Replisome bypass of a protein-based R-loop block by Pif1

Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5′–3′ direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstituted Saccharomyces cerevisiae replisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.

Efficient and faithful replication of the genome is essential to maintain genome stability and is carried out by a multiprotein complex called the replisome (1–4). There are numerous obstacles to progression of the replisome during the process of chromosome duplication. These obstacles include RNA-DNA hybrids (R-loops), DNA secondary structures, transcribing RNA polymerases, and other tightly bound proteins (5–9). Failure to bypass these barriers may result in genome instability, which can lead to cellular abnormalities and genetic disease. Cells contain various accessory helicases that help the replisome bypass these difficult barriers (10–20). A subset of these helicases act on the opposite strand of the replicative helicase (1, 2, 14, 19).All eukaryotes contain an accessory helicase, Pif1, which tracks in a 5′–3′ direction on single-stranded DNA (ssDNA) (11–16). Pif1 is important in pathways such as Okazaki-fragment processing and break-induced repair that require the removal of DNA-binding proteins as well as potential displacement of R-loops (11–13, 21, 15–18, 22–25). Genetic studies and immunoprecipitation pull-down assays indicate that Pif1 interacts with PCNA (the DNA sliding clamp), Pol ε (the leading-strand polymerase), the MCMs (the motor subunits of the replicative helicase CMG), and RPA (the single-stranded DNA-binding protein) (15, 26, 27). Pif1 activity in break-induced repair strongly depends on its interaction with PCNA (26). These interactions with replisomal components suggest that Pif1 could interact with the replisome during replication. In Escherichia coli, the replicative helicase is the DnaB homohexamer that encircles the lagging strand and moves in a 5′–3′ direction (20). E. coli accessory helicases include the monomeric UvrD (helicase II) and Rep, which move in the 3′–5′ direction and operate on the opposite strand from the DnaB hexamer. It is known that these monomeric helicases promote the bypass of barriers during replication such as stalled RNA polymerases (5). The eukaryotic replicative helicase is the 11-subunit CMG (Cdc45, Mcm2–7, GINS) and tracks in the 3′–5′ direction, opposite to the direction of Pif1 (25, 28). Once activated by Mcm10, the MCM motor domains of CMG encircle the leading strand (29–32). We hypothesized that, similar to UvrD and Rep in E. coli, Pif1 interacts with the replisome tracking in the opposite direction to enable bypass of replication obstacles.In this report, we use an in vitro reconstituted Saccharomyces cerevisiae replisome to study the role of Pif1 in bypass of a “dead” Cas9 (dCas9), which is a Cas9 protein that is deactivated in DNA cleavage but otherwise fully functional in DNA binding. As with Cas9, dCas9 is a single-turnover enzyme that can be programmed with a guide RNA (gRNA) to target either strand. The dCas9–gRNA complex forms a roadblock consisting of an R-loop and a tightly bound protein (dCas9), a construct that is similar to a stalled RNA polymerase. This roadblock (hereafter dCas9 R-loop) arrests replisomes independent of whether the dCas9 R-loop is targeted to the leading or lagging strand (30). Besides its utility due to its programmable nature (33), the use of the dCas9 R-loop allows us to answer several mechanistic questions. For example, the ability to program the dCas9 R-loop block to any specific sequence enables us to observe whether block removal is different depending on whether the block is on the leading or lagging strand. Furthermore, the inner diameter of CMG can accommodate double-stranded DNA (dsDNA) and possibly an R-loop, but not a dCas9 protein. Using the dCas9 R-loop block allows us to determine the fate of each of its components.Here, we report that Pif1 enables the bypass of the dCas9 R-loop by the replisome. Interestingly, dCas9 R-loops targeted to either the leading or lagging strand are bypassed with similar efficiency. In addition, the PCNA clamp is not required for bypass of the block, indicating that Pif1 does not need to interact with PCNA during bypass of the block. We used a single-molecule fluorescence imaging to show that both the dCas9 and the R-loop are displaced as an intact nucleoprotein complex. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.     

DDX11 loss causes replication stress and pharmacologically exploitable DNA repair defects

DDX11 encodes an iron–sulfur cluster DNA helicase required for development, mutated, and overexpressed in cancers. Here, we show that loss of DDX11 causes replication stress and sensitizes cancer cells to DNA damaging agents, including poly ADP ribose polymerase (PARP) inhibitors and platinum drugs. We find that DDX11 helicase activity prevents chemotherapy drug hypersensitivity and accumulation of DNA damage. Mechanistically, DDX11 acts downstream of 53BP1 to mediate homology-directed repair and RAD51 focus formation in manners nonredundant with BRCA1 and BRCA2. As a result, DDX11 down-regulation aggravates the chemotherapeutic sensitivity of BRCA1/2-mutated cancers and resensitizes chemotherapy drug–resistant BRCA1/2-mutated cancer cells that regained homologous recombination proficiency. The results further indicate that DDX11 facilitates recombination repair by assisting double strand break resection and the loading of both RPA and RAD51 on single-stranded DNA substrates. We propose DDX11 as a potential target in cancers by creating pharmacologically exploitable DNA repair vulnerabilities.

Faithful DNA replication and DNA repair processes are essential for genome integrity. Inherited mutations in BRCA1 or BRCA2 genes predispose to breast and ovarian cancer, among other types of malignancies such as pancreatic cancers and brain tumors (1). Mechanistically, BRCA1 and BRCA2 are critical for double strand break (DSB) repair by homologous recombination (HR) and for the protection of stalled replication forks by facilitating RAD51 filament formation (2).Tumors with mutations in HR factors, the most widespread being those harboring mutations in BRCA1 and BRCA2, are sensitive to chemotherapeutic drugs that block replication and cause DSBs (3). Platinum drugs, such as cisplatin, create intra- and interstrand adducts that require HR activities for DNA repair during replication and therefore are effective in killing HR-defective cancers. Analysis of the plateau of the survival curve of different cancers revealed that patients often develop resistance, and thus, alternative strategies are needed. The advent of PARP (poly ADP ribose polymerase) inhibitors (PARPi), including olaparib, which exhibit synthetic lethal effects when applied to cells and tumors defective in HR (4, 5), holds significant promise. PARP1, 2, and 3 are required to repair numerous DNA single-strand breaks (SSBs) resulting from oxidative damage and during base excision repair. When PARP enzymes are locally trapped at SSBs, they prevent fork progression and generate DSBs (6), which need to be repaired by BRCA1/2 and other HR factors (4, 5). While the synthetic lethality of PARPi and HR deficiency is being exploited clinically, many BRCA-mutated carcinomas acquire resistance to PARPi (2). Identifying key factors that are functionally linked with BRCA1/2 and/or PARP during replication stress response may indicate useful alternative or combinatorial chemotherapeutic strategies.DDX11 is a conserved iron–sulfur (Fe–S) cluster 5′ to 3′ DNA helicase facilitating chromatin structure and DNA repair in manners that are not fully understood. Biallelic DDX11 mutations in humans cause the developmental disorder Warsaw breakage syndrome (WBS), which presents overlaps with Fanconi anemia in terms of chromosomal instability induced by intra- and interstrand crosslinking (ICL) agents and with cohesinopathies in terms of sister chromatid cohesion defects (7, 8). DDX11 has also strong ties to cancer. Specifically, DDX11 is highly up-regulated or amplified in diverse cancers, such as breast and ovarian cancers, including one-fifth of high-grade serous ovarian cancers (cBioPortal and The Cancer Genome Atlas [TCGA]). Moreover, DDX11 is required for the survival of advanced melanomas (9), lung adenocarcinomas (10), and hepatocellular carcinomas (11). In terms of molecular functions, DDX11 interacts physically with the replication fork component Timeless to assist replisome progression and to facilitate epigenetic stability at G-quadruplex (G4) structures and sister chromatid cohesion (12–16). Notably, DDX11 also contributes along 9–1-1, Fanconi anemia factors, and SMC5/6 to prevent cytotoxicity of PARPi and ICLs (17–20). However, if the DNA damage tolerance functions of DDX11 are relevant for tumorigenesis or cancer therapies remains currently unknown.Here, we find that targeting DDX11 sensitizes ovarian and other cancer cell lines to drug therapies involving cisplatin and the PARP inhibitor olaparib. We established DDX11 knockout (KO) in HeLa uterine and U2OS osteosarcoma cancer cell lines and uncovered via chemical drug screens and immunofluorescence of DNA damage markers that they show typical hallmarks of increased replication stress. DDX11 helicase activity and the Fe–S domain are critical to prevent cellular sensitization to olaparib and ICLs and to avert accumulation of DSB markers. Mechanistically, we uncover that DDX11 facilitates homology-directed repair of DSBs and RAD51 focus formation downstream of 53BP1. Importantly, DDX11 is required for viability in BRCA1-depleted cells that are resistant to chemotherapy by concomitant depletion of 53BP1, REV7, and other shieldin components (21, 22), indicating roles for DDX11 in the activated BRCA2-dependent HR pathway, often accounting for the resistance of BRCA1-mutated tumors (2). DDX11 DNA repair function is nonredundant with BRCA1 and BRCA2 pathways, facilitating resection and loading of both RPA and RAD51 on single-stranded DNA substrates. Altogether, our results define a DDX11-mediated DNA repair pathway that creates pharmaceutically targetable vulnerabilities in cancers.     

A limited role of NKCC1 in telencephalic glutamatergic neurons for developing hippocampal network dynamics and behavior

NKCC1 is the primary transporter mediating chloride uptake in immature principal neurons, but its role in the development of in vivo network dynamics and cognitive abilities remains unknown. Here, we address the function of NKCC1 in developing mice using electrophysiological, optical, and behavioral approaches. We report that NKCC1 deletion from telencephalic glutamatergic neurons decreases in vitro excitatory actions of γ-aminobutyric acid (GABA) and impairs neuronal synchrony in neonatal hippocampal brain slices. In vivo, it has a minor impact on correlated spontaneous activity in the hippocampus and does not affect network activity in the intact visual cortex. Moreover, long-term effects of the developmental NKCC1 deletion on synaptic maturation, network dynamics, and behavioral performance are subtle. Our data reveal a neural network function of NKCC1 in hippocampal glutamatergic neurons in vivo, but challenge the hypothesis that NKCC1 is essential for major aspects of hippocampal development.

Intracellular chloride concentration ([Cl⁻]_i) is a major determinant of neuronal excitability, as synaptic inhibition is primarily mediated by chloride-permeable receptors (1). In the mature brain, [Cl⁻]_i is maintained at low levels by chloride extrusion, which renders γ-aminobutyric acid (GABA) hyperpolarizing (2) and counteracts activity-dependent chloride loads (3). GABAergic inhibition in the adult is crucial not only for preventing runaway excitation of glutamatergic cells (4) but also for entraining neuronal assemblies into oscillations underlying cognitive processing (5). However, the capacity of chloride extrusion is low during early brain development (6, 7). Additionally, immature neurons are equipped with chloride uptake mechanisms, particularly with the Na⁺/K⁺/2Cl⁻ cotransporter NKCC1 (8–12). NKCC1 contributes to the maintenance of high [Cl⁻]_i in the developing brain (13), favoring depolarization through GABA_A receptor (GABA_AR) activation in vivo (14, 15).When GABA acts as a depolarizing neurotransmitter, neural circuits generate burst-like spontaneous activity (16–20), which is crucial for their developmental refinement (21–24). In vitro evidence indicates that GABAergic interneurons promote neuronal synchrony in an NKCC1-dependent manner (10, 12, 25–28). However, the in vivo developmental functions of NKCC1 are far from understood (29, 30). One fundamental question is to what extent NKCC1 and GABAergic depolarization supports correlated spontaneous activity in the neonatal brain. In the neocortex, GABA imposes spatiotemporal inhibition on network activity already in the neonatal period (14, 25, 31, 32). Whether a similar situation applies to other brain regions is unknown, as two recent chemo- and optogenetic studies in the hippocampus yielded opposing results (25, 33). Manipulations of the chloride driving force are potentially suited to resolve these divergent findings, but pharmacological (34–36) or conventional knockout (10, 11, 37) strategies suffer from unspecific effects that complicate interpretations.Here, we overcome this limitation by selectively deleting Slc12a2 (encoding NKCC1) from telencephalic glutamatergic neurons. We show that chloride uptake via NKCC1 promotes synchronized activity in acute hippocampal slices, but has weak and event type-dependent effects in CA1 in vivo. Long-term loss of NKCC1 leads to subtle changes of network dynamics in the adult, leaving synaptic development unperturbed and behavioral performance intact. Our data suggest that NKCC1-dependent chloride uptake is largely dispensable for several key aspects of hippocampal development in vivo.     

A conserved Ctp1/CtIP C-terminal peptide stimulates Mre11 endonuclease activity

The Mre11-Rad50-Nbs1 complex (MRN) is important for repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The endonuclease activity of MRN is critical for resecting 5′-ended DNA strands at DSB ends, producing 3′-ended single-strand DNA, a prerequisite for HR. This endonuclease activity is stimulated by Ctp1, the Schizosaccharomyces pombe homolog of human CtIP. Here, with purified proteins, we show that Ctp1 phosphorylation stimulates MRN endonuclease activity by inducing the association of Ctp1 with Nbs1. The highly conserved extreme C terminus of Ctp1 is indispensable for MRN activation. Importantly, a polypeptide composed of the conserved 15 amino acids at the C terminus of Ctp1 (CT15) is sufficient to stimulate Mre11 endonuclease activity. Furthermore, the CT15 equivalent from CtIP can stimulate human MRE11 endonuclease activity, arguing for the generality of this stimulatory mechanism. Thus, we propose that Nbs1-mediated recruitment of CT15 plays a pivotal role in the activation of the Mre11 endonuclease by Ctp1/CtIP.

DNA double-strand breaks (DSBs) are potentially lethal lesions that threaten genomic integrity and cell viability. DSBs can occur spontaneously as a result of faulty DNA metabolism or by exposure to genotoxins. In eukaryotes, these DSBs have “dirty ends” that lack ligatable 3ʹ-hydroxyl/5ʹ-phosphate groups and are often firmly attached to proteins such as the Ku70-80 heterodimer and topoisomerases (1, 2). During meiosis, the topoisomerase-like Spo11 protein generates DSBs and remains covalently attached to the 5ʹ DNA ends (3). To enable further processing, these DSB ends must be converted to “clean” ends with 3ʹ-hydroxyl/5ʹ-phosphate groups properly exposed. This step is achieved by endonucleolytic cleavage, or clipping, by Mre11 (4–7).In mammals, the MRE11, RAD50, and NBS1 complex (Mre11-Rad50-Nbs1 [MRN]), together with CtIP, is involved in the clipping reaction. MRE11 is the nuclease subunit that has both endonuclease and 3′-to-5′ exonuclease activities, but only the former is essential for clipping (4, 8–12). RAD50, a member of the Structural Maintenance of Chromosomes protein family, binds to MRE11 to form an (MRE11)₂-(RAD50)₂ ring structure (MR complex) (13–15). NBS1 binds to the MR complex via MRE11 to form the MRN complex (16). Homologs of CtIP include Ctp1 in Schizosaccharomyces pombe and Sae2 in Saccharomyces cerevisiae (17–19). Upon phosphorylation, these proteins physically interact with their cognate MRN complex via the N-terminal forkhead-associated domain of NBS1, leading to activation of the MRE11 endonucleolytic clipping activity (20–24). However, the mechanistic details underlying this activation have not yet been determined.Through biochemical reconstitution using fission yeast proteins, we made three key findings regarding how Ctp1 activates MRN. First, MRN activation is mediated by Ctp1 phosphorylation, which promotes the direct association of Ctp1 with the Nbs1 subunit of MRN. Second, the highly conserved extreme C terminus of Ctp1 retains the ability to promote the endonuclease activity of MRN. Strikingly, a synthetic polypeptide comprising the 15 amino acids from the extreme C terminus of Ctp1 was sufficient for the full activation of MRN. Third, we verified the evolutionary significance of these findings by demonstrating that the conserved C-terminal polypeptide of CtIP can also stimulate the endonuclease activity of human MRN. Together, our results strongly suggest that the Ctp1-promoted MRN activation mechanism consists of at least two fundamentally separable elements: phosphorylation-induced Ctp1-MRN association and activation of MRN by the C-terminal peptide of Ctp1. Thus, recruitment of the Ctp1 C terminus to MRN is likely pivotal in this activation mechanism.     

IDO1 scavenges reactive oxygen species in myeloid-derived suppressor cells to prevent graft-versus-host disease

Tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) also has an immunological function to suppress T cell activation in inflammatory circumstances, including graft-versus-host disease (GVHD), a fatal complication after allogeneic bone marrow transplantation (allo-BMT). Although the mononuclear cell expression of IDO1 has been associated with improved outcomes in GVHD, the underlying mechanisms remain unclear. Herein, we used IDO-deficient (Ido1^−/−) BMT to understand why myeloid IDO limits the severity of GVHD. Hosts with Ido1^−/− BM exhibited increased lethality, with enhanced proinflammatory and reduced regulatory T cell responses compared with wild type (WT) allo-BMT controls. Despite the comparable expression of the myeloid-derived suppressor cell (MDSC) mediators, arginase-1, inducible nitric oxide synthase, and interleukin 10, Ido1^−/− Gr-1⁺CD11b⁺ cells from allo-BMT or in vitro BM culture showed compromised immune-suppressive functions and were skewed toward the Ly6C^lowLy6G^hi subset, compared with the WT counterparts. Importantly, Ido1^−/−Gr-1⁺CD11b⁺ cells exhibited elevated levels of reactive oxygen species (ROS) and neutrophil numbers. These characteristics were rescued by human IDO1 with intact heme-binding and catalytic activities and were recapitulated by the treatment of WT cells with the IDO1 inhibitor L1-methyl tryptophan. ROS scavenging by N-acetylcysteine reverted the Ido1^−/−Gr-1⁺CD11b⁺ composition and function to an MDSC state, as well as improved the survival of GVHD hosts with Ido1^−/− BM. In summary, myeloid-derived IDO1 enhances GVHD survival by regulating ROS levels and limiting the ability of Gr-1⁺CD11b⁺ MDSCs to differentiate into proinflammatory neutrophils. Our findings provide a mechanistic insight into the immune-regulatory roles of the metabolic enzyme IDO1.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-binding metabolic enzyme that catalyzes the conversion of tryptophan (Trp) into kynurenine (Kyn). In addition to Trp catabolism, IDO1 has long been recognized to have immune-regulatory roles, preventing excessive inflammation (1). IDO1 is up-regulated in response to inflammatory stimuli, including Toll-like receptor (TLR) and type I/II interferon (IFN) signaling (1, 2). The induction of IDO1 after TLR9 stimulation has been demonstrated to mitigate experimental colitis (3). Catalytic function blockade in mice by pharmacological inhibition or genetic ablation of IDO1 (Ido1^−/−) enhanced inflammation and aggravated autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE) (4). The enhanced immune responses induced by IDO1 deficiency were associated with increased T helper (Th)1/Th17 responses; in contrast, regulatory T cell (Treg) responses were repressed (4–6). Consistently, IDO1 inhibition enhanced antitumor immune responses (7–9). The immune-regulatory effects of IDO1 have been ascribed to the depletion of Trp (10, 11) and the production of toxic catabolites along the Kyn pathway (4, 12–14). However, it remains unclear whether additional mechanisms are involved in IDO1-mediated immune suppression.Graft-versus-host disease (GVHD) is a severe inflammatory disease for which IDO1 has been shown to play a protective role (2, 14, 15). GVHD often develops as an adverse systemic complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is induced by activation of donor T cells reactive to the recipient’s major histocompatibility complexes (MHCs) and/or minor histocompatibility antigens (MiHAs) (16). Allo-reactivity of the activated donor T cells promotes tissue inflammation in the host, leading to morbidity and mortality. IDO1 deficiency in the bone marrow (BM) of the donor or the recipient has been linked to increased lethality (2, 14, 15), indicating a crucial role of IDO1 expression in the parenchymal and hematopoietic compartments in preventing GVHD. Kyn produced in IDO1-expressing lung epithelial cells and tissue macrophages suppressed T cell activation by binding to and activating immunomodulatory aryl hydrocarbon receptors (AhRs), which could explain the GVHD aggravation in Ido1^−/− recipients (14). Nevertheless, the mechanisms behind GVHD exacerbation by Ido1^−/− BM transfer remain obscure. Wild-type (WT) donor antigen-presenting cells prolonged survival in GVHD regardless of epithelial cell expression of IDO1, and IDO1 up-regulation after treatment of donor BM with TLR ligands reduced GVHD severity (2). These findings suggest an important role of IDO1 expressed by donor-derived myeloid cells in preventing severe GVHD. However, the immune-regulatory roles of IDO1 expressed in myeloid cells (termed myeloid IDO1 hereafter) remain elusive.Myeloid-derived suppressor cells (MDSCs) are innate cells that have immune-suppressive functions (17). Conventionally, MDSCs are identified as Gr-1⁺CD11b⁺ cells and can be further classified into Ly6C^hiLy6G^low monocytic (M) or Ly6C^lowLy6G^hi polymorphonuclear (PMN) subsets. MDSCs produce various immune-suppressive mediators, including arginase-1 (Arg-1), inducible nitric oxide synthase (iNOS), and interleukin 10 (IL-10) (17, 18). Their ability to enhance Treg responses has also been reported (19, 20). As immature cells, MDSCs maintain the ability to differentiate into dendritic cells (DCs), macrophages, or neutrophils (21, 22). In GVHD, MDSCs derived from donor BM are the major population of myeloid cells expanding in the host (23), and along with Tregs they suppress GVHD (24–26). We previously reported that transplantation of MyD88-deficient (Myd88^−/−) BM suppressed Gr-1⁺CD11b⁺ cell expansion and polarized the differentiation of Gr-1⁺CD11b⁺ cells into DCs, aggravating GVHD (27, 28). These findings indicate that increasing the number of undifferentiated Gr-1⁺CD11b⁺ cells is essential for MDSC-mediated immune suppression in GVHD. Additionally, the finding that IDO1 expression in mononuclear cells, rather than in parenchymal cells, correlated positively with the survival of GVHD patients (29) suggested that IDOl expression in myeloid cells might be involved in the MDSC-mediated suppression of GVHD. Understanding the role of IDO1 in the function of MDSCs derived from the donor BM could lead to novel therapeutic strategies for the treatment of GVHD.In this study, we investigated the mechanisms underlying GVHD aggravation in hosts transplanted with IDO1-deficient BM. We found that IDO1 deficiency in donor BM did not affect the expansion of Gr-1⁺CD11b⁺ cells in GVHD hosts but polarized them toward a Ly6C^lowLy6G^hi phenotype, reducing their immune-regulatory potential. This phenomenon was ascribed to increased reactive oxygen species (ROS) generation in the Ido1^−/− Gr-1⁺CD11b⁺ cells and their skewing to neutrophil differentiation. Treatment of ROS-scavenging chemical reversed this phenomenon. Our findings suggest that the immune-regulatory roles of IDO1 are mediated by ROS scavenging and suppression of the differentiation of Gr-1⁺CD11b⁺ cells.     

4E-BP2–dependent translation in parvalbumin neurons controls epileptic seizure threshold

The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) integrates multiple signals to regulate critical cellular processes such as mRNA translation, lipid biogenesis, and autophagy. Germline and somatic mutations in mTOR and genes upstream of mTORC1, such as PTEN, TSC1/2, AKT3, PIK3CA, and components of GATOR1 and KICSTOR complexes, are associated with various epileptic disorders. Increased mTORC1 activity is linked to the pathophysiology of epilepsy in both humans and animal models, and mTORC1 inhibition suppresses epileptogenesis in humans with tuberous sclerosis and animal models with elevated mTORC1 activity. However, the role of mTORC1-dependent translation and the neuronal cell types mediating the effect of enhanced mTORC1 activity in seizures remain unknown. The eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and 2 (4E-BP2) are translational repressors downstream of mTORC1. Here we show that the ablation of 4E-BP2, but not 4E-BP1, in mice increases the sensitivity to pentylenetetrazole (PTZ)- and kainic acid (KA)–induced seizures. We demonstrate that the deletion of 4E-BP2 in inhibitory, but not excitatory neurons, causes an increase in the susceptibility to PTZ-induced seizures. Moreover, mice lacking 4E-BP2 in parvalbumin, but not somatostatin or VIP inhibitory neurons exhibit a lowered threshold for seizure induction and reduced number of parvalbumin neurons. A mouse model harboring a human PIK3CA mutation that enhances the activity of the PI3K-AKT pathway (Pik3ca^H1047R-Pvalb) selectively in parvalbumin neurons shows susceptibility to PTZ-induced seizures. Our data identify 4E-BP2 as a regulator of epileptogenesis and highlight the central role of increased mTORC1-dependent translation in parvalbumin neurons in the pathophysiology of epilepsy.

Epilepsy is a prevalent (0.5 to 1% of the general population) (1) heterogeneous neurological disorder affecting all age groups and is characterized by seizures and associated psychological and social stigmas (2–4). Hyperactivation of the mechanistic/mammalian target of rapamycin (mTOR) pathway has been reported in brain lesions of epileptic patients with neurodevelopmental disorders (5, 6), and human genetic studies have shown that mutations in mTOR (7, 8) and other components of its pathway are linked to epileptogenesis (6, 9–13). mTOR is a highly conserved serine/threonine protein kinase that forms two distinct complexes: mTORC1 and mTORC2. mTORC1 integrates multiple environmental and intracellular signals to modulate brain functions by controlling key cellular processes such as mRNA translation, nucleotide, lipid and mitochondrial biogenesis, and autophagy (14, 15). Germline or somatic mutations, which result in enhanced mTORC1 activity, including in PIK3CA, PTEN, AKT3, TSC1/2, RHEB, and MTOR, are associated with neurodevelopmental disorders with epilepsy (16–23). Recent studies have also identified mutations in mTORC1 upstream amino acid–sensing GATOR1-KICSTOR-Rag GTPase pathways as a common cause of epilepsy (24), revealing that mutations in GATOR1 (DEPDC5, NPRL2, and NPRL3) (25, 26) and KICSTOR (ITFG2, KPTN, SZT2, and C12ORF66) (6, 7, 27) genes are often found in epileptic pathologies. The link between the mTORC1 and epilepsy has been recapitulated in animal models with enhanced mTORC1 activity (e.g., Pten^+/−, TSC1/2^+/−, and activating mutations in Pik3ca Nestin-Cre knockin (KI), Akt3 KI, MTOR, and Rheb KI) (18, 20, 23, 28–31) while inhibition of mTORC1 reversed epileptogenesis in TSC1^GFAP-Cre and Pten^+/− mice (20, 31). Notably, the mTORC1 rapalog, everolimus, has been approved by the US Food and Drug Administration (FDA) for the treatment of epilepsy in tuberous sclerosis complex (TSC) patients (32, 33).mTORC1 is a master regulator of mRNA translation. Upon activation, mTORC1 phosphorylates S6 protein kinases 1 and 2 (S6K1/2) and 4E-binding proteins (4E-BPs) (34, 35). In the hypophosphorylated form, 4E-BPs bind and prevent the association of the cap-binding protein eIF4E with the large scaffolding protein eIF4G, thereby inhibiting the formation of the eIF4F complex (composed of eIF4E, eIF4G, and an mRNA helicase eIF4A), which is essential for the initiation of cap-dependent translation. Phosphorylation of 4E-BPs by mTORC1 results in the release of eIF4E from 4E-BPs, allowing eIF4F complex formation and initiation of translation (36, 37). Among the three 4E-BP family members (4E-BP1, 4E-BP2, and 4E-BP3), 4E-BP2 is the most abundant paralog in the mammalian brain (38, 39). A recent study (5) has identified aberrant activation of eIF4E as a major mechanism for translational changes in focal malformations of cortical development (FMCD), a condition that is often caused by brain somatic activating mutations in MTOR and presents with intractable epilepsy in children, accompanied by developmental abnormalities and autism spectrum disorder (ASD) (5, 40–42). Increased eIF4E activity has a pathogenic role in inducing epileptic seizures in FMCD as eIF4E knockdown prevented spontaneous seizures in mTOR Cys1483Tyr and Leu2427Pro mutant mice (5), which show mTORC1 hyperactivation.Despite the progress in understanding the causal link between enhanced mTORC1 activity and epilepsy, the mTORC1-downstream molecular mechanisms promoting epileptogenesis and the cell types mediating the effect on seizure threshold and severity remain poorly understood. In this work, we investigated the role of two main mTORC1-downstream effectors, 4E-BP1 and 4E-BP2, in regulating seizure susceptibility and studied the neuronal cell types mediating epileptogenic effects. We report that mice with whole-body or parvalbumin neuron–specific deletion of 4E-BP2 exhibit reduced threshold and increased severity of epileptic seizures. Moreover, we show that Pik3ca^H1047R-Pvalb mutant mice harboring a conditional parvalbumin neuron–specific KI gain-of-function mutation (H1047R) in the PIK3CA kinase domain are prone to seizures. Collectively, these findings demonstrate a central role of 4E-BP2 and parvalbumin neurons in mediating mTORC1-dependent epileptogenesis, thus expanding our understanding of cell type–specific molecular mechanisms of translation dysregulation in epilepsy and other neurodevelopmental disorders.     

T-cell responses to hybrid insulin peptides prior to type 1 diabetes development

T-cell responses to posttranslationally modified self-antigens are associated with many autoimmune disorders. In type 1 diabetes, hybrid insulin peptides (HIPs) are implicated in the T-cell–mediated destruction of insulin-producing β-cells within pancreatic islets. The natural history of the disease is such that it allows for the study of T-cell reactivity prior to the onset of clinical symptoms. We hypothesized that CD4 T-cell responses to posttranslationally modified islet peptides precedes diabetes onset. In a cohort of genetically at-risk individuals, we measured longitudinal T-cell responses to native insulin and hybrid insulin peptides. Both proinflammatory (interferon-γ) and antiinflammatory (interluekin-10) cytokine responses to HIPs were more robust than those to native peptides, and the ratio of such responses oscillated between pro- and antiinflammatory over time. However, individuals who developed islet autoantibodies or progressed to clinical type 1 diabetes had predominantly inflammatory T-cell responses to HIPs. Additionally, several HIP T-cell responses correlated to worsening measurements of blood glucose, highlighting the relevance of T-cell responses to posttranslationally modified peptides prior to autoimmune disease development.

Type 1 diabetes (T1D) is a prototypical organ-specific autoimmune disease that develops in stages (1, 2). The stages are marked by the presence of islet autoantibodies directed against insulin and other β-cell proteins, followed by impaired glucose tolerance, and finally clinical diabetes marked by hyperglycemia and the need for insulin treatment (3). The T1D disease course provides a defined preclinical period and the ability to measure immune responses prior to clinical symptoms.Self-reactive T cells target pancreatic β-cells in both murine models of spontaneous autoimmune diabetes and human T1D (4), with a number of antigens implicated as T-cell epitopes (5, 6). Recently, posttranslationally modified (PTM) epitopes have been characterized as novel autoantigens that may lead to a break in tolerance, thus resulting in T-cell–mediated immunity to pancreatic islets. PTM of antigens is well-described in autoimmune diseases, such as celiac disease (gluten sensitivity), in which tissue transglutaminase mediates deamidation of glutamine to glutamic acid within gliadin to create immunogenic CD4 T-cell epitopes (7–10). In rheumatoid arthritis, citrullinated peptides form epitopes from cartilage proteins that both elicit antibody responses and activate CD4 T cells (11). Similarly, a novel class of epitopes within T1D are hybrid insulin peptides (HIPs) that are formed within lysozymes of β-cells through a covalent bond between an insulin peptide fragment and another β-cell peptide, thereby generating a neo-epitope (12, 13).Recent studies provide strong evidence for the role of HIPs in the development of diabetes in the nonobese diabetic (NOD) mouse model of spontaneous autoimmune diabetes (12, 14–16). Notably, the antigen for the well-studied “diabetogenic” BDC2.5 T-cell clone and transgenic mouse model is a HIP formed between a peptide fusion of C-peptide and a cleavage product of chromogranin A, termed WE14 (12, 17, 18). C-peptide is cleaved from the A and B chains of insulin prior to secretion from the β-cell. In the NOD mouse, HIP-reactive CD4 T cells have a proinflammatory phenotype, can be detected prior to the onset of diabetes, and their frequency increases as the disease progresses (15). Another CD4 T-cell epitope critical for NOD diabetes development is a fragment of the insulin B chain, consisting of amino acids 9 to 23 (B:9–23) (19–21). A strongly stimulating T-cell epitope is very likely a HIP consisting of a fragment of this insulin B-chain peptide with a portion of C-peptide fused to the C-terminal end (22). HIP-reactive CD4 T cells have also been studied in the context of human T1D, with multiple CD4 T-cell clones and lines grown from the residual pancreatic islets of T1D organ donors subsequently responding to these neo-epitopes (12, 23, 24). A number of HIP-reactive T cells have also been measured from the peripheral blood in newly diagnosed T1D patients (25–30); however, the timing of when these T cells appear in the disease course and whether these responses directed at PTM peptides precede those toward native antigens remains to be addressed. We hypothesized that HIP T-cell responses precede clinical diabetes development and are more robust than responses to native insulin peptides.In this study, we longitudinally collected peripheral blood mononuclear cells (PBMCs) from genetically at-risk individuals and measured reactivity to a panel of HIPs and native antigens using sensitive enzyme-linked immunospot (ELISPOT) assays, which have previously been used to identify CD4 T-cell responses in T1D (31). We show that PBMCs respond to native insulin peptides, but the cells respond more robustly to specific HIPs, including the insulin B chain B:9–23 HIP (B22E) and two C-peptide–derived HIPs (C-peptide/islet amyloid polypeptide-2 [C:IAPP-2] and C:A chain). We demonstrate that T-cell responses fluctuate between pro- and antiinflammatory during the preclinical phase prior to T1D development. Interestingly, individuals who progressed to clinical disease or who seroconverted to islet autoantibody positivity during the course of the study had a distinct polarization toward proinflammatory responses to specific HIPs. Remarkably, the T-cell response to the C:IAPP-2 HIP correlated with worsening measures of blood glucose. Overall, the data support a pathogenic role for PTM epitopes in the preclinical stage of T1D, and the fluctuating nature of the T-cell responses has implications for timing therapies to prevent T1D and potentially other autoimmune disorders.