Chien-Ping Ko - US grants

The long term goals of my research are to study the basic mechanisms of formation, elimination and plasticity of synapses. One of the most important structures at the synapses is the site of transmitter release: the active zone. Under this proposal frog neuromuscular junctions will be studied in various situations using intracellular recording and freeze-fracture electron microscopy. The major objectives are to study: (I) Cytochemistry and differentiation of the active zone membranes. Filipin has been used as a cytochemical probe for membrane cholesterol. Distinctive filipin-sterol complexes are seen in most areas of freeze-fractured presynaptic membranes but are absent from active zones. Differentiation of this membrane heterogeneity, especially in relation to active zone particles and junctional folds, will be studied by treating neuroomuscular junctions with filipin during degeneration, regeneration and development. Changes in membrane heterogeneity at multiply-innervated endplates will also be examined, particularly during elimination of synapses in young frogs. Effects of cholesterol on transmitter release and active zone cytochemistry will be studied by applying cholesterol-rich liposomes to normal junctions. (II) Influence of postjunctional folds on the formation of active zones (1) Ectopic junctions will be induced de novo by removing the original endplate zone and implanting original nerves to enplate-free areas. Formation of active zones at ectopic sites with no previous junctional folds will be examined and compared with regeneration of active zones at original endplates with persisting junctional folds. (2) Vagus-Muscle junctions will be formed by implanting vagus nerves to denervated skeletal muscles. At these junctions it will be examined whether active zones regenerate to their original structures found at normal vagal terminals or differentiate into two double rows of particles as seen in motor nerve terminals. The proposed research will elucidate how the unique organization and cytochemistry of active zones is induced, differentiated and maintained. It will also provide a better understanding of the fundamental process of neurotransmitter release. This basic knowledge will have a strong impact on understanding the mechanisms of learning, intelligence development, and certain developmental neurological disorders.

The long-term objectives are to elucidate cellular and molecular mechanisms of development, maintenance, and plasticity of synapses. Recent studies have shown that peanut agglutinin (PNA) recognizes synapse-specific molecules in the extracellular matrix, and its fluorescent conjugates stains living frog neuromuscular junctions. This proposal aims to characterize further the PNA-binding and to investigate the possible roles of PNA-binding molecules (PNA-BM) in frog endplates. (1) Postembedding staining with gold-conjugates PNA will be used to examine the ultrastructural localization of PNA-BM in neuromuscular junctions. (2) The effects of sialic acid removal by neuraminidase on PNA binding will be examined in normal and developing junctions. (3) Changes in the distribution of PNA-BM in relation to innervation will be examined with electron microscopy and epi-fluorescence light microscopy of whole mounts and cryosections of denervated, reinnervated, and developing neuromuscular junctions. (4) PNA will be applied to adult muscles during reinnervation of PNA-BM and acetylcholine receptors in culture also will be examined. (5) Nerve terminals and synaptic matrix in normal junctions stained with 4-di-2-Asp and rhodamine- PNA will be examined in situ repeatedly with video-enhanced microscopy. Dynamics changes in synaptic matrix during synapse formation and elimination will also be examined in situ. (6) Synaptic extracellular matrix of normal, degenerating, and developing endplates will be examined by gel electrophoresis and probed with peroxidase-conjugated PNA for biochemical characterization of PNA-BM. This research will characterize a new molecular probe for living neuromuscular junctions and provide further insights into cellular and molecular mechanisms of development, maintenance and plasticity of synapses. Elucidation of these basic mechanisms will enhance fundamental understanding of certain developmental and neuromuscular disorders.

The long-term goals of this research are to elucidate the cellular and molecular mechanisms of transmitter release and differentiation of the presynaptic nerve terminal. The present proposal will focus on the active zone, where transmitter release is believed to occur. It has been postulated that Ca 2+ channels are strategically located at active zones. This hypothesis will be tested using w-conotoxin (CTX) as a probe for Ca 2+ channels at the frog neuromuscular junction . In addition, CTX will be used to study the correlation between active zone morphometry and synaptic efficacy as well as the mechanisms of active zone differentiation. (1) Thin-section electron microscopy will be used to reveal the distribution of Ca 2+ channels in the nerve terminal and to examine if Ca 2+ channels are located preferentially at the active zone. (2) Freeze-fracture cytochemical techniques, using CTX tagged with gold particles, will be applied to study whether Ca 2+ channels coincide with the large intramembrane particles seen at the active zone. (3) Quantal contents of identified neuromuscular junctions will be measured with intracellular recording. Active zones of the same junctions will be stained with rhodamine-labeled CTX and visualized with a confocal microscope. The correlation between quantal contents and active zone sizes will be analyzed. (4) Neuromuscular junctions, during reinnervation in the frog and during synaptogenesis in the tadpole, will be double-labeled with rhodamine-CTX and fluorescein alpha bungarotoxin. Whether Ca 2+ channels are initially distributed evenly throughout the nerve terminal and later accumulated at active zones will be investigated. In addition, the temporal and spatial relationship between clusters of Ca 2+ channels and acetylcholine receptors during synaptogenesis will be examined. The proposed research on two of the most important elements at the synapse, active zones and Ca 2+ channels, will provide new insights into the mechanisms on how the synapse works and forms. These results may lead to further understanding on Lambert-Eaton myasthenic syndrome and may provide morphological correlate of learning and memory which are thought to involve changes in synaptic efficacy.

The long-term goals of this research are to elucidate the cellular and molecular mechanisms of transmitter release and differentiation of the presynaptic nerve terminal. The present proposal will focus on the active zone, where transmitter release is believed to occur. It has been postulated that Ca 2+ channels are strategically located at active zones. This hypothesis will be tested using w-conotoxin (CTX) as a probe for Ca 2+ channels at the frog neuromuscular junction . In addition, CTX will be used to study the correlation between active zone morphometry and synaptic efficacy as well as the mechanisms of active zone differentiation. (1) Thin-section electron microscopy will be used to reveal the distribution of Ca 2+ channels in the nerve terminal and to examine if Ca 2+ channels are located preferentially at the active zone. (2) Freeze-fracture cytochemical techniques, using CTX tagged with gold particles, will be applied to study whether Ca 2+ channels coincide with the large intramembrane particles seen at the active zone. (3) Quantal contents of identified neuromuscular junctions will be measured with intracellular recording. Active zones of the same junctions will be stained with rhodamine-labeled CTX and visualized with a confocal microscope. The correlation between quantal contents and active zone sizes will be analyzed. (4) Neuromuscular junctions, during reinnervation in the frog and during synaptogenesis in the tadpole, will be double-labeled with rhodamine-CTX and fluorescein alpha bungarotoxin. Whether Ca 2+ channels are initially distributed evenly throughout the nerve terminal and later accumulated at active zones will be investigated. In addition, the temporal and spatial relationship between clusters of Ca 2+ channels and acetylcholine receptors during synaptogenesis will be examined. The proposed research on two of the most important elements at the synapse, active zones and Ca 2+ channels, will provide new insights into the mechanisms on how the synapse works and forms. These results may lead to further understanding on Lambert-Eaton myasthenic syndrome and may provide morphological correlate of learning and memory which are thought to involve changes in synaptic efficacy.

This proposal is for funding for a BioRad DVC-250 laser-scanning confocal microscope, together with associated camera, computer and printer equipment. This equipment will be used for imaging both living and fixed biological specimens, at high resolution, and in three dimensions. This rapidly maturing technology is now in very wide use, and is particularly suited to visualizing multiple antigens or stains, simultaneously, and their digital analysis. With the equipment system requested, we will be able to acquire microscopic images, process them with a computer, and print them out, at publication quality. While there are a number of other microscopes available on this campus (compound microscopes, Electron microscopes, etc.), there is no confocal microscope. The USC medical school houses a confocal system, but this is not available to labs on this campus. Two of the proposed major users now use confocal systems at other sites. Ko uses a system at the City of Hope Hospital (20 miles distant), which is not available to others. Warrior uses a system at U.C. Irvine (50 miles distant), which is available, but charges a $100/hour user fee. In short, the biological researchers here do not have regular, or open access to this important technology. We have examined three different confocal microscopes: the Nikon K2Bio, BioRad DVC-250, and the Meridian, and we have determined that the BioRad offers the best compromise of performance and cost, for our purposes. This institution (USC), has undertaken to provide 30% of the total cost of this equipment, as a cost share. This equipment system will be used by a group of Biological research labs at the University Park campus, of the University of Southern California (in Los Angeles). This group consists of five major users (Moses, Warrior, Tower, Herrera and Ko, the principal investigators listed), and five minor users (Bottjer, Manahan, McFall-Ngai, Thompson and Watts). All of the major users are currently supported by federal gr ants. While our research interests are somewhat disparate we all share a strong interest in using the confocal microscope to visualize structures and antigens in three dimensions in biological specimens. The Moses lab studies pattern formation and cell fate determination in the Drosophila compound eye. They will use the confocal system to colocalize gene products (such as Drosophila homologs of Transforming Growth Factor a, and its putative receptor), and to examine double and triple stained genetic mosaic tissues. The Warrior lab studies inter and intra-cellular movements in the developing Drosophila embryo. They will use the equipment to study germ cell migration, and the functions of the schnurri and DnudC genes in early development. The Tower lab studies development of the Drosophila chorion, and the regulation of DNA replication. They will use the system to quantitate DNA levels in the developing follicle cells. The Herrera lab studies developmental plasticity in frog neuromuscular junctions, and the action of steroid hormones on this structure. They will use the system to study the junction structures in living specimens, in which their function, and response to hormones, can be tested. The Ko lab studies neuro transmitter release and the function of the presynaptic terminal. They will use this equipment to explore the behavior and morphology of voltage coupled Calcium channels, using specifically binding toxins.

The long-term goals are to elucidate the mechanisms of transmitter release and differentiation o the presynaptic nerve terminal. The present proposal will focus on voltage-sensitive calcium channels (VSCCs) and the active zone (site of transmitter release) at the neuromuscular junction (NMJ). Novel synthetic omega conopeptides and dihydropyridine (DHP) will be used as probes to characterize VSCCs in relation to transmitter release at developing, regenerating and diseased NMJs. (I) To examine the ontogeny of VSCC subtypes at developing mammalian NMJs. Two hypotheses will be tested: (A) L-type VSCCs modulate transmitter release at developing. but not mature NMJs. The effect of DHP on synaptic potentials, and the possible involvement of Ca2+-gated K+ channels and/or somatostatin in L-type VSCC-modulated transmitter release will be studied. The presence of L-type VSCCs at developing nerve terminals will be confirmed with immunocytochemistry. (B) N-type VSCCS mediate transmitter release at developing, but not at mature NMJs. The hypothesis will be tested by physiological and morphological approaches with omega conopeptides. In addition, the notion that developing NMJs also use PIQ-type VSCCs to mediate transmitter release as in adult muscles will be examined. (II) To examine the change in VSCC subtypes at regenerating NMJs in adult muscles. The hypothesis that re-formation of adult NMJs following injury mimics embryonic development with respect to the switch in VSCC subtypes will be tested. (III) To test the hypothesis that Lambert-Eaton Myasthenic Syndrome (LEMS) antibodies cause a reduction of VSCCs from the motor nerve terminal. LEMS antibodies will be passively transferred to mice. The effect on the number of VSCCs will be studied with fluorescence microscopy and autoradiography. The proposed research would provide the first study on the ontogeny of VSCCs at developing and regenerating NMJs. Due to the lack of specific probes for VSCCs in the past, our knowledge of presynaptic differentiation has considerably lagged behind that of postsynaptic differentiation. Thus, the proposed research would yield new insights into the mechanisms on how the synapse works, forms and is repaired. The proposed work may also provide a better understanding of the etiology of human neuromuscular diseases.

DESCRIPTION (provided by applicant): Proximal Spinal Muscular Atrophy (SMA), a leading genetic cause of infant mortality, is an autosomal recessive disease characterized by the loss of spinal motoneurons, muscle atrophy, and motor impairments with varying disease onset and severity (type I is severe;type II, moderate;type III, mild). Currently, there is no cure for this devastating neurological disease, and the mechanisms of the pathogenesis of SMA are not well understood. In this application, type II SMA-like mouse models (SMN 7 SMA and SMN-Hung SMA) will be used to test a novel concept that reduction of synaptic inputs to motoneurons in the spinal cord, instead of degeneration of neuromuscular junctions, is the key event contributing to motor impairments. Aim 1 will test the hypothesis that the neuromuscular junction is not the major site of defects in type II SMA mice. Light and electron microscopy, as well as electrophysiological analyses, will be applied to examine whether neuromuscular junctions in type II SMA mice at various ages are innervated and function normally, as compared with age- and gender-matched non-SMA littermates. Aim 2 will test the hypothesis that synaptic inputs onto spinal motoneurons are reduced in type II SMA mice. Morphological and biochemical analyses will be used to compare numbers of synaptic puncta on spinal motoneurons and the expression of synaptic vesicle proteins in the spinal cord in type II SMA with those in age- and gender-matched non-SMA littermates. Whether the synapse loss involves synaptic stripping by microglia also will be examined. In addition, whether the synaptic defects are attributed to a decrease or degeneration of synaptic inputs from proprioceptive sensory neurons in the dorsal root ganglion will be investigated. The findings of the proposed research will provide a new concept that SMA is a disease of synapse loss in the spinal motoneurons, rather than degeneration of neuromuscular junctions, as suggested by the prevailing thinking. The proposed research is thus relevant to the development of novel therapies for SMA by targeting synaptic defects in the spinal cord. The new therapeutic concept could be applied to treat other types of motoneuron diseases. PUBLIC HEALTH RELEVANCE: The proposed research is highly relevant to Spinal Muscular Atrophy (SMA), a leading genetic cause of infant death characterized by motor impairments and the loss of motor neurons in the spinal cord. We will use mouse models mimicking type II (moderate) SMA to test a novel concept that synapse loss in spinal motoneurons is a key event contributing to motor impairments. The proposed research would lead to future development of novel therapies for SMA by targeting synaptic defects in the spinal cord.