Marianne Wessling-Resnick - US grants

DESCRIPTION: Dr. Wessling-Resnick's overall aim is to understand the mechanism of iron transport across membranes. This is a very important problem both from the clinical and basic biology point of view. She has identified a protein called SFT, which is required for the Transferrin independent transport of Fe in Xenopus oocytes and in a preerythroid cell-line K562. SFT has some similarities with the ABC or ATP-Binding Cassette transporters. The three major aims of this proposal are: (1) to characterize the role of SFT in Fe transport; (2) to understand how SFT is localized to both the plasma membrane and endosomes; and (3) to identify the cis-acting elements that regulate the intracellular levels of SFT.

Iron is absolutely essential to sustain life and maintain growth of mammalian cells. Despite our fundamental knowledge of the utilization and storage of iron, however, relatively little is known about the translocation of the cation across biological membranes. The import of iron across cellular membranes must somehow be tightly controlled to guard against excessive assimilation while enabling the entry of adequate amounts of this essential nutrient. Project #3 focuses on a human K562 cell Fe transport protein, SFT, which we recently cloned by functional expression of non-transferrin-bound iron transport activity in Xenopus oocytes, and seeks to further our understanding of the function and mechanism of action of this novel factor. Interestingly, SFT not only mediates non-transferrin-bound iron uptake, but can also stimulate the acquisition of iron from transferrin as well. We will elucidate the mechanistic basis for SFT's effects on iron uptake mediated by the transferrin receptor. Possible interactions of SFT in the transferrin receptor-independent pathway also will be explored in Project #1. Our preliminary data further demonstrate that copper depletion blocks non-transferrin-bound iron uptake as well as transferrin-mediated transport stimulated by SFT. We seek to unravel the molecular basis for this effect. In this project, we focus our efforts on the requirement of copper for SFT-mediated uptake; it is anticipated that the synergy with Project #4 will reveal critical relationships between copper and various iron transport processes, including activities mediated by SFT. Finally, our preliminary results describe the high-affinity binding of extracellular iron by SFT. We will undertake a spectroscopic analysis of extra-membranous peptides of SFT and their interactions with iron. These studies will complement scanning mutagenesis approaches to identify functional domains of SFT.

Hereditary hemochromatosis is a genetic disorder that promotes increased intestinal absorption and progressive tissue deposition of iron resulting in cirrhosis of the liver, hepatic carcinoma, congestive heart failure, endocrinopathies and premature death. It is estimated that 1 in 200-to-400 people in the US are homozygous for this disease which is the most common defective genetic trait known in humans, more prevalent than cystic fibrosis, phenylketonuria and muscular dystrophy combined. Iron assimilation is a tightly regulated process that is limited to prevent harmful effects due to overload of this toxic metal and therefore a reciprocal relationship exists between body iron stores and dietary iron absorption, although the molecular basis for ion homeostasis remains unknown. Many studies of the molecular basis for hemochromatosis have evaluated the expression of factors involved in iron metabolism , including transferrin, transferrin receptor, ferritin and IRPs, but strong evidence to support their abnormal regulation in this disease is lacking. We recently identified SFT (Stimulator of Fe Transport) as a facilitator of non-transferrin-bound iron uptake. Our preliminary results demonstrate that SFT expression is down- regulated at both the mRNA and protein level in response to iron-loading. However, in the course of these studies, we made the significant discovery that SFT mRNA is 5-fold higher in liver from hemochromatosis patients despite the deposition of iron that occurs in this tissue. Thus, our working hypothesis is that malregulated expression of SFT contributes to the etiology of hemochromatosis. The proposed research will specifically evaluate our hypothesis through the following goals: 1) determination of SFT activity in iron transport by hepatocytes and intestinal enterocytes; 2) examination of interactions of interactions with the hemochromatosis protein Hfe that may modulate SFT expression and function in these cells; and 3) characterization of the mechanism that regulates SFT expression.

DESCRIPTION (provided by applicant): Except for members of the Lactobacillus family and Borrelia burgdorferi, virtually all studied organisms from Archaea to man depend on iron for survival. Iron deficiency remains the most prevalent nutritional problem in our country, yet recent identification of the gene responsible for hereditary hemochromatosis indicates that 1 in 20 Caucasians carry the defective allele and thus 1 in 400 may be susceptible to iron overload. Increased knowledge about the factors protecting against iron deficiency and overload is essential to address these significant health problems. The ultimate goal of this project is to utilize the multi-cellular organism C. elegans to elucidate the homeostatic mechanisms regulating the transport and assimilation of iron in man. C. elegans provides a useful multi-cellular model system with the power of genetic manipulation to study iron transport and its homeostasis. Furthermore, the completed sequence of the C. elegans genome allows for the prediction of key genes involved in the transport process and for the rapid identification of those that remain unknown. The purpose of the R21 application is to establish sufficient preliminary data from the study of iron transport and its regulation in C. elegans to generate R01 funding to enable the translation of this information to mammalian systems. The specific aims of the proposal are straightforward: 1) Define the relationships between proteins identified by homology to mammalian counterparts to play a role in iron uptake and its regulation in the nematode; 2) Perform global expression analysis to profile the iron-regulated genome of C. elegans; 3) Use genetic selection to establish transport mutants useful for the elucidation of additional proteins involved in iron import and/or export and the factors that regulate these processes.

DESCRIPTION (provided by applicant): The focus of the proposed research is on DMT1 (Divalent Metal Transporter 1) and its role in Mn ingestion and inhalation. We have chosen the Belgrade rat for these studies since its DMT1 gene contains glycine-to-arginine substitution at amino acid codon 185 (G185R). This defective allele encodes a protein with little or no activity in iron uptake assays and transfection studies suggest that the mutant protein is rapidly degraded. Functionally, DMT1 mediates the uptake of many different divalent metal cations including Fe+2 and Mn+2. Not only does the Belgrade rat suffer from profoundly impaired Fe metabolism, these animals also display significant defects in Mn metabolism. Dietary Fe absorption is modulated by iron status, and DMT1 expression is up-regulated by Fe deficiency. Dietary Fe overload diminishes Mn accumulation in the brain while Fe deficiency is conversely associated with increased Mn in the central nervous system. These lines of evidence further suggest that Mn absorption is modulated by Fe status due to the regulation of DMT1 expression. However, while the role for this transporter in dietary Fe absorption has been established, and its function in intestinal Mn absorption is predicted, whether DMT1 is also involved in metal absorption in the lung remains unknown. Thus, it is possible that the absorption of inhaled Fe and Mn is also affected by iron status via DMT1 regulation. The major underlying hypothesis of this grant proposal is that Fe and Mn utilize the same carrier transport system in the respiratory and gastrointestinal tracts such that absorption of both metals is up-regulated upon iron-deficiency. If so, neurological complications of poor Fe status could be compound by an increased vulnerability to the toxic effects of Mn exposure. If these hypotheses are correct, children suffering from Fe deficiency may be particularly at risk for Mn neurotoxicity because of the relationship between carrier systems for these two metals. The Belgrade rat provides the model system to address test our hypothesis.

A grant has been awarded to the Harvard School of Public Health (HSPH) under the direction of Dr. Dieter Wolf to purchase instrumentation which will open new opportunities in the emerging field of system-wide analysis of protein expression and function ("proteomics"). A detailed understanding of the information contained within the many sequenced genomes available in public databases today requires state-of-the-art means of analyzing quantitative and qualitative differences in protein expression and function in a high-throughput format. Peptide mass spectrometry is a relatively new technology that is ideal for these applications and is widely expected to remain the most important core technology of proteomics research for many years to come.
There are two major areas of proteomics research that are addressed with this equipment: 1. protein expression profiling, and 2. identification of protein assemblies purified from complex samples. The research activities enabled by this NSF grant make extensive use of these applications in order to answer major questions in basic biology. The projects use samples derived from organisms across the evolutionary ladder. The scope of research activities is wide-ranging, and includes large scale analysis of the ubiquitin-dependent proteolysis system, analyzing determinants of cellular responses to radicals and oxidative stress, determining factors controlling membrane transport of metals and peptide growth factors, analyzing signaling and transcriptional activation pathways in cell lineage determination, determining protein interactions involved in establishing cell polarity, and many others.
The grant awarded by NSF provides funds for the acquisition of a combined liquid chromatography tandem mass spectrometer system (LC/MS/MS). This instrument adds an essential component of modern proteomics technology to preexisting tools HSPH has committed to this initiative by enabling rapid and highly accurate protein identification from diverse biological samples. The mass spectrometer is integrated with a high performance liquid chromatography system required for sample fractionation. Sophisticated software assists in protein identification and database searching.
Apart from major scientific advances through proteomics technology, the main impact of this grant lies with the enhancement of the training environment at HSPH. The new research instrumentation provides students and postgraduates with hands-on experience in cutting edge proteomics technology, thereby optimally preparing them for the demanding mission of prevailing in the rapidly changing landscape of the post-genomic era. Through the Minority Internship Program administered by the Division of Biological Sciences at HSPH underrepresented groups will be actively recruited to the burgeoning filed of proteomics research.

Our training grant proposal "Interdisciplinary Training in Genetics and Complex Diseases" directly meets the need to develop pre- and post-doctoral training in an integrative approach to meet the challenges of today's public health science. Our goal is to develop a cadre of young scientists who can participate at the intersection of molecular biology, epidemiology, and biostatistics to become leaders in integrative and team approaches to understanding genetics and complex diseases in the public health arena. In the post-genomic era we are beginning to comprehend and compile the breadth of genetic variation within the human population. Refined use of this information requires the development of advanced methods of biostatistical analyses. In addition, modern epidemiological studies have evolved an enhanced view of health risk exposures to include factors such as diet, lifestyle, metabolic alterations, socioeconornic status along with environmental exposures (e.g., pollutants, toxicants). The latter expanded view provides more meaningful and precise studies of environmental contributions to complex disease. Finally, to make significant progress in disease prevention, a hallmark of public health, there is a pressing need to translate genetic advances into programs and policies focused on preventing common and costly chronic diseases. Only by understanding the importance of genetic profile can we identify who will truly benefit from public interventions. A new science and new scientific toolbox will be needed if we are to truly understand the nature of common genetic modifiers that interact with multiple environmental factors. We seek to establish a new track for interdisciplinary education that intersects the boundaries of molecular biology, epidemiology and biostatistics with a core foundation in cell physiology and metabolism that will develop key concepts focused on context-dependent gene-environment interactions in complex diseases. Our specific focus for trainees is on gene-environment interactions in the broadest possible sense, and on a generalized set of complex diseases rather than on an individual syndrome, with the recognition that science today is becoming substantially predicated in several "core" areas that intersect across disciplines. We will develop integrated coursework, sponsor workshops, establish a new seminar series, and foster interactive "cores" to enable our trainees to undertake the challenges that lie ahead to define the molecular signatures of disease patterns.

DESCRIPTION (provided by applicant): Our training grant proposal "Interdisciplinary Training in Genetics and Complex Diseases" directly meets the need to develop pre- and post-doctoral training in an integrative approach to meet the challenges of today s public health science. Our goal is to develop a cadre of young scientists who can participate at the intersection of molecular biology, epidemiology, and biostatistics to become leaders in integrative and team approaches to understanding genetics and complex diseases in the public health arena. In the post-genomic era we are beginning to comprehend and compile the breadth of genetic variation within the human population. Refined use of this information requires the development of advanced methods of biostatistical analyses. In addition, modern epidemiological studies have evolved an enhanced view of health risk exposures to include factors such as diet, lifestyle, metabolic alterations, socioeconomic status along with environmental exposures (e.g., pollutants, toxicants). The latter expanded view provides more meaningful and precise studies of environmental contributions to complex disease. Finally, to make significant progress in disease prevention, a hallmark of public health, there is a pressing need to translate genetic advances into programs and policies focused on preventing common and costly chronic diseases. Only by understanding the importance of genetic profile can we identify who will truly benefit from public interventions. A new science and new scientific toolbox will be needed if we are to truly understand the nature of common genetic modifiers that interact with multiple environmental factors. We seek to establish a new track for interdisciplinary education that intersects the boundaries of molecular biology, epidemiology and biostatistics with a core foundation in cell physiology and metabolism that will develop key concepts focused on context-dependent gene-environment interactions in complex diseases. Our specific focus for trainees is on gene-environment interactions in the broadest possible sense, and on a generalized set of complex diseases rather than on an individual syndrome, with the recognition that science today is becoming substantially predicated in several "core" areas that intersect across disciplines. We will develop integrated coursework, sponsor workshops, establish a new seminar series, and foster interactive "cores" to enable our trainees to undertake the challenges that lie ahead to define the molecular signatures of disease patterns.

DESCRIPTION (provided by applicant): Chemical genetics is an emerging field that takes advantage of combinatorial chemical and small molecule libraries to dissect complex biological processes. Small molecules can act very fast, can be very specific, and can help to distinguish the temporal order of molecular steps and the hierarchical regulation of biological processes. Because small molecules can alter the function of a specific gene product, they can be used in a manner analogous to the use of inducible dominant or homozygous recessive genetic mutations. A large body of biochemical literature is based on the past use of small molecule antagonists that were employed in "reverse chemical genetics" approaches to conditionally eliminate protein function, and on that basis to subsequently identify the target, its mechanism of action, and its regulation. Thus, ouabain helped to define the catalytic cycle of the NaK-ATPase, cytochalasin B was instrumental in defining the molecular basis for insulin's action to stimulate glucose uptake, and analogs of amiloride were used to purify and define the epithelial Na channel. There is a need to develop "forward chemical genetics" in order to discover small molecules that partner with key elements in a pathway of interest. This proposal is supported by preliminary data that establish a fluorescence-based assay to screen for inhibitors of iron uptake by mammalian cells. Iron deficiency remains the most prevalent nutritional problem in our country, yet recent identification of the gene responsible for hereditary hemochromatosis indicates that 1 in 20 Caucasians carry the defective allele and thus 1 in 400 may be susceptible to iron overload. Increased knowledge about the transport factors and how they protect against iron deficiency and overload is essential to more broadly address these significant health problems. Using the cell-based fluorescence assay, we propose to: 1) Perform chemical genetic screens for selective inhibitors of different pathways of iron transport using combinatorial libraries;2) Characterize the compounds identified to block iron uptake with highest potency;3) Develop structure-activity profiles on compounds of interest and identify their targets. The goals of this project are to discover small molecule inhibitors of iron transport using chemical genetics and to use these reagents to advance our understanding of the factors, mechanisms, and regulation of different pathways of iron uptake.

Our training grant proposal "Interdisciplinary Training in Genetics and Complex Diseases" directly meets the need to develop pre- and post-doctoral training in an integrative approach to meet the challenges of today's public health science. Our goal is to develop a cadre of young scientists who can participate at the intersection of molecular biology, epidemiology, and biostatistics to become leaders in integrative and team approaches to understanding genetics and complex diseases in the public health arena. In the post-genomic era we are beginning to comprehend and compile the breadth of genetic variation within the human population. Refined use of this information requires the development of advanced methods of biostatistical analyses. In addition, modern epidemiological studies have evolved an enhanced view of health risk exposures to include factors such as diet, lifestyle, metabolic alterations, socioeconornic status along with environmental exposures (e.g., pollutants, toxicants). The latter expanded view provides more meaningful and precise studies of environmental contributions to complex disease. Finally, to make significant progress in disease prevention, a hallmark of public health, there is a pressing need to translate genetic advances into programs and policies focused on preventing common and costly chronic diseases. Only by understanding the importance of genetic profile can we identify who will truly benefit from public interventions. A new science and new scientific toolbox will be needed if we are to truly understand the nature of common genetic modifiers that interact with multiple environmental factors. We seek to establish a new track for interdisciplinary education that intersects the boundaries of molecular biology, epidemiology and biostatistics with a core foundation in cell physiology and metabolism that will develop key concepts focused on context-dependent gene-environment interactions in complex diseases. Our specific focus for trainees is on gene-environment interactions in the broadest possible sense, and on a generalized set of complex diseases rather than on an individual syndrome, with the recognition that science today is becoming substantially predicated in several "core" areas that intersect across disciplines. We will develop integrated coursework, sponsor workshops, establish a new seminar series, and foster interactive "cores" to enable our trainees to undertake the challenges that lie ahead to define the molecular signatures of disease patterns.

DESCRIPTION (provided by applicant): Our training grant proposal "Interdisciplinary Training in Genetics and Complex Diseases" directly meets the need to develop pre- and post-doctoral training in an integrative approach to meet the challenges of today s public health science. Our goal is to develop a cadre of young scientists who can participate at the intersection of molecular biology, epidemiology, and biostatistics to become leaders in integrative and team approaches to understanding genetics and complex diseases in the public health arena. In the post-genomic era we are beginning to comprehend and compile the breadth of genetic variation within the human population. Refined use of this information requires the development of advanced methods of biostatistical analyses. In addition, modern epidemiological studies have evolved an enhanced view of health risk exposures to include factors such as diet, lifestyle, metabolic alterations, socioeconomic status along with environmental exposures (e.g., pollutants, toxicants). The latter expanded view provides more meaningful and precise studies of environmental contributions to complex disease. Finally, to make significant progress in disease prevention, a hallmark of public health, there is a pressing need to translate genetic advances into programs and policies focused on preventing common and costly chronic diseases. Only by understanding the importance of genetic profile can we identify who will truly benefit from public interventions. A new science and new scientific toolbox will be needed if we are to truly understand the nature of common genetic modifiers that interact with multiple environmental factors. We seek to establish a new track for interdisciplinary education that intersects the boundaries of molecular biology, epidemiology and biostatistics with a core foundation in cell physiology and metabolism that will develop key concepts focused on context-dependent gene-environment interactions in complex diseases. Our specific focus for trainees is on gene-environment interactions in the broadest possible sense, and on a generalized set of complex diseases rather than on an individual syndrome, with the recognition that science today is becoming substantially predicated in several "core" areas that intersect across disciplines. We will develop integrated coursework, sponsor workshops, establish a new seminar series, and foster interactive "cores" to enable our trainees to undertake the challenges that lie ahead to define the molecular signatures of disease patterns.

Our training grant proposal "Interdisciplinary Training in Genetics and Complex Diseases" directly meets the need to develop pre- and post-doctoral training in an integrative approach to meet the challenges of today's public health science. Our goal is to develop a cadre of young scientists who can participate at the intersection of molecular biology, epidemiology, and biostatistics to become leaders in integrative and team approaches to understanding genetics and complex diseases in the public health arena. In the post-genomic era we are beginning to comprehend and compile the breadth of genetic variation within the human population. Refined use of this information requires the development of advanced methods of biostatistical analyses. In addition, modern epidemiological studies have evolved an enhanced view of health risk exposures to include factors such as diet, lifestyle, metabolic alterations, socioeconornic status along with environmental exposures (e.g., pollutants, toxicants). The latter expanded view provides more meaningful and precise studies of environmental contributions to complex disease. Finally, to make significant progress in disease prevention, a hallmark of public health, there is a pressing need to translate genetic advances into programs and policies focused on preventing common and costly chronic diseases. Only by understanding the importance of genetic profile can we identify who will truly benefit from public interventions. A new science and new scientific toolbox will be needed if we are to truly understand the nature of common genetic modifiers that interact with multiple environmental factors. We seek to establish a new track for interdisciplinary education that intersects the boundaries of molecular biology, epidemiology and biostatistics with a core foundation in cell physiology and metabolism that will develop key concepts focused on context-dependent gene-environment interactions in complex diseases. Our specific focus for trainees is on gene-environment interactions in the broadest possible sense, and on a generalized set of complex diseases rather than on an individual syndrome, with the recognition that science today is becoming substantially predicated in several "core" areas that intersect across disciplines. We will develop integrated coursework, sponsor workshops, establish a new seminar series, and foster interactive "cores" to enable our trainees to undertake the challenges that lie ahead to define the molecular signatures of disease patterns.

[unreadable] DESCRIPTION (provided by applicant): A significant relationship exists between iron nutrition and cognition and behavior. Behavioral problems are also observed in children with high manganese and manganese neurotoxicity resulting in a Parkinson-like disorder that is widely recognized in workers employed in mining and manganese ore processing. Our recent work has established that iron deficiency enhances olfactory uptake of manganese and promotes accumulation of this toxic metal in the basal ganglia. Thus, the major underlying hypothesis of this proposal is that absorption of inhaled manganese is up-regulated upon iron-deficiency such that neurological complications of poor iron status are compounded by an increased vulnerability to the toxic effects of manganese exposure. The proposed research will contribute fundamental understanding of physiological risks associated with metal-induced toxicity and, more specifically, the interactions between iron status and manganese neurotoxicity. To accomplish this goal we will: 1) Determine the distribution of intranasally instilled manganese in the brain of control and iron-deficient rats using magnetic resonance imaging; 2) Determine motor coordination and learning/memory capacity of exposed and non-exposed cohorts through use of rotorod and bridge-walking as well Morris water maze tests; and 3) Examine CNS damage due to manganese intoxication in control and iron-deficient rats by determining indices of oxidative stress (3- nitrotyrosine and 8-hydroxy-2'deoxyguanosine levels), identifying neuronal degeneration (Fluoro-Jade staining) and evaluating astrocyte viability using anti-fibrillary acidic protein (GFAP) and microglia status using anti-isolectin B4. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The final step of dietary iron assimilation - the exsorption of iron from the mucosa into circulation - involves the iron transport protein ferroportin. Macrophages of the reticuloendothelial system also play a major role in iron metabolism by recycling iron from damaged or senescent erythrocytes. Ferroportin also plays a major role in the release of iron from macrophages, and thus provides the major mechanism for the export of iron from the cells of our body. Body iron stores, hypoxia, the rate of erythropoiesis, pregnancy and inflammation all have dramatic effects on dietary iron assimilation and iron recycling by the reticuloendothelial system. How such factors influence the activity of ferroportin to modify iron export is an active area of interest in the field. The most important recent discovery has been the role of the liver-derived peptide hepcidin. Hepcidin is produced under iron- loading and inflammatory conditions to suppress dietary iron absorption and macrophage iron recycling, and its synthesis is diminished in response to iron deficiency, pregnancy or enhanced erythropoiesis to promote iron uptake from the diet and enhance iron recycling. This peptide therefore provides a direct link between the liver, bone marrow and intestine to adjust metabolism to meet the body's demand for iron. Hepcidin regulates ferroportin protein levels through binding interactions that induce its internalization and lysosomal degradation. The model that ferroportin is a receptor for hepcidin implicates a homeostatic mechanism that allows systemic regulation of intestinal iron absorption and macrophage iron recycling directly in response to the body's iron demands targeting ferroportin function. This model is borne out by clinical studies of hemochromatosis, an inherited disorder leading to abnormal iron accumulation in different tissues. This disease is associated with mutations in genes encoding HFE, hepcidin, hemojuvelin, transferrin receptor-2 or FPN. Although the molecular pathogenesis has not been precisely elucidated in all cases, abnormal regulation of iron metabolism by hepcidin has been proposed as a common, unifying feature of the different forms of this disease. The specific aim of this R21 proposal is to develop a high throughput assay to screen for small molecule inhibitors of hepcidin-ferroportin binding interactions. Our strategy is to utilize Sfp-catalyzed site-specific protein labeling to establish a rapid, sensitive, and reproducible fluorescent-based screen for small molecules that block ligand-induced degradation of the hepcidin receptor, ferroportin. The project has broader applicability in the development of HTS to assay ligand-receptor interactions as well as representing a major advance towards the discovery of small molecule probes of iron transport.

DESCRIPTION (provided by applicant): Our training grant proposal "Interdisciplinary Training in Genetics and Complex Diseases" directly meets the need to develop pre- and post-doctoral training in an integrative approach to meet the challenges of today s public health science. Our goal is to develop a cadre of young scientists who can participate at the intersection of molecular biology, epidemiology, and biostatistics to become leaders in integrative and team approaches to understanding genetics and complex diseases in the public health arena. In the post-genomic era we are beginning to comprehend and compile the breadth of genetic variation within the human population. Refined use of this information requires the development of advanced methods of biostatistical analyses. In addition, modern epidemiological studies have evolved an enhanced view of health risk exposures to include factors such as diet, lifestyle, metabolic alterations, socioeconomic status along with environmental exposures (e.g., pollutants, toxicants). The latter expanded view provides more meaningful and precise studies of environmental contributions to complex disease. Finally, to make significant progress in disease prevention, a hallmark of public health, there is a pressing need to translate genetic advances into programs and policies focused on preventing common and costly chronic diseases. Only by understanding the importance of genetic profile can we identify who will truly benefit from public interventions. A new science and new scientific toolbox will be needed if we are to truly understand the nature of common genetic modifiers that interact with multiple environmental factors. We seek to establish a new track for interdisciplinary education that intersects the boundaries of molecular biology, epidemiology and biostatistics with a core foundation in cell physiology and metabolism that will develop key concepts focused on context-dependent gene-environment interactions in complex diseases. Our specific focus for trainees is on gene-environment interactions in the broadest possible sense, and on a generalized set of complex diseases rather than on an individual syndrome, with the recognition that science today is becoming substantially predicated in several "core" areas that intersect across disciplines. We will develop integrated coursework, sponsor workshops, establish a new seminar series, and foster interactive "cores" to enable our trainees to undertake the challenges that lie ahead to define the molecular signatures of disease patterns.

[unreadable] DESCRIPTION (provided by applicant) [unreadable] [unreadable] The Interdisciplinary Training in Genes and the Environment program at the Harvard School of Public Health (HSPH) will address the critical need for well-trained scientists who have an understanding of, and commitment to, cutting-edge research at the intersection of molecular and environmental exposure biology, and statistical and computational methods. The training program will involve active participation by 30 accomplished and experienced multidisciplinary faculty members, including environmental health scientists, molecular biologists, molecular epidemiologists, computational biologists, biostatisticians and bioinformaticians. The two interrelated goals of our proposed training program are: To train true collaborative partners able to pursue methodological research that is motivated by, and helps to solve, difficult analytic issues that arise in studies of human environmental exposures and genetic susceptibility to complex diseases; To encourage interdisciplinary research, especially in genetics and the various "omics" arising from new methodologies for characterizing biological activity associated with environmental exposures in laboratory and population sciences. Trainees will be pre-doctoral students and post-doctoral fellows at HSPH in the Departments of Environmental Health, Epidemiology, Biostatistics, and Genetics and Complex Diseases, which will jointly administer the grant. The program proposes initial support for 3 pre-doctoral students with 2 additional trainees in year 2 and 3 additional trainees in year 3 to bring an annual total of 8 pre-doctoral students into the program (year 3-8). The program also plans to support training of one post-doctoral fellow per year. These post-doctoral trainees will have an advanced degree (PhD, MD, MD-PhD or other doctoral degree) relevant to exposure biology/environmental health sciences or computational areas of genomics/proteomics and will have a specific interest in cross-training research experience. All trainees in the Interdisciplinary Training in Genes and Environment program will be provided an outstanding opportunity to become equally skilled in genomics, environmental health sciences and quantitative methods in order to attain leadership roles in interdisciplinary studies of human genes and the environment, with the ultimate goal of serving public health interests in developing effective disease prevention and intervention strategies. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): ?9-tetrahydrocannabinol (?9-THC) is the major psychoactive component of the marijuana plant Cannabis sativa, and it produces a number of behavioral and pharmacological effects mediated through interactions with the central nervous system cannabinoid receptor CB1 and the peripheral receptor CB2. Endogenous cannabinoids also activate these G-protein coupled receptors to negatively regulate adenylate cyclase activity and positively regulate inward rectifying K+ channels. Receptor-independent effects of cannabinoids have also been reported to negatively regulate a number of ion channels, including T-type Ca2+ channels, TASK-1 channels, and Na+ channels. In a recent screen of small molecule libraries containing known bioactive molecules, it was discovered that ?9-THC also potently blocks iron uptake by Divalent Metal Transporter-1 (DMT1). DMT1 is responsible for dietary iron absorption across the intestine as well as the delivery of iron to peripheral tissues after receptor-mediated uptake of the serum iron-binding protein transferrin. This completely unexpected and novel finding raises immediate and profound questions about the relationships between iron metabolism and cannabinoid action. To develop new insights to explain how cannabinoids perturb iron transport by DMT1 and to determine what physiological consequences ensue from this activity, the proposed project will: 1) Examine changes in the iron status of weanling rats resulting from chronic ?9-THC administration; 2) Explore G protein-coupled regulation of DMT1 activity through CB receptor interactions; and 3) Study binding of cannabinoids to DMT1 and changes in the phosphorylation state of the transporter that may be elicited through such interactions. PUBLIC HEALTH RELEVANCE: This project will determine how cannabinoids interfere with iron uptake by Divalent Metal Transporter-1. There is significant interest in identifying targets of cannabinoid action since drugs that modify endogenous cannabinoid activity are being developed to control obesity (Rimonabant), to prevent osteoporosis (HU-308) and to treat multiple sclerosis (Sativex), and are in current use for AIDS and cancer patients (Marinol). [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Genetic variants of the HFE gene are the leading cause of adult onset hereditary hemochromatosis (HH), the most common Mendelian genetic disease in the North American Caucasian population. The missense variants C282Y and H63D promote increased intestinal absorption and progressive tissue deposition of iron. HH has also been associated with derangements in the metabolism of other divalent metals, and it is thought these effects arise due to malregulation of divalent metal transporter (DMT1). Work in our laboratory has demonstrated that manganese can enter the central nervous system directly across the olfactory epithelium by a mechanism that involves DMT1. We therefore hypothesize that carriers of C282Y and/or H63D HFE alleles may be more susceptible to manganese exposure through the olfactory pathway, and consequently may have impaired olfactory function. Our model further suggests that Hfe-/- knockout mice would have increased 54Mn absorption through the olfactory system and that these animals would be more sensitive to manganese exposures causing impaired olfaction. This pilot project will test the hypothesis that HFE acts as a genetic determinant of olfactory manganese absorption and toxicity. PUBLIC HEALTH RELEVANCE: Genetic variants of the HFE gene are the leading cause of adult onset hereditary hemochromatosis (HH), the most common Mendelian genetic disease in the North American Caucasian population. The missense variants C282Y and H63D promote increased intestinal absorption and progressive tissue deposition of iron. Our hypothesis is that HFE acts as a genetic determinant of olfactory manganese absorption and toxicity. To test this hypothesis, we will study a mouse model of HFE-associated hemochromatosis to test whether absorption of inhaled manganese is altered, and whether HFE therefore promotes a greater susceptibility to damage to olfaction - the sense of smell.

DESCRIPTION (provided by applicant): This application addresses the broad Challenge Area 15, Translational Sciences, and specific challenge topic 15-DK-103: Translate discovery of new molecules and pathways in pathogenesis of NIDDK diseases into potential therapies, diagnostics, or research tools. The biochemistry of iron transport is not thoroughly understood. Although iron deficiency is the most prevalent nutritional problem in the U.S., 1 in 20 Caucasians carry genetic variants of HFE alleles that promote susceptibility to iron overload. Thus, there is a need to develop new therapeutic strategies for diseases of both iron deficiency and overload. Through high-throughput fluorescence-based screening, our lab recently discovered that ferristatin (NSC306711) inhibits both of the major iron transport processes that maintain homeostasis: transferrin- mediated iron uptake and non-transferrin-bound iron uptake by Divalent Metal Transporter 1 (DMT1). Chlorazol black (NSC8679) is structurally similar and has comparable effects on transport. This project will further investigate the impact of these two small molecules and related compounds in vivo, on a) pharmacokinetics of intestinal iron uptake to the vasculature;b) iron uptake into erythroid cells and hepatic non-transferrin bound iron uptake;and c) iron homeostasis and reversal of overload. These efforts will further our goals to elucidate the biochemical processes regulating iron homeostasis, and to provide a foundation for the development of targeted small-molecule therapies for states of anemia and hemochromatosis. PUBLIC HEALTH RELEVANCE: Iron deficiency remains the most prevalent nutritional problem in our country, yet recent identification of the gene responsible for hereditary hemochromatosis indicates that 1 in 20 Caucasians carry the defective allele and thus 1 in 400 may be susceptible to iron overload. Increased knowledge about the transport factors and how they protect against iron deficiency and overload is essential to more broadly address these significant health problems.

DESCRIPTION (provided by applicant): The proposed R25 Summer Intern Program (SIP) in Environmental Health Sciences seeks to offer summer research training in environmental health sciences for 10 outstanding under-represented minority undergraduate students from across the nation. These interns will participate in the investigators well-established and comprehensive 9-week laboratory training program at Harvard School of Public Health in conjunction with its Ph.D. Program in Biological Sciences in Public Health. HSPH has pledged to match NIH support to enhance the experience offered by its nationally recognized summer internship. The investigators will provide hands-on training in bench research, develop lasting mentoring relationships between summer undergraduate interns and ten outstanding HSPH faculty, and promote interest in environmental health graduate science careers by organizing curricular and extracurricular activities for interns enrolled in their program. All training program faculty are experienced in serving as mentors for minority students and have worked with their summer program for several years. Additionally, these faculty members have active research programs in a broad spectrum of research areas, including environmental health, genetics, molecular epidemiology and biomarkers, toxicology, and mechanistic disease research. This breadth of interest will benefit their program as each of the faculty will speak to the students about their research and professional development to foster careers in environmental sciences. Prospective applicants from across the nation will be evaluated for their potential based on statement of purpose, coursework and grades at the undergraduate level, and letters of reference. Those accepted into the program will begin their summer experience the second or third week in June. The SIP includes an individualized supervised research project in HSPH faculty laboratories, laboratory safety training, responsible conduct of science, participation in a weekly seminar series focusing on environmental health sciences, formal and informal mentoring lunches with HSPH faculty and graduate students, university-wide lectures bringing together summer interns across the university, afternoon and evening socials, practical preparation for the graduate school admission process, and Harvard University-sponsored Science Symposium for trainees to present the results of their research. The program evaluation will assess how well the SIP enhanced the motivation and academic preparation and participation in environmental health careers. The specific aim is to motivate and enhance the pipeline of underrepresented minority students seeking graduate level training in the environmental health sciences.

DESCRIPTION (provided by applicant): We advance the hypothesis that hepcidin regulates manganese metabolism. Accumulating evidence from in vitro studies suggests that ferroportin, the target of hepcidin regulation, plays a role in the transport of manganese. Our in vivo studies of Hfe-/- mice strongly support this idea. We will directly test its function in manganese metabolism using flatiron (ffe+/-) mice as a genetic model of ferroportin deficiency. While ferroportin function in iron export by intestinal enterocytes and macrophages of the reticuloendothelial system has been established, its activity has yet-to-be fully explored in hepatocytes where the exporter also is highly expressed. We hypothesize that in the liver, ferroportin functions in biliary excretion of manganese, a known homeostatic pathway that clears excess metal from the body. Based on our model, we speculate that circulating manganese levels are suppressed during inflammation by hepcidin, an idea that is well-supported by known host-pathogen interactions. Finally, we will determine the mechanism responsible for increased olfactory manganese absorption we have observed in Hfe knockout mice. The original new ideas forming the basis of our research will have a powerful sustaining influence on the field of metal metabolism by creating new paradigms to explain the molecular basis for manganese homeostasis.

Neurodegenerative diseases of human aging are significant US health burdens due to the growing number of people living with dementia. Many disorders like Alzheimer's disease are associated with excess brain iron that accumulates with age. Such observations have led to the metal theory of dementia, which suggests that over time, environmental exposure to iron promotes neurodegeneration. We have discovered that ?9-THC and other cannabinoids inhibit the iron transporter divalent metal transporter-1 (DMT1) through cannabinoid receptor-2 (CB2). CB2 is an immunomodulatory receptor and its anti-inflammatory neuroprotective effects confer suppression of microglia activation. Microglial cells act as the immune cells of the brain and spinal cord, becoming activated by changes in their local microenvironment. Microglia polarize between reactive and repair states to actively transition from an immune-stimulating antimicrobial phenotype to one that supports tissue repair and resolution of inflammation. SPECIFIC AIM 1: Determine mechanisms of microglial iron transport and metabolism. We have determined that IMG cell iron uptake corresponds to the M1/M2 activation state of IMG cells. While more non-transferrin (Tf) bound iron (NTBI) is taken up by LPS-treated cells, Tf-mediated transport is increased by IL-4. Based on our data, we hypothesize that microglial cell polarization directs iron trafficking. We will: a) Test the hypothesis that microglial cell polarization controls uptake and metabolic partitioning of iron into subcellular compartments; and b) Test the hypothesis that IMG cell activation by A? alters microglia metabolism by up- regulating DMT1 activity and dysregulating energy metabolism. SPECIFIC AIM 2: Determine influence of CB2 on microglial iron transport and metabolism. We have determined the CB2 selective agonist JWH102 reduces the pro-inflammatory activation state of IMG cells promoted by A?. We have also found that CB2 promotes dephosphorylation of DMT1 to block its activation. Based on our data, we hypothesize that CB2 redirects iron trafficking and cellular metabolism by regulating DMT1 phosphorylation. We will: a) Test the hypothesis that the CB2 selective agonist JWH102 reduces NTBI iron transport and shifts energy metabolism in LPS and A?-activated IMG cells; and b) Test the hypothesis that CB2 regulates DMT1 phosphorylation to control is activity in IMG cells.