Rae Nishi - US grants

nervous system regeneration; axon; extracellular matrix; epithelium; submandibular gland; ganglions; monoclonal antibody; salivary glands; embryo /fetus tissue /cell culture; immunofluorescence technique; histochemistry /cytochemistry; embryo /fetus;

A fundamental process in the development of a functional nervous system is the accurate connection of neurons with the target tissues they control. The overall objective of this project has been to identify molecules important in guiding the outgrowth of axons from embryonic neurons. A bioassay system has been developed which tests the ability of axons from embryonic mouse or rat neurons to extend on modified forms of the pathway which they normally use in vivo. This has been possible by using an explant culture system that reproduces the patterned outgrowth of submandibular ganglion axons over the salivary gland epithelium. Monoclonal antibodies are being generated using a novel immunological technique that biases the immune response towards highly conserved epitopes such as the functional sites of molecules. Initially, the primary focus of the study was to raise antibodies against substrates and to use these to identify the molecules involved. This is still the case; however, a new approach has also been undertaken to identify the complimentary molecules present in axonal membranes that mediate recognition of substrate-associated molecules. The specific objectives for the final year of support will be two-fold: 1) To continue to refine the immunization technique to produce monoclonal antibodies specific for the epithelial basement membrane upon which submandibular ganglion neurons travel; and to screen these antibodies by using the in vitro bioassay to test for the ability of these antibodies to block axonal outgrowth; 2) To isolate receptors in the neuronal membrane that recognize substrate molecules such as laminin, a potent inducer of neurite outgrowth, by affinity purification. The affinity purified material will be used to raise antibodies. If such molecules actually mediate outgrowth then antibodies directed against their functional sites should interfere with outgrowth. These antibodies will provide powerful tools for the further study of the regulation of axonal outgrowth during development and regeneration. They will be especially important tools not only in further understanding how these processes may go awry to produce birth defects and neurological malfunction but also in defining conditions necessary for the recovery of function after trauma to the nervous system.

The development and maintenance of connections between motor neurons and muscle are critical to normal physiological function. Nowhere is the result of disrupting these connections more devastating than in patients with degenerative motor neurons diseases such as amyotrophic lateral sclerosis (ALS). During ALS many motor neurons die, leaving the muscles without ability to function. Little is known about why they die. During development there is a period of normal cell death for many neurons including motor neurons; however not all the neurons die as they do in ALS. What are the mechanisms that regulate and restrict this cell death? Coincident with this period of cell death is a period of rapid induction of proteins required for normal motor neurons function such as choline acetyltransferase (ChAT). Do the same molecules that regulate cell death also regulate the differentiation of the surviving neurons? Could these mechanisms be disrupted in ALS? the objectives of this study are to isolate molecules that may be involved in regulating motor neuron death and development, and to understand the molecular mechanisms by which they act. These objectives will be accomplished by using cultures of pure motor neurons form the chick ciliary ganglion (CG) as a bioassay for trophic molecules. Our previous work has identified two distinct biological activities that affect the development of CG neurons: ChAT- stimulating activity (CSA) and growth promoting activity (GPA). CSA stimulate specific induction of ChAT, which is essential for functional neural transmission, and GPA supports survival and long-term growth of CG neurons. The specific aims of this proposal are: 1. To purify the molecules responsible for GPA and CSA and to obtain sufficient amino acid sequence information to clone their cDNAs; 2. To raise monoclonal antibodies against GPA and CSA by utilizing partially purified fractions of the activities; and 3. To test the specificity of action of GPA and CSA on other neuronal cell types. Future work will involve using the purified molecules and the antibodies obtained in this study to test the hypothesis that survival, growth and differentiation of motor neurons are controlled by the GPA and CSA released by neuronal target tissues such as muscle.

Many neurological diseases are due to the degeneration of specific populations of neurons. An important contribution of basic research towards the understanding of such disorders would be the understanding of how postmitotic neuron survival and function is maintained in the normal organism. A prominent hypothesis is that adult neuron survival and function is dependent upon the availability of trophic factors produced by target tissues. The long term goal of this project is to identify and isolate such growth factors, and to determine the mechanisms by which they regulate neural development and maintenance. Our model system is the innervation of ocular tissues by neurons of the ciliary ganglion, thus this work also has application to the further understanding of how innervation of the eye is maintained by growth factors produced in the choroid layer, pigmented epithelium, iris, ciliary body and neural retina. We have purified, cloned and expressed a factor that supports the survival of chick ciliary ganglion (CG) neurons called growth promoting activity (GPA). Activities in eye extract and conditioned medium from choroid layer cells that control cholinergic and peptidergic transmitter phenotype expression in CG neurons termed ChAT stimulating activity (CSA) and somatostatin stimulating activity (SSA) have also been identified. Preliminary data indicates that SSA is activin. The primary goal of this application is to test the hypothesis that GPA, CSA, and SSA are all target-derived molecules that regulate the development of CG neurons. In order to establish that a molecule is a target-derived trophic molecule a number of criteria must be fulfilled. Thus, the specific aims of this proposal are: 1) To test whether the temporal and tissue specific pattern of the expression of GPA and GPA mRNA is consistent with that of a target derived trophic factor; 2) To use immunoprecipitation and HPLC to test whether GPA is secreted by a target tissue of CG neurons, and if GPA is secreted to determine the mechanism of secretion; 3) To test whether addition of GPA to developing chicken embryos is able to rescue CG neurons from dying and to test whether antibodies directed against GPA are able to increase the number of neurons dying; 4) To use iodinated recombinant GPA to test whether a saturable high affinity binding site for GPA is found on CG neurons; 5) To isolate and clone SSA and CSA, and to produce specific antibodies in order to test whether SSA and CSA are target derived factors that influence transmitter status of CG neurons.

The long term goal of this study is to understand the molecular and cellular mechanisms of target- neuron interactions during development. We will accomplish this goal by applying a variety of methods for manipulating gene and protein expression during chicken embryo development in vivo. These studies are expected to advance the understanding of the development and maintenance of the nervous system, providing insights into the processes of neural maintenance and neurodegenerative disease. Our studies will use the avian ciliary ganglion (CG) because of its simple neuronal composition, the accessibility of the neurons and their targets throughout development, and the coincidence of synapse formation, cell death, and transmitter expression within a well-defined window of development. The CG contains two populations of neurons: ciliary neurons that innervate iris and ciliary body and choroid neurons that innervate arterial smooth muscle in the choroid layer of the eye. We have identified three molecules by their actions on CG neurons in cell culture: growth promoting activity (GPA), which supports neuronal survival; activin A, which induces the expression of somatostatin; and follistatin, an inhibitor of activin. Our working model is that availability and levels of these three target-derived molecules regulate survival and neuropeptide phenotype of CG neurons. We will determine the validity of this model by testing the following hypotheses: l). Cell death in the CG during development is controlled by GPA expressed in the iris, ciliary body, and choroid layer; 2) Specific expression of somatostatin in choroid neurons of the CG is induced by activin expressed in the choroid layer, whereas follistatin blocks the effects of activin expressed in the iris/ciliary body. The specific aims are: l) to overexpress GPA in order to determine if CG neurons are rescued from cell death; 2) to use cell cultures of CG target tissues as well as Co-cultures to assay the effectiveness of GPA blocking agents; 3) to block GPA during embryonic development in vivo to determine if CG cell death is exacerbated; 4) to identify the mechanism by which alpha bungarotoxin increases CG neuron survival; 5) to quantify and characterize GPA receptor and receptor associated molecules during development; 6) to test the effectiveness of activin and follistatin blocking agents on co-cultures of CG neurons with choroid smooth muscle and iris/ciliary body; 7) to block to the action of activin in vivo to determine if somatostatin expression in choroid neurons is prevented; 8) to inject activin in the anterior chamber of the eye to induce somatostatin in ciliary neurons; 9) to block the effects of follistatin in the iris/ciliary body to determine if somatostatin is induced in ciliary neurons.

DESCRIPTION (Adapted from the applicant's abstract): The etiology of neurodegenerative orders has been linked to damage caused by oxidative stress. One mechanism by which oxidative stress can be mediated is an excess of reactive iron, which catalyzes the formation of free radicals. The mechanism by which iron is stored in the brain can induce neuronal death has not been clear because cells are normally protected against the deleterious effects of iron. The investigator have purified an activity that induces apoptosis in embryonic day 8 (E8) chick ciliary ganglion neurons. In contrast more mature (>E10) ciliary ganglion neurons were not killed. N-terminal sequencing revealed that this activity was ovotransferrin. Death inducing activity required that iron be bound to the transferrin. The EC50 of diferric recombinant transferrin in inducing apoptosis was 5nM, well within the levels found in embryonic extracellular fluid and blood (30-40 uM). This effect of FeTr was not limited to ciliary ganglion neurons: lumbar sympathetic ganglia contain two populations of neurons, one which survives in nerve growth factor and does not die when exposed to FeTf, and another which survives in clilary neurotrophic factor (CNTF) and is killed by FeTf. The developmental switch in CG neuron sensitivity to FeTf suggests that susceptibility of neurons to transferrin-mediated apoptosis is likely to be a normal developmental event that is regulated by cell-cell interactions. The investigator proposes to study the molecular basis for the differential sensitivity of neurons to killing caused by transferrin-mediated iron transport. These studies are likely to lead to important clues as to how the process may go awry in neurodegenerative disease. The specific aims are: (1) to test the hypothesis that neurons become sensitive to killing by FeTf when they fail to downregulate transferrin receptor, human transferrin receptor will be overexpressed in insensitive neuronal populations in order to test whether it confers sensitivity to killing by human FeTf; (2) to test the hypothesis that susceptibility to FeTf is mediated by reduced levels of ferritin, the intracellular iron binding protein, ferritin heavy chain will be overexpressed in order test whether neurons are protected from death induced by FeTf; (3) to test the hypothesis that intracellular iron induces apoptosis by accumulating in mitochondria through reduced levels of frataxin, a protein that stimulates iron transport out of mitochondria, frataxin will be overexpressed in neurons in order to determine if they can be protected from killing by FeTf; and (4) to test the hypothesis that intracellular iron kills cells by generating excess free radicals which damage mitochondria, it will be determined if: (1) FeTf generates free radicals; (2) anti-oxidants rescue neurons from FeTf; (3) inhibitors of oxidative phosphorylation induce apoptosis with the same characteristics as FeTf; (4) cytochrome c is released by FeTf treated neurons; (5) overexpression of the mitochondrial anti-apoptotic protein Bcl-2 protects cells from FeTf; and (6) caspase inhibitors rescue neurons from FeTf.

DESCRIPTION (provided by applicant): In addition to their traditional function of mediating rapid cholinergic transmission, nicotinic acetylcholine receptors (nAChRs) are poised to serve non-traditional functions such as regulating gene transcription through calcium-dependent signaling. Therefore, studying the role that nicotinic signaling plays in guiding the development of the nervous system provides a means for assessing the potential adverse effects of prenatal exposure to drugs such as nicotine. We propose to use the chicken ciliary ganglion as a simple model system in which the role of nicotinic signaling in programmed cell death in vivo can be studied in detail. Programmed cell death normally reduces the total number of neurons in the ciliary ganglion by 50% between embryonic days (E)8 and E14, and embryonic neurons express two functionally distinct populations of nAChRs: homomeric alpha7 containing nAChRs that become extrasynaptically localized by E14, and heteromeric nAChRs containing alpha3, alpha5, Beta2 and Beta4 subunits (alpha3 * nAChRs). Our previous studies suggested that activation of neuronal alpha7 subunit containing nAChRs caused neuronal cell death. We propose that between E6-9, neurons that express a high density of alpha7 nAChRs on their surfaces allow calcium influx that exceeds the set point for survival, thereby inducing apoptosis. After E9, target interactions act to prevent cell death through alpha7 by upregulating an endogenous abtx-like molecule, lynx, that "silences" alpha7 nAChRs. Thus, the central hypothesis of this proposal is that an overabundance of signaling through alpha7 subunit containing nAChRs induces calcium-dependent cell death during normal ciliary ganglion development. To test this hypothesis, the aims of our project are: 1. To determine whether embryonic neurons express more heterogeneity with respect to the levels of alpha7 mRNA prior to cell death and whether neurons expressing elevated alpha7 remain after rescue by MLA and abtx;2. To determine whether overexpression of alpha7 nAChR subunits causes enhanced levels of intracellular calcium in response to nicotinic stimulation and exacerbates cell death in vivo;3. To determine whether reducing alpha7 nAChR expression or locally blocking alpha7 nAChRs in ciliary ganglion neurons promotes survival;4. To test the hypothesis that target- interactions prevent cell death by inducing the expression of lynx, a cell surface molecule with homology to abtx that silences cell surface alpha7 nAChRs. These studies will make a significant contribution towards the detailed understanding of how nicotinic signaling contributes to the development of the nervous system.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (15) Translational Science and specific Challenge Topic, 15- DA-110: Determining if and how Adolescent Behaviors Affect Connections in the Developing Brain. The adverse impact of smoking on health is a world-wide epidemic that contributes to four million deaths a year, with an expected increase to 10 million per year world-wide by 2030. Tobacco dependence is viewed as a "pediatric disease" because most people begin smoking during adolescence. Adolescents display a greater sensitivity to the addictive effects of nicotine, becoming dependent upon smoking more rapidly than adults. Studies also show that smokers who began as adolescents smoke more frequently with a lower rate of quitting. Despite the many reports establishing a greater sensitivity of adolescent brains to nicotine, the fundamental basis of this greater sensitivity together with the long-term consequences of nicotine exposure on the wiring and neurochemistry of the brain is unknown. Most animal studies on adolescents have limited outcome measures of nicotine exposure to a single aspect such as behavior, agonist binding, or electrophysiological outcomes. Furthermore, none of the studies have examined plastic changes at the level of individual neurons with respect to the normal endogenous cholinergic system of the basal forebrain, and we know of no studies that integrate information from animal systems with human behavior. Thus, this Challenge Grant is a multidisciplinary approach that involves investigators from the fields of animal behavior, human cognition, genetic epidemiology, and molecular cell biology to test the validity of the following model: that the enhanced sensitivity of adolescents to nicotine is due to an imbalance between the endogenous cholinergic system and the function of nicotinic acetylcholine receptors (nAChRs). We predict that the cholinergic pathway from the basal forebrain to the cortex and hippocampus is not yet functionally mature in adolescents, yet an overabundance of terminals with nAChRs combined with a reduced level of prototoxin modulators of nAChRs allows exogenously administered nicotine to lead to long term changes that enhance reward pathways, while preventing full cholinergic maturation, leading to a beneficial effect of nicotine in cognitive performance that reinforces the use of nicotine. The specific aims of this application are: 1) to use adolescent and adult transgenic Thy-1-eYFP mice that demonstrate nicotine-induced conditioned place to determine whether changes in arborization of eYFP- labeled dendrites in layer 5 as well as changes in eYFP-terminals in nucleus accumbens occur;to determine if correlative changes in cholinergic innervation or dopaminergic interaction with eYFP-containing structures occur;2) to determine whether mice that lack or carry only one copy of the prototoxin gene psca exhibit a greater physiological and behavioral response to nicotine and to determine whether this correlates with parameters examined in Aim 1;3) to genotype human adolescent subjects that have been screened and tested for impulsivity, nicotine exposure, and drug abuse to determine whether specific alleles of prototoxin genes are correlated with increased impulsivity or nicotine dependence. PUBLIC HEALTH RELEVANCE: The adverse impact of smoking on health is a world-wide epidemic that contributes to four million deaths a year. Studies show that smokers who began as adolescents smoke more frequently with a lower rate of quitting. Despite the many reports establishing a greater sensitivity of adolescent brains to nicotine, the fundamental basis of this greater sensitivity together with the long-term consequences of nicotine exposure on the wiring and neurochemistry of the brain is unknown. A greater understanding of these will lead to possible preventive measures and therapies, having a significant long-term impact on human health.

DESCRIPTION (provided by applicant): Neuroblastoma is a cancer arising from autonomic neuron progenitor cells that occurs in the adrenal medulla or as paraspinal tumors in the abdomen or thorax of infants and young children. If it is identified in children over the age of 1, it is nearly always a highly malignant, aggressive cancer that kills the child within 4-5 years, even after a strenuous chemotherapeutic regimen combined with radiation and a bone marrow transplant. This project will determine whether an unusual nicotinic acetylcholine receptor is a novel target for therapeutics in treating neuroblastoma. We have isolated cells from bone marrow aspirates obtained from patients with stage 4 neuroblastoma. When tested in xenograft assays in immunodeficient mice, the cells can be categorized into tumor-initiating cells (TICs) and non-tumor initiating cells (non-TICs). We have discovered that the gene encoding the a5 nicotinic acetylcholine receptor (nAChR) subunit, CHRNA5, is expressed at 7- 19-fold higher levels in TICs than in non-TICs. Furthermore, CHRNA5 is 2-10-fold higher in TICs than in normal neurogenic neural crest stem cells, and CHRNA5 is elevated in primary neuroblastoma tumors and cell lines derived from tumors (SH-SY5Y, SKN-KCN, SKN-KCNR) above levels found in normal sympathetic ganglia. In a high throughput drug screen for compounds that would reduce proliferation of TICs, MG 624, a nAChR antagonist, was identified. Based upon our expression data, we hypothesize that nicotinic receptors containing a5 contribute to the malignant phenotype of neuroblastoma and therefore may be a new target for drugs because these receptors are uncommon in the normal autonomic nervous system. The proposed aims will test this hypothesis by: 1) using RNAi to determine whether one or more nAChR subunits is necessary for the malignant phenotype of neuroblastoma cells (TICs and cell lines from primary tumors) and their sensitivity to MG 624; 2) determining whether overexpression of CHRNA5 and/or other subunits in normal skin-derived precursors is sufficient to produce a transformed phenotype.