Single-molecule dynamics of amyloid precursor protein with pooled genetic perturbations

The National Institute on Aging awarded The Broad Institute, Inc. $698,718 on September 3, 2026, under the Aging Research program (CFDA 93.866) to develop an innovative live-cell imaging platform for functional genomics screening of amyloid precursor protein (APP) processing relevant to Alzheimer's disease.

The award funds development and validation of SPT-OPS, a multiplexed imaging technology integrating single-particle tracking (SPT) with optical pooled screening (OPS). The platform uses two acquisition modes: conventional dyes for genome-scale hit-finding via short-duration movies, and bright, photostable upconverting nanoparticles (UCNPs) for long-duration, multicolor tracking at sub-genome scale to capture rare events and protein-protein interactions. After live-cell imaging, in situ sequencing decodes each cell's CRISPR perturbation, and fixed-cell immunofluorescence provides cell-state context. Initial validation occurs using the EGFR family in HeLa cells to establish cross-microscope registration and automated feature extraction. The research addresses the gap between bulk biochemical assays and single-molecule behaviors in live neurons, targeting APP's encounters with B-secretase (BACE1) for amyloidogenic cleavage versus A-secretase (ADAM10) for protective shedding—a mechanism poorly understood despite decades of research and the burden of Alzheimer's disease affecting over 6 million Americans.

Performance occurs in Cambridge, Massachusetts from September 3, 2026, through August 31, 2029.

Abstract

Alzheimer's disease (ad) affects over 6 million americans, with amyloid-beta (AΒ) plaques stemming from amyloid precursor protein (APP) misprocessing as a central pathological driver. Despite decades of research, the dynamic molecular mechanisms regulating APP's encounters with β-secretase (BACE1) for amyloidogenic cleavage versus α-secretase (ADAM10) for protective shedding remain poorly understood, hindered by bulk biochemical assays that obscure single-molecule behaviors in live neurons. Current anti-AΒ therapies, such as monoclonal antibodies, achieve modest slowing of early cognitive decline but fail to reverse established impairments, underscoring the need for upstream interventions targeting APP processing. This R21 proposal develops an innovative live-cell imaging platform that performs functional genomics screens using single- molecule dynamic readouts. We will integrate single-particle tracking (SPT) with optical pooled screening (OPS) to develop a transformative multiplexed imaging technology called SPT-OPS that further sharpens OPS sensitivity and greatly exponentiates SPT throughput. We will use two SPT acquisition modes balancing breadth and depth: mode 1 uses conventional dyes to collect short movies at genome scale for HIT-finding; mode 2 uses bright, photostable, multicolor upconverting nanoparticles (ucnps) to enable long-duration, multiplexed tracking at sub-genome scale that captures rare events and protein-protein interactions, ideal for detailed mechanistic studies. After live-cell imaging, in situ sequencing decodes each cell's crispr perturbation, and fixed-cell immunofluorescence adds cell-state context. Aim 1 will develop and validates SPT-OPS using the well-characterized egfr family in hela cells, establishing robust cross-microscope registration, automated feature extraction (e.g., diffusive state fractions, transition rates, confinement, endocytosis), and throughput benchmarks. Aim 2 will apply SPT-OPS to human ipsc-derived neurons to discover genetic modulators of APP dynamics, quantifying diffusion, internalization, and encounter kinetics among APP, ADAM10 (α-secretase), and BACE1 (β-secretase) and along endosomal pathways (RAB5/RAB7). Unsupervised clustering, network analysis, and multimodal integration with AΒ readouts and other ad hallmarks will prioritize actionable pathway nodes. This program directly measures how genotype reshapes live-cell molecular behavior, supplying mechanistic traction that ensemble assays cannot provide. Expected outcomes include (i) a first-in-class, scalable platform that maps functional genomics through single-molecule dynamics, and (II) a prioritized, mechanism-grounded set of ad targets that reduce APP-BACE1 encounters and/or favor non-amyloidogenic processing, potentially advancing safer, more precise therapeutics.