Scott T. Brady, PhD - US grants

This proposal focuses on two major goals: 1) characterization of axonal tubulin and microtubules; and 2) evaluation of the roles played by tubulin and axonal growth and regeneration. Tubulin is one of the major proteins of brain and knowledge about the properties of neuronal tubulin and microtubules is essential for understanding the roles played by tubulin in neuronal function. Axonal transport studies have shown that the bulk of axonal tubulin is cold insoluble and biochemically distinct from the tubulin in microtubules prepared by cycling from whole brain. The morphological correlate of cold insoluble tubulin was shown to be stable segments of microtubules. Such stable microtubule segments would affect the dynamics of the axonal cytoskeleton and could function as local regulators of microtubule polymerization in the axon. The plan of research will involve development of procedures for purification of cold insoluble tubulin from nervous tissue. Both axonal tubulin labeled by axonal transport and purified cold insoluble tubulin will be analyzed to determine the biochemical basis of cold stability in axonal microtubules. Particular emphasis will be placed on possible posttranslation modification and differences in microtubule associated proteins. The properties and biological function of cold stable microtubules will be evaluated by examination of the effects of insoluble tubulin on microtubule assembly in vitro. The amount, morphological form, and changes in cold insoluble tubulin in different neurons and regenerating neurites will be evauated biochemically and immunochemically. These studies will provide a better understanding of the roles that tubulin and microtubules play in the function of neurons as well as identifying molecular mechanisms for modulating the properties of neuronal tubulin.

DESCRIPTION: The processes of axonal transport are essential for neuronal function and underlie neuronal growth, maintenance and regeneration. Understanding axonal transport is a key to understanding the dynamics of the nervous system. Functions as diverse as conduction of the action potential, release of neurotransmitter, generation and maintenance of the presynaptic terminal, neuronal development and regeneration, and maintenance of neuronal architecture depend critically on fast axonal transport of membrane bounded organelles along microtubules. Similarly, a wide range of neuropathological conditions, including diabetic and toxic neuropathies, motor neuron diseases and degenerative diseases of the nervous system have characteristics expected from a disruption of fast axonal transport. Based on studies of fast axonal transport in isolated axoplasm from the squid giant axon, a new family of mechanochemical ATPases, the kinesins, has been defined. Kinesins are motors for the movement of membrane bounded organelles in the anterograde direction of fast axonal transport. Previous work by the applicant has answered a number of questions about the biochemistry, molecular biology, cell biology, and neurobiology of kinesin. The applicant proposes that different kinesin transcripts and subunits in the neuron reflect distinct physiological roles. The proposed experiments constitute a multidisciplinary effort that will use methods from both cellular and molecular biology to define the functional architecture of neuronal kinesins. These experiments will provide a molecular basis for kinesin isoforms and test hypotheses about their physiological roles. Kinesin is subject to posttranslational modifications in the neuron and experiments are proposed that will determine the functional significance of posttranslational modifications to kinesin heavy and light chains. The applicant proposes that they may affect kinesin interactions with specific classes of membrane bounded organelles and be important for regulation of kinesin function. Factors and functional domains which form the molecular basis for interactions of kinesin with defined neuronal organelles will be defined in vitro and in vivo. The applicant proposes that kinesins are targeted to specific classes of membrane bounded organelles by unique biochemical motifs. Experiments proposed in this application are an extension of current studies by the applicant on the molecular mechanisms of fast axonal transport.

DESCRIPTION: (Adapted from Applicant's Abstract) Long-term changes in neuronal function have been reported in connection with extended stays in a microgravity environment. Although some correlations may be made with metabolic effects of extended stays in space, these mechanisms cannot readily be used to explain specific changes in nervous system function. A systematic examination of neuronal dynamics at the cellular and molecular level is needed to understand how extended exposure to microgravity can result in compromised neuronal function. The experiments in this application will provide a basis for defining the molecular basis for changes in neuronal plasticity, connectivity, and function. Particular attention will be paid to identifying regulatory pathways which may be used to ameliorate or reduce potentially deleterious changes in the nervous system associated with extended stages in space. Two specific aims will be addressed: 1) To determine the effects of space flight on the dynamics, organization, and composition of the neuronal cytoskeleton. Long-term changes in neuronal function have been reported following extended stays in a space environment. Cytoskeletal elements form the structural basis for neuronal architecture and dynamics. Since changes in the composition and organization of the neuronal architecture and regeneration, the plasticity of a neuronal population is closely linked to dynamics of the cytoskeleton. Specific properties appear to be locally modulated by the microenvironment of the axon and interactions with target cells, so the conditions of space flight may adversely affect neuronal connectivity and plasticity through several mechanisms, including changes in patterns of synaptic activity, and alterations in the axonal microenvironment associated with microgravity or stress. Experiments in this aim are designed to characterize the effects of space flight on the axonal cytoskeleton and identify underlying mechanisms. 2). To evaluate molecular mechanisms of vesicle trafficking in the presynaptic terminal important for neuronal plasticity and synaptic transmission. Sustained release of neurotransmitter requires precise coordination of vesicle movements, targeting of organelles, sorting of membrane proteins, turnaround of fast axonal transport, and recycling of synaptic vesicle constituents. While considerable progress has been made toward understanding some of the associated molecular mechanisms such as fast axonal transport, relatively little is known about the molecular signals, motors, or sorting machinery associated with vesicle trafficking in the presynaptic terminal. Alterations in vesicle recycling may affect maintenance of synaptic terminals and stability of connections through a loss of trophic interactions or disruption of signalling pathways mediated through axonal transport. Experiments in this aim are designed to define molecular mechanisms that control vesicle trafficking and to identify intermediates that might be affected by the conditions of space flight.

Diabetic neuropathies and their associated neurological complications represent one of the least tractable problems encountered in the clinics during long term management of the disease. Although the consequences and costs are clear, the underlying pathogenic mechanisms remain obscure. One proposed mechanism is alteration of axonal transport processes. Evidence that changes in axonal transport do occur in diabetic nerves exists, but the data do not establish whether this plays a primary or secondary role in development of diabetic neuropathy and the biochemical basis for these changes has been unclear. Recent studies in our laboratory have identified a series of kinase activities that inhibit or modulate fast axonal transport. Unexpectedly, several of these kinase activities are known to be altered in diabetic tissues, including protein kinase C and glycogen synthase kinase 3b. Preliminary data suggest that misregulation of kinase and phosphatase activities in nervous tissue associated with inappropriate levels of insulin may affect kinesin-based motility and targeting of specific neuronal proteins. This may provide a critical link between metabolic changes in diabetic patients and the mechanisms of fast axonal transport. In this application, we propose to analyze kinesin phosphorylation in normal and diabetic nerves in a rat model of type 1 diabetes. These experiments will determine the extent to which kinesin is altered in diabetic nerve and facilitate identification of kinase/phosphatase pathways involved. As specific phosphorylation patterns relevant to diabetes are identified, we will characterize biochemical and biophysical effects of kinesin phosphorylation at sites altered in diabetes. These studies will determine how kinesin phosphorylation may affect kinesin motor activities. Finally, we will evaluate changes in kinesin function in diabetic nerves. These experiments will determine the extent to which diabetes induced alterations in phosphorylation affect metabolic turnover of kinesin, interaction of kinesin with other motor proteins and kinesin based motility of membrane bounded organelles. Studies of the effects that insulin levels and diabetes may have on kinesin-based transport processes have the potential to identify promising protective strategies that will minimize or eliminate diabetic neuropathies in clinically controlled diabetics.

DESCRIPTION (provided by applicant): Support is requested for the next Annual Meeting of the American Society for Neurochemistry to be held in Palm Beach, FL from 22-26 June 2002 and for the next three ASN annual meetings in the series. Funds provided for two previous meeting of the ASN have been invaluable for support of the scientific programs and for enhancing our ability to reach graduate students and postdoctoral researchers. To accommodate the breadth of neurochemistry and provide in depth analyses of particular topics, a series of Symposia, Colloquia and Workshops on four interwoven, but distinct themes are proposed to increase our understanding of the cellular and molecular bases of neural development and disease. These are ongoing themes in our meeting that several of the sessions intentionally contain elements from more than one theme to enhance scientific interactions.BUILDING THE NERVOUS SYSTEM. The mechanisms that facilitate the emergence of multiple neural cell types and their specific connections are just beginning to be understood. Sessions in this theme address the generation of cell diversity, elaboration of neuronal cytoarchitecture, specificity of synapse formation and the role of growth factors during development. CELL AND MOLECULAR NEUROSCIENCE. The fundamental molecular mechanisms that underlie cellular function in the nervous system provide a foundation for our understanding of disease and injury. Sessions under this theme deal with basic mechanisms applicable in a wide range of health issues, ranging from metabolism to neurotransmitter function from cell motility to cell structure. GLIAL MECHANISMS AND INJURY. The crucial role of non-neuronal cells in neural development and pathogenesis will be explored in sessions that address basic glial biology as well as the role of the glia in diseases such as Multiple Sclerosis, Alzheimer's Disease and CNS injury. This has long been a strength of the ASN. NEURONAL DEGENERATION AND DISEASE. This theme continues the focus neurodegenerative diseases sustained at recent ASN meetings. Sessions will address the mechanisms of neurodegeneration, the role of inflammation and the contributions of neurotransmitters to the disease state. Three major goals will be met at the ASN 2002 meeting and future ASN meetings: To provide the strongest scientific program possible; To bring together investigators from diverse disciplines to facilitate integration of scientific information; and to attract and support the active participation of young investigators.

DESCRIPTION (provided by applicant): Taupathology is a prominent feature of multiple neurological diseases known collectively as tauopathies. These include Alzheimer's disease (AD), Progressive Supranuclear Palsy (PSP), Cortical Basal Degeneration (CBD), Pick's disease, and Frontotemporal Dementia with Parkinsonism linked to chromosome 17. Some of these diseases are hereditary, associated with mutations in the tau gene, but normal tau may also be pathological. Although each tauopathy has a disease specific phenotype, histological presentation, morphology, and neurological presentation, all of them are associated with misfolded tau and altered phosphorylation of tau. The search for a common pathogenic mechanism has been hindered by this clinical diversity. Two recent findings provide new insight into tau pathology. The first is identification of conformation specific tau antibodies that recognize some, but not all, pathological forms of tau, suggesting conformational diversity within the tauopathies. Second, our recent demonstration of a biologically active motif in the tau amino terminus that activates a signaling pathway involving protein phosphatase 1 (PP1) and glycogen synthase kinase 3b (GSK3b): 17 amino acids comprising a Phosphatase Activation Domain (PAD) provides a molecular basis for altered kinase activities in tauopathies. The central hypothesis of this application is that pathogenic forms of tau represent a misregulation of a normal biological function for tau as a scaffold for localization and regulation of microtubule based kinases and phosphatases. This PAD region is aberrantly displayed in all pathological forms of tau examined to date and is a necessary component of at least two forms of tau toxicity: inhibition of fast axonal transport and cell toxicity in culture. We propose that pathological forms of tau in different tauopathies are structurally distinct with variable degrees f toxicity. Experiments in this application will characterize the conformations of tau from different tauopathies and evaluate their relative toxicity in affecting the PP1/GSK3b pathway and axonal transport using authentic and synthetic aggregates. We further hypothesize that toxicity of different tau conformers may be modulated by disease specific patterns of tau phosphorylation and conformation. Disease specific patterns of these alterations will be determined for AD, PSP and CBD. Normal and pathological functions of tau will be analyzed to test the hypothesis that tau serves as a scaffold for localizing and regulating specific kinases and phosphatases to microtubules. We will focus on the role of tau in the normal regulation of PP1 and GSK3b in microtubule rich domains of the axon and identify interaction domains with tau for these phosphotransferases. The localization of the PP1/GSK3b pathway by tau allows for spatial and temporal control of these activities and we propose that presentation of PAD is restricted to specific subcellular compartments in normal neurons and deregulated in pathological states. Consistent with this model, tau, PP1 and GSK3b have all been implicated in neuronal development. Developmental regulation of tau isoforms, conformation, and phosphorylation may play critical roles in neuronal development. We suggest that the regulated presentation of PAD is important for neurite outgrowth and targeting of axonal proteins during normal neuronal development and function, allowing us to understand the relationship between the toxicity of misfolded tau and normal tau function.