Thomas A. Jongens - US grants

The long term objective of this application is to gain a molecular understanding of how the germ cell lineage of Drosophila is established during embryogenesis. Proper establishment and maintenance of cell fate is essential for normal growth and development of an organism. Defects in this fundamental process leads to cancers and birth defects. This proposal focuses on the genetic, molecular and biochemical examination of one gene known to be required for the establishment of the germ cell lineage, the germ cell-less gene. The germ cell-less gene encodes an mRNA that is localized to the germ plasm which contains the germ cell determinants. The function of germ cell-less, based on antisense, over-expression and ectopic localization studies is required for, and is capable of initiating some of the events of germ cell precursors, or ~pole cell~ formation. Analysis of the subcellular distribution of germ cell-less protein indicates that it is localized to the nuclear pores of the pole cell nuclei prior to, during and after germ cell fate is established. The germ cell- less gene therefore plays a central role in the establishment of the germ cell lineage. The initial focus of this project will be to verify the specificty of germ cell-less function for he establishment of the germ cell lineage by isolating strong and null mutants. Germ cell-less mutants will be obtained by a n lecular based, P-element reversion screen and a standard ems mutageneis based gentic screen. Germ cell- less mutants will be molecularly, genetically and phenotypically characerized to precisely define its role in germ cell formation and other developmental pathways. To explore its biochemical function, the germ cell-less protein will be mutated to make sub-cellular localization mutants to test if nucelar pore association is required for function. It will also be ectopically expressed in Drosophila tissue culture celis to assay for a role in regulating nuclear trafficking. To identify other genes which are required for germ cell specification, a yeast two-hybrid screen will be performed to identify proteins which physically interact with the germ cell-less protein. Genes identified by this screen will be evaluated by several criteria to determine if they have a role in the establishment of the germ cell lineage. In addition, a genetic interaction screen will be performed also to identify additional genes in this pathway.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Fragile X syndrome is one of the most commonly inherited forms of human mental retardation with an incidence rate of 1 in 4000 males and 1 in 6000 females. It is caused by the loss of FMR1 gene function. Patients with Fragile X syndrome suffer from a variety of symptoms including; mental retardation, attention deficit, hyperactivity, sleep disorders, anxiety, unstable mood and autistic-like behaviors. Physical defects include macroorchidism and irregular dendritic spine morphology. In previous studies, our lab developed a Fragile X model in Drosophila. This model is based on the dfmr1 (also called dfxr) gene, which has a high degree of sequence identity/similarity to the FMR1 gene. The dFMR1 protein has similar RNA binding properties, developmental expression pattern and subcellular distribution to the FMR1 protein (FMRP). In recent studies, we have shown that dfmr1 null mutants display several behavioral defects that bear similarity to symptoms of Fragile X patients. The relevant phenotypes in Drosophila include arrhythmic circadian behavior, attention deficit during courtship, memory defects and subtle defects of neuronal morphology. The similarities in the biochemical properties of dFMR1 and FMRP and their loss of function phenotypes suggest that these two proteins have conserved function in similar behavioral and developmental processes. Thus the Drosophila dfmr1 mutants are a relevant model to study aspects of Fragile X syndrome. To ameliorate Fragile X syndrome it is imperative that we understand when and how FMR1 activity functions to prevent cognitive and behavioral defects. The temporal requirements and molecular role of FMR1 are currently not known. We propose to use the Drosophila model of Fragile X to determine when dfmr1 activity is required to determine if the behavioral defects are due to developmental or physiological defects. We are also investigating possible physiological pathways affected by loss of dfmr1 function. Through these studies we have identified a pharmacological treatment that rescues the courtship and memory defects displayed in our dfmr1 mutants. In this proposal we will determine and verify a route of action of this drug to identify potential targets for the treatment of Fragile X syndrome. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Studies of the Fragile X Mental Retardation Protein 1 (FMRP) in humans and its mouse and fly homologs have shown that its proper expression is important for proper development and behavior. Loss, or very low levels of FMRP expression, leads to Fragile X syndrome in humans and analogous behavioral and neuro-anatomical defects in mice and flies. Over-expression studies in the mouse and fly have identified behavioral and neuro-anatomical defects as well. In addition to the importance of proper steady state levels of FMRP, studies in the mice have shown that transient modulation of FMRP levels in response to synaptic activation are crucial for proper synaptic plasticity. Given the importance of the proper regulation of FMRP levels, very little is known about how the levels of this protein are regulated. Recently we discovered that expression of the fly homologue of FMRP, called dFMR1, is regulated by components of the siRNA pathway. Loss of the core members of this pathway (AGO2, R2D2 and Dicer-2) leads to significant upregulation of dFMR1 protein levels in the germline and nervous system that cause specific germ line and neuronal defects due to dFMR1 misregulation. Although recent studies have identified that there is a functioning endogenous siRNA pathway in flies, our results indicate that this pathway is not being used to regulate dFMR1 expression through its canonically defined mechanism. In fact we have found genes, involved in other small RNA pathways, but outside of the canonical siRNA pathway, that also regulate dFMR1 expression. In the first subaim we will screen a candidate set of genes that includes genes in the piRNA pathway, genes that act in multiple small RNA pathways as well as genes that interact with small RNA pathways for a role in regulating dfmr1 expression. In the second subaim we will define the dfmr1 cis-elements required for its regulation by the siRNA pathway members. In the third subaim we will define the basic mechanism by which the siRNA pathway components regulate dfmr1 expression. Results from these studies will more precisely define a novel regulatory pathway that controls dfmr1 expression, providing valuable information as to how this medically important gene is regulated. PUBLIC HEALTH RELEVANCE: The proper regulation of the Fragile X Mental Retardation is fundamentally important for normal cognition and the prevention of at least three human diseases (Fragile X mental retardation, FXTAS and premature ovarian failure). In this application we put forth experiments to further define and characterize a novel regulatory pathway that we have found to be important for the proper regulation dfmr1 expression and the prevention of ovarian and neuronal defects due to dfmr1 misexpression. These studies will provide a better understanding about how this medically important gene is regulated, as well as may identify other genes that lead to other human diseases due to FMR1 misregulation.

DESCRIPTION (provided by applicant): Fragile X syndrome is one of the most commonly inherited forms of mental retardation and is caused by the loss of FMR1 gene function. Patients with fragile X syndrome suffer from a variety of symptoms including;mental retardation, attention deficit, hyperactivity, sleep disorders, anxiety, unstable mood and autistic-like behaviors. In previous studies, we have characterized a Drosophila model for Fragile X, based on dfmr1 loss of function mutations. Surprisingly, dfmr1 mutants display several phenotypes that bear similarity to Fragile X symptoms. These phenotypes include courtship defects, memory deficits, lack of proper circadian regulation and neuroanatomical defects. Recent observations have indicated that enhanced metabotropic glutamate receptor (mGluR) signaling is a cause of a large number of the Fragile X symptoms. In support of this model, we previously demonstrated that treatment with mGluR antagonists rescues several of the dfmr1 mutant phenotypes, including the courtship defects, memory and some neuroanatomical defects. We now are poised to examine the biological relevance of a reported deficit in cAMP regulation. We will also map the requirements of dfmr1 in the brain for normal circadian behavior. In another study we have been investigating the mechanism by which dFMR1 functions with a key member of the small RNA pathways to regulate circadian behavior. These studies will impact our basic understanding of the primary defects that lead to fragile X, as well a improve our general knowledge of the processes of cognition and circadian behavior. PUBLIC HEALTH RELEVANCE: This proposal investigates a Drosophila model for Fragile X Syndrome at multiple levels, including examination of defects in physiology and neuronal circuits that cause relevant phenotypes in memory and circadian behavior. Findings from this research will both increase our understanding of the underlying defects that cause fragile X as well as increase our basic knowledge on the requirements for proper cognition and circadian behavior, as well as how the small RNA pathway is linked to the FMR1.

Project Summary As people age, their risk for developing dementia increases. This risk is enhanced for those with type II diabetes. In fact, individuals with type II diabetes are more than twice as likely to suffer from dementia, either through the development of Alzheimers disease (AD), Vascular cognitive impairment (CVI) or dementia in general. Examination of brains from AD patients also reveals this correlation as most brains from AD patients display insulin resistance in the hippocampus, even in patients that have not been clinically diagnoses with type II diabetes. This brain form of insulin resistance is referred to as type III diabetes. In previous studies we examined the effect of reduced presenilin activity utilizing known loss of function mutations of Drosophila presenilin (psn). We found that flies with reduced psn activity (psn-hets) displayed an age-onset loss-of-learning and memory in the classic courtship learning and memory paradigm. In more recent studies of the psn-het brains we have found that they develop brain insulin resistance with age. We find that when the psn-het flies are young (day 5 of adulthood) and display normal cognition, their brains display increased insulin signaling and increased sensitivity to insulin stimulation. Old psn-het brains (day 30 of adulthood) that display loss of learning and memory fail to respond to insulin stimulation. We hypothesize that the establishment of insulin resistance in the brains of the old psn-hets causes the cognitive deficits displayed by this model. In the first aim of this proposal we will determine if psn mutations linked to familial Alzheimers disease (FAD) also lead to altered insulin signaling in the brain, brain insulin resistance and age onset cognitive loss. We will then explore if the reduction of insulin signaling in the brain can rescue the formation of brain insulin resistance and cognitive loss. In the second aim of this proposal we will determine if psn mutations lead to alterations in peripheral insulin signaling and peripheral insulin resistance. These studies will determine if loss or alteration of psn activity preferentially induce insulin resistance in the brain. We will also test if treatments that are known to induce the development of peripheral insulin resistance in flies also cause brain insulin-resistance and cognitive deficits. These studies will be performed with control flies, psn-hets and flies heterozygous for FAD mutations, allowing for an examination of the interaction of diet and reduced psn activity. These studies will explore the role that reduction of, or alteration in psn activity has on insulin signaling and the establishment of insulin resistance in the brain and whether this can cause cognitive impairment. These studies will also provide a useful model to explore the dementia due to the development of brain insulin resistance in general.

Abstract Despite a century of research and countless clinical trials, Alzheimer's Disease (AD) etiology is still poorly understood and treatment options are incredibly limited. The histopathology of the disorder, has generally focused on the accumulation of neurotoxic plaques in the brain which are composed of the A? peptide, a cleavage product of the Amyloid Precursor Protein (APP). This peptide has received much attention in the AD field due to its role in Familial Alzheimer's Disease (FAD), a genetic form of the disease with rapid but similar disease progression. Importantly, FAD is caused by the autosomal dominant inheritance of mutations, in APP and the presenilin genes. This is significant because presenilin is the catalytic component of the ?-secretase complex that cleaves APP, releasing A?. Since anti-amyloidogenic and anti-?-secretase agents have been largely ineffective in treating AD, and often even seem to worsen the condition in AD patients, alternative mechanisms need to be considered. One such mechanism is altered insulin signaling. There are significant links between AD and Type II diabetes (T2D), and signs of altered insulin resistance have been identified in the brains of AD patients. This is important as insulin signaling also plays a role in memory formation. We believe that these two distinct areas of AD research may actually be closely linked. We believe that it is irrefutable that A? plays a pivotal role in AD pathogenesis, however we also believe that this role has not been fully illustrated. It has been suggested in select publications that A? competes with insulin for binding to both the insulin receptor and the insulin degrading enzyme. We believe that this activity is responsible for regulating insulin signaling in the brain. When A? levels are altered, insulin signaling may proceed unregulated resulting in brain insulin resistance with age, as occurs in the periphery of T2D patients. Our hypothesis is that A? plays a positive biological role in insulin signaling regulation, and that this regulation is altered in AD. To test this hypothesis, we will generate animal models that expresses both A? and insulin at physiologically accurate levels. In Aim 1, we propose to use the newly developed CRISPR technology to generate a physiologically relevant fly model of FAD. In Aim 2, we will use in vitro techniques to test the concept that A? and insulin work in tandem to modulate insulin signaling levels. Finally, in Aim 3, we will use the new FAD fly model to determine if A? is required in the brain to regulate insulin signaling, and if this balance is altered in AD. Upon conclusion of the experiments proposed, we will have determined if A? plays a positive biological role in the regulation of insulin signaling, and if alterations in this regulatory role may contribute to AD pathogenesis. Further, we will have developed a new, physiologically accurate fly model of AD which will be an invaluable tool in both our future studies and in the work of other groups investigating the cause of this devastating disorder.