Cytoskeletal Networks Driving Presynaptic Plasticity

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2021

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Synapses – the delicate connections between our neurons – adjust and refine their strength to shape our brains, our thoughts, and our memories. Proteomic and genetic techniques have revealed that this process, known as synaptic plasticity, is tightly controlled by signaling cascades that ultimately expand or contract actin networks within postsynaptic sites. In this dissertation, I advance the field of synaptic plasticity by focusing on presynaptic terminals, which are equal partners with their postsynaptic counterparts. To date, the study of presynaptic plasticity has been difficult due to the limited number of presynaptic signaling molecules currently identified (particularly those regulating the cytoskeleton), as well as the lack of tools to manipulate these molecules specifically within presynaptic terminals. I therefore developed new experimental approaches to tackle both of these hurdles. After mapping presynaptic cytoskeletal signaling pathways in the mouse brain, I discovered a new mechanism of presynaptic plasticity that is driven by action potential-coupled actin remodeling.

Presynaptic terminals cannot be biochemically purified away from postsynaptic sites. This has restricted previous presynaptic proteomic studies to isolated synaptic vesicles or other fractions, which have only identified a few actin signaling molecules. I thus turned to a new proteomic method called in vivo BioID. This approach is based on proximity-based biotinylation, which labels proteins in a compartment of interest as defined by a bait protein. My choice of presynaptic bait worked beautifully, leading to the mass spectrometry-based identification of 54 cytoskeletal regulators, most of which were previously not known to be presynaptic. The networks of presynaptic actin signaling molecules turn out to be just as richly diverse as those of the postsynapse. Many proteins also converge on a Rac1-Arp2/3 signaling pathway that leads to the de novo nucleation of branched actin filaments. This reveals that the presynaptic cytoskeleton consists of a dynamic, branched actin network.

This finding was unexpected because Rac1 and Arp2/3 have long-established roles in the development and plasticity of the postsynapse. This also makes it difficult to isolate the presynaptic functions of these proteins. I thus created optogenetic tools and electrophysiological strategies to acutely and bidirectionally manipulate their activity specifically within presynaptic terminals. I showed that presynaptic Rac1 and Arp2/3 negatively regulate the recycling of synaptic vesicles, thereby driving a form of plasticity known as short-term depression. I also showed that this mechanism is conserved between excitatory and inhibitory synapses, demonstrating it is a fundamental aspect of presynaptic function. Finally, I conducted a series of experiments using two-photon fluorescence lifetime imaging (2pFLIM) with a FRET-based biosensor of Rac1 activity. I discovered that calcium entry during action potential firing activates Rac1 within presynaptic terminals. This establishes a new mechanism of short-term depression that is driven by an action potential-coupled signal to the presynaptic cytoskeleton.

This dissertation thus combines proteomics, optogenetics, electrophysiology, and 2pFLIM-FRET to gain new insights into presynaptic plasticity. These findings have three significant implications. First, they challenge the prevailing view that the Rac1-Arp2/3 pathway functions largely at excitatory postsynaptic sites. This compels re-evaluation of how mutations in Rac1 and Arp2/3 cause neurological diseases such as intellectual disability and schizophrenia. Second, the genetic and optogenetic tools I developed are the first way to specifically modulate short-term depression, finally allowing for the exact functions of this form of plasticity to be determined in vivo. This has particular relevance for working memory, which has been theorized to be controlled by short-term presynaptic plasticity. Finally, this study provides a proteomic framework and blueprint of experimental strategies to conduct a systematic genetic analysis of the presynaptic cytoskeleton, which may finally unify the controversial theories about presynaptic actin function. In sum, the experimental strategies and resources that I developed highlight the multifaceted, sophisticated signaling that occurs in presynaptic terminals. This may yet shed light on how we remember our experiences, and why we are who we are.

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O'Neil, Shataakshi Dube (2021). Cytoskeletal Networks Driving Presynaptic Plasticity. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/23788.

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