Research Interest: Molecular Mechanisms of Synaptic Function

Background
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 the transport mechanisms that bridge the large distance between synaptic terminals and the cell body.

Research Description
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.
Emerging evidence suggests that chaperone systems are pivotal to protect neurons from genetic and/or environmental insults that lead to abnormally folded proteins, which in turn jeopardize neuronal and/or synaptic function, and often result in neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease and related poly-Q diseases. However, how particular chaperone systems specifically maintain and protect synaptic function from use-dependent failure are unsolved problems in cellular neurobiology.

I originally discovered the evolutionary conserved synaptic vesicle-associated cysteine-string protein (CSP) in Drosophila and our initial analysis revealed its critical neuroprotective role. Studies from my laboratory and others provided biochemical and genetic evidence suggesting that CSP acts in cooperation with Hsc70 as a highly-specialized molecular chaperone facilitating transmitter release by controlling the assembly of synaptic multi-protein complexes like the SNARE complex. Work from my laboratory and many others has implicated CSP in the regulation of presynaptic calcium channels, presynaptic calcium clearance, filling of vesicles with neurotransmitter, vesicle docking, and vesicle fusion. In addition, we and others have suggested that CSP may maintain synaptic function by acting as a general use-dependent synaptic chaperone renaturing presynaptic proteins during continuous operation of the synaptic vesicle cycle. Finally, we have recently demonstrated that CSP is also required for synaptic growth. Currently, we are examining interactions of CSP with small glutamine-rich tetratricopeptide repeat protein (SGT) and with various proteins that have been linked to neurodegenerative diseases in humans.

B) Axonal transport and presynaptic function 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 ways: 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. In addition, we are generating novel transgenes that express mitochondria- and ER-specific calcium indicators to comprehensively analyze calcium homeostasis at synaptic terminals.

C) Identification of novel proteins mediating synaptic function.
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 synaptogenesis and synaptic function. Despite these advances, many molecular mechanisms facilitating synaptic function remain unknown. Since a long-term understanding will probably only come from a systematic analysis of all the protein components, we have exploited a genetic screen to identify some of these missing key components.

Our screen for mutations affecting synaptic structure and function was based on the simple assumption that blindness is caused either by a loss of phototransduction, nerve excitation, or synaptic transmission. Hence, we screened genetically mosaic flies in which only the eye is mutant for 'blindness'. In a second round, we screened for mutations that impair neurotransmission at larval NMJs. The result of this screen exceeded our expectations. We screened more than 13,000 mutagenized chromosomes (flies) and recovered 89 genes that are required for vision but not eye morphology. Further analysis suggests that roughly 70% of these mostly novel genes may be required for synaptic function at larval neuromuscular junctions (NMJs). Out of this collection, we initially focused our analysis on a group of mutations that affect the mitochondrial GTPase dMiro (see B). Now, we have shifted our attention to some of the remaining 61 genes still to go.