Tallant, Lauren E.

Abstract INTRODUCTION Anti‐amyloid beta (Aβ) monoclonal antibodies are effective at lowering amyloid in Alzheimer's disease (AD). However, whether Fc‐mediated effector function is absolutely required for efficacy is not completely understood. This is important for optimizing therapeutic efficacy and mitigating side effects such as amyloid‐related imaging abnormalities (ARIA). Antibodies lacking Fc effector function, like single‐domain antibodies (nanobodies), offer a unique tool to dissect these mechanisms, as their small size facilitates blood–brain barrier (BBB) penetration and allows Fc‐mediated functions to be studied independently. METHODS We immunized a llama with Aβ aggregates and constructed a phage display library to screen for aggregate‐specific nanobodies. Lead candidates were characterized by epitope mapping and binding affinity to amyloid plaques in both murine and human AD brain tissues. We further assessed their BBB permeability and evaluated their efficacy in clearing pre‐existing plaques in amyloid precursor protein (APP)/presenilin 1 (PS1) mice. RESULTS We identified two lead nanobodies, 3A11 and 2D10, that bind distinct epitopes and specifically bind Aβ plaques in murine and human AD brain tissues. Following systemic administration, the monovalent, unmodified (Fc‐less) 2D10 nanobody, but not 3A11, successfully crossed the BBB and engaged amyloid plaques in APP/PS1 mice. However, despite robust target engagement, the Fc‐less 2D10 failed to recruit microglia or reduce plaque burden. In contrast, an engineered 2D10‐Fc fusion antibody potently cleared amyloid plaques, achieving a reduction in pathology comparable to aducanumab treatment. This efficacy was directly correlated with Fc‐mediated microglial recruitment and activation, demonstrating that the Fc domain is essential for phagocytic plaque removal. DISCUSSION Our findings demonstrate that Fc effector function is indispensable for microglial‐mediated amyloid clearance in vivo. By clarifying this fundamental mechanism, this study provides a framework for the rational design of next‐generation immunotherapies. Furthermore, 2D10‐Fc represents a promising therapeutic candidate, combining the high‐affinity targeting of nanobodies with the effector power necessary for robust plaque clearance.

Abstract INTRODUCTION Neurodegenerative diseases often involve overlapping alpha‐synuclein (asyn), amyloid beta, and tau proteinopathies, yet the mechanisms, impact, and directionality of their interactions remain unclear. METHODS We induced brain‐wide neuronal asyn/tau pathologies via viral expression of wild‐type asyn, mutant asyn E46K , mutant tau A152T , or both asyn E46K /tau A152T in adult amyloidosis knock‐in mice and controls, either post‐plaque deposition (6 months old) or pre‐plaque (3 months old). Open‐field behavior was assessed baseline and 3 and 6 months post‐transduction, followed by neuropathology and neuroinflammation analyses. RESULTS Post‐plaque induction in amyloid mice increased asyn/tau total and phosphorylated levels and exacerbated amyloid‐related hyperlocomotion/anxiety. Pre‐plaque induction produced robust phosphorylated pathologies irrespective of amyloid, while causing similar amyloid‐dependent behavioral synergy. Tau pathology drove LGALS3 + inflammatory glial responses in white‐matter fibers. DISCUSSION Amyloid context gates vulnerability, with certain synergies manifesting across stages. White‐matter gliosis is a novel mechanism of tau A152T risk. Together, our data argue for the development of stage‐aware, multitarget interventions and biomarkers.

One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1200 transgenic zebrafish strains made with the gene-break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression shows previously undocumented expression of 35% and 90% of cloned genes at 2 and 4 days post-fertilization, respectively. Further, investigated alleles regularly show 99% gene-specific mRNA knockdown. Homozygous GBT animals in ryr1b , fras1 , tnnt2a , edar and hmcn1 phenocopied established mutants. 204 cloned lines trapped diverse proteins, including 64 orthologs of human disease-associated genes with 40 as potential new disease models. Severely reduced skeletal muscle Ca 2+ transients in GBT ryr1b homozygous animals validated the ability to explore molecular mechanisms of genetic diseases. This GBT system facilitates novel functional genome annotation towards understanding cellular and molecular underpinnings of vertebrate biology and human disease.