Clarissa L. Waites, Phd - US grants

DESCRIPTION (provided by applicant): A central question in neuroscience is how individual neurons connect with one another during development. However, mechanisms of synapse formation are still poorly understood. Many of the proteins and organelles present at mature synapses have been identified, but how these components arrive at newly forming synapses is not known. Two recent studies, including one from this laboratory, have implicated dense core vesicles (DCVs) in the transport of proteins to the presynaptic active zone during synapse formation. DCVs are a major type of secretory vesicle involved in signaling between neurons. They secrete neuropeptides, hormones, neurotrophins and certain neurotransmitters. These studies now suggest an additional role for DCVs in the transport of presynaptic proteins to newly forming synapses. The goals of this proposal are to determine whether the DCVs involved in protein transport differ from those involved in secretion, and to understand the precise role of DCVs in synapse formation. In the first two specific aims, we will use immunostaining and optical imaging to describe the properties of DCVs involved in protein transport. In the third specific aim, we will use antisense RNAs to reduce or eliminate DCVs in developing neurons, then examine how synapse formation is affected.

DESCRIPTION (provided by applicant): Synapses not only support stable signaling between neurons over the course of months to years, but they also have the remarkable capacity to rapidly change their response properties based on inputs from other neurons. These functions depend upon the exquisite regulation of protein synthesis, trafficking, and degradation, collectively termed 'protein homeostasis' or 'proteostasis'. A critical aspect of synaptic proteostasis is the maintenance of synaptic vesicle (SV) pools within presynaptic boutons ('SV-stasis'). SV pools support the sustained release of neurotransmitter by maintaining a local reservoir of proteins to facilitate vesicle recycling [3, 4]. Moreover, SV loss precedes synapse degeneration and cell death in many forms of neurodegeneration [5- 8], suggesting that disruption of SV-stasis triggers more widespread degenerative processes. Understanding how SV-stasis is maintained and regulated will therefore provide critical insights into the etiology of neurodegenerative diseases such as Alzheimer's and Parkinson's. However, while the molecules that regulate SV exo/endocytosis have been extensively studied, those that regulate SV maintenance and degradation remain almost entirely unknown. The overall goal of this proposal is to elucidate the molecular pathway that mediates SV protein degradation in mammalian glutamatergic neurons. Our previous studies have identified three potential components of this pathway (the E3 ubiquitin ligase Siah1, the endosomal sorting complex required for transport (ESCRT) system, and the small GTPase Rab35), and this project will evaluate their roles in facilitating SV protein degradation. We will further assess whether pathological activation of this pathway disrupts SV-stasis and triggers synapse degeneration. In Aim 1, we will test whether Siah1 is a key mediator of SV protein ubiquitination. We propose that its over-activation induces hyper-ubiquitination and degradation of SV proteins, followed by SV loss and synapse degeneration, while its inhibition or knockdown leads to increased SV pool size and stability. To test this hypothesis, we will use biochemistry, immunofluorescence microscopy, live imaging, and electron microscopy to assess effects of Siah1 gain- or loss-of-function on SV protein abundance and turnover. In Aim 2, we will determine whether ubiquitinated SV proteins are targeted to lysosomes via the ESCRT pathway. Here, we will use the techniques from Aim 1 together with knockdown of key ESCRT proteins to examine whether the ESCRT pathway is essential for SV protein degradation under normal and pathological conditions. In Aim 3, we will evaluate whether Rab35 mediates SV protein degradation and functions upstream of the ESCRT pathway. We will again use techniques from Aim 1, together with Rab35 gain- and loss-of-function, to reveal whether Rab35 sorts SV membrane proteins into endosomal intermediates, promoting their entry into an ESCRT-dependent degradative pathway. Together, these studies will provide novel insights into how SV-stasis is maintained, and how its dysregulation contributes to synapse degeneration and the etiology of neurodegenerative disease.

DESCRIPTION (provided by applicant): Synapses are the essential components of neural circuits in the brain. It is widely accepted that a causal factor in many neuropsychiatric disorder is synaptic dysfunction, including abnormal synapse formation or defects in synaptic transmission. However, few tools are available for imaging synapse density and function in the living mammalian brain. Although viral delivery of genetically encoded probes (e.g. fluorescent proteins, channel rhodopsins) is widely used for live imaging/functional assays of synapses in the rodent brain, this approach is highly invasive, requires specialized techniques and reagents, and has had limited success in non-human primates, let alone humans. We therefore propose an alternative approach based upon the molecular recognition of native synaptic components by organic small molecules, thereby facilitating their selective localization to synapses. This chemical approach has the potential to extend the use of synaptic markers from rodent models into non-human primates and humans via non-invasive administration routes. Such synaptic markers could also serve as molecular targeting devices for the delivery of sensors and therapeutics to synapses in the living mammalian brain, affording transformative tools for neuroscience research, as well as for the diagnosis and treatment of neuropsychiatric disorders. As a first step toward these long-term goals, we propose in this application to develop a high-throughput screening (HTS) platform for discovery of small molecule fluorescent synaptic markers. We have obtained a library of ~8,000 novel fluorescent compounds based on diverse fluorophore structural cores, spanning a wide structural and spectroscopic range. This unique resource is a valuable tool for the discovery and development of synaptic markers. In Aim 1, we will develop a HTS assay using cultured cortical neurons in 96-well plate format. Synaptic labeling will be assessed based on colocalization with fluorescent protein (FP)-tagged synaptic vesicle-associated proteins that label presynaptic boutons. The screening, imaging, data mining, and hit selection protocols will be optimized with a preliminary screen of ~1,000-2,000 fluorescent compounds. In Aim 2, we will use protocols optimized in Aim 1 to screen the entire library of ~8,000 fluorescent dyes, and identify hit compounds that label synapses. We will subsequently determine their optimal concentrations and selectivity for glutamatergic versus GABAergic synapses. In Aim 3, we will perform a series of post-screening assays to eliminate toxic compounds and false positives. Select compounds will be resynthesized, structurally confirmed, and re-tested to validate their synaptic labeling. Remaining advanced hits will subsequently be classified as stable (fluorescence is stable during synaptic stimulation), ratiometric (light emission properties change during stimulation), or dynamic (compound is lost from synapses during exocytosis/synaptic activity) synaptic markers. Finally, their impact on synaptic function will be assessed using live imaging methods.

DESCRIPTION (provided by applicant): Synapses not only support stable signaling between neurons over the course of months to years, but they also have the remarkable capacity to rapidly change their response properties based on inputs from other neurons. These functions depend upon the exquisite regulation of protein synthesis, trafficking, and degradation, collectively termed 'protein homeostasis' or 'proteostasis'. A critical aspect of synaptic proteostasis is the maintenance of synaptic vesicle (SV) pools within presynaptic boutons ('SV-stasis'). SV pools support the sustained release of neurotransmitter by maintaining a local reservoir of proteins to facilitate vesicle recycling [3, 4]. Moreover, SV loss precedes synapse degeneration and cell death in many forms of neurodegeneration [5- 8], suggesting that disruption of SV-stasis triggers more widespread degenerative processes. Understanding how SV-stasis is maintained and regulated will therefore provide critical insights into the etiology of neurodegenerative diseases such as Alzheimer's and Parkinson's. However, while the molecules that regulate SV exo/endocytosis have been extensively studied, those that regulate SV maintenance and degradation remain almost entirely unknown. The overall goal of this proposal is to elucidate the molecular pathway that mediates SV protein degradation in mammalian glutamatergic neurons. Our previous studies have identified three potential components of this pathway (the E3 ubiquitin ligase Siah1, the endosomal sorting complex required for transport (ESCRT) system, and the small GTPase Rab35), and this project will evaluate their roles in facilitating SV protein degradation. We will further assess whether pathological activation of this pathway disrupts SV-stasis and triggers synapse degeneration. In Aim 1, we will test whether Siah1 is a key mediator of SV protein ubiquitination. We propose that its over-activation induces hyper-ubiquitination and degradation of SV proteins, followed by SV loss and synapse degeneration, while its inhibition or knockdown leads to increased SV pool size and stability. To test this hypothesis, we will use biochemistry, immunofluorescence microscopy, live imaging, and electron microscopy to assess effects of Siah1 gain- or loss-of-function on SV protein abundance and turnover. In Aim 2, we will determine whether ubiquitinated SV proteins are targeted to lysosomes via the ESCRT pathway. Here, we will use the techniques from Aim 1 together with knockdown of key ESCRT proteins to examine whether the ESCRT pathway is essential for SV protein degradation under normal and pathological conditions. In Aim 3, we will evaluate whether Rab35 mediates SV protein degradation and functions upstream of the ESCRT pathway. We will again use techniques from Aim 1, together with Rab35 gain- and loss-of-function, to reveal whether Rab35 sorts SV membrane proteins into endosomal intermediates, promoting their entry into an ESCRT-dependent degradative pathway. Together, these studies will provide novel insights into how SV-stasis is maintained, and how its dysregulation contributes to synapse degeneration and the etiology of neurodegenerative disease.

PROJECT SUMMARY Synaptic vesicles (SVs) are highly specialized organelles that store and release neurotransmitters. The accumulation of old or damaged proteins on SVs compromises neurotransmission and can lead to dysfunctional neural circuits and networks. Indeed, recent studies have shown that mutations in genes that regulate SV protein degradation are associated with neurological and neurodegenerative disorders, demonstrating the critical importance of SV protein turnover for nervous system health. Yet the molecular mechanisms responsible for SV turnover and degradation remain poorly understood. The overall goal of this project is to elucidate these mechanisms, providing critical insights into the etiology of diseases that afflict millions of Americans. Our recent work has shown that the ESCRT pathway mediates the activity-dependent degradation of SV membrane proteins. The ESCRT pathway comprises a series of protein complexes that sequentially recruit ubiquitinated cargo and catalyze the formation of multivesicular bodies (MVBs) for delivery of these cargo to lysosomes. Intriguingly, we find that increased neuronal firing stimulates the activation of de/ubiquitinating enzymes at the synapse, as well as the motility of axonal transport vesicles carrying initial ESCRT protein Hrs, and their recruitment to SV pools. We hypothesize that these events are critical rate- limiting steps for activity-dependent turnover of SV membrane proteins. We will test this hypothesis with three aims. In Aim 1, we will evaluate the role of de/ubiquitination in the recycling of SV membrane proteins. Here, we will use biochemical and fluorescence imaging assays to evaluate how ubiquitination regulates SV protein recycling vs. degradation in hippocampal neurons. We will also investigate whether the deubiquitinating enzyme UCHL1 is necessary for maintaining SV proteins on recycling SVs, counteracting their degradative sorting. In Aim 2, we will characterize Hrs vesicles and the impact of Hrs on downstream ESCRT protein recruitment to SV pools. We will use super-resolution fluorescence/electron microscopy and proximity biotinylation to characterize the morphology and molecular composition of these vesicles, and Hrs gain- and loss-of-function combined with live imaging to determine whether the recruitment of downstream ESCRT proteins to SV pools requires Hrs. In Aim 3, we will investigate the mechanisms of activity-dependent Hrs recruitment to SV pools. We will test the roles of specific kinesins in the axonal transport of Hrs, and test whether its recruitment to SV pools requires the lipid PI(3)P, the presence of ubiquitinated proteins, and/or the small GTPase Rab35. Together, these studies will uncover fundamental mechanisms underlying SV proteostasis in neurons.