Biology subjects

Sullivan, E. E.

Publications and source records attributed to Sullivan, E. E..

5 recordsLinked to original sources

bioRxiv2026

ESCRT-0 regulates AMPA receptor currents and Ca2+- dependent signaling

Membrane protein trafficking is essential for synaptic growth, maintenance, function, and plasticity, and involves the regulated exocytosis and endocytosis of proteins to and from the pre-and post-synaptic membranes. Defects in the clearance of membrane proteins can lead to the accumulation of ubiquitinated membrane proteins and contribute to neurodegenerative disease. The ESCRT (endosomal sorting complexes required for transport) machinery binds and sorts ubiquitinated membrane proteins into lysosomes for degradation, yet the presence and function of ESCRTs in sorting ubiquitinated AMPA and other receptors at the post-synapse remain unclear. Here we show that the ubiquitin-binding ESCRT-0 protein, Hrs, localizes to both pre- and post-synapses, and levels are modulated by neuronal activity, increasing and decreasing with higher and lower neuronal activity, respectively. Phosphoproteomic profiling of Hrs-depleted post-synaptic membranes revealed a role for Hrs in glutamatergic synaptic transmission, including long-term potentiation. In addition, Hrs-depleted neurons showed faster AMPAR current kinetics and reduced amplitude in whole-cell patch-clamp recordings. Genetic deletion of neuronal Hgs in mice led to reductions in phosphorylated CaMKII- and -{beta} (T286/T287) and structural proteins, PSD-95 and gephyrin, suggestive of LTD (long-term depression)-like synaptic depression. In contrast, Hrs overexpression led to increases in Ca2+-dependent signaling, including protein kinase C (PKC) and PKC substrate, AMPAR subunit GluA1-S831, a site which increases conductance. Together, these findings identify a dynamic, bidirectional role for Hrs at the post-synapse as it both senses and is modulated by neuronal activity, ultimately impacting excitatory synaptic strength. Significance StatementSynaptic plasticity relies on dynamic trafficking and turnover of membrane proteins, including AMPA-type glutamate receptors (AMPARs), yet how receptor trafficking intersects with ubiquitin-mediated sorting pathways at synapses remains unclear. We show that the ubiquitin-binding ESCRT-0 protein, Hrs, localizes to both pre- and post-synapses, and its abundance is bidirectionally regulated by neuronal activity. Genetic depletion of Hrs in mice reduces CaMKII phosphorylation and impacts AMPAR channel surface localization. In contrast, neuronal-specific Hrs overexpression led to enhanced GluA1 and protein kinase C substrate phosphorylation, suggesting altered AMPAR trafficking, subunit composition, and/or function. Thus, Hrs emerges as a modulator of glutamatergic signaling, coupling ubiquitin-mediated receptor sorting to the fine-tuning of synaptic transmission, with direct implications for learning and memory in health and disease.

neuroscience↗

bioRxiv2025

AAVs Targeting Human Carbonic Anhydrase IV Enhance Gene Delivery to the Brain

Clinically approved gene therapies based on natural adeno-associated virus (AAV) serotypes have restricted applications, particularly in the brain, due to their poor targeting, high dose requirements, and resulting safety concerns. Directed evolution of enhanced AAV capsids in mice or non-human primates (NHPs) has resulted in markedly improved performance in those species, but inter-species differences present a serious challenge for translating these vectors into human therapies. Here, we engineer AAVs to target human carbonic anhydrase IV (CA-IV), a recently identified blood-brain barrier (BBB) transcytosis receptor. Among known transcytosis receptors, CA-IV is notable for its relatively specific expression in brain endothelial cells and the potency of AAVs that target it in mice. CA-IVs AAV binding site, and thus the mouse vectors enhanced brain potency, is not conserved across species, so we employed a two-phase engineering strategy to identify AAVs optimized for human CA-IV-dependent gene delivery to the brain. We first used in vitro receptor-based selection of a vast AAV library to exclude capsids that do not bind human CA-IV, followed by in vivo selection in "humanized" mice expressing human CA-IV in brain endothelial cells. Notably, we find that human CA-IV binding capsid variants that were poorly enriched in the pull-down selection outperform strong binders in vivo. The most promising vector, AAV-hCA4-IV77, engages human CA-IV to achieve 100-fold greater brain transduction than AAV9, with robust neuronal and astrocytic coverage throughout multiple brain regions. These results advance our understanding of receptor-targeted capsid design and support the therapeutic potential of human CA-IV-engaging AAVs.

bioengineering↗

bioRxiv2024

Human cell surface-AAV interactomes identify LRP6 as blood-brain-barrier transcytosis receptor and immune cytokine IL3 as AAV9 binder

Adeno-associated viruses (AAVs) are foundational gene delivery tools for basic science and clinical therapeutics. However, lack of mechanistic insight, especially for engineered vectors created by directed evolution, can hamper their application. Here, we adapted an unbiased human cell microarray platform to determine the extracellular and cell surface interactomes of natural and engineered AAVs. We identified a naturally-evolved and serotype-specific interaction between the AAV9 capsid and human interleukin 3 (IL3), with possible roles in host immune modulation, as well as lab-evolved low-density-lipoprotein-receptor-related-protein 6 (LRP6) interactions specific to engineered capsids that cross the blood-brain barrier in non-human primates after intravenous administration. The unbiased cell microarray screening approach also allowed us to identify off-target tissue binding interactions of engineered brain-enriched AAV capsids that may inform vectors peripheral organ tropism and side effects. These results allow confident application of engineered AAVs in diverse organisms and unlock future target-informed engineering of improved viral and non-viral vectors for non-invasive therapeutic delivery to the brain.

bioengineering↗

bioRxiv2023

APPRAISE: Fast, accurate ranking of engineered proteins by receptor binding propensity using structural modeling

Deep learning-based methods for protein structure prediction have achieved unprecedented accuracy. However, the power of these tools to guide the engineering of protein-based therapeutics remains limited due to a gap between the ability to predict the structures of candidate proteins and the ability to assess which of those proteins are most likely to bind to a target receptor. Here we bridge this gap by introducing Automated Pairwise Peptide-Receptor AnalysIs for Screening Engineered proteins (APPRAISE), a method for predicting the receptor binding propensity of engineered proteins. After generating models of engineered proteins competing for binding to a target using an established structure-prediction tool such as AlphaFold-Multimer or ESM-Fold, APPRAISE performs a rapid (under 1 CPU second per model) scoring analysis that takes into account biophysical and geometrical constraints. As a proof-of-concept, we demonstrate that APPRAISE can accurately classify receptor-dependent vs. receptor-independent adeno-associated viral vectors and diverse classes of engineered proteins such as miniproteins targeting the SARS-CoV-2 spike, nanobodies targeting a G-protein-coupled receptor, and peptides that specifically bind to transferrin receptor or PD-L1. APPRAISE is accessible through a web-based notebook interface using Google Colaboratory (https://tiny.cc/APPRAISE). With its accuracy, interpretability, and generalizability, APPRAISE promises to expand the utility of protein structure prediction and accelerate protein engineering for biomedical applications.

bioengineering↗

bioRxiv2023

Primate-conserved Carbonic Anhydrase IV and murine-restricted Ly6c1 are new targets for crossing the blood-brain barrier

The blood-brain barrier (BBB) presents a major challenge to delivering large molecules to study and treat the central nervous system (CNS). This is due in part to the scarcity of effective targets for BBB crossing, the identification of which is the crucial first step of drug development. Here, we leveraged a panel of adeno-associated viruses (AAVs) previously identified through directed evolution for improved BBB transport to reverse engineer protein targets for enhanced BBB crossing. We identify both murine-restricted Ly6c1 and primate-conserved carbonic anhydrase IV (Car4; CA4) as novel receptors for crossing the BBB. We demonstrate how these receptors can unlock new experimental and computational target-focused engineering strategies by creating the enhanced Ly6c1-binding vector AAV-PHP.eC and by applying AlphaFold2-enabled in silico methods to rank capsids against identified receptors and generate capsid-receptor binding models. Here, with Car4, we add a completely new receptor to the very short list currently available for crossing the BBB in humans and, with Ly6c1, we validate a pipeline for receptor-targeted engineering. The identification of Car4/CA4 and structural insights from computational modeling provide new paths toward human brain-penetrant chemicals (drugs) and biologicals (including gene delivery).

bioengineering↗