Stephen L. Helfand - US grants

This proposal is for an individual Physician Scientist Award to receive intensive training in basic biomedical research in the field of developmental neurobiology. Training will be carried out in Dr. Corey Goodman's laboratory at Stanford University. Phase I of the proposal includes both didactic study and intense laboratory experience. The applicant will be involved in courses, seminars, extensive literature reviews, and tutorials in advanced developmental biology, developmental neurobiology, molecular neurobiology, and eukaryotic gene expression. At the same time, the program will include extensive research training in monoclonal antibody technology, Drosophila embryology, biochemistry, and cDNA expression cloning technology. Phase II will be fully devoted to intensive research on the embryogenesis of neuronal specificity. The long term goal of this research program is to understand the molecular mechanism underlying cell recognition during the embryonic development of the central nervous system. An ideal model system for such molecular genetic studies is the Drosophila embryo. Previous studies provided evidence for the presence of molecular differences on the surfaces of axon fascicles during embryonic development. In order to isolate and characterize these surface molecules involved in cell recognition during neuronal development, monoclonal antibody and cDNA expression cloning technologies will be applied to the Drosophila embryo. The specific aims are: (i) to generate monoclonal antibodies that recognize surface molecules on specific subsets of embryonic neurons; and (ii) to screen these MAbs on a cDNA expression library in an attempt to isolate the genes which encode these molecules.

Understanding the biological mechanisms underlying the aging process is one of the most important and urgent problems of modern biomedical science. As the demographics of the industrialized countries have changed, age-related diseases such as cancer, cardiovascular disease, stroke, osteoporosis, and Alzheimer's syndrome have taken on epidemic proportions. It is only through a thorough understanding of the aging process itself that we can begin to design rational therapeutic interventions for the alleviation of the pain of old age and its associated disorders. This proposal concerns the development and use of an animal model system for examining the relationship between gene expression and aging. Using our model system we have been able to demonstrate the existence of age-dependent gene regulation in the adult. Documentation of genes showing age-dependent regulation supports the importance of gene regulation in the aging process, confirms the existence of genetic mechanisms for its control, and provides a model system for aging which is amenable to direct genetic and molecular analysis. Through the use of our molecular biomarkers and a combination of new and traditional molecular genetic approaches we will be able to identify and isolate genetic elements that are important in setting up and controlling gene expression during the aging process.

The long-term goal of our studies is to understand the molecular and genetic elements that underlie the process of aging and determine longevity. The aim of this proposal is to examine how mutations in a single gene, Indy, lead to a dramatic increase in life span in Drosophila melanogaster. We present data on the isolation and initial characterization of three independent P-element insertional mutations in the same gene, Indy, which cause a large increase in life span, and show that excision of the insert leads to reversion to a normal life span. The distribution of expression of the Indy gene, primarily in the fat body and oenocytes, and predicted sequence of the Indy protein, suggest that Indy may be playing a role in intermediary metabolism. The molecular and genetic analyses indicate that a partial reduction in the activity of this gene is responsible for an extension in life span, while a more severe reduction is detrimental to long life. The molecular, genetic, and physiological studies in this proposal will help provide an understanding of how an alteration in this single gene can lead to such a profound effect on increasing longevity.

DESCRIPTION (provided by applicant): The focus of this proposal is to determine how changes in chromatin with age contribute to the deleterious consequences of aging, and to develop new genetic and pharmacological interventions that extend healthy life span by effecting a more youthful chromatin state. With age, repressive heterochromatin decreases, and both expression and mobilization of transposable elements (TEs) increase. We hypothesize that expression and mobilization of TEs contributes to aging and age-related disorders, and propose to exploit one of the cellular systems that normally suppresses TE expression and expansion, constitutive repressive heterochromatin, in order to maintain silencing of TEs and promote a longer, healthier life. We will use genome-wide approaches and interventions that affect life span, including Dietary Restriction (DR), and interventions predicted to maintain repressive heterochromatin, in order to establish the relationship between chromatin states, TE expression and transposition, and longevity. We will utilize the esiRNA RISC pathway that naturally suppresses TE expression and mobilization, in order to test directly whether repression of TE activity is an important component of longevity. The aims of this proposal are to test the hypotheses that: (i) age-related decreases in repressive heterochromatin lead to increased TE activity; (ii) interventions that extend life span maintain repressive heterochromatin, thereby preventing increases in TE activity; (iii) interventions that increase repressive heterochromatin reduce TE activity and extend life span; and (iv) repression of TE expression and TE mobilization are important elements in longevity determination. To better understand the relationship between TE activity, the cellular mechanisms that repress it, and longevity, as well as to identify new chromatin-related pathways that can extend healthy life span, we will: (1) use RNA-seq, genomic deep-sequencing, ChIP-seq (H3K9me2, H3K9Me3, HP1) and FAIRE-seq, to test that with age there is a loss of repressive heterochromatin that is associated with increased TE expression, and that life span-extending interventions, including DR, restore repressive heterochromatin and suppress TE activity; (2) use molecular genetic manipulations known to maintain or increase repressive heterochromatin with age to test that a reduction of TE expression and mobilization will extend life span; (3) determine how the activity of the esiRNA pathway (Dicer-2, Ago2 and R2D2) is altered with age and in life span-extending interventions; and (4) use molecular genetic tools to directly alter the activity of the esiRNA pathway to affect TE expression and mobilization, in order to determine the effects of these manipulations on normal aging and on the outcome of life span-extending interventions.

DESCRIPTION (provided by applicant): The long-term goal of our studies is to understand the molecular and genetic elements that underlie the process of aging and determine longevity. The aim of this proposal is to understand how mutations in a single gene, Indy, result in a dramatic increase in life span in Drosophila melanogaster without a concomitant loss of reproduction, physical activity or metabolic rate. In particular we will seek to determine where and when Indy mutations act to extend life span. The function of the INDY protein as a transporter of Krebs cycle intermediates and its preliminary localization to regions of the fly important in uptake, utilization and storage of nutrients, indicate that reductions in the level of INDY protein alters the metabolic state of the fly in a way that favors life span extension. INDY's similarity in sequence, function, and tissue expression to mammalian and human dicarboxylate transporters suggests that knowledge of how Indy mutations extend life span in flies may be useful for the development of therapeutic interventions for extending healthy life in humans. We will first examine the tissues and times during life INDY expression is altered in the long-lived Indy mutant animals. Using molecular genetic approaches we will restore Indy function to directly determine where and when Indy mutations act to extend life span. Finally we will determine which of the several possible human Indy-like genes can functionally rescue the Indy mutation. A more complete understanding of how mutations in Indy lead to life span extension should yield valuable insights into general mechanisms of life span extension relevant to a variety of organisms including humans.

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of our studies is to understand the molecular and genetic elements that underlie the process of aging and determine longevity. One of the most prominent hypotheses explaining the aging process is the oxidative stress hypothesis, which states that the rate of aging and life span is directly related to the accumulation of oxidative damage to organelles and macromolecules. Comparative studies between species having different life spans have been one of the foundations of the oxidative stress hypothesis, predicting a direct relationship between the accumulation of oxidative damage, the rate of aging and life span. We propose to use the fruit fly model system, Drosophila melanogaster, to compare the relationship between life span and the accrual of oxidative damage within a single, genetically well defined species, using a variety of strains and environmental and genetic conditions known to alter life span. These studies will better define the precise relationship between oxidative damage and life span, laying the foundation for a map detailing the specific quantitative relationship between oxidative damage, aging and life span. An additional advantage of using Drosophila to perform these comparative studies is that the powerful molecular and genetic techniques available in Drosophila can be used to directly identify and test specific physiological systems important in aging. [unreadable] [unreadable] [unreadable]

PROJECT SUMMARY (PROJECT 2) The focus of this project is to exploit the Drosophila model system to study the role of retrotransposable elements (RTEs) in the progression of cellular dysfunction that occurs during aging, and to develop inter- ventions that suppress RTE activity and extend healthy life span. The maintenance of repressive hetero- chromatin declines with age, resulting in increased expression and mobilization of RTEs across species as diverse as fruitflies and mice. We hypothesize that the increase in the expression and mobilization of RTEs leads to an accumulation of damage to the genome of somatic cells, causing loss of cellular and organismal homeostasis, and thus promoting aging. We will use the powerful molecular and genetic tools and the short life span of Drosophila to determine the effects of RTEs on aging, and in particular, we will do this over the entire life span of the organism, something that would not be possible in humans, and would be prohibitively time- consuming and expensive in any mammalian model system on a large scale. Confirmation of a mechanistic relationship between RTE activity and aging will provide the groundwork for developing interventions that diminish RTE activity and genomic damage during aging, and should extend healthy life span. The aims of this proposal are to test the hypotheses that: (i) loss of chromatin-regulated suppression of RTE activity with age is associated with increased DNA damage and genome instability; (ii) decreases in sirtuins with age lead to elevated RTE activity that can be reversed by increasing sirtuin activity; and (iii) interventions that suppress RTE activity in somatic cells can extend healthy life span. To better understand the relationship between RTE activity, genomic damage and longevity, as well as to develop new interventions that can extend healthy life span, in Aim 1 we will develop high throughput DNA sequencing methods (collaboration with Project 1, Core B), deploy available and new RTE reporters (collaboration with Core B) to measure mobilization of RTEs in aging Drosophila, and carry out a forward genetic screens to identify new genes and pathways involved in the suppression of RTEs. We will examine the effects of (i) age, (ii) dietary restriction (DR), and (iii) genetic interventions that stabilize heterochromatin, such as increasing Su(var)3-9, HP1a, Dicer-2, Piwi or decreasing Adar expression, on RTE activity and mobilization. In Aim 2, to determine the role of Sir2 and Sirt6 in the repression of RTE activity during aging, we will combine the tools developed and utilized in Aim 1 to monitor RTE activity with genetic manipulations that increase or decrease the expression of Sir2 and of Sirt6 activity (collaboration with Project 3 and Core B). In Aim 3, we will (i) determine the effects of the genetic interventions developed in Aims 1 and 2 on the life span of flies (collaboration with Projects 1 and 3 and Core B), and (ii) test the effects of nucleoside reverse transcriptase inhibitors (nRTIs) and RNAi knockdowns of specific retrotransposons on RTE mobilization and lifespan (collaboration with Projects 1 and 3 and Core B).

PROJECT SUMMARY/ABSTRACT The focus of this proposal is to generate ?humanized? models of AD in Drosophila with robust AD-relevant phenotypes and utilize them to identify proximal causes of AD, reliable biomarkers of disease progression, and discover novel avenues to treatments. Unlike transgenic over-expression models, our ?humanized? genetic model will convert the endogenous fly dAPPL, psn, and dBACE genes to their human counterparts. This will preserve all of the complexities of human APP metabolism in the fly by recapitulating key aspects of human AD in vivo in this experimentally malleable model organism, providing a unique and valuable platform for the discovery of new and novel genetic and pharmacological interventions for the treatment of AD. A systems biology approach will be employed to integrate gene expression and metabolomics with relevant AD phenotypes using these genetic models to facilitate our understanding of the proximate causes of AD and for the development of biomarkers of disease progression. Finally, these new ?humanized? AD models will be used in powerful forward genetics screens in Drosophila for suppressors of AD disease to directly identify novel proteins and pathways that reveal new avenues to effective therapies for the treatment of AD. The aims of this proposal are to: (i) develop new ?humanized? models of AD in the fly by humanizing the fly cognate genes to be dh(drosophila-to-human)APP, dhPSN and dhBACE, and introducing a series of known human familial early onset AD mutations in these humanized genes to perturb the endogenous production of human A? in flies to create fly models with robust AD-relevant phenotypes; (ii) assess genetic interactions between humanized loci for dhAPP, dhPSN and dhBACE to determine whether certain combinations of human AD mutant alleles achieve maximal A? production and accelerate phenotypes; (iii) utilize systems biology approaches to integrate transcriptomics and metabolomics with disease phenotypes to determine the molecular and genetic pathways associated with disease progression and to develop reliable biomarkers of disease progression; and (iv) make use of the enormous power of fly genetics to perform an unbiased forward genetic suppressor screen on the most robust AD genetic model to identify new targets for treatment of AD. To better understand the proximal molecular events leading to the progression to neurodegeneration and dementia in AD and to identify new interventions for its treatment , we will develop: (i) new humanized fly models for AD in which flies will express normal and mutated human proteins from AD-relevant cognate fly genes in order to replicate the important pathological processes of human AD in Aim 1; (ii) utilize RNA-seq and metabolomics to measure the transcriptome and metabolome of the humanized AD models and employ systems biological approaches to integrate these data with AD phenotypes in Aim 1 and 2; (iii) perform an unbiased forward genetic screen for suppressors of the robust human AD genetic models in the fly in order to identify proteins and pathways involved in AD in Aim 3.

Project Summary/Abstract: (30 lines maximum) The overall goal of this proposal is to develop genetic and pharmacological interventions that will delay the onset and progression of Alzheimer?s Disease (AD). AD and other Related Dementias (ADRD) affect well over 5 million people in the United States. By mid-century, these numbers are predicted to at least triple. Despite this, there are no effective treatments for these devastating illnesses. The onset and progression of AD has been linked to age-related changes in several critical physiological pathways including cellular and mitochondrial metabolism and genomic stability. We propose the age-related decline in the efficacy of these pathways promotes AD-related neurodegeneration and interventions which improve and stabilize physiological pathways leading to extension of healthy life span will delay the onset and progression of AD. In this proposal we will use the fly and mouse AD models in combination to determine whether potential genetic and pharmacological geroprotectors known to extend life span or health span in flies and mice, through an enhancement of the activity of cellular and mitochondrial metabolism or improvement in genomic stability, delay the onset and progression of neurodegeneration and other AD-related phenotypes in AD models in mice and flies. The goal of these studies is to identify new and novel genetic and pharmacological geroprotectors that can be translated for use in the treatment of AD. We will use specific molecular genetic and pharmacological interventions known to extend life span in flies or to extend health span in mice in the context of molecular genetic models of fly and mouse AD. The effect of each intervention will be evaluated in two different fly AD models (neuronal-specific expression of human Aß42 or human tau) and the 5XFAD mouse AD model. We will test the hypotheses that: (i) genetic interventions known to improve cellular and mitochondrial metabolism or genomic stability and to extend life span in flies or health span in mammals serve as geroprotectors that can delay the onset and progression of the neurodegeneration associated phenotypes in the fly and mouse models of AD and (ii) pharmacological interventions known to improve cellular and mitochondrial metabolism or genomic stability and extend life span in flies or health span in mammals can delay the onset and progression of the neurodegeneration associated phenotypes in the fly and mouse AD models. We will determine whether these interventions delay the onset and progression of a series of AD-related neurodegeneration phenotypes including: (i) decline in whole organismal health (life span in flies, frailty in mice); (ii) loss of neurobehavioral robustness or resilience (mobility and circadian rhythms in flies, a series of neurobehavioral tests in mice); (iii) microscopic histological examination of neurodegeneration in the brain and the retina of flies and gross and microscopic neuro-pathological examination of the relevant regions of the mouse brain; and (iv) molecular changes in mice.