Mark M. Black, BS, PhD - US grants

Studies during the coming year will extend our previous work on the composition of the axonal and dendritic cytoskeletons. We will focus on microtubules (MT), which are composed of tubulin and microtubule-associated proteins (MAPs). Tubulin, which is the major subunit of MT, consists of a-tubulins and B-tubulins, each of which are composed of several isoforms. We will determine whether the tubulins of axonal MT differ from those of cell body + dendritic MT. Explant cultures of rat sympathetic neurons will be used for these experiments because they permit the ready preparation of pure axons and fractions enriched in cell bodies and dendrites. Tubulin will be immunoprecipitated from pure axon and cell body + dendrite-enriched fractions and analyzed by isoelectric focusing. These experiments will show which tubulin isoforms are present in axons as well as cell bodies and dendrites, and also whether any isoforms are uniquely present or greatly enriched in one region of the neuron compared to others. We have previously shown that axonal MT differ from dendritic MT with respect to their MAPs, and have hypothesized that these differences contribute to the well documented differences seen in the spacing between MT of axons compared to dendrites. In vitro experiments will be carried out to test this hypothesis. MT will be reconstituted from pure tubulin plus varying amounts of axonal or dendritic MAPs. The resulting MT will be centrifuged and the packing density of the MT will be quantified biochemically and electron microscopically. The results will reveal whether axon specific MAPs and dendrite specific MAPs have differing effects on the lateral spacing of microtubules under these controlled in vitro conditions. Finally, we will extend our recent work on the phosphorylation of coated membrane proteins of cultured neurons. Coated membranes are involved in a variety of physiological processes including membrane re-cycling following depolarization and stimulation with various hormones. We have recently shown that specific proteins of the coat structure of coated membranes are phosphorylated in intact neurons. Biochemical and immunochemical methods will be used to determine whether the phosphorylation state of these proteins changes in association with depolarization or stimulation with hormones such as nerve growth factor.

The experiments of this proposal are part of a long-term goal to define the cellular processes that regulate the organization of the neuronal cytoskeleton. These experiments focus on neurofilaments (NF) and neurofilament proteins (NFP). NF are prominent components of the axonal cytoskeleton which contribute to its volume, stability, and form, and thereby the radial dimension of axons. NFP are phosphorylated in vivo. It is generally assumed that phosphorylation regulates various properties of NF, although the nature of these properties and how phosphorylation regulates them are unknown. For example, little information is available concerning where in the neuron NFP phosphorylation occurs, whether NFP phosphorylation is coordinated with dephosphorylation, or when phosphorylation occurs relative to the synthesis and assembly of NFP. Experiments are proposed in this application to directly address all of these issues. These experiments will use well defined culture systems coupled with electrophoretic, immunoprecipitation, and immunoblotting assays of NFP to analyze NFP phosphorylation (and dephosphorylation) in the neuron as a whole and in preparations of cell bodies or pure axons. An important parameter of NF organization is the lateral spacing between NF. Experiments are also proposed to evaluate the contribution of the various NFP to the packing of NF and to test the hypothesis that phosphorylation of NFP regulates the spacing between NF. These experiments will make use of a recently published procedure for quantifying the effect of various factors on the spacing of linear polymers in vitro. NF of varying composition and phosphorylation state will be prepared from purified NFP or by enzymatic treatment of native NF. The NF will be pelleted by centrifugation, and the spacing of NF in the pellets will be determined directly by electron microscopy and also by measuring the volume of the pellet. Completion of the proposed experiments will clarify several issues concerning the regulation of NF organization, and thus will provide a cellular and molecular framework for understanding the regulation of neuronal geometry. In addition, many degenerative and toxic disorders of the nervous system are manifest at a cellular level in dramatic alterations in the NF network. By defining normal mechanisms for the control of NF organization, the proposed experiments will provide information that is essential for understanding the cellular bases of these disorders.

Identifying the mechanisms for producing and maintaining neuronal morphology is central to studies of brain development and aging. Several lines of evidence indicate that the pattern of cytoskeletal organization defines neuronal morphology. The experiments of this proposal are aimed at defining mechanisms for elaborating and maintaining cytoskeletal patterns in neurons. The relative plasticity of neuronal shape declines during maturation. This decrease is an important event in neuronal maturation because it ensures that the morphological features produced early in development are maintained during subsequent life. We propose that this decrease in plasticity reflects a corresponding decrease in the plasticity of the cytoskeleton. Microtubules (MT), which are major components of the neuronal cytoskeleton, have labile and stable components; stable MT are not depolymerized by standard MT depolymerizing conditions, while labile MT are. Preliminary studies suggest that the balance between labile and stable MT changes during neuronal maturation. The proposed project will use in vivo and in vitro systems to (1) determine whether the balance between labile and stable MT changes during neuronal development and (2) identify the molecular bases for changes that occur in MT properties during neurite growth. MT are polymers of tubulin and MT-associated proteins (MAPs). Several properties of MAPs suggest that they influence the organization of MT and thereby the cytoskeleton as a whole. The composition of MAPs appears to vary in axons and dendrites of individual neurons. This regional heterogeneity of MAPs has implications for the ability of neurons to generate morphologically distinct domains such as axons and dendrites. The proposed project will use a variety of biochemical and immunological procedures to more fully define the (1) extent of selective partitioning of MAPs in individual neurons and (2) cellular bases for selective partitioning of MT proteins in neurons. The results of these and the above-mentioned studies will relate to the broader issue of how neurons elaborate and maintain the detailed shapes that are so crucial to their functional specialization within the nervous system.

Neurons contain elaborate cytoskeletons which consist of microtubules, neurofilaments, and microfilaments. The neuronal cytoskeleton provides the architectural framework that defines the external shape of the neuron, and also organizes the cytoplasm to carry out motile and metabolic processes essential to life. Thus, the mechanisms for regulating cytoskeletal structure are directly involved in generating and maintaining neuronal form and function. This application proposes direct experiments to determine how cytoskeletal organization is regulated in neurons. These experiments focus on microtubules (MT) and MT proteins. The specific experiments proposed are based on previous work demonstrating that the MT system of axons differs from that of dendrites with respect to composition and organization. These findings indicate neurons generate two distinct MT systems, one for the axon and on for the dendrites. The proposed experiments will (i) identify the structures responsible for assembling and organizing the MT systems of the axon and dendrites; (ii) determine the relative roles of co-translational and posttranslation mechanisms in the compartmentation of MT proteins between the axonal and somatodendritic compartments; (iii) define how the compartmentation of MT components specializes the MT systems of the axon and dendrites. All of these experiments combine structural, immunological, and biochemical analyses of MT in intact neurons with in vitro studies of MT and MT proteins. We have already used this approach to understand how specific MT components influence the structure of the microtubule framework in neurons. Successful completion of the proposed experiments will resolve several issues concerning the regulation of MT structure and organization, and thereby further current understanding of the cellular and molecular bases of neuronal form and function. In addition, many pathologies of the nervous system are manifest at a cellular level in dramatic alterations in the neuronal cytoskeleton. By defining normal mechanisms for the control of MT organization, the proposed research will contribute toward a better understanding of these pathologies.

DESCRIPTION: Axons contain an elaborate cytoskeleton that consists of microtubules, neurofilaments, and microfilaments. The cytoskeleton comprises an architectural framework that defines the external shape of the axon and also organizes the intracellular motility necessary to grow and maintain the axon. Thus the mechanisms that establish the cytoskeleton in neurons contribute directly to the elaboration and maintenance of neuronal form and thereby function. This application proposes direct experiments on the dynamic processes that generate the cytoskeleton in growing axons. These experiments focus on the class of microtubule proteins known as microtubule-associated proteins (MAPs). MAPs promote the assembly and stabilization of microtubules in vitro, and also integrate microtubules with other cytoskeletal elements. The goal of the proposed experiments is to define the functional specializations of MAPs in axon growth. The applicant hypothesizes that MAPs coordinate the assembly and stabilization of microtubules with the motile events that underlie the initiation and continued growth of the axon. To test this hypothesis, he has developed a novel approach for evaluating the functional specialization of MAPs in axonal growth with involves microinjection of anti-MAP antibodies into cultured neurons to selectively inactivate specific MAPs. The consequences of MAP inactivation on axonal growth and axonal microtubules are then evaluated. The proposed experiments combine microinjection of anti-MAP antibodies into cultured neurons with high resolution video and fluorescence microscopy to evaluate the effects of MAP inactivation on axon structure, growth cone motility, and the organization, assembly dynamics and stability of neuronal microtubules. He has already used these approaches to demonstrate that MAP1b, a major MAP in growing axons, is apparently essential for the initiation and continued elongation of axons in culture, Successful completion of the proposed experiments will define essential mechanisms involved in generating the microtubule arrangements required for the normal growth of axons. In addition, many pathologies of the nervous system are characterized by abnormalities of cytoskeletal organization. By defining normal mechanisms for generating and maintaining microtubule arrangements in neurons, the proposed research will contribute toward a better understanding of these pathologies.

DESCRIPTION (Adapted from applicant's abstract): Axons contain an elaborate cytoskeleton that consists of microtubules, neurofilaments, and microfilaments. The cytoskeleton comprises an architectural framework that defines the external shape of the axon and also organizes the intracellular motility necessary to grow and maintain the axon. Thus, the mechanisms that establish the cytoskeleton in neurons contribute directly to the elaboration and maintenance of neuronal form and thereby function. This application proposes direct experiments on the dynamic processes that generate the cytoskeleton in growing axons. These experiments focus on microtubules, with the goal of defining the mechanisms that modulate microtubule assembly and dynamics in growing axons. These mechanisms are essential to the generation of the microtubule array required for axon elongation and to aspects of growth cone motility involved in axonal pathfinding. The proposed experiments focus on three proteins that are excellent candidates to modulate the properties of the microtubule array in growing axons; these proteins are tau, microtubule-associated protein 1b (MAP1b), and stable-tubule-only-polypeptide (STOP). All three proteins promote microtubule assembly and stability in vitro. We hypothesize that these proteins establish the dynamic behavior of microtubules required for the initiation and continued growth of the axon. To test this hypothesis, we propose to selectively inactivate these proteins using antibody injection approaches, or to introduce excess amounts of these proteins into neurons, and then to use high resolution video and fluorescence microscopy as well as electron microscopy to evaluate the consequences of these manipulations on axon structure, growth cone motility, and the organization, assembly dynamics, and stability of axonal microtubules. We have already made progress on tau and MAP1b functions in developing neurons using these approaches. Successful completion of the proposed experiments will define essential mechanisms involved in generating the microtubule arrangements required for the normal growth of axons. In addition, many pathologies of the nervous system are characterized by abnormalities of cytoskeletal organization. By defining normal mechanisms for generating and maintaining microtubule arrangements in neurons, the proposed research will contribute toward a better understanding of these pathologies.