autophagy regulates the levels of neurological disease-related proteins, many of which form oligomers/aggregates that are neurotoxic; examples include polyQ-containing proteins such as huntingtin in Huntington disease, α-synuclein in Parkinson's disease, tau in tauopathies, and Aβ/APP metabolites in Alzheimer's disease. I will present our work that shows intersection of autophagy with cellular pathways that regulate disease protein homeostasis, and therefore, autophagy is an important disease-modifying mechanism. I will also discuss our effort in developing autophagy-enhancing therapeutics for the purpose of treating neurodegenerative diseases.

Damage Control — How the Pink1/Parkin Pathway can Regulate Removal of Impaired Mitochondria by Autophagy
Richard J. Youle, NINDS, NIH

The products of two genes mutated in autosomal recessive forms of Parkinson's disease, Pink1 and Parkin, have been identified in Drosophila to work in the same pathway to maintain healthy flight muscles and dopaminergic neurons. PINK1 is a kinase located on mitochondria whereas Parkin is an E3 ubiquitin ligase normally located in the cytosol. Upon mitochondrial damage Pink1 recruits cytosolic Parkin to mitochondria to mediate mitophagy suggesting in mammalian cells that Pink1 can work in the same pathway as Parkin to mediate mitochondrial quality control. PINK1 appears to be constitutively imported to the inner mitochondrial membrane of healthy mitochondria and degraded by PARL and downstream proteases. When import is impaired by mitochondrial damage PINK1 accumulates on the outer mitochondrial membrane where it can recruit Parkin from the cytosol. This sensing mechanism allows the detection and selective removal of damaged mitochondria within a cell. We have found that PINK1 on the outer mitochondrial membrane is stably bound to the TOM complex. However, the TOM complex does not appear crucial for PINK1 to recruit Parkin as inducible targeting of PINK1 to peroxisomes that lack the TOM complex mediates Parkin recruitment to peroxisomes and induces pexophagy. If mitochondria recover membrane potential PINK1 is rapidly reimported into mitochondria and degraded suggesting that PINK1 binding to TOM allows rapid downregulation of PINK1 and salvage of mitochondria from the mitophagy pathway.

Role of Autophagy in Lung Cancer
Eileen P. White, Rutgers University

Autophagy, or cellular self-digestion, degrades and recycles proteins and organelles and is an adaptive stress response that supports cellular metabolism and survival. Oncogenic Ras upregulates basal autophagy, and Ras-transformed cell lines require autophagy to maintain mitochondrial health, survive stress, and efficiently form engrafted tumors. To explore the role of autophagy in initiation and progression of spontaneously occurring Ras-driven tumors, the essential autophagy gene, autophagy-related-7, atg7, was deleted concurrently with K-ras^G12D activation in mouse lung in a model of non-small-cell lung cancer (NSCLC). Here we show that deficiency in atg7 did not alter early tumor growth, but led to the accumulation of autophagy substrates and dysfunctional mitochondria, and resulted in eventual tumor atrophy. Although atg7 deletion slowed tumor growth, it did not delay death, which resulted from fulminant pulmonary inflammation rather than ablation of the lung by tumor. To explore the cause of the impaired tumor growth and enhanced inflammation in atg7-deficient mice, we analyzed the metabolome of Ras-driven tumors, finding specific depletion of adenosine in the absence of autophagy. Tumor-produced ATP is degraded extracellularly to adenosine, which interacts with receptors to promote tumor growth by acting as a paracrine signal that suppresses the anti-tumor immune response and activates oncogenic signal transduction. Adenosine receptor antagonism suppressed growth of wild type but not autophagy-deficient tumors, indicating that autophagy promotes tumor growth in part by supporting extracellular levels of the anti-inflammatory compound adenosine. Blocking autophagy or adenosine signaling are novel approaches to lung cancer treatment.

Identification of a Potent Autophagy-Inducing Peptide with Potential Therapeutic Benefits
Beth Levine, University of Texas Southwestern Medical Center

The lysosomal degradation pathway of autophagy plays a crucial role in defense against infection, neurodegenerative disorders, cancer, and aging, and impairment of autophagy may partially contribute to host susceptibility to these diseases. Accordingly, novel pharmacological agents that increase autophagy may have broad clinical applications. One approach to developing such agents is to learn from the strategies employed by microbial virulence proteins to manipulate host autophagy. Previously, the HIV pathogenic protein, Nef, was shown to interact with an essential autophagy protein, Beclin 1, and modulate autophagy. Our laboratory mapped the region of Beclin 1 required for binding to Nef, and identified 18 amino acids within the evolutionarily conserved domain of Beclin 1 that is essential for the Nef-Beclin 1 interaction. We found that a fusion peptide, Tat-Beclin 1, consisting of the Tat protein transduction domain and a derivative of this region of Beclin 1, was a potent inducer of autophagy in cultured mammalian cells as well as in vivo in mice. We identified a cellular target of the Tat-Beclin 1-peptide that interacts with full-length Beclin 1 and functions as a newly identified negative regulator of autophagy. Consistent with the known role of autophagy in clearing protein aggregates and restricting the replication of some intracellular pathogens, the Tat-Beclin 1 autophagy-inducing peptide decreased huntingtin protein aggregates in cells expressing mutant huntingtin and decreased the in vitro replication of chikungunya virus, West Nile virus, HIV, and Listeria monocytogenes. Moreover, Tat-Beclin 1 treatment improved clinical outcomes in mice infected with chikungunya and West Nile virus. Thus, through characterizing a domain of Beclin 1 that interacts with HIV-1 Nef, we have identified a novel regulator of the autophagy-inducing Beclin 1/Class III PI3K complex and have identified an autophagy-inducing peptide that has potential efficacy in the treatment of certain clinical diseases.

Autophagy Dysfunction in Alzheimer's Disease
Ralph A. Nixon, MD, PhD, Nathan Kline Institute

Neurons are particularly vulnerable to dysfunction within the endocytic and autophagic pathways (the "lysosomal network") because of their extreme polar shapes and high levels of vesicular trafficking. In the extensive neuritic dystrophy of Alzheimer's disease (AD), which is a pathological hallmark of the disease, autophagic vacuoles containing incompletely digested proteins selectively accumulate in focal axonal swellings, reflecting defects in both autophagy and axonal transport. Growing evidence indicates that this massive "storage" of waste proteins in neurons, reminiscent of lysosomal storage diseases, mainly reflects a failure of lysosomal proteolytic clearance of autophagic substrates. Compounding the problem is a moderate induction of autophagy, as evidenced by elevated neuronal expression of autophagy genes measured in CA1 hippocampal neurons of the AD brain in mRNA profiling analyses.

Impaired lysosomal proteolysis is the likely basis for defects in both autophagy and axonal transport leading to the neuritic dystrophy of AD. In living primary cortical neurons expressing fluorescence-tagged markers, LC3-positive autophagosomes in axons rapidly acquired endo-lysosomal markers (Rab7 and LAMP1), underwent retrograde movement, and fused with bi-directionally moving lysosomes that are increasingly numerous at proximal axon levels and in the perikaryon. Disrupting lysosomal proteolysis by either inhibiting cathepsins directly or suppressing lysosomal acidification slowed the axonal transport of autophagy-related organelles but not other organelles and caused their selective accumulation within dystrophic axonal swellings that display various AD-like characteristics. Restoration of lysosomal proteolysis cleared accumulated autophagic substrates and reversed the axonal dystrophy.

Defective lysosomal proteolysis is one facet of a continuum of lysosomal system deficits in AD that begin to be evident even prior to amyloid-ß deposition and are driven in part by AD-related genes. The AD-related gene presenilin1 (PS1) is essential for lysosomal proteolysis and autophagy and plays a novel role in lysosome acidification required for protease activation. In cells lacking PS1, including neurons in PS1 mice, a failure to deliver the proton pump vATPase to lysosomes results in autophagy failure. PS1 mutations causing familial AD also confer partial loss of these same functions in fibroblasts from PS-FAD patients and in neurons of AD model mice. Lysosomal and autophagy dysfunction also develops in sporadic AD and in AD mouse models driven in part by other AD-related genes, including amyloid precursor protein and apolipoprotein Ε. Mutations or polymorphisms of these genes that increase AD risk interfere with cell signaling, markedly upregulate endocytosis, placing stress on autophagy/lysosomal mechanisms by increasing delivery of substrates to this system. Supporting the pathogenic significance of lysosomal system dysfunction in AD, we found that partially restoring deficient autophagy in the CRND8 mouse model of AD by genetically manipulating lysosomal protease activities substantially ameliorates lysosomal pathology, amyloid burden, neuritic dystrophy, and memory deficits.

These findings and others indicate that the same AD genetics that implicate Aß in AD also implicate antecedent lysosomal system dysfunction as a mechanism that both cripples neuronal functions critical for synaptic plasticity and neuron survival and promotes accumulation of toxic proteins, including Aß and tau.

This work is supported by the National Institute on Aging P01 AG01 7617

Drug Discovery Efforts in Autophagy - Therapeutic Targets for Alzheimer's and
Parkinson's Diseases.
Zdenek Berger, PhD, Pfizer

Autophagy represents a major route for degradation of cellular proteins and is important for turnover of aggregated proteins and organelles. Previous studies demonstrated that enhanced autophagy is associated with both decreased levels of toxic aggregate-prone proteins and reduced toxicity in the context of various models of neurodegeneration. The most commonly used enhancer of autophagy is rapamycin, an allosteric mTOR inhibitor. Recent screens have identified new putative autophagy enhancers, potentially offering a wealth of different targets. However, many of these different compounds and targets remain to be validated. In addition, their relative potencies have not been comprehensively determined, in part due to a lack of reliable and quantitative autophagy assays. We have generated cell based autophagy assays that measure autophagy flux. I will present an analysis of previously suggested autophagy enhancers and compare their relative potencies. This allows one to prioritize promising targets and small molecules that have robust effects.

In order to achieve up-regulation of autophagy in human subjects, it is important to understand any potential alterations of this pathway in the human disease. Changes in the autophagy pathway have been observed in brain tissues from patients with Alzheimer's and Parkinson's diseases, raising the possibility that the aggregate-prone proteins may alter autophagy. However, there are conflicting results from prior studies examining effects of a-synuclein on autophagy. In addition, while autophagy pathology has been carefully examined in APP transgenics, little is known about effects of tau or of tau pathology on autophagy. I will present analysis of effects of both mutant a-synuclein and mutant tau on autophagy flux, both in primary neurons and transgenic animals. Our results suggest that aggregate-proteins likely have complex effects on autophagy and that these should be carefully considered prior to any therapeutic intervention and also target selection.

Role of Chaperone Mediated Autophagy in Protein Quality Control
Ana Maria Cuervo, Albert Einstein College of Medicine

All types of autophagy fulfill two important functions in mammalian cells, serving both as an alternative source of energy, when nutrients are scarce, and as an efficient mechanism for the removal of any intracellular damaged structures. In this talk, I will focus on a selective form of autophagy, known as chaperone-mediated autophagy (CMA) and the connections between this pathway and cellular quality control.
 
CMA mediates selective targeting of soluble cytosolic proteins to lysosomes for their degradation. CMA is active in most cell types in mammalians but its activity varies depending on cellular conditions. Maximal activation of this pathway occurs during stress or in conditions leading to increased amount of misfolded/damaged proteins. Degradation via this pathway requires a set of cytosolic and lysosomal chaperones and a receptor protein at the lysosomal membrane, the lysosome-associated membrane protein type 2A (LAMP-2A). The limiting step of this type of autophagy is the binding of substrates to LAMP-2A. We have found that changes in the levels and organization of LAMP-2A at the lysosomal membrane underlie the molecular basis for the regulation of CMA. Two lysosomal chaperones, hsc70 and hsp90, are critical regulators of the higher order organization of LAMP-2A and the assembly of the translocation complex.
 
In recent years, the better molecular characterization of CMA has considerably advanced our understanding of the physiological role of this pathway and the consequences of its malfunctioning in the pathogenesis of detrimental human pathologies, such as cancer, neurodegenerative and metabolic diseases.
 
In this talk, I will describe some of the recent findings on the interplay between pathogenic proteins and CMA. We are addressing this interaction as a two-side relationship: 1) the contribution of CMA to the removal of pathogenic proteins and 2) the effect that pathogenic proteins can exert on CMA activity. Lastly, I will comment on the functional decline of CMA with age and the reasons behind this decline.

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