Luis Parada, Ph.D. - US grants

This proposal concerns the development and analysis of mouse models for the study of Trk protooncogene receptors and their cognate ligands, the NGF-related neurotrophins. Neurotrophic factors perform critical functions in the development and survival of diverse neurons and glia during vertebrate embryonic development. Recent evidence indicates that neurotrophins continue to support nerve cell survival and regeneration in adults CNS and in peripheral motor neurons. These results imply that neurotrophins may have pharmacological potential in nerve regeneration. The correlation between neuronal NGF dependence, NGF (trk ) receptor expression and neuronal death observed in Alzheimer's patients, suggests that neurotrophins may also have potential therapeutic value in neurodegenerative diseases. The availability of reliable in vivo models to study neurotrophin function is of critical importance for further evaluation of neurotrophin sites and mechanisms of activity. The Trk receptor tyrosine kinase gene family encodes the functional receptors for the neurotrophins. This observation has opened new areas of signal transduction and biochemistry to the field. However several complexities remain unanswered and the potential for neurotrophin therapy via intervention of their cognate receptor awaits further understanding of the system. Thus, while the different receptor/ligand interactions for the different Trk//neurotrophin pairings can be assessed in cell culture systems, the ultimate significance of these interactions is best addressed in the organism. We intend to generate mouse lines harboring targeted null mutations in the following genes: trk, trkB, trkC, BDNF, NT-3 and NT4/5 through ES cell technology. The consequences of these mutations, in the homozygous state, for neuroblast development and survival will be analyzed during development of the nervous system. Direct comparison of the different mutations, and genetic intercrossing of the various mutations, will provide critical information concerning the importance and developmental timing of the different receptor/ligand interactions that have been observed in vitro. The spontaneous neurological mutations spastic and pcd map to the same chromosomal regions as do trk and trkB respectively. Genetic crosses between the receptor mutants and the spontaneous mutants will allow us to determine whether spa and pcd are alleles of the trk and trkB genes. Trk family genes encode various isoforms including receptors lacking the kinase domains and receptors harboring kinase domain insertions. The functions of these isoforms are currently unknown. We propose to generate targeted mutations in the trkB and trkC genes that specifically ablate these isoforms as a means of creating a system that will permit study of their functions. Recognizing the potential value of the mouse strains developed in our study, to the fields of cancer biology, aging, nerve disease, the and spinal cord injury , among others, we will propagate our stocks and distribute them to investigators in the basic research community.

This proposal concerns the development and analysis of mouse models for the study of the neurofibromatosis gene product and its role in the development of the nervous system. Von Recklinghausen's neurofibromatosis type l (NF-I) is a dominant autosomal disorder that strikes one in three thousand individuals. It is characterized by anomalies of diverse cell types, many of which are of the neural crest lineage. The severity of these phenotypes can vary greatly. However, schwannomas often mature into malignant neurofibrosarcomas. The NF-I gene product, neurofibromin, is a cytoplasmic protein of approximately 3000 amino acids that exhibits structural and functional homology to the super family of GTPase activating (GAP) proteins that function as negative regulators of ras oncoproteins. We have generated a mouse model that is specifically and exclusively mutated in the NF-I gene thus enabling us to investigate the action of NF- I in the development of the nervous system. Our results indicate that sensory and sympathetic neurons which normally require neurotrophins (NGF, BDNF, NT-3 & NTA/5) for their normal development and survival, escape this requirement when the NF-I gene has been ablated. This exciting finding implicates neurofibromin as a functional intermediary in neurotrophin signalling and provides a unique system for testing the role of this tumor suppressor protein in the regulation of neuronal signals that are transmitted from neurotrophins to maintain neurons alive. We propose four specific aims: 1) to characterize the consequences of neurofibromin loss for neurotrophin-dependent neuronal survival in primary neuronal culture assays. 2) We will use genetic approaches to dissect the signalling intermediates that function through the neurotrophin/NF-l pathway. This will be accomplished with the use of adenovirus gene delivery vectors of oncogenes such as ras and raf. 3) We will intercross the NF-l mutation into each of the neurotrophin (Trk) receptor and neurotrophin mutation knockouts that were generated in our laboratory. Analysis of the neurons from double mutants should allow us to determine the location of NF-I in the neurotrophin signalling pathway and the direct effect of this gene on trk neurotrophin mediated signalling. 4) NF-1 transgenic mice will be generated and crossed into the NF knockouts in the effort to obtain partial rescue of the embryonic lethality to enable analysis of mutant neurons in late embryonic stages. The availability of reliable in vivo models to study the role of neurofibromin in neurotrophin function is of critical importance for further evaluation of this suppressor oncogene in human disease, in programmed cell death, and in neural development.

This proposal concerns the continued study of neurotrophins (NTFs) and their receptors, via the analysis of diverse mouse models as a means to understand neural development. Trk family receptor tyrosine kinases and their ligands, the NTFs, have been implicated in neuronal survival and differentiation. NTFs have been considered as potential therapeutic agents for neurodegenerative diseases including Alzheimer's & ALS as well as for spinal cord injury. In the previous period, we generated a variety of Trk receptor & NTF mutant mice. Study of these mice has provided valuable information regarding the in vivo function of NTFs in neuronal survival. We proposed to continue these studies &in the First Aim will continue to study NTF function in sensory neuron development and survival. We will further exploit these mutant mice to continue preliminary analysis of NTF requirement in development of the olfactory, and gustatory systems. We will also perform a detailed comparative analysis of our null trkC & NT-3 mutants with a Kinase null trkC mutant. These proposed studies for the first time directly address the in vivo function of truncated receptors. In the Second Aim we will identify the determinants that regulate the NGF receptor, TrkA. This enhancer analysis will provide novel information about the upstream molecules that regulate neurotrophin receptors & the developing nervous system. In the third aim, we will exploit the existence of a TrkA enhancer, to perform structure/function analysis of the TrkA receptor in vivo. We will in vitro mutate intracellular signaling determinants of TrkA and introduce mutated transgenic receptors into the TrkA-/- mouse. Transgenic embryos will be analyzed for rescue of several defined parameters. This approach will reveal physiologically relevant signaling effectors for survival and differentiation of neurons in vivo. A limitation of NTF knockout studies has been early postnatal lethality. This precludes analysis of function in the adult. In the Fourth Aim, we propose to complete our ongoing conditional Knock Out strategies for TrkB and BDNF which will enable us to expand our studies.

DESCRIPTION(Applicant's abstract verbatim): Type I neurofibromatosis (NF1) is an autosomal dominant disorder that strikes 1 in 3000 individuals. It is characterized by anomalies of diverse cell types, many of which are derived from the neural crest. The severity of these phenotypes can vary greatly even among individuals from the same family who carry the same mutation. The NF1 gene product, neurofibromin, is a cytoplasmic protein that exhibits structural and functional homology to the family of GTPase activating proteins that function as regulators of Ras oncoproteins. The group has generated a mouse model of NF1 that permits inactivation of the Nf1 gene in specific cells and tissues. This model affords a unique system for testing the role of this tumor suppressor protein in the regulation of signals in a variety of cell types including the neural crest, Schwann cells, fibroblasts, neurons, astrocytes and oligodendrocytes. This application concerns the further development and analysis of conditional mutant Nf1 mouse strains to analyze the role of neurofibromin in normal and malignant cells. The specific aims are: 1) To characterize the consequences of neurofibromin loss for growth properties in primary neuronal and Schwann cell culture assays. 2) The group has applied genetic approaches to cross a neuron-specific synapsin-cre into the conditional Nf1 mutant. The consequences of Nf1 loss will be analyzed in a variety of neuronal systems. 3) A P0-cre mouse has been crossed with the Nf1 mutants. This P0 strain expresses mutant Nf1 in the entire neural crest and the resultant mice are embryonic lethal. The group plans to perform a battery of cellular and molecular assays similar to those performed in analysis of the original null Nf1 mutation to determine the extent of neural crest involvement. Additional P0-cre mice will be obtained to further study Nf1 loss in more confined neural crest derived compartments. 4) The group has intercrossed the Nf1 mutation into a GFAP-cre transgenic mouse. These mice exhibit dramatic behavioral abnormalities and die prematurely. Mice lacking neurofibromin function in GFAP-expressing cells will be studied for the properties of astrocytes in vivo and the consequences of neurofibromin loss. The availability of reliable in vivo models to study the role of neurofibromin in neurotrophin function is of critical importance for further evaluation of this suppressor oncogene in human disease, in programmed cell death, and in neural development.

DESCRIPTION(Applicant's abstract verbatim): Type I neurofibromatosis (NF1) is an autosomal dominant disorder that strikes 1 in 3000 individuals. It is characterized by anomalies of diverse cell types, many of which are derived from the neural crest. The severity of these phenotypes can vary greatly even among individuals from the same family who carry the same mutation. The NF1 gene product, neurofibromin, is a cytoplasmic protein that exhibits structural and functional homology to the family of GTPase activating proteins that function as regulators of Ras oncoproteins. The group has generated a mouse model of NF1 that permits inactivation of the Nf1 gene in specific cells and tissues. This model affords a unique system for testing the role of this tumor suppressor protein in the regulation of signals in a variety of cell types including the neural crest, Schwann cells, fibroblasts, neurons, astrocytes and oligodendrocytes. This application concerns the further development and analysis of conditional mutant Nf1 mouse strains to analyze the role of neurofibromin in normal and malignant cells. The specific aims are: 1) To characterize the consequences of neurofibromin loss for growth properties in primary neuronal and Schwann cell culture assays. 2) The group has applied genetic approaches to cross a neuron-specific synapsin-cre into the conditional Nf1 mutant. The consequences of Nf1 loss will be analyzed in a variety of neuronal systems. 3) A P0-cre mouse has been crossed with the Nf1 mutants. This P0 strain expresses mutant Nf1 in the entire neural crest and the resultant mice are embryonic lethal. The group plans to perform a battery of cellular and molecular assays similar to those performed in analysis of the original null Nf1 mutation to determine the extent of neural crest involvement. Additional P0-cre mice will be obtained to further study Nf1 loss in more confined neural crest derived compartments. 4) The group has intercrossed the Nf1 mutation into a GFAP-cre transgenic mouse. These mice exhibit dramatic behavioral abnormalities and die prematurely. Mice lacking neurofibromin function in GFAP-expressing cells will be studied for the properties of astrocytes in vivo and the consequences of neurofibromin loss. The availability of reliable in vivo models to study the role of neurofibromin in neurotrophin function is of critical importance for further evaluation of this suppressor oncogene in human disease, in programmed cell death, and in neural development.

The Administrative Core of the Center serves a straightforward function. First, it maintains active oversight of the scientific priorities and directions of the research through regular conference calls of the Pi's that serve as an Executive Committee. Second, the Core will be responsible for administering the day-to-day activities of the Center. This includes the appropriate disbursement and expenditure of funds for personnel, equipment, supplies, and miscellaneous expenses, according to the established budget. Oversight over preparation of progress reports, shipments between sites (including, reagents, mice, tissue samples, etc.).

Neurofibromatosis type 1 is a genetic disease with wide ranging consequences on the afflicted individuals ranging from potential intellectual and cognitive deficits to appearance of idiopathic tumors in the peripheral nervous system collectively called neurofibromas. Over the past 12 years we have developed mouse models of NFl with the objective of recapitulating a variety of the pathologic features seen in patients. This proposal continues and expands upon our experience in generating faithful genocopies of NFl-associated neurofibromas. In preceding work funded by this award, our scientific teams joined forces to test the hypothesis that NFl haploinsufficiency outside the Schwann cell lineage provided critical contribution to plexiform neurofibroma development. This hypothesis emerged from studies with our mouse models and the outcome has revealed the importance of mast cells in contributing to the tumor phenotype and ultimately leading to clinical trials to block mast cell activity in patients. In the present application we propose to extend our mouse modeling capabilities to further understand the etiology of plexiform neurofibromas, to identify the source and etiology of dermal neurofibromas, and to use our MPNST models to seek out therapeutic opportunities. In Specific Aim 1, we will employ tamoxifen-inducible ere driver lines and alternative approaches to better define the source of the cell of origin for plexiform neurofibromas and to define the temporal window of competence for tumor development. In Specific Aim 2, we will expand on our recent development of a murine model for dermal neurofibromas and on the discovery that skin-derived precursors (SKPs) are the cell of origin for these tumors. We will use multiple techniques including the use of chick/quail embryo transplantation of neural tubes to examine whether the neural crest is the original source of these tumor-competent cells. We will also develop new valuable tamoxifeninducible transgenic Cre driver lines to probe the neural crest-derived tissues for tumor potential. Finally, in Specific Aim 3, we will screen primary MPNST-derived tumor cells to undertake small chemical and RNAi highthroughput screens. These screens aim to identify small molecule compounds and genes that are required for tumor cell proliferation and growth and that can become targets for therapeutics.

The Transgenic Core provides state-of-the-art generation, maintenance, and breeding of animals to Project Investigators. Centralizing these functions within a single Core derives substantial cost savings and ensures the proper, consistent and rigorous use of these approaches. First, numerous lines of transgenic and knockout mice, obtained from other laboratories, are required for the studies proposed in this Center. The Core is responsible for breeding, genotyping, and maintaining the progeny for use by the individual Projects, as well as providing the appropriate control mice. Second, new lines of transgenic and knockout mice are required for the Center Projects, will be made as part of this Core. Among these new lines are those that manipulate the expression of specific genes both temporally and spatially, in other words, inducible and cell-targeted mutations in both brain and peripheral nervous system. Our group has significant experience with these methods, which include the Estrogen Receptor/tamoxifen gene regulation system and the Cre-loxP system. A major goal of the Core is to now adapt these methods to generate state of the art targeted knockouts that will permit the most ample and controlled experimentation.

DESCRIPTION (provided by applicant): Malignant astrocytomas are brain tumors that are locally infiltrative and incurable, with poor prognosis for the patient. Despite profound therapeutic implications, the identity of the cell(s) of origin of these tumors has not been rigorously determined. In addition, glioblastoma multiforme, the most prevalent and deadly form of brain tumor, can either progress from lower grade astrocytic gliomas or arise de novo, the mechanisms by which are not well understood. We previously reported mouse models based on conditional inactivation of human astrocytoma-relevant tumor suppressors p53, Nf1, and Pten, wherein through somatic loss of heterozygosity, mutant mice develop tumors that histologically and molecularly resemble human astrocytomas with 100% penetrance. In the present application, we propose experiments designed to investigate the cell(s) of tumor origin and to extend our analyses of tumorigenesis in these mouse models. To this end, Specific Aim 1 rigorously tests our hypothesis that gliomas originate in stem/progenitor cells. Our research design utilizes genetic and stereotactic methods to mutate the tumor suppressor genes specifically in the stem cell niche. We will also use a genetic method to ablate these neural stem cells in our tumor models and then analyze the effect on tumor formation. Specific Aim 2 will exploit our ability to culture fresh tumor tissue as self-renewable neurospheres to further characterize our Nf1;p53;Pten de novo glioma mouse model. We will evaluate their growth and differentiation properties, as well as their tumorigenic potential via transplantation techniques. Additionally, using a stem cell-specific GFP transgenic mouse, we will analyze the expression of candidate cell surface markers with the aim of identifying signature markers that will allow us to prospectively isolate the cancer stem cells. Specific Aim 3 will employ microarray analyses to identify the gene expression profiles that correlate with de novo vs. progressive glioma, using neurospheres derived from Nf1;p53;Pten and Nf1;p53 tumors, respectively. We also propose to analyze tissue from pre-symptomatic mice in order to gain insight into the early molecular events of tumor initiation. Potentially interesting genes will be functionally pursued using RNAi and overexpression techniques. As microRNAs have recently been implicated in glioma, we will also use microarray analysis to identify microRNAs that are differentially regulated in our tumor models and also investigate the role of candidate microRNAs in glioma. Our fully penetrant glioma mouse models are clinically relevant and powerful tools for identifying and functionally characterizing novel genes and pathways that may be therapeutically tractable in human glioma.

The Administrative Core of the Center will continue to function as it did in the previous funding period. The Core's primary function is to maintain active oversight of the scientific priorities and directions of the research through regular conference calls of the Pis that serve as an Executive Committee. Second, the Core will be responsible for administering the day-to-day activities of the Center. This includes the appropriate disbursement and expenditure of funds for personnel, equipment supplies, and miscellaneous expenses, according to the established budget This has proved to be a critical task given the complexities of administering funds from NIH that must be distributed through UT Southwestern to Indiana University in a timely fashion. The administrative core also provides centralized oversight of preparation of progress reports and shipments between sites (including, reagents, mice, tissue samples, etc.).

DESCRIPTION (provided by applicant): In this application, entitled: NF: from animal models to therapeutics, we are requesting funds for continued support of the NF Center. We build on our preceding success in exploiting animal models to gain novel insight into tumor etiology and revealing the mast cell as a therapeutic target that is now in clinical trials. The Center supports a series of highly interactive and multidisciplinary studies aimed at (1) delineating the mechanisms underlying the two most common tumors in NF1: dermal and plexiform neurofibromas and (2) dissecting the unknown functions of neurofibromin in regulating adult endothelial and vascular smooth muscle function - two cell lineages critical for establishment of the tumor vasculature in neurofibromas, and in the development of NF1 vasculopathies. The Center is organized into a small Administrative Core, a Transgenic/Animal Core, and four Projects. The Transgenic/Animal Core (PI, Luis F. Parada) is responsible for breeding transgenic and knockout mice, genotyping, and for developing or importing additional mouse strains. Project 1 (PI, Luis F. Parada) is a continuation of a productive effort to study the molecular and cellular mechanisms by which NF1 mutation engenders plexiform neurofibromas and malignant peripheral nerve sheath tumors. Project 2 (PI, D. Wade Clapp) focuses on the signaling mechanisms that promote interplay between Nf1-/- Schwann cells and different heterozygous cell lineages identified in the tumor microenvironment in neurofibroma development and targeting these cell-cell interactions with FDA-approved therapeutics. Project 2 will also utilize human cells isolated from NF1 patients to verify that the observations in the murine model are faithfully recapitulated in the human system as a preclinical platform to identify therapeutic targets within the neurofibroma microenvironment. Project 3 (PI, David Ingram) will study the role of Nf1 in controlling endothelial and vascular smooth muscle/pericyte function. Project 4 (PI, Lu Q. Le) will investigate the etiology of dermal neurofibromas in mouse and proposes a clinical trial for treatment of human patients. Project 4 also proposes the establishment of a comprehensive NF1 clinic. Together, the proposed program of research promises to contribute to a better understanding of NF1-associated tumorigenesis at the molecular, cellular, and systems levels. PUBLIC HEALTH RELEVANCE: Neurofibromatosis type 1 is an incurable disease with an incidence of 1 in 3,500 live births. Hallmark features of NF1 include cafe au lait macules, and axillary and groin freckling, along with multiple peripheral and central nerve tumors including dermal and plexiform neurofibromas. The current research of the NF Center investigators will lead to a better understanding of the underlying molecular and cellular events that occur in NF1. 2P50NS052606-06/Project 1 PARADA, LUIS DESCRIPTION (provided by applicant): Neurofibromatosis type 1 is a genetic disease with wide ranging consequences on the afflicted individuals ranging from potential intellectual and cognitive deficits to appearance of idiopathic tumors in the peripheral nervous system collectively called neurofibromas. Over the past 12 years we have developed mouse models of NF1 with the objective of recapitulating a variety of the pathologic features seen in patients. This proposal continues and expands upon our experience in generating faithful genocopies of NF1-associated neurofibromas. In preceding work funded by this award, our scientific teams joined forces to test the hypothesis that NF1 haploinsufficiency outside the Schwann cell lineage provided critical contribution to plexiform neurofibroma development. This hypothesis emerged from studies with our mouse models and the outcome has revealed the importance of mast cells in contributing to the tumor phenotype and ultimately leading to clinical trials to block mast cell activity in patients. In the present application we propose to extend our mouse modeling capabilities to further understand the etiology of plexiform neurofibromas, to identify the source and etiology of dermal neurofibromas, and to use our MPNST models to seek out therapeutic opportunities. In Specific Aim 1, we will employ tamoxifen-inducible ere driver lines and alternative approaches to better define the source of the cell of origin for plexiform neurofibromas and to define the temporal window of competence for tumor development. In Specific Aim 2, we will expand on our recent development of a murine model for dermal neurofibromas and on the discovery that skin-derived precursors (SKPs) are the cell of origin for these tumors. We will use multiple techniques including the use of chick/quail embryo transplantation of neural tubes to examine whether the neural crest is the original source of these tumor-competent cells. We will also develop new valuable tamoxifen-inducible transgenic Cre driver lines to probe the neural crest-derived tissues for tumor potential. Finally, in Specific Aim 3, we will screen primary MPNST-derived tumor cells to undertake small chemical and RNAi high throughput screens. These screens aim to identify small molecule compounds and genes that are required for tumor cell proliferation and growth and that can become targets for therapeutics. PUBLIC HEALTH RELEVANCE: Neurofibromas and MPNSTs are incurable cancers that arise in Neurofibromatosis Type 1. We have created mutations in the NF1 gene in mice that allow us to generate faithful models of many of the disease pathologies. In this application, we use our genetic mouse models to study plexiform and dermal neurofibroma tumors to derive critical information for therapy development. We also propose strategies to discover novel potential therapeutic molecules for the treatment of MPNSTs.