Ana M. Cuervo - US grants

The candidate is a junior researcher with considerable experience in intracellular protein degradation and some training in aging research. In order to become a fully independent researcher studying changes in protein metabolism with age, an additional supervised training period will be very important. Dr. J. Fred Dice, due to his extensive research experience in protein degradation in aging, would be the ideal mentor for this training. The research project will be carried out at Tufts University which offers a stimulating scientific environment and a well- established research program in cellular metabolism and physiology. The proposed four year career development plan is oriented to: i) confer the candidate strong background in aging, ii)improve her research experience, including learning new experimental approaches, iii)facilitate productive collaborations with well established researchers and iv)develop her teaching, training and group leader skills. The main goal of the research project is to identify the cause(s) of the decrease in the specific degradation of cytosolic proteins in lysosmers with age. The reduction of this degradative system may contribute to the cytosolic accumulation of abnormal proteins in senescent cells and the subsequent generalized failure of many cellular functions. The selective lysosomal pathway is induced in response to nutrient deprivation, and it is mediated by a cytosolic heat shock protein of 73KDa (hsc73), that stimulates binding of substrates to the lysosomal membrane, and by a lysosomal hsc73, essential for the import of substrate proteins. The characterization of the different components involved in this lysosomal uptake, and the further comparison of their levels in young and old rats, will be decisive in identifying the origin of the lysosomal failure with age. The steps followed by the substrates of this pathway will be analyzed in an in vitro system with isolated rat liver lysosomes. Immunological procedures and protein interaction assays will be use to determine changes in levels or activity of the components of the system with age. Overexpression of some of those components in cultured cells will be used to determine their participation in the selective degradation of proteins in lysosomes and to analyze the contribution of their age-dependent changes to some of the characteristics of senescent cells. These studies will also evaluate the possible reversibility of the lysosomal defect with age.

A genetically altered mouse will be generated in which the selective degradation of specific cytosolic proteins in lysosomes could be modulated at wish. This mouse model will allow further analysis of the role of this lysosomal protein degradation pathway in preventing the intracellular accumulation of damaged proteins with age. Under stress conditions, such as nutrient deprivation, the selective degradation of cytosolic proteins in lysosomes by chaperone-mediated autophagy (CMA) is activated. Degradation of proteins by CMA requires their binding in a rate-limiting fashion to a receptor, the lysosome-associated membrane protein type 2a (lamp2a), in the lysosomal membrane. Activity of CMA is modulated by changes in the levels of lamp2a in the lysosomal membrane. CMA decreases with age in cultured cells and in different rodent tissues. Decreased levels of the receptor in the lysosomal membrane have been found in lysosomes from old tissues. We intend to prevent the decrease in receptor levels by conditionally overexpressing lamp2a in specific mouse tissues using the reverse tetracycline-controlled transactivator system (Tet-on). If, as demonstrated for cells in culture, overexpression of lamp2a in mouse increases CMA, the consequences of maintaining that increased CMA activity in several tissues on their ability to respond to different oxidative challenges will be analyzed. We will compare levels and rates of degradation of abnormally modified proteins in the cytosol of mice overexpressing or not lamp2a for different periods of time. If accumulation of damaged proteins after different challenges is prevented by improving their removal, the lamp2a-transgenic mice will become a very useful animal model to determine the contribution of the accumulation of abnormal proteins to the loss of cellular function with age. Using this system in old animals will allow us to test the reversibility of the protein accumulation and whether or not that results in improved function in old organisms.

DESCRIPTION (provided by applicant): Rates of protein degradation decrease with age, contributing to the cytosolic accumulation of altered proteins in old cells. We have identified a decline with age in chaperone-mediated autophagy (CMA), one of the mechanisms responsible for the degradation of both normal and damaged cytosolic proteins inside lysosomes. CMA substrates are targeted by cytosolic chaperones to a receptor protein in the surface of the lysosomes. Assisted by an intralysosomal chaperone, substrates are then translocated and completely degraded by lysosomal proteases. We have found that substrate binding and uptake are the CMA steps primarily altered during aging, and have identified an age-related decrease in the levels of the lysosomal receptor. However, because restoration of normal receptor levels in senescent cells only recovers CMA activity partially, it is likely that other still unidentified CMA components, might also change with age and contribute to this impaired function. We will analyze age-related qualitative and quantitative changes in lysosomal membrane and matrix protein components and will identify the ones responsible for the declined CMA activity during aging. Because no single separation technology is applicable to all proteins, we will use a complementary dual approach to generate the CMA-related lysosome subproteome and to track changes with age in this subproteome: 1) differential two-dimensional electrophoresis of lysosome associated membrane proteins and of lysosomal matrix proteins isolated from different age rodents, and 2) multidimensional chromatography and mass spectrometry of trypsinized lysosomal membranes isolated from early and late passage fibroblasts, differentially labeled in culture with stable isotope- labeled amino acids (SlLAC). We have previously defined six different conditions, other than aging, resulting in changes in CMA activity. Therefore, we expect to be able to discriminate changes in the lysosomal components that may affect CMA activity from the other age-related changes. Changes altering CMA activity will be the target of future corrective interventions aimed to restore normal lysosomal activity in aging and to thus, facilitate the elimination of abnormal proteins accumulated in old tissues.

DESCRIPTION (provided by applicant): The Gordon Research Conference on Autophagy in Stress, Development, and Disease will be held June 22-27, 2003 at Colby College in Waterville, Maine and at biannual intervals thereafter. This is a new Gordon Research Conference and represents the first meeting ever to be held in the United States on autophagy. Autophagy is a cellular pathway involving the bulk degradation of long-lived proteins and organelles by autophagolysosomes, and has recently emerged as an exciting novel area of biomedical research. The regulation of autophagy plays an essential role in the cellular response to nutrient deprivation, in development, in normal physiologic processes such as aging, and in the host response to microbial infection; the deregulation of autophagy has also been implicated in diseases such as heart disease, neurodegenerative diseases, and most notably, cancer. Until recently, very little has been known about the genetics, biochemistry, and molecular cell biology of autophagy or about the precise role of autophagy in normal physiology and pathophysiology. However, there have been several major scientific advances in the past decade that are revolutionizing the field, including the identification of evolutionarily conserved genes that mediate autophagy, elucidation of the biochemical pathways of autophagy, and elucidation of the role of autophagy in normal physiology and disease pathophysiology. These recent advances have created a fertile scientific foundation that is likely to lead to an explosion of research in the next decade that will further elucidate the molecular mechanisms of autophagy and the role of autophagy in diverse biologic processes as well as identify important new targets for the treatment of cancer, neurodegenerative diseases, heart disease, and infectious diseases. This meeting will bring together established leading international scientists as well as promising junior investigators who have expertise in diverse aspects of autophagy. The conference will focus on the biochemistry and genetics of autophagy, the role of autophagy in development, signaling pathways that regulate cell growth control and autophagy, the interrelationship between autophagy and apoptosis, and the role of autophagy in cancer and other disease pathologies. By creating a scientific forum for established and junior investigators to exchange ideas related to autophagy, it is anticipated that this meeting will make a landmark contribution to the development of this critically important, emerging area of biomedical research.

DESCRIPTION (provided by applicant): The selective packaging of peptide hormone precursors into secretory vesicles is essential for hormone biosynthesis. In the trans Golgi network (TGN), the precursors are sorted into nascent secretory granules where they are processed to generate bioactive polypeptides. Data from our laboratory has demonstrated that the maintenance of Golgi structure and release of hormone containing vesicles from the TGN requires phosphatidic acid (PA), the product of phospholipase D (PLD)-mediated phosphatidylcholine hydrolysis, as well as synthesis of the inositol phospholipids phosphatidylinositol (4) phosphate, PI(4)P, and phosphatidylinositol (4,5) bisphosphate, PI(4,5)P2. The goal of this proposal is to understand the mechanisms whereby these lipids regulate Golgi structure, budding of secretory vesicles from the TGN and hormone processing. Aim 1: The role of PI(4)P and PI(4,5)P2 in maintaining Golgi structure and function in endocrine cells: We will test the hypothesis that in pituitary GH3 cells both PI(4)P and PI(4,5)P2 are required to maintain Golgi architecture whereas Pi(4,5)P2 also mediates release of post-Golgi vesicles and trafficking to the plasma membrane. Aim I1: The Regulation of Golgi PI(4,5)P2 synthesis: Surprisingly, the Golgi apparatus appears to possess little PI(4,5)P2 and its level maybe tightly controlled, in part, by a potent Pl(4,5)P2-5-phosphatase activity that we have identified. We will test the hypothesis that PI(4,5)P2 synthesis is regulated locally by recruitment of specific PI(4)P 5-kinases to the Golgi apparatus and by the activity of the Pl(4,5)P2-5-phosphatase. Aim III. To determine the role of PLD isoforms and PA in maintaining Golgi structure: PA has been implicated in vesicle trafficking, signal transduction and regulation of PI(4,5)P2 synthesis. We demonstrated in endocrine cells that PLD1 and -2, which generate PA, localize to different regions of the Golgi apparatus. We will determine the mechanism of PLD recruitment to the Golgi apparatus and use novel PA reporter proteins to demonstrate PA itself localizes to the Golgi apparatus. Although multiple enzymes involved in phospholipid biosynthesis have been characterized, in endocrine cells relatively little is known whereby the different activities are coordinated and integrated in the Golgi apparatus to control the formation of nascent secretory vesicles. Consequently, understanding the mechanism of secretory vesicle budding will facilitate the rational design of drugs that could be used to enhance or diminish hormone secretion in a variety of pathological conditions including diabetes.

DESCRIPTION (provided by applicant): The Gordon Research Conference (GRC) on Biology of Aging has been held every 18 months since 1962 and is part of the over 170 conferences that will be organized in 2010 by GRC, an organization known world-wide because of the high-quality cutting-edge nature of its conferences. The program for the 2010 GRC on Biology of Aging described in this application has been developed around the theme "Determinants of Health-span: from cell to humans" which will serve as a basis for discussion of the recently emerging changes in the way in which aging is perceived, measured and studied. Aging is a multifactorial and complex process that results from changes at the genetic, biochemical and physiological level. Our understanding of those changes in cellular and animal models of aging and age-related disorders has advanced vertiginously during recent years. The current challenge of this field is two-fold: 1) to continue and complete the molecular dissection of the factors that contribute to aging and 2) to promote the translation of these novel findings into interventions to improve the health-span of our aging human population. This Gordon Research Conference will bring together leading international experts with a broad range of interests related to diverse aspects of biology of aging, and will create a high quality scientific forum for discussion of findings on the basic mechanisms of aging and their implementation into interventions aimed at increasing quality of heath in the aging population. Furthermore, for the first time in the history of this conference, a pre-conference Gordon Research Seminar will be organized by junior investigators (student and postdoctoral level). The activities during the Seminar are completely oriented to junior investigators and are intended to 1) provide them with the basic background on Biology of Aging necessary to maximize their understanding of the science discussed in the conference, 2) receive feedback on their ongoing research projects from experts in the field and 3) facilitate their interaction with senior members of this scientific community and promote networking in between the junior generation of aging researchers. Significance: We fully anticipate that the scientific discussions, research talks and informal interactions between the participants in this conference will contribute to advance our understanding of the molecular mechanism behind the functional decline associated to aging and will set the basis for the development of collaborative interventions aimed at promoting healthy human aging. PUBLIC HEALTH RELEVANCE: The Gordon Research Conference on Biology of Aging will bring together major leaders in different basic scientific disciplines (biochemistry, cell biology, genetics, etc.) and in clinical areas of relevance to gerontology (neuropathology, endocrinology, experts in metabolism, etc.) along with junior investigators that will constitute the future generation of researchers with interest in the field of aging and who will participate in addition in the pre-meeting Gordon Research Seminar. The scientific presentations, discussions and workshops during this conference will contribute to advance our understanding of the reasons for the functional decline that occurs in aging individuals and the increased incidence of certain pathologies in elders. It is anticipated that the collegial and cooperative atmosphere that has traditionally characterized this conference will provide the perfect setting for the intellectual development and future implementation of interventions to assure healthy aging.

SUMMARY Lysosomal function is crucial for cell homeostasis, autophagy, nutrient sensing, apoptosis and tissue remodeling. In lysosomal storage disorders (LSDs), characterized by genetic defects leading to anomalous accumulation of metabolites in lysosomes, cells are affected by lysosomal malfunction frequently leading to cell death. Cystinosis is a lysosomal storage disorder resulting from defects in the cystine transporter cystinosin (CTNS). Increased levels of intra-lysosomal cystine lead to cell malfunction and progressive tissue deterioration, which is especially manifested in kidneys. As with most LSDs, this leads to a slow but irreversible deterioration, organ dysfunction and early death. Patients with nephropathic cystinosis develop proximal tubule cell dedifferentiation, Fanconi syndrome and progressive renal injury, which are not corrected by the current therapy, cysteamine. Thus, cell malfunction and tissue failure occur despite cystine depletion, suggesting that cystine accumulation is not the only cause of all the defects observed in cystinosis. We recently revealed a defective mechanism of chaperone- mediated autophagy (CMA) in cystinosis. Defective CMA is directly linked to human disease, including kidney pathologies and neurological disorders. CMA defects in cystinosis are caused by mislocalization and downregulation of the only lysosomal CMA receptor, LAMP2A. Defective CMA activity correlates with high susceptibility to cell death in cystinosis. Importantly, the defect was not rescued by cystine depleting therapies supporting that it is independent of lysosomal overload. Our data highlight that CMA impairment is an important contributor to the pathogenesis of cystinosis and underline the need for new treatments to complement cystine- depletion therapies. Our research plan aims to elucidate the molecular and cellular mechanisms leading to abnormal CMA activity in cystinosis. We also propose translational approaches that utilize small-molecule activators of CMA to improve cellular function in cystinosis. Our Specific Aims are: Aim 1: To elucidate the molecular basis of the regulation of LAMP2A function in cystinosis. To this end, we will study the interplay between the CTNS protein and the CMA receptor LAMP2A and elucidate the mechanisms that mediate LAMP2A trafficking and destabilization at the lysosomal membrane in cystinosis. Aim 2: To determine the molecular basis of the regulation of CMA activity in cystinosis. We will study the mechanisms mediated by CTNS to regulate CMA function and will test the hypothesis that the rescue of CMA activity improves the function of proximal tubule cells from cystinotic patients. Aim 3: To utilize small-molecule CMA activators, in vivo, to improve renal function in cystinotic mice. We will correct cellular and renal function in cystinotic mice using CMA activators, alone, or in combination with cysteamine. Our research is highly significant because it aims to elucidate molecular mechanisms associated with a devastating human pathology and will help develop new therapies for the treatment of cystinosis and other human diseases.