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<p>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.</p><p>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.</p><p>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.</p><p>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.</p>
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