Specialized cell-cell contact sites, called synapses, facilitate communication and computation of information in the brain on a sub-millisecond scale. The accuracy of this process is vital as even subtle changes in synaptic function can disturb neuronal circuits and cause pathological abnormalities that lead to neurological and/or psychiatric disorders.
Synaptic terminals are specialized secretory machines that release neurotransmitters by synaptic vesicle (SV) exocytosis in response to an action potential. Neurotransmitter release requires a complex molecular machinery including calcium channels, calcium sensors, the core machineries for SV exocytosis and endocytosis, and large number of regulatory proteins. Remarkably, despite their complexity, synaptic terminals exhibit not only a extraordinary speed and precision as secretory machines but also an autonomy and durability that is unusual for a membrane-trafficking compartment.
Neurons can form thousands of synaptic terminals that are usually far away from the major sites of biogenesis in the cell body. To maintain operations, synaptic terminals require a) special mechanisms to be functionally autonomous from the cell body like the local recycling of SVs, and b) effective long-distance transport mechanism supplying them with newly synthesized proteins, vesicles, and mitochondria. To gain a comprehensive understanding of synaptic function, it is consequently necessary to understand not only mechanisms of synaptic function per se but also mechanisms that maintain synaptic function and prevent synaptic failure.
My laboratory focuses on molecular mechanisms that mediate, modulate, and/or maintain synaptic function by employing synapses of genetically modified Drosophila as a model system. Forward and reverse genetics are used to examine effects on synaptic function that are induced by mutations in critical components of the release machinery. Abnormal synaptic function is assayed by a variety of techniques including electrical recordings, electron microscopy, confocal microscopy, live imaging of intracellular calcium, endo- and exocytosis.
A) The neuroprotective role of cysteine-string protein
The necessity of understanding causes of neurodegenerative diseases and developing potential treatments is increasing as life expectancy is extending. The progressive appearance of protein aggregates is a common pathological feature of many degenerative diseases, Emerging evidence suggests that synaptic dysfunction often precedes degeneration, and that maintaining synaptic function may be critical preventing neurodegeneration. Cysteine-string protein (CSP) and a-synuclein are emerging as critical neuroprotective factors maintaining synaptic function.
CSP-alpha and alpha-synuclein have partially overlapping synaptic roles and are critical neuroprotective factors maintaining synaptic function. Mutations in each cause neurodegenerative disease, Parkinson’s disease (PD) in alpha-synuclein’s case and Adult-onset Neuronal Ceroid Lipofuscinosis (ANCL) in CSP’s. CSP is also markedly reduced in brains of Alzheimer’s patients. Finally, CSP has a critical role for secretion of disease-causing protein aggregates, likely through exosome-like mechanism.
Studies from my laboratory and others provided biochemical and genetic evidence suggesting that CSP acts in cooperation with Hsc70 as a specialized molecular chaperone facilitating transmitter release by controlling the assembly of synaptic multi-protein complexes like SNARE or dynamin complexes. Currently, we are examining how dominant mutations in CSP-alpha cause lysosomal failure and neurodegeneration in ANCL patients by using a “humanized” Drosophila ANCL model.
B) Axonal biology of mitochondria
This work is based on the necessity to understand not only mechanisms of synaptic function per se but also the transport mechanisms that bridge the large distance between synaptic terminals and the major sites of biogenesis in the cell body. Mitochondria are critical for aerobic respiration, calcium homeostasis, aging and apoptosis, and modulate synaptic function in four fundamental mitoways: by producing ATP, by acting as a calcium sink buffering cytosolic calcium, by acting as a calcium source that slowly releases calcium, and by preventing oxidative damage. The need to properly distribute mitochondria is underscored by diverse pathological conditions, including muscular dystrophy, neurodegeneration and paraplegia.
Our genetic analysis identified the Drosophila mitochondrial Rho-like GTPase (dMiro ) as a mitochondrial sensor that integrates subcellular signals to control the subcellular distribution of mitochondria by activating their long-distance transport to synaptic terminals. Specifically, loss of dMiro function prevents mitochondrial transport into axons and dendrites while gain of dMiro function leads to an abnormal accumulation of mitochondria in distal synaptic terminals of NMJs. In addition, our study shed new light on the role of mitochondria for synaptic structure and function. Specifically, our analysis of dMiro mutant synaptic terminals that entirely lack mitochondria identified the specific subcellular events that are most dependent on mitochondria. Our initial study included the first estimates of mitochondrial calcium uptake at presynaptic terminals of Drosophila expanding, once again, the analytical possibilities of this genetic model system. Currently, we are investing how dMiro protein may link mitochondria to microtubules-based motors and control their activity.
Yong Zhou, C.O. Wong, K. Cho, D. van der Hoeven, H. Liang, D.P. Thakur, J. Luo, M. Babic, K.E. Zinsmaier, M.X. Zhu, H. Hu, K. Venkatachalam and J.F. Hancock (2015). Membrane potential modulates plasma membrane phospholipid dynamics and K-Ras signaling. Science 349, 873.
Babic, M. G.J Russo, A.J. Wellington, R. Sangston, M. Gonzalez, K.E. Zinsmaier (2015). Miro's N-terminal GTPase domain is required for transport of mitochondria into axons and dendrites. J Neurosci. 35, 5754.
Imler, E. and K.E. Zinsmaier (2014). TRPV1 Channels: Not So Inactive on the ER. Neuron 84, 659.
Tsai, P., M.M. Course, J.R. Lovas, C. Hsieh, M. Babic, K.E. Zinsmaier, X.Wang (2014) PINK1-mediated Phosphorylation of Miro Inhibits Synaptic Growth and Protects Dopaminergic Neurons in Drosophila. Scientific Reports 4: 6962.
Liu, Y.C., M.W. Pearce, T. Honda, T.K. Johnson, S. Charlu, K. R. Sharma, M. Imad, R.E. Burke, K.E. Zinsmaier, A. Ray, A. Dahanukar, M. de Bruyne, C.G. Warr (2014). The Drosophila phospholipid flippase dATP8B is required for odorant receptor function. PLOS Genetics 10.
Babic, M. and K.E. Zinsmaier (2011). Memory, synapse stability, and β-Adducin. Neuron, 69:1039.
Zinsmaier, K.E. and M. Imad (2010). Cysteine-string protein’s role at the synapse. In “Folding for the synapse” (eds. A. Wyttenbach and V. O'Connor), Springer New Yord, pp. 145-176.
Zinsmaier KE (2010). Cysteine-string protein's neuroprotective role. J Neurogenet 24:120-132. doi: 10.3109/01677063.2010.489625. PMID: 20583963.
Chouhan AK, Zhang J, Zinsmaier KE, Macleod GT. 2010. Presynaptic mitochondria in functionally different motor neurons exhibit similar affinities for Ca2+ but exert little influence as Ca2+ buffers at nerve firing rates in situ, J Neurosci. 30(5):1869-1881.
Zarnescu DC, Zinsmaier KE. (2009). Ferrying wingless across the synaptic cleft. Cell, 139(2):229-231.
Zinsmaier KE, Babic M, Russo GJ. (2009). Mitochondrial transport dynamics in axons and dendrites, Results Probl Cell Differ. 48:107-139.
Cziko AM, McCann CT, Howlett IC, Barbee SA, Duncan RP, Luedemann R, Zarnescu D, Zinsmaier KE, Parker RR, Ramaswami M. (2009). Genetic modifiers of dFMR1 encode RNA granule components in Drosophila. Genetics. 182(4):1051-1060.
Russo, G.J., K. Louie, A. Wellington, G.T. Macleod, F. Hu, S. Panchumarthi, and K.E. Zinsmaier, (2009). Drosophila Miro is required for both anterograde and retrograde axonal mitochondrial transport. J Neuroscience 29, 5443-5455.
Louie, K., G.J. Russo, D.B. Salkoff, A.Wellington, and K.E. Zinsmaier (2008) Effects of imaging conditions on mitochondrial transport and length in larval motor axons of Drosophila. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 151, 159-172.
Dawson-Scully, K, Y. Lin, M. Imad, J. Zhang, L. Marin, J.A. Horne, I.A. Meinertzhagen, S. Karunanithi, K.E. Zinsmaier and H.L. Atwood (2007). Morphological and Functional Effects of Altered Cysteine String Protein at the Drosophila Larval Neuromuscular Junction. Synapse 61, 1-16.
Macleod, G.T., and K.E. Zinsmaier (2006) Synaptic homeostasis on the fast-track. Neuron 52, 569-571.
Guo, X., G.T. Macleod, A. Wellington, F. Hu, J. Zhang, S. Panchumarthi, M. Schoenfield, L. Marin, M.P. Charlton, H. L. Atwood, and K. E. Zinsmaier (2005). The GTPase dMiro is required for axonal transport of mitochondria to Drosophila synapses. Neuron 47, 379-393.
Bronk, P., Z. Nie, M.K. Klose, K. Dawson-Scully, J. Zhang, R.M. Roberston, H. L. Atwood, and K. E. Zinsmaier. (2005) The multiple functions of cysteine-string protein analyzed at Drosophila nerve terminals. J Neuroscience 25, 2204-2214.
Song, W. and K. E. Zinsmaier (2003). Endophilin and Synaptojanin hook up to promote synaptic vesicle exocytosis. Neuron 40, 665-667.
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.
Zinsmaier, K.E. & P. Bronk. (2001). Molecular chaperones and the regulation of neurotransmitter exocytosis. Biochemical Pharmacology 62, 1-11.
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.
Dawson-Scully, K., P. Bronk, H. L. Atwood, and K. E. Zinsmaier (2000). Cysteine-string protein increases the calcium-sensitivity of neurotransmitter exocytosis in Drosophila. J. Neuroscience 20, 6039-6047.
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.
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.
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 vesicle recycling. J. Neuroscience 18: 956-964.
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., 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.
Reifegerste, R., S. Grimm, S. Albert, N. Lipski, G. Heimbeck, A. Hofbauer, G.O. Pflugfelder, D. Quack, C. Reichmuth, K.E. Zinsmaier, S. Buchner, and E. Buchner (1993). An invertebrate Calcium-binding Protein of the Calbindin Subfamily: Protein structure, Genomic Organization and Expression Pattern of the Calbindin-32 Gene of Drosophila. J. Neuroscience 13(5): 2186-2198.
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.