XPF-ERCC1 acts in Unhooking DNA interstrand crosslinks in cooperation with FANCD2 and FANCP/SLX4.

DNA interstrand crosslinks (ICLs), highly toxic lesions that covalently link the Watson and Crick strands of the double helix, are repaired by a complex, replication-coupled pathway in higher eukaryotes. The earliest DNA processing event in ICL repair is the incision of parental DNA on either side of the ICL ("unhooking"), which allows lesion bypass. Incisions depend critically on the Fanconi anemia pathway, whose activation involves ubiquitylation of the FANCD2 protein. Using Xenopus egg extracts, which support replication-coupled ICL repair, we show that the 3' flap endonuclease XPF-ERCC1 cooperates with SLX4/FANCP to carry out the unhooking incisions. Efficient recruitment of XPF-ERCC1 and SLX4 to the ICL depends on FANCD2 and its ubiquitylation. These data help define the molecular mechanism by which the Fanconi anemia pathway promotes a key event in replication-coupled ICL repair.

The role of SLX4 and its associated nucleases in DNA interstrand crosslink repair.

A key step in the Fanconi anemia pathway of DNA interstrand crosslink (ICL) repair is the ICL unhooking by dual endonucleolytic incisions. SLX4/FANCP is a large scaffold protein that plays a central role in ICL unhooking. It contains multiple domains that interact with many proteins including three different endonucleases and also acts in several other DNA repair pathways. While it is known that its interaction with the endonuclease XPF-ERCC1 is required for its function in ICL repair, which other domains act in this process is unclear. Here, we used Xenopus egg extracts to determine ICL repair specific features of SLX4. We show that the SLX4-interacting endonuclease SLX1 is not required for ICL repair and demonstrate that all essential SLX4 domains are located at the N-terminal half of the protein. The MLR domain is crucial for the recruitment of XPF-ERCC1 but also has an unanticipated function in recruiting SLX4 to the site of damage. Although we find the BTB is not essential for ICL repair in our system, dimerization of SLX4 could be important. Our data provide new insights into the mechanism by which SLX4 acts in ICL repair.

CHEK2 variants: linking functional impact to cancer risk.

Protein-truncating variants in the breast cancer susceptibility gene CHEK2 are associated with a moderately increased risk of breast cancer. By contrast, for missense variants of uncertain significance (VUS) in CHEK2 the associated breast cancer risk is often unclear. To facilitate their classification, functional assays that determine the impact of missense VUS on CHK2 protein function have been performed. Here we discuss these functional analyses that consistently reveal an association between impaired protein function and increased breast cancer risk. Overall, these findings suggest that damaging CHEK2 missense VUS are associated with a risk of breast cancer similar to that of protein-truncating variants. This indicates the urgency of expanding the functional characterization of CHEK2 missense VUS to further understand the associated cancer risk.

Functional Analysis Identifies Damaging CHEK2 Missense Variants Associated with Increased Cancer Risk.

Heterozygous carriers of germline loss-of-function variants in the tumor suppressor gene checkpoint kinase 2 (CHEK2) are at an increased risk for developing breast and other cancers. While truncating variants in CHEK2 are known to be pathogenic, the interpretation of missense variants of uncertain significance (VUS) is challenging. Consequently, many VUS remain unclassified both functionally and clinically. Here we describe a mouse embryonic stem (mES) cell-based system to quantitatively determine the functional impact of 50 missense VUS in human CHEK2. By assessing the activity of human CHK2 to phosphorylate one of its main targets, Kap1, in Chek2 knockout mES cells, 31 missense VUS in CHEK2 were found to impair protein function to a similar extent as truncating variants, while 9 CHEK2 missense VUS resulted in intermediate functional defects. Mechanistically, most VUS impaired CHK2 kinase function by causing protein instability or by impairing activation through (auto)phosphorylation. Quantitative results showed that the degree of CHK2 kinase dysfunction correlates with an increased risk for breast cancer. Both damaging CHEK2 variants as a group [OR 2.23; 95% confidence interval (CI), 1.62-3.07; P < 0.0001] and intermediate variants (OR 1.63; 95% CI, 1.21-2.20; P = 0.0014) were associated with an increased breast cancer risk, while functional variants did not show this association (OR 1.13; 95% CI, 0.87-1.46; P = 0.378). Finally, a damaging VUS in CHEK2, c.486A>G/p.D162G, was also identified, which cosegregated with familial prostate cancer. Altogether, these functional assays efficiently and reliably identified VUS in CHEK2 that associate with cancer. SIGNIFICANCE: Quantitative assessment of the functional consequences of CHEK2 variants of uncertain significance identifies damaging variants associated with increased cancer risk, which may aid in the clinical management of patients and carriers.

Functional Characterization of PALB2 Variants of Uncertain Significance: Toward Cancer Risk and Therapy Response Prediction.

In recent years it has become clear that pathogenic variants in PALB2 are associated with a high risk for breast, ovarian and pancreatic cancer. However, the clinical relevance of variants of uncertain significance (VUS) in PALB2, which are increasingly identified through clinical genetic testing, is unclear. Here we review recent advances in the functional characterization of VUS in PALB2. A combination of assays has been used to assess the impact of PALB2 VUS on its function in DNA repair by homologous recombination, cell cycle regulation and the control of cellular levels of reactive oxygen species (ROS). We discuss the outcome of this comprehensive analysis of PALB2 VUS, which showed that VUS in PALB2's Coiled-Coil (CC) domain can impair the interaction with BRCA1, whereas VUS in its WD40 domain affect PALB2 protein stability. Accordingly, the CC and WD40 domains of PALB2 represent hotspots for variants that impair PALB2 protein function. We also provide a future perspective on the high-throughput analysis of VUS in PALB2, as well as the functional characterization of variants that affect PALB2 RNA splicing. Finally, we discuss how results from these functional assays can be valuable for predicting cancer risk and responsiveness to cancer therapy, such as treatment with PARP inhibitor- or platinum-based chemotherapy.

Functional analysis of genetic variants in the high-risk breast cancer susceptibility gene PALB2.

Heterozygous carriers of germ-line loss-of-function variants in the DNA repair gene PALB2 are at a highly increased lifetime risk for developing breast cancer. While truncating variants in PALB2 are known to increase cancer risk, the interpretation of missense variants of uncertain significance (VUS) is in its infancy. Here we describe the development of a relatively fast and easy cDNA-based system for the semi high-throughput functional analysis of 48 VUS in human PALB2. By assessing the ability of PALB2 VUS to rescue the DNA repair and checkpoint defects in Palb2 knockout mouse embryonic stem (mES) cells, we identify various VUS in PALB2 that impair its function. Three VUS in the coiled-coil domain of PALB2 abrogate the interaction with BRCA1, whereas several VUS in the WD40 domain dramatically reduce protein stability. Thus, our functional assays identify damaging VUS in PALB2 that may increase cancer risk.

Neuronal activity enhances tau propagation and tau pathology in vivo.

Tau protein can transfer between neurons transneuronally and trans-synaptically, which is thought to explain the progressive spread of tauopathy observed in the brain of patients with Alzheimer's disease. Here we show that physiological tau released from donor cells can transfer to recipient cells via the medium, suggesting that at least one mechanism by which tau can transfer is via the extracellular space. Neuronal activity has been shown to regulate tau secretion, but its effect on tau pathology is unknown. Using optogenetic and chemogenetic approaches, we found that increased neuronal activity stimulates the release of tau in vitro and enhances tau pathology in vivo. These data have implications for disease pathogenesis and therapeutic strategies for Alzheimer's disease and other tauopathies.

Wnt signaling in Alzheimer's disease: up or down, that is the question.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder, neuropathologically characterized by amyloid-beta (Abeta) plaques and hyperphosphorylated tau accumulation. AD occurs sporadically (SAD), or is caused by hereditary missense mutations in the amyloid precursor protein (APP) or presenilin-1 and -2 (PSEN1 and PSEN2) genes, leading to early-onset familial AD (FAD). Accumulating evidence points towards a role for altered Wnt/beta-catenin-dependent signaling in the etiology of both forms of AD. Presenilins are involved in modulating beta-catenin stability; therefore FAD-linked PSEN-mediated effects can deregulate the Wnt pathway. Genetic variations in the low-density lipoprotein receptor-related protein 6 and apolipoprotein E in AD have been associated with reduced Wnt signaling. In addition, tau phosphorylation is mediated by glycogen synthase kinase-3 (GSK-3), a key antagonist of the Wnt pathway. In this review, we discuss Wnt/beta-catenin signaling in both SAD and FAD, and recapitulate which of its aberrant functions may be critical for (F)AD pathogenesis. We discuss the intriguing possibility that Abeta toxicity may downregulate the Wnt/beta-catenin pathway, thereby upregulating GSK-3 and consequent tau hyperphosphorylation, linking Abeta and tangle pathology. The currently available evidence implies that disruption of tightly regulated Wnt signaling may constitute a key pathological event in AD. In this context, drug targets aimed at rescuing Wnt signaling may prove to be a constructive therapeutic strategy for AD.