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David B. Ascher

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Open access Aug 2026

Copine-6 integrates calcium, phosphoinositide and Rab11 signals to coordinate glutamate receptor recycling

Endosomal trafficking is a major pathway that delivers cell-surface proteins, including glutamate receptors, to support neurotransmission and normal brain functions. Activity-dependent insertion of glutamate receptors is essential for synaptic plasticity, learning and memory. Copine-6 is a neuronal-specific calcium (Ca2+) binding protein that mediates activity-induced exocytosis of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors. The activation of N-methyl-D-aspartate (NMDA) receptors triggers Ca2+-dependent translocation of Copine-6 to intracellular endosomal compartments. However, the mechanisms underlying the activity-dependent accumulation of Copine-6 in endosomes remain unknown. Here, we show that Copine-6 exhibits Ca2+-dependent binding to phosphatidylinositol-3-phosphate (PI(3)P) through the C2B domain and displays enhanced interaction with active Rab11a in a Ca2+-independent manner via the vWA domain. Mutations in the C2B that inhibit binding to PI(3)P not only block the activity-induced translocation of Copine-6 to early endosomes, but it also causes an aberrant accumulation of Copine-6 in recycling endosomes. Consequently, loss of Copine-6 expression impairs the efficient coupling of early and recycling endosomes and blocks activity-dependent delivery of both AMPA and NMDA receptors onto the neuronal plasma membrane. These defects can be restored by re-expressing wild-type Copine-6, but not the C2B phospholipid-binding mutant. Together, our findings establish Copine-6 as a molecular bridge that enhances coupling between the early and recycling endosomal membranes, thereby facilitating the activity-dependent forward trafficking of glutamate receptors to the neuronal plasma membrane to maintain synaptic potentiation. Significance Statement Activity-dependent trafficking of glutamate receptors is essential for synaptic plasticity, learning, and memory, but the mechanisms coordinating receptor transport through endosomal compartments remain unclear. This study identifies the neuronal calcium-binding protein Copine-6 as a molecular bridge that couples early and recycling endosomes during receptor trafficking. Copine-6 integrates calcium-dependent phospholipid binding and Rab11-mediated endosomal interactions to promote activity-dependent delivery of both AMPA and NMDA receptors to the neuronal surface. Loss of Copine-6 or disruption of its phospholipid-binding activity uncouples endosomal trafficking and impairs receptor insertion during synaptic potentiation. These findings reveal a key mechanism linking neuronal activity, endosomal organization, and glutamate receptor delivery to support synaptic function.

J. A. Tan, Ana Batallas-Borja, Mintu Chandra et al. · bioRxiv · 0 citations

#gene editing Review Open access Sep 2026

LDLR variant classification through activity-normalized prime editing screening

Background: Inherited variants in the LDL receptor (LDLR) gene are the most common cause of familial hypercholesterolemia (FH), significantly increasing coronary artery disease risk. Early identification of pathogenic LDLR variants enables prompt lipid-lowering therapy and cascade testing of at-risk relatives; however, most LDLR variants observed in the population have uncertain or absent clinical classifications, leaving many patients without actionable information. Methods: We developed the first activity-normalized prime editing screening pipeline to measure the impact of 5,184 LDLR coding variants on LDL-cholesterol (LDL-C) uptake. Each prime editing guide RNA (pegRNA) is paired with a genotypic outcome reporter to correct for variable editing efficiency, overcoming a key limitation of prior pooled genome editing screens. A statistical framework further improves variant effect estimates by jointly analyzing all missense variants at each amino acid position. Results: We show that prime editing of the reporter construct correlates with endogenous variant installation frequency, validating the activity normalization approach. The resulting scores capture a continuous spectrum of functional effects, robustly separate pathogenic vs. benign ClinVar variants, and show concordance with LDL-C levels in UK Biobank participants. We calibrate functional evidence strengths to the ACMG/AMP variant interpretation framework, enabling integration into a clinical variant classification workflow. By combining functional, computational, population, and contextual evidence, 322 of 434 LDLR variants currently classified as VUS, conflicting, or absent from ClinVar appears to meet evidence thresholds for reclassification and can be prioritized for expert review, substantially expanding the pool of actionable variant classifications. The screen also reveals a cluster of gain-of-function variants in LDLR class A repeat 5, at least some of which enhance LDL-C uptake through increased apolipoprotein B interaction, with implications for therapeutic genome editing. Finally, prime editing uniquely detects splice-altering coding variants missed by cDNA-based screens and pathogenicity predictors, revealing an advantage of endogenous variant installation. Conclusions: Altogether, activity-normalized prime editing provides a scalable framework for LDLR variant classification that substantially expands the proportion of variants with evidence for genetic diagnosis and reveals novel biology with therapeutic relevance.

P. Zhou, Minja Velimirovic, Tian Yu et al. · Circulation · 0 citations