Dennis A. Steindler - US grants

The experiments proposed here will determine and compare the organization of divergent axonal projections which arise from the midbrain raphe nuclei, locus coeruleus, and trigeminal nuclei. Recently developed, sensitive double retrograde axonal tracing techniques will be employed using the tracers wheat germ agglutinin (WGA), N-[acetyl-3H] and WGA conjugated to horseradish peroxidase (HRP) as well as fluorescent tracers in paired injection strategies within projection targets of these brainstem neuronal groups to reveal the order of their collateralized and unbranched axonal projections. Light and fluorescence microscopic analysis of tissue sections from these studies will determine differences or similarities that exist in the organization of divergent axonal projections arising from brainstem monoaminergic neurons versus those which arise from sensory relay neurons of the midbrain and medullary trigeminal nuclei. Preliminary experiments have revealed that each CNS structure receiving midbrain raphe projections has its own unique representation within a topographically distinct portion of the raphe. It also appears that raphe-innervated structures possessing interconnections with each other are interrelated by a topographically distinct group of collaterilized raphe projection neurons. Correlative anterograde axonal tracing experiments using small injections of lectins within portions of the raphe, locus coeruleus, and trigeminal nuclei will reveal different patterns of topographical organization and geometric orderliness within target structures of these divergently projecting brainstem neurons. Locus coeruleus, raphe, and trigeminal neurons all project to the forebrain (e.g., thalamus and cerebral cortex), midbrain (e.g., tectum and mesencephalic reticular formation), hindbrain (e.g., cerebellum) and spinal sites. Studies proposed here which attempt to resolve the different plans for organizing brainstem neurons and their axonal projections to various central nervous system structures will reveal the ways in which brainstem neurons interrelate neuronal circuitries in such structures as the cerebral and cerebellar cortices. When these circuits are altered, usually a consequence of neuronal loss, central nervous system disease states such as Parkinson's Disease or Cerebral Palsy can follow. Understanding the development, maintenance, and interrelative schemes of brainstem-target circuitries will shed light on the cause and possible treatments for such diseases.

During brain development and again following injury, glial cells produce a variety of molecules that affect the positioning and growth of neurons and their processes. Recently, glial and glycoconjugate (glycoproteins, glycolipids, and glycosaminoglycans) boundaries have been discovered during brain pattern formation, and these structures cordon off developing groups of functionally distinct neurons and their neurites. In the developing caudate-putamen (neostriatum), these boundaries surround different compartments of a neostriatal mosaic, the patch and matrix, and most likely serve to separate growing processes of cells in the two different compartments during a critical period of their formation. The following proposal will test possible roles for astrocytes and glycoconjugates during normal and abnormal development of a clinically important area of the brain, the nigrostriatal circuit. In Aim 1, the time course of appearance and disappearance of astrocyte- derived extracellular matrix (ECM) molecules will be determined in this circuit. Specific Aim 2 will focus on the potential reappearance of boundaries following different lesions within the nigrostriatal circuit during development and in the adult, and antibody perturbation experiments versus studies on a tenascin-knockout mouse will provide complementary data on possible functions of particular ECM boundary molecules on neuritic growth. A third set of experiments will exploit two in vitro bioassays to affect the functions of certain boundary molecules during cell-boundary interactions in the developing nigrostriatal circuit, as well as in the lesioned adult circuit that results in the appearance of another type of glial/glycoconjugate boundary - the astroglial scar. These studies will be performed in normal as well as tenascin-deficient animals where other ECM molecules (e.g. DSD-1) can now be studied and manipulated in a nigrostriatal circuit that, e.g., has always lacked tenascin. In Specific 4, the potential roles of astrocytes and ECM in neurodegenerative diseases that affect the human basal ganglia will be explored in an extensive collection of Huntington's, Parkinson's, and other disease and control specimens that allow a thorough correlation of boundary elements in relation to neuronal loss that occurs in nigrostriatal circuit in these diseases. In all, the studies proposed here will establish functional roles for astrocytes and associated, developmentally-regulated molecules in shaping normal basal circuitry during development, and perhaps how they adversely affect neurons and possible neurite regeneration following traumatic injury or chronic disease. The normal developing and injured nigrostriatal circuit is amenable to studies of functions of glial/glycoconjugate boundary elements. Glia and glycoconjugates may play important roles during brain pattern formation, but the recapitulation of cell and molecular interactions that might occur during normal development may have deleterious effects on neuron survival and neuritic regrowth in the compromised, mature brain.

Stem and precursor cells have now been identified in the adult rodent and human brain, and there is a growing interest in manipulating these cells to proliferate and differentiate along particular lines that might favor their use in cell replacement therapies for neurological disease. Whereas most groups have studied these cells and their in vitro generation of neurospheres from the embryonic or early postnatal brain, our lab has focused on the presence and proliferation of stem/precursor cells in the adult brain - so called neuropoiesis . The presence of developmentally regulated cell adhesion and extracellular matrix (ECM) molecules on and around these cells in vivo in the subependymal zone, a region we have begun to refer to as brain marrow , suggests that manipulating the adhesive interactions of stem/precursor cells might enhance their proliferation and also offer a level of control over their differentiation into, e.g., particular types of neurons. This proposal is founded on the premise that in vitro ECM perturbation experiments, use of novel cell feeder layers, and transplantation experiments will afford the isolation and characterization of distinct types of adult brain stem/precursor cells, and at the same time help us direct the growth and differentiation of these cells into progenitors and fully differentiated cells (e.g. neurons) that are able to integrate into established and compromised neuronal circuitries. Tissue culture and brain grafting experiments will be performed using normal and ECM-deficient transgenic mice to: 1) prove that manipulating ECM molecules can lead to an ex vivo expansion of stem/precursor cell populations, and also restrict cell lineage; 2) test feeder layers derived from non-neural tissues to release growth factors and other molecules that help to discriminate and sustain the most primitive classes of stem/precursor cells for extended periods of time in culture to allow more extensive analyses of their self-renewal, proliferation and differentiation; and 3) use feeder layer and other growth- inducing substrates to prime stem/precursor cell populations from the adult brain to better survive and integrate as grafts into the adult brain. Thus, using novel culture approaches, and immunocytochemical and molecular analyses of cell phenotype and developmental genes expressed by adult brain stem/precursor cells, these studies will elucidate factors that may be crucial to the survival and growth of these potentially clinically important, resident cells of the mature human brain.

DESCRIPTION (provided by applicant): The notion of stem cell plasticity has been recently supported by numerous examples of so-called tissue specific stem calls being coaxed into other tissue phenotypes. Altering fate of tissue-specific stem cells, inducing transdifferentiation, has been reported in many different bioassays, using a variety of cell culture and in vivo conditions and manipulations, without a systematic and direct comparison of different stem cell populations from different tissues in the same study. This proposal sets up a "head-to-head" comparison of transdifferentiation abilities of hematopoietic and neuropoietic stem cells in three sets of experiments that will establish the ability to alter the fate of hematopoietic and neuropoietic stem cells In vitro, embryonic and adult mouse in vivo studies will test hypotheses related to epigenetic factors that control the choice of fate as well as terminal differentiation of stem cells isolated from hematopoietic and neuropoietic structures Their ability to transdifferentiate, both in vitro and in vivo, will be evaluated using sensitive phenotypic and functional analyses, to confirm their full conversion and integration into their new host environments Specific Aim 1 will determine whether a candidate stem cell from the postnatal and adult mouse brain in fact exhibits true stem cell behaviors, looking at established attributes of hematopoietic stem cells including self-renewal and long-term clonal, multilineage reconstitution. This starting population will be sorted and enriched using specific surface antigens in order to determine the affects of culturing on developmental potential. Specific Aim 2 will look at the ability of hematopoietic stem cells to respond to neural cues in vitro and in vivo, by contributing functional cells that integrate within the developing, and repopulating adult nervous system Finally, Aim 3 will test the hypothesis that neural stem cells are plastic enough to respond to hematopoietic cues, following their transplantation into embryoid bodies, fetal mice, and radiation-ablated bone marrow The "head-to-head" assay systems to be used here will determine the potential usefulness of transdifferentiation approaches for establishing new cell therapeutics for many different human diseases, including cell loss as well as hyperplasia in a variety of blood and brain disorders Before we can understand the potential of blood ceils to become brain cells, a profound example of transdifferentiation that offers numerous alternative approaches to current cell replacement therapies, it is necessary to establish sternness of starting cell populations, and also understand factors involved in blood/brain stem cell proliferation, commitment to fate, and differentiation.

[unreadable] DESCRIPTION (provided by applicant): There is a need to develop cell and molecular protection approaches for at-risk neurons in neurodegenerative diseases, and regenerative medicine offers designer cells to be used for factor-delivery in diseases like Parkinson's (PD) where we know that certain growth factors might, if delivered properly, have the ability to rescue a discrete population of nigrostriatal cells and hence slow or halt the progression of the disease. This proposal will use a novel cellular transplant approach to deliver glial cell line derived neurotrophic factor (GDNF) in comparable in vitro and in vivo bioassays of dopamine neuron degeneration. Three specific aims focus on which astrocytes, from different sources, can be used to deliver GDNF to at risk dopamine neurons in a culture model of PD as well as a well-accepted rodent model (6-hydroxydopamine, 6- OHDA lesions of the neostriatum) of the disease. Specific Aim 1 will establish the potential for autologous cellular repair by focusing on newly-generated "immature" astrocytes derived from the mouse brain (e.g. adult cerebral cortex and striatum), and bone marrow versus mouse embryonic stem cells. These candidate astrocyte populations will be transduced with an eGFP-GDNF lentiviral construct to determine which population(s) is most amenable to stable growth factor transduction and release of the growth factor, for subsequent testing in an in vivo lesion-protection model (Aim 3). Specific Aim 2 will look at adult human brain astrotypic and bone marrow-derived cells and their ability to be stably transduced with lenti-GDNF and release the growth factor as the mouse cells do in Aim 1. Specific Aim 3 will assay the potential protective actions of GDNF-releasing mouse and human cells in DA depleted mice. These studies will incorporate methods already developed and tested in our laboratories, including cell culture, electrophysiology, gene therapy, cell grafting, immunophenotyping, biochemical and behavioral testing at PD models and repair, with the goal of developing a new therapeutic protocol for at-risk neuron protection and rescue in human PD. The possibility of using autologous cells derived from brain or bone marrow offers a tremendous therapeutic advantage for exploiting stem/progenitor as well as differentiated cells to slow and even halt the course of devastating neurological disorders. It is hypothesized that astrotypic cells, derived either from brain or bone marrow, have the ability to be engineered to release therapeutic factors in PD. [unreadable] [unreadable] [unreadable] [unreadable]