Konrad E. Zinsmaier, Ph.D. Associate Professor ARL Division of Neurobiology 627 Gould-Simpson Bldg. Phone (office): (520) 626-1343 Phone (lab): (520) 626-8612 Fax: (520) 621-8282 Email: kez@neurobio.arizona.edu

RESEARCH INTERESTS
Our research focuses on the molecular mechanisms of excitation-dependent neurotransmitter exocytosis at nerve terminals. A tight linkage of Ca²⁺ signaling with the synaptic vesicle cycle facilitates regulated neurotransmitter release. Ca²⁺ ions, entering the cell through voltage-gated Ca²⁺ channels, trigger vesicle fusion and synaptic vesicle cycling supplies releasable vesicles. In addition to these obligatory steps, regulatory mechanisms are equally important as they confer upon synapses their characteristic plasticity, adaptability, and individuality. However, many obligatory as well regulatory components of this molecular machinery remain to be identified. A long-term understanding of regulated release will probably only come from a systematic identification of all the protein components and interactions.

KEYWORDS
Synaptic transmission, neurotransmitter exocytosis, presynaptic calcium signaling, membrane fusion, synaptic vesicle recycling, synaptic vesicle genesis.

RESEARCH TECHNIQUES
We are employing a multidisciplinary approach based on Genetics, Molecular Biology, Biochemistry, Electrophysiology, and Imaging. Forward and reverse genetics are exploited to genetically modify our "model synapse", the neuromuscular junction of Drosophila. Mutant effects on synaptic function are examined by a variety of techniques including electrical recordings, electron microscopy, confocal microscopy, Ca²⁺ imaging, pH imaging, and FM1-43 dye imaging.

RESEARCH PROJECTS

(A) Molecular chaperones regulating vesicle fusion and G protein-mediated inhibition of Ca²⁺ entry.

High fidelity coupling of nerve signaling and neuroexocytosis requires sequential transitions of numerous protein-protein interactions through transient states. These delicate and fast transitions are supervised by highly specialized molecular chaperones, which, at nerve terminals, regulate many key components of transmitter release including Ca²⁺channels and proteins of the fusion complex.

We originally discovered one of these synapse-specific cofactors, cysteine string protein (CSP). CSP acts in concert with Hsc70 and SGT protein (Fig. 1). This chaperone complex apparently serves three mechanisms: Firstly, it reduces the probability of release by inhibiting Ca²⁺ entry through interactions with Gß subunits and Ca²⁺ channels. Secondly, the complex increases the probability of Ca²⁺ triggered vesicle fusion at a step downstream from Ca²⁺ entry. Although the molecular mechanism for this function is not clear yet, possible protein targets for this function are syntaxin and synaptotagmin. Thirdly, the complex protects a mechanism of Ca²⁺ homeostasis at nerve terminals against thermal stress at elevated temperatures.

Figure 1. The trimeric synaptic vesicle-associated chaperone complex consisting of CSP, Hsc70 and SGT gains its substrate specificity from interactions of CSP and/or SGT and may mediate three functions: First, CSP is likely to reduce the probability of release by inhibiting Ca2+ entry through its interactions with Gbß and Ca2+ channels (Function I). Second, CSP increases the probability of Ca2+ triggered vesicle fusion at a step downstream from Ca2+ entry (Function II). The molecular mechanism of this function is not known. Third, CSP apparently regulates Ca2+ clearance at elevated temperatures (Function III) because mutations in particular domains of CSP can separate the Ca2+ entry and Ca2+ clearance function of CSP (unpublished).

(B) G protein-mediated regulation of synaptic vesicle exocytosis.

Activity-dependent adjustments of the efficacy of synaptic transmission are critical for the processing of information in neural circuits. Presynaptically, these modulated variables include all steps of the vesicle cycle and the Ca²⁺ signaling pathway. Activation of G protein-coupled receptors (GPCRs) is often key to the modulation of neurotransmitter exocytosis and of general interest because most therapeutic or abusive drugs act mostly through GPCRs, often inducing profound synaptic and behavioral changes.

Recently, we discovered that the putative GPCR Methuselah (Mth) controls neurotransmitter release at Drosophila neuromuscular junctions (NMJs). Mth is closely related to GPCRs of the secretin-like receptor family, specifically with the "Ca²⁺ independent receptor for a-latrotoxin", which has also been implicated in regulating transmitter release. Our studies demonstrate that presynaptically localized Mth acutely up-regulates the efficiency of transmitter release by controlling the number of docked and clustered vesicles at synaptic release sites. Paradoxically, loss of mth function not only decreases neurotransmitter release but also increases life span and stress resistance (Lin et al Science 282, 943). However, the connection between the synaptic deficit and the behavioral gain in mth mutants remains a puzzle. Additional components must be identified before we can fully understand how Mth regulates the size of the docked vesicle pool, and whether and how these functions may relate to a modulation of life span and stress resistance.

Figure 3. Mutations the G protein coupled receptor Methuselah (Mth) uncover a perplexing relationship between excitatory neurotransmission and aging. Behaviorally, decreasing Mth function extends life span in adult Drosophila (Lin et al, Science 282, 943-946). Synaptically, decreasing or increasing Mth function depresses or facilitates neurotransmitter release, respectively.

(C) Identification of novel proteins mediating regulated neurotransmitter release.

A combination of biochemical and genetic approaches by many laboratories has lead to the identification of many synaptic proteins and the elaboration of molecular models describing exocytotic and endocytotic mechanisms. However, despite these recent advances many steps are poorly understood. Since these missing components have eluded identification by biochemical approaches, we have exploited a genetic screen to identify some of these components.

Why use a genetic approach to identify mutations affecting neurotransmitter release and subsequently the underlying genes? The crucial methodological advantage is the absence of any limits on detection, and learning about the role of a protein prior to its molecular identification. The major problem associated with this approach was that most mutations affecting transmission cause premature death during development. To bypass this major hurdle, we used the EGUF/Hid method developed by Stowers & Schwarz (Genetics 152: 1631-1639), which produces genetically mosaic flies that are otherwise heterozygous, but in which the eye is composed exclusively of cells homozygous for the targeted chromosome.

Screening for mutations affecting synaptic transmission was based on the simple assumption that blindness is caused either by a loss of phototransduction, nerve excitation, or synaptic transmission. Subsequently, we recorded from mutant eyes or in most cases, from larval NMJs of homozygous individuals to identify physiological defects of neurotransmission. The result of this screen exceeded our expectations. We screened more than 13,000 mutagenized chromosomes (flies). Of these, a total of 173 "blind" mutations were recovered. The ongoing electrophysiological analysis at the larval NMJ is revealing a high rate of "blind" mutations that disrupt synaptic transmission.

KEY REFERENCES
Zinsmaier, K.E., A. Hofbauer, G. Heimbeck, G.O. Pflugfelder, S. Buchner, E. Buchner (1990). A cysteine-string protein is expressed in retina and brain of Drosophila. J. Neurogenetics 7, 15-29.

Zinsmaier, K.E., K.K. Eberle, E. Buchner, Walter, N., and S. Benzer. (1994) Paralysis and Early Death in Cysteine-String Protein Mutants of Drosophila. Science 263: 977-990.

Umbach, J.A., K.E. Zinsmaier, K.K. Eberle, E. Buchner, S. Benzer, and C. B. Gundersen (1994) Presynaptic dysfunction in Drosophila csp mutants. Neuron 13: 899-907.

Zinsmaier, K.E. (1997) Cysteine-String Proteins. In "Guidebook to Molecular Chaperones and Protein Folding Sequences". (ed. Gething, M.J.), Oxford University Press, pp115-117.

Ranjan, R., P. Bronk, K.E. Zinsmaier (1998) Cysteine string protein is required for calcium secretion coupling of evoked neurotransmitter release, but not for vesicle recycling. J. Neuroscience 18: 956-964.

Eberle K. K., K. E. Zinsmaier, S. Buchner, M. Gruhn, M. Jenni, C. Arnold, C. Leibold, D. Reisch, N. Walter, E. Hafen, A. Hofbauer, G.O. Pflugfelder, E. Buchner (1998) Wide distribution of the cysteine string proteins in Drosophila tissues revealed by targeted mutagenesis. Cell. Tiss. Res. 294: 203-217.

Nie, Z., R. Ranjan, J. Wenniger J., S. Hong, P. Bronk, and K. E. Zinsmaier (1999) Overexpression of cysteine-string protein in Drosophila reveals interactions with syntaxin. J. Neuroscience 19: 10270-10279.

Dawson-Scully, K., P. Bronk, H. Atwood, K.E. Zinsmaier (2000) Cysteine-string protein increases the calcium sensitivity of neurotransmitter exocytosis in Drosophila. J Neuroscience 20: 6039-6047.

Bronk, P., J. J. Wenniger, K. Dawson-Scully, X. Guo, S. Hong, H. L. Atwood, and K. E. Zinsmaier (2001). Drosophila Hsc70-4 is critical for neurotransmitter exocytosis in vivo. Neuron 30, 475-488.

Zinsmaier, K.E. & P. Bronk. (2001). Molecular chaperones and the regulation of neurotransmitter exocytosis. Biochemical Pharmacology 62, 1-11.

Song, W., R. Ranjan, K. Dawson-Scully, P. Bronk, L. Marin, L. Seroude, Y. Lin, Z. Nie, H. L. Atwood, S. Benzer, and K. E. Zinsmaier. (2002) Presynaptic regulation of neurotransmission in Drosophila by the G protein-coupled receptor Methuselah. Neuron 36, 105-119.

Song, W. and K. E. Zinsmaier (2003). Endophilin and Synaptojanin hook up to promote synaptic vesicle exocytosis. Neuron 40, 665-667.

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