1985 — 1992 |
Carlson, Steven Scott |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Extracellular Matrix Nerve Terminal Anchorage Proteins @ University of Washington
No presynaptic membrane proteins which anchor nerve terminals to the extracellular matrix (ECM) are known. The goal of my proposed research is to identify and characterize some of these proteins involved with anchoring nerve terminals. Such proteins could mediate the well-known trophic interactions between the nerve terminal and basal lamina during nerve regeneration. Of special interest, therefore, will be those proteins that are specific for a subgroup of synapses. We have identified a proteoglycan from elasmobranch electric organ which has some of the characteristics of such an anchoring protein. It is found on the nerve terminal surface restricted to the synaptic region. It is tightly bound to and enriched in an extracellular matrix (ECM) fraction. Intriguingly, this proteoglycan contains an antigenic determinant which is only associated with the electric organ neurons. Finally, synaptic vesicles also contain a transmembrane proteoglycan which shares this antigenic determinant. Thus, synaptic vesicles also might contain this same proteoglycan. From this data we hypothesize that this proteoglycan is presynaptic membrane protein which links the nerve terminal to the ECM. In addition, the synaptic vesicles may act as vehicles for shuttling this molecule to and from the nerve terminal surface. This research proposal has two basic goals: 1) to determine the validity of the above hypothesis; 2) to identify and characterize other presynaptic membrane proteins which anchor the synaptic ECM. To accomplish the first goal several steps are required. We will determine whether the proteoglycan is an integral membrane protein by the presence or absence of a hydrophobic tail. We will identify the ECM components which bind the proteoglycan. If the proteoglycan is an integral membrane protein and binds ECM components, it must link the nerve terminal to the matrix. If this linkage is through the pathway specific antigenic determinant, this linkage could be specific to this synapse. A biochemical comparison of the synaptic vesicle and ECM proteoglycan protein cores will determine whether these two molecules could share a precursor-product relationship. To achieve the second goal we will use the ECM fraction, in which the nerve terminal proteoglycan was originally found. We will isolate a subfraction containing the integral membrane proteins contained in this ECM material. If presynaptic membrane proteins (other than the proteoglycan) bind to the ECM, they should be present in this subfraction. By making monoclonal antibodies against this subfraction, we should be able to identify them.
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1995 — 1998 |
Carlson, Steven Scott |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Extracellular Matrix Nerve Terminal Anchorage Protein @ University of Washington
During nerve regeneration, extracellular matrix (ECM) directs the rebuilding of the neuromuscular junction. Active zones for neurotransmitter release in the presynaptic plasma membrane appear to be rebuilt following cues of the synaptic ECM. Presumably, transmembrane nerve terminal ECM receptors (nerve terminal anchorage proteins) bind these synaptic ECM cues and form a link to the cytosolic, exocytotic machinery of the nerve terminal. We have identified a potential nerve terminal ECM receptor, a protein complex of 4 proteins and a transmembrane proteoglycan, SV2pg. We call this protein complex the SV2pg complex. The SV2pg complex is a component of the synaptic vesicle membrane and has the properties expected of an ECM receptor. 1) In addition to being part of the synaptic vesicle, this complex is found on the nerve terminal surface in the presynaptic plasma membrane. 2) Subcellular fractionation of electric organ demonstrates that, although most SV2pg is in synaptic vesicles, a significant amount is tightly bound to ECM. 3) We find that the purified SV2pg complex binds to a component of the electric organ ECM extracts. 4) The SV2pg complex binds to laminin. Laminin is a known component of the synaptic ECM and is abundant in electric organ ECM extracts. We propose to test the hypothesis that the SV2pg complex acts as an ECM receptor on the nerve terminal surface: l) We will fully characterize the SV2pg complex and determine how to isolate the components from one another. 2) We will determine whether the SV2pg becomes tightly associated with synaptic ECM during synaptogenesis at the neuromuscular junction. 3) We will determine whether we can block this association during synaptogenesis with antibodies to the SV2pg complex and look at the effects of this blockage on the resulting synaptic structure. 4) We will attempt to isolate the ECM ligand for the SV2pg complex and determine which component of the complex binds the ligand. As mentioned above, this ligand may be laminin. From successful completion of our research we hope to gain some understanding of the molecular events involved in building the synapse during synaptogenesis. In addition, since the SV2pg complex is a synaptic vesicle component and perhaps an ECM receptor, we may elucidate an activity-dependent adhesion mechanism of the synapse.
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2003 — 2006 |
Carlson, Steven Scott |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Synaptic Laminin and the Calcium Channel @ University of Washington
DESCRIPTION (provided by applicant): Our proposal is aimed at elucidating how the extracellular matrix localizes the neurosecretory apparatus in the nerve terminal during synaptogenesis at the neuromuscular junction (NMJ). Nerve regeneration studies at NMJ suggest that synaptic basal lamina components tell the retuming axon where to locate neurotransmitter release machinery, including synaptic vesicle clusters and active zones, the sites of neurotransmitter release. Genetic evidence suggests that synaptic laminins containing alpha4 or Beta2 chains are the critical basal lamina ligands which localize the neurosecretory apparatus. Electron microscopic studies suggest that the transmembrane anchor for the neurotransmitter release machinery is a voltage-gated calcium channel. We have found a transmembrane link between a synaptic laminin, containing alpha4, Beta2, and gamma1chains, and the voltage-gated calcium channel at the electric organ synapse, which is homologous to the NMJ. Using detergent-solubilized electric organ synaptosomes, we have immuno-isolated a protein complex containing this laminin, as well as the calcium channel, the cytoskeletal protein spectrin, and several unidentified proteins. Cross-linking studies suggest that the alpha4Beta2gamma1 laminin is directly bound to the calcium channel in the protein complex. Further, we find that this laminin binds preferentially to cultured cells expressing the calcium channel. We hypothesize that alpha4Beta2gamma1 laminin in the synaptic basal lamina attaches to calcium channel, which in turn joins to the cytosolic neurosecretory apparatus. The alpha4Beta2gamma1 laminin might act to organize the presynaptic secretory apparatus the way agrin organizes the postsynaptic membrane. To test our hypothesis we will ask the following questions: Does the calcium channel act as the receptor for the alpha4Beta2gamma1 laminin? Is it the only receptor? Are there other alpha4Beta2gamma1 laminin-binding proteins in the complex? What kinds of proteins are the unidentified components of the calcium channel/ alpha4Beta2gamma1 laminin protein complex ? Could they be signal transduction proteins, or new active zone components? Will clustering of calcium channels on the surface of motor neuron axons cause the clustering of other components of the neurosecretory apparatus, such as synaptic vesicles? This work investigates a fundamental question of neurobiology and may have clinical relevance to nerve regeneration.
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