PROJECT SUMMARY
Alzheimer’s disease (AD) is a devastating neurodegenerative disorder with no definitive treatment that reverses
the course of the disease, and we still lack a firm grasp on how older people develop AD. Bridging Integrator 1
(BIN1) is the second-largest genetic risk factor for late-onset AD. At least 12 different alternatively spliced BIN1
isoforms are expressed in the brain, including the neuron-specific BIN1 isoform 1 (BIN1iso1) and the ubiquitously
expressed BIN1 isoform 9 (BIN1iso9). In the brain gray matter of patients with AD, there is a decrease in neuronal
BIN1iso1 and an increase in BIN1iso9 compared to healthy controls. Thus far, evidence has shown that neuronal
BIN1 isoforms participate in clathrin-mediated endocytosis, endocytic recycling, and synaptic vesicle release and
retrieval. However, the mechanisms by which BIN1 contributes to these functions and the neighborhood of
proteins that BIN1 interacts with to accomplish these cellular tasks remain largely undefined. Therefore, a
fundamental gap in the field is an unbiased characterization of BIN1iso1 interacting proteins and
proximal neighbors. Closing this gap will help define BIN1’s biological functions in healthy and diseased brain
neurons. Utilizing the highly innovative proximity biotin ligase, TurboID, fused to BIN1iso1, will allow the
identification of all proteins within a 10-nm radius. TurboID-based proximity labeling coupled with the most recent
advanced quantitative mass spectrometry and data analysis methods represents a powerful strategy for
discovery research. My preliminary in vitro studies using this approach in mouse N2a neuroblastoma cells
resulted in the discovery of 234 proteins as BIN1-associated (proximal) or interacting proteins. These
results identified several known BIN1 interactors such as tau, dynamin, synaptojanin, and many previously
unknown proximal proteins. The following Specific Aims will translate these findings in vivo and dramatically
advance the field. Aim 1. Identify neuronal BIN1iso1 interacting proteins in vivo using wild-type mice under
homeostatic conditions. Aim 2. Establish neuronal BIN1iso interactome in mouse models of AD (5XFAD and
PS19) before and after the onset of pathology. This project will not only advance the field by providing those
studying AD and BIN1 with a list of BIN1iso1 proximal proteins to generate novel hypotheses but will also support
the applicant’s pre-doctoral research training in AD pathophysiology, advanced methods such as in vivo AAV
transduction, proximity-based labeling in the context of AD pathology, large-scale proteomics data analysis, and
bioinformatics analysis of BIN1 functional pathways. The dynamic and highly collaborative research environment
at the USF Health Byrd Alzheimer’s Institute will enhance the learning opportunities in cell biology and molecular
pathology within an established AD laboratory led by a committed mentor. Furthermore, research training in
proteomics approaches offered by the collaborator’s lab and the Proteomics Core facilities will complement the
applicant’s interdisciplinary research training in age-related neurodegenerative disease.