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.

Public Health Relevance

Neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's, are devastating age- related disorders that affect nearly 15% of Americans over the age of 65, and cost upwards of $200 billion/year in lost productivity and care. This proposal seeks to understand how altered protein degradation within synapses, the sites of communication between neurons, could contribute to neurodegenerative disease etiology.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Gubitz, Amelie
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Columbia University
Schools of Medicine
New York
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Zhu, Mei; Cortese, Giuseppe P; Waites, Clarissa L (2018) Parkinson's disease-linked Parkin mutations impair glutamatergic signaling in hippocampal neurons. BMC Biol 16:100
Vaz-Silva, João; Gomes, Patrícia; Jin, Qi et al. (2018) Endolysosomal degradation of Tau and its role in glucocorticoid-driven hippocampal malfunction. EMBO J 37:
Birdsall, Veronica; Waites, Clarissa L (2018) Autophagy at the synapse. Neurosci Lett :
Sheehan, Patricia; Zhu, Mei; Beskow, Anne et al. (2016) Activity-Dependent Degradation of Synaptic Vesicle Proteins Requires Rab35 and the ESCRT Pathway. J Neurosci 36:8668-86
Cortese, Giuseppe P; Zhu, Mei; Williams, Damian et al. (2016) Parkin Deficiency Reduces Hippocampal Glutamatergic Neurotransmission by Impairing AMPA Receptor Endocytosis. J Neurosci 36:12243-12258
Ackermann, Frauke; Waites, Clarissa L; Garner, Craig C (2015) Presynaptic active zones in invertebrates and vertebrates. EMBO Rep 16:923-38