Sidney Strickland - US grants

We have developed a hormonal treatment that allows the induction of differentiation in teratocarcinoma stem cells in two stages: retrinoic acid treatment yields an altered, stable phenotype and further treatment with dibutyryl cAMP results in the generation of cell type indistinguishable from definitive embryonic parietal endoderm. these differentiated cells produce copious quantities of Type IV collagen, laminin, and plasminogen activator, in contrast to the stem cells which produce little if any on these proteins. We propose to use this system to begin investigations of gene regulation in early embryonic cells. We have prepared to cDNA library from the differentiated cells, and will isolate from this library clones containing cDNAs related to these 3 proteins. We will then use the probes to: 1) explore the regulation of expression of these during differentiation; 2) examine the gene and chromatin structure for the proteins in the stem cells and differentiated cells; 3) analyze the early mouse embryo for the nature and regulation of expression of these genes. Other colonies present in the cDNA library may also be useful both for exploring how the synthesis of certain RNA sequences present most abundantly in the stem cells in regulated, and also for investigating the function of these RNA molecules.

Plasminogen activators are serine proteases that have been implicated in a wide variety of normal and abnormal processes. The experiments proposed address two broad questions concerning these enzymes: how is the synthesis of tissue plasminogen activator (tPA) controlled, and what are the respective roles for tPA and the urokinase-type plasminogen activator (uPA). Regulation of tPA synthesis will be studied in F9 teratocarcinoma cells, in which enzyme production can be conveniently controlled by manipulation of the culture conditions. The fact that tPA can feedback inhibit its own synthesis will also be examined in these cells by classical techniques and oligonucleotide-mediated mutagenesis followed by transfection and expression of specifically modified enzymes. The tumorigenic properties of F9 cells producing these modified tPAs will also be determined. In the rat ovary, granulosa cells produce tPA and thecal cells produce uPA. With both cells, enzyme synthesis is maximal at the time of ovulation and is induced by gonadotropins. This system will be used in vivo and in vitro to investigate the function of the two enzymes in the ovulatory process. The possibility that tPA is instrumental in degrading follicular proteoglycans, and that proteoglycans can stimulate the activity of tPA, will be examined.

Plasminogen activators are serine proteases that have been implicated in a wide variety of normal and abnormal processes. The experiments proposed address two broad questions concerning these enzymes: how is the synthesis of tissue plasminogen activator (tPA) controlled, and what are the respective roles for tPA and the urokinase-type plasminogen activator (uPA). Regulation of tPA synthesis will be studied in F9 teratocarcinoma cells, in which enzyme production can be conveniently controlled by manipulation of the culture conditions. The fact that tPA can feedback inhibit its own synthesis will also be examined in these cells by classical techniques and oligonucleotide-mediated mutagenesis followed by transfection and expression of specifically modified enzymes. The tumorigenic properties of F9 cells producing these modified tPAs will also be determined. In the rat ovary, granulosa cells produce tPA and thecal cells produce uPA. With both cells, enzyme synthesis is maximal at the time of ovulation and is induced by gonadotropins. This system will be used in vivo and in vitro to investigate the function of the two enzymes in the ovulatory process. The possibility that tPA is instrumental in degrading follicular proteoglycans, and that proteoglycans can stimulate the activity of tPA, will be examined.

In oocytes, the translation of many dormant mRNAs is regulated by the length of the poly (A) tail. The poly (A) status is controlled by a conserved sequence present in the 3' untranslated region (UTR), designated the adenylation control element (ACE). We will exploit this regulatory scheme to study the control of mouse oogenesis and early development as follows: 1. Using the ACE sequence, we will selectively clone a set of mRNAs whose expression is under poly (A)-dependent translational control. Using a variety of approaches, we will systematically address the significance and function of these mRNAs in early mouse development. 2. We will perform a mutagenic analysis of the ACE element and surrounding sequences to investigate the exact requirements for both shortening and elongation of the poly (A) tail. These experiments may shed light on the mechanism by which different dormant mRNAs are activated for translation at various times in development. 3. We will investigate the molecular aspects of cytoplasmic regulation of poly (A) tail length in mouse oocytes. Using probes directed against molecules that participate in nuclear polyadenylation from other species, we will attempt to identify the mouse oocyte factors which interact with the cytoplasmic dormant mRNAs and control their poly (A) status. These factors could then be used to isolate other novel components of the system.

The overall aim of this proposal is to explore methods for blocking gene expression in the mouse in vivo. Initially, we will use as a test system the production of the protease tissue plasminogen activator (t-PA). This enzyme is produced in high amounts in maturing mouse oocytes, early embryos, and various somatic tissues. However, its physiological role is not defined. We will use two strategies for interrupting t-PA expression: 1. We will produce transgenic mice that express antisense RNAs specifically in the oocyte. Included will be approaches that exploit recent information about the control of transcription in an attempt to design new strategies for the use of antisense RNA. 2. We will disrupt one or both copies of the t-PA gene in mice via homologous recombination. These studies should give new information about the role(s) of t-PA in oogenesis, early embryogenesis, and mammalian physiology. They may also identify better methods for gene disruption in mammals that can be extended to other gene products.

The overall aim of this proposal is to explore methods for blocking gene expression in the mouse in vivo. Initially, we will use as a test system the production of the protease tissue plasminogen activator (t-PA). This enzyme is produced in high amounts in maturing mouse oocytes, early embryos, and various somatic tissues. However, its physiological role is not defined. We will use two strategies for interrupting t-PA expression: 1. We will produce transgenic mice that express antisense RNAs specifically in the oocyte. Included will be approaches that exploit recent information about the control of transcription in an attempt to design new strategies for the use of antisense RNA. 2. We will disrupt one or both copies of the t-PA gene in mice via homologous recombination. These studies should give new information about the role(s) of t-PA in oogenesis, early embryogenesis, and mammalian physiology. They may also identify better methods for gene disruption in mammals that can be extended to other gene products.

In the preceding grant period, we have identified two genes that are essential for meiosis in Drosophila oocytes, and that influence the translational activation of maternal mRNAs. Drosophila offers a very attractive system to study this regulatory pathway, since we can use both genetics and biochemistry to explore the molecules involved. We therefore propose to use a combined genetic and biochemical approach to analyze the meiotic cell cycle and translational activation in Drosophila oocytes/embryos: 1. Oocytes from females homozygous for either a grauzone or cortex mutation do not complete meiosis, and the embryos have defects in translational activation of certain maternal mRNAs. These results indicate that grauzone and cortex function in the meiotic cell cycle and translational regulation pathways. We will clone and characterize the grauzone and cortex genes, and explore their function during meiosis, oocyte maturation, and early development. 2. Translational activation of many maternal mRNas is controlled by cytoplasmic polyadenylation. This polyadenylation is regulated by proteins that interact with the 3' UTR of the mRNAs. We will characterize the transacting factors that bind to the 3' UTR of translationally activated Drosophila mRNAs, and determine the role of these proteins in regulating poly (A) tail length. Our strategy will include both a biochemical and a genetic approach for isolating the factors. These studies should identify key regulatory features of translation activation during development. These two aims are intimately linked. It is possible that either grauzone or cortex regulates the activity or expression of factors that bind to the 3' UTR of maternal mRNAs and regulate their translation. It is also possible that one of the 3' UTR binding factors is grauzone or cortex. We are confident that attacking the problem on both fronts will yield interesting new information about the meiotic cell cycle and how translational control participates in its regulation.

Laminin is a heterotrimeric, extracellular matrix (ECM) protein that has been implicated in mediating various aspects of cell function and survival. We have recently found that laminin is highly expressed in the mouse hippocampus, and that proteolytic degradation of this protein can promote the death of neurons. In this application, we propose a systematic exploration of the expression and function of laminin in the central nervous system (CNS). Specifically, we will determine the isoform(s) of laminin that is expressed in the hippocampus, identify the proteases directly responsible for its degradation and their sites of cleavage, and establish which region of laminin interacts with neurons to promote survival. Armed with this information, we will produce conditional knockout mice that lack the specific CNS laminin isoforms only in the hippocampus. These experiments will define the molecular characteristics and functional role of a critical ECM component in the mammalian CNS.

DESCRIPTION (provided by applicant): Schwann cells require the formation of an organized basal lamina in order to properly ensheath and myelinate axons. Laminins are a group of heterotrimeric extracellular proteins and are critical components within the basal lamina responsible for these processes in peripheral nervous system (PNS). Laminin gamma1 is a component of most laminin isoforms identified to date and is a subunit of laminin-2 (alpha2 beta1 gamma1), the major laminin isoform present in Schwann cell basal lamina. To investigate the function of laminin gamma1 in the PNS, we generated mice homozygous for a laminin gamma1 gene that has a critical exon flanked by IoxP recombination sites (fLAM gamma1) that also carry a Cre recombinase transgene under the control of a Schwann cell-specific promoter, the myelin protein zero (P0) promoter. These mice have disruption of the laminin gamma1 gene in their peripheral nerves, resulting in a severe defect in myelination. The objectives of this proposal are to take advantage of these mice with Schwann cell-specific disruption of laminin gamma1 gene to investigate the role of laminin in the development, myelination, and maintenance of the PNS. To accomplish this goal, we propose three specific aims. First, we will investigate the mechanisms of action of laminin gamma1 in signaling pathways involved in Schwann cell proliferation and apoptosis. Second, we will investigate the mechanisms of action of laminin gamma1 in radial sorting and myelination of PNS. Third, we will investigate the role of laminin gamma1 in the maintenance of peripheral nerves. These experiments will allow us to understand the mechanisms of Schwann cell-basal lamina interactions and may suggest possible new approaches to the therapy of peripheral neuropathies.

[unreadable] DESCRIPTION (provided by applicant): The overall hypothesis of this proposal is that the tissue plasminogen activator/plasminogen (tPA/plg) fibrinolytic cascade, a proteolytic system which has been implicated in the clearance of amyloid-beta (A-beta) peptide, is an important pathway to investigate for developing possible therapeutic agents against Alzheimer's disease (AD). The abnormal deposition of A-beta in the parenchyma and blood vessels of the brain is a pathological hallmark of AD, the most common cause of dementia and cognitive decline in the aged. Additionally, epidemiological studies indicate that diseases that compromise the circulatory system are risk factors for the development of AD, and imply that AD has a cerebrovascular component. The objectives of this proposal are to investigate the role of the tPA/plg system in the progression of AD-like pathology in transgenic mice overexpressing the amyloid-beta precursor protein (A-betaPP). The tPA/plg system activity is depressed in AD transgenic mice and in individuals with AD, due to the expression of plasminogen activator inhibitor-1 (PAl-l), a protein overexpressed during inflammation, commonly seen in AD. To accomplish this goal, we propose three specific aims. First, we plan to investigate the effects of the loss of tPA, plg, or PAl-1 expression in A-betaPP transgenic mice in a C57/BI6 background. Second, we plan to investigate the role of fibrin deposition in exacerbating the pathology and cerebrovascular dysfunction in AD. Third, we plan to identify new compounds that block the interaction of PAl-1 and tPA, and to test these, along with known PAl-1 inhibitors, for their effects in AD mouse models. These experiments will take advantage of transgenic and knockout mouse lines as in vivo paradigms for the development of possible therapeutic intervention strategies, targeting the tPA/plasmin cascade, against AD progression [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This application is a renewal for a pre-doctoral training program in genetics and cell biology at The Rockefeller University, an institution with a rich history in these areas. Since the previous application, we have added four new faculty, and the training faculty would be 54 strong investigators, ranging from full Professors, with extensive experience in training students, to newly hired Assistant Professors. The faculty has expertise in a very broad range of genetics and cell biology, ranging from the most basic to epidemiological studies on humans, and the program encourages students to perform collaborative work in various areas with different faculty. The applicant pool is outstanding, containing a large number of undergraduate students who have superb academic and research accomplishments, and who are primarily interested in genetics and cell biology. To train the students, we have devised a unique program of study, including a specialized course exclusively for the trainees;a defined, comprehensive curriculum;superb research possibilities;and an interactive and supportive environment. To provide structure, the Program incorporates careful faculty oversight to ensure a comprehensive formal education and expeditious progress towards the Ph.D. degree. Finally, the University provides strong support for the graduate program in general, which benefits the training program. The confluence of these attributes defines a training program that equips students with the educational background, analytical abilities, and experimental expertise to forge future advances in genetics and cell biology.

DESCRIPTION (provided by applicant): It has been shown in various laboratories that the tissue plasminogen activator (tPA)/plasmin system is critical for excitotoxin-mediated seizures and neurodegeneration in the hippocampus. Upon excitotoxic challenge, tPA activates plasminogen to produce excess plasmin, which then degrades a critical component of the extracellular matrix (ECM), laminin. The loss of the neuron-matrix connection facilitates neuronal death as it does with other cell types. We have found that tPA activity during ethanol treatment and EW is up-regulated in the mouse hippocampus and the amygdala, and that tPA-/- mice are protected from EW-induced seizures and neurodegeneration, as they are from these pathologies induced by excitotoxin injection. Therefore, the mechanism(s) of excitotoxinand EW-induced seizures and neurodegeneration have at least one molecule in common, tPA. To understand better the mechanism of EW-induced seizures and neurodegeneration, we propose to systematically explore the role of tPA in these processes

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a neurodegenerative disorder that leads to profound cognitive decline and eventually death. There are no effective long- term treatments or preventative measures available, and the prevalence of the disease is increasing. New insights and therapeutic targets are sorely needed. Genetic evidence indicates that a major cause of AD is the production of the amyloid-[unreadable] (A[unreadable]) peptide, which is proteolytically derived from the amyloid-b precursor protein. The A[unreadable] peptide can oligomerize and be deposited as extracellular plaques in the brain and blood vessels, but the mechanism of how it leads to neuronal death is not known. There is increasing evidence of a vascular contribution in AD: patients suffer from brain hypoperfusion, the cerebral vasculature is damaged, and abnormal hemostasis is present. Circulatory deficiencies could therefore play an important role in the pathogenesis of this disease. During the last grant period, we demonstrated an increase in blood brain barrier (BBB) permeability and neurovascular damage in AD mice, and we showed that fibrin deposition potentiates these processes. We have now found that A[unreadable] binds to fibrinogen and has a dramatic effect on fibrin clot formation. Clots formed in the presence of A[unreadable] have an abnormal structure and are resistant to degradation by fibrinolytic enzymes. Therefore, in the presence of A[unreadable], any fibrin clots formed would be more persistent and exacerbate BBB damage, neuroinflammation, and neuronal death. In keeping with the known genetic interaction between AD and the ApoE genotype, we have also demonstrated that the different ApoE isoforms differentially affect the fibrinogen/Ab interaction and the structure of the fibrin clot formed. To further study the role of A[unreadable] in fibrin clot formation, we will combine in vitro and in vivo techniques to analyze and characterize the interaction between A[unreadable] and fibrin(ogen). The role of the various ApoE isoforms in this process will be also examined. The proposed experiments will define the role of fibrin(ogen) in AD and possibly lead to new therapeutic strategies for preventing or retarding progression of the disease. PUBLIC HEALTH RELEVANCE: Alzheimer's disease (AD) affects a large and growing portion of the population and has been studied for over a century, yet there are few available therapies to aid in the loss of cognition and no effective preventative measures. We have found that the build-up of fibrin in the brains of AD mouse models is deleterious and contributes to their cognitive decline. The proposed studies will provide a new way to understand how this disease progresses from the perspective of disrupted blood flow to the brain, which will also explain how the nature of the fibrin clot is altered in the presence of amyloid-[unreadable] to allow for this persistence and how other genes can influence progression.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a neurodegenerative disorder that leads to profound cognitive decline and eventually death. There are no effective treatments or preventative measures available, and the incidence and prevalence of the disease are increasing. New insights and tractable therapeutic targets are sorely needed. Genetic evidence indicates that a major cause of AD is the production of the [unreadable]-amyloid (A[unreadable]) peptide. The A[unreadable] peptide can oligomerize and be deposited as extracellular plaques in the brain and blood vessels, but the mechanism of how it leads to neuronal death is not known. There is increasing evidence of a vascular contribution in AD: patients suffer from brain hypoperfusion, the cerebral vasculature is damaged, and abnormal hemostasis is present. Circulatory deficiencies could therefore play an important role in the pathogenesis of this disease. We have demonstrated an increase in blood brain barrier (BBB) permeability and neurovascular damage in AD mice, and we showed that fibrin(ogen) deposition potentiates these processes. We have also found that A binds to fibrinogen and has a dramatic effect on fibrin clot formation. Clots formed in the presence of A[unreadable] have an abnormal structure and are resistant to degradation by fibrinolytic enzymes. Therefore, in the presence of A[unreadable], any fibrin deposits formed would be more persistent and would exacerbate BBB damage, neuroinflammation, and neuronal death. In keeping with the known genetic interaction between AD and the ApoE genotype, we have also demonstrated that ApoE affects the interaction between fibrinogen and A[unreadable] and the isoforms differentially influence fibrinogen deposition in the human brain. To further study the role of A[unreadable] in fibrin clot formation, we will combine in vitro and in vivo techniques to analyze and characterize the interaction between A[unreadable] and fibrinogen. The role of the various ApoE isoforms in this process will also be examined. Finally, we will examine the effects of fibrinogen deposition on the brain in the absence of A[unreadable] to deduce the specific contribution of fibrin(ogen) to AD-related pathologies. The proposed experiments will define the role of fibrin(ogen) in AD and could lead to new therapeutic strategies for preventing or retarding progression of the disease. PUBLIC HEALTH RELEVANCE: Alzheimer's disease (AD) affects a large and growing portion of the population and has been studied for over a century, yet there are few available therapies to aid in the loss of cognition and no effective preventative measures. We have found that the buildup of fibrin in the brains of AD mouse models is deleterious and contributes to their cognitive decline. The proposed studies will provide a new way to understand how this disease progresses from the perspective of disrupted blood flow to the brain, which will also explain how the nature of the fibrin clot is altered in the presence of [unreadable]-amyloid to allow for this persistence and how other genes can influence progression.

PROJECT SUMMARY In addition to neuronal degeneration, many Alzheimer?s disease (AD) patients suffer from vascular abnormalities and inflammation. The contact activation system may contribute to both of these pathologies since it can launch both pro-thrombotic and pro-inflammatory pathways. We have shown that the contact system is significantly more activated in AD patients and AD mouse models compared to control groups. The beta-amyloid peptide (A?), a driver of AD pathology, can activate Factor 12 (F12), the initiator of the contact system. We have recently shown that depletion of F12 ameliorates pathology in AD mice at early stages of disease. These results indicate that excess contact system activation may promote AD pathology and cognitive decline. There is no effective treatment for AD. A link between F12 activation and the pathogenesis of AD provides a possible novel approach to treatment. The contact system is an attractive target for AD therapy; humans deficient in F12 and mice with knockout of different contact system pathway genes have normal hemostasis. If F12 activation is indeed deleterious in AD pathology, therapies designed to block the contact system might slow disease progression while not affecting normal hemostasis. Thus, our studies may reveal new targets to suppress both thrombotic and inflammatory contributions to AD progression. Positive results might be able to be applied to AD patients rapidly as already FDA-approved drugs targeting the contact system already exist.