Michael, W. | Search bioRxiv

bioRxiv2026

An NHEJ-independent Role for DNA-PKcs in ATR activation at DNA Double-Strand Breaks

DNA double-strand breaks (DSBs) are threats to genome integrity, and to mitigate this risk cells activate the Ataxia Telangiectasia and Rad3-related (ATR) kinase, which halts cell cycle progression to allow time for repair. While ATR signalling during replication stress is well understood, how ATR is activated at DSBs remain unclear. Topoisomerase 2-Binding Protein 1 (TOPBP1) is a key activator of ATR, and activation is mediated by phosphorylation of TOPBP1 at Serine 1131 (S1131). Previous work showed that the Ataxia Telangiectasia Mutated (ATM) kinase phosphorylates TOPBP1 at S1131. ATM is primarily linked to the homologous recombination (HR)-based repair of DSBs, however the majority of cellular DSBs are repaired via the Non-Homologous End-Joining (NHEJ) repair pathway, raising the question of how (or if) ATR is activated in an ATM-independent manner. Here, using Xenopus egg extracts, we demonstrate that DNA-PKcs controls a pathway acting in parallel to ATM that promotes ATR signalling at DSBs. We show that, like ATM, DNA-PKcs phosphorylates TOPBP1 at S1131. DNA-PKcs is best known for orchestrating NHEJ, however we find that its roles in NHEJ and ATR signalling are separable. Our findings reveal an alternative pathway for ATR activation in which DNA-PKcs directly couples DSB recognition to ATR signalling. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/733426v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@4eb847org.highwire.dtl.DTLVardef@244193org.highwire.dtl.DTLVardef@4d4f4forg.highwire.dtl.DTLVardef@191de27_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cell Biology↗

bioRxiv2025

Phagocytes as Plaque Catalysts: Human Macrophages Actively Generate Pathogenic Aβ42 Fibrils with Seeding and Cross-Seeding Potency

The prevailing view frames microglia and macrophages as guardians against amyloid beta (A{beta}) accumulation in Alzheimers disease (AD). Here, we overturn this paradigm by demonstrating that human phagocytic cells--including differentiated THP-1 macrophages and iPSC-derived microglia--are not merely passive responders but active producers of extracellular, seeding-competent A{beta}42 fibrils, the amyloid species most strongly linked to parenchymal plaque formation and neurodegeneration. These cell-generated aggregates differ structurally and functionally from synthetic fibrils, exhibiting heightened seeding activity and the ability to cross-seed tau aggregation, a key driver of AD progression. Notably, A{beta}42 fibril formation in this system requires active cellular processes and is exacerbated by loss of TREM2, a major AD risk gene. Transcriptomic profiling reveals an early inflammatory response resembling microglial states observed in human AD models, positioning this system as a tractable, human-relevant platform to dissect the interplay between A{beta} aggregation, innate immunity, and genetic susceptibility. Our findings suggest that macrophages and microglia play a dual role in AD, acting both as responders and inadvertent catalysts of pathogenic amyloid formation, with implications for early therapeutic intervention. Significance StatementHow amyloid plaques emerge and spread in Alzheimers disease remains a critical unanswered question. Here, we show that human immune cells--including brain-resident microglia--can actively generate A{beta}42 fibrils, the form of amyloid most strongly linked to neurodegeneration. These cell-produced fibrils not only seed further amyloid buildup but also trigger tau aggregation, a key event in disease progression. We further demonstrate that genetic risk factors like TREM2 amplify this process. Our findings reveal a direct link between immune cell activity, genetic susceptibility, and the earliest stages of Alzheimers pathology, offering new insights into disease mechanisms and potential intervention points.

biochemistry↗