The NMDA receptor subunit GluN3A regulates synaptic activity-induced and myocyte enhancer factor 2C (MEF2C)-dependent transcription.

N-Methyl-d-aspartate type glutamate receptors (NMDARs) are key mediators of synaptic activity-regulated gene transcription in neurons, both during development and in the adult brain. Developmental differences in the glutamate receptor ionotropic NMDA 2 (GluN2) subunit composition of NMDARs determines whether they activate the transcription factor cAMP-responsive element-binding protein 1 (CREB). However, whether the developmentally regulated GluN3A subunit also modulates NMDAR-induced transcription is unknown. Here, using an array of techniques, including quantitative real-time PCR, immunostaining, reporter gene assays, RNA-Seq, and two-photon glutamate uncaging with calcium imaging, we show that knocking down GluN3A in rat hippocampal neurons promotes the inducible transcription of a subset of NMDAR-sensitive genes. We found that this enhancement is mediated by the accumulation of phosphorylated p38 mitogen-activated protein kinase in the nucleus, which drives the activation of the transcription factor myocyte enhancer factor 2C (MEF2C) and promotes the transcription of a subset of synaptic activity-induced genes, including brain-derived neurotrophic factor (Bdnf) and activity-regulated cytoskeleton-associated protein (Arc). Our evidence that GluN3A regulates MEF2C-dependent transcription reveals a novel mechanism by which NMDAR subunit composition confers specificity to the program of synaptic activity-regulated gene transcription in developing neurons.

Systematic analysis of DNA crosslink repair pathways during development and aging in Caenorhabditis elegans.

DNA interstrand crosslinks (ICLs) are generated by endogenous sources and chemotherapeutics, and pose a threat to genome stability and cell survival. Using Caenorhabditis elegans mutants, we identify DNA repair factors that protect against the genotoxicity of ICLs generated by trioxsalen/ultraviolet A (TMP/UVA) during development and aging. Mutations in nucleotide excision repair (NER) components (e.g. XPA-1 and XPF-1) imparted extreme sensitivity to TMP/UVA relative to wild-type animals, manifested as developmental arrest, defects in adult tissue morphology and functionality, and shortened lifespan. Compensatory roles for global-genome (XPC-1) and transcription-coupled (CSB-1) NER in ICL sensing were exposed. The analysis also revealed contributions of homologous recombination (BRC-1/BRCA1), the MUS-81, EXO-1, SLX-1 and FAN-1 nucleases, and the DOG-1 (FANCJ) helicase in ICL resolution, influenced by the replicative-status of the cell/tissue. No obvious or critical role in ICL repair was seen for non-homologous end-joining (cku-80) or base excision repair (nth-1, exo-3), the Fanconi-related proteins BRC-2 (BRCA2/FANCD1) and FCD-2 (FANCD2), the WRN-1 or HIM-6 (BLM) helicases, or the GEN-1 or MRT-1 (SNM1) nucleases. Our efforts uncover replication-dependent and -independent ICL repair networks, and establish nematodes as a model for investigating the repair and consequences of DNA crosslinks in metazoan development and in adult post-mitotic and proliferative germ cells.

Eviction notice: new insights into Rad51 removal from DNA during homologous recombination.

In this issue of Molecular Cell, Ward et al. (2010) identify two genes whose products act redundantly to clear Rad51 from DNA after successful strand invasion, thereby enabling the downstream events of homologous recombination to go smoothly.

Proximity to boundaries reveals spatial context representation in human hippocampal CA1.

Recollection of real-world events is often accompanied by a sense of being in the place where the event transpired. Convergent evidence suggests the hippocampus plays a key role in supporting episodic memory by associating information with the time and place it was originally encountered. This representation is reinstated during memory retrieval. However, little is known about the roles of different subfields of the human hippocampus in this process. Research in humans and non-human animal models has suggested that spatial environmental boundaries have a powerful influence on spatial and episodic memory, as well as hippocampal representations of contexts and events. Here, we used high-resolution fMRI to investigate how boundaries influence hippocampal activity patterns during the recollection of objects encountered in different spatial contexts. During the encoding phase, participants viewed objects once in a naturalistic virtual reality task in which they passively explored two rooms in one of two houses. Following the encoding phase, participants were scanned while they recollected items in the absence of any spatial contextual information. Our behavioral results demonstrated that spatial context memory was enhanced for objects encountered near a boundary. Activity patterns in CA1 carried information about the spatial context associated with each of these boundary items. Exploratory analyses revealed that recollection performance was correlated with the fidelity of retrieved spatial context representations in anterior parahippocampal cortex and subiculum. Our results highlight the privileged role of boundaries in CA1 and suggest more generally a close relationship between memory for spatial contexts and representations in the hippocampus and parahippocampal region.

An optimized live bacterial delivery vehicle safely and efficaciously delivers bacterially transcribed therapeutic nucleic acids.

There is an unmet need for delivery platforms that realize the full potential of next-generation nucleic acid therapeutics. The in vivo usefulness of current delivery systems is limited by numerous weaknesses, including poor targeting specificity, inefficient access to target cell cytoplasm, immune activation, off-target effects, small therapeutic windows, limited genetic encoding and cargo capacity, and manufacturing challenges. Here we characterize the safety and efficacy of a delivery platform comprising engineered live, tissue-targeting, non-pathogenic bacteria (Escherichia coli SVC1) for intracellular cargo delivery. SVC1 bacteria are engineered to specifically bind to epithelial cells via a surface-expressed targeting ligand, to allow escape of their cargo from the phagosome, and to have minimal immunogenicity. We describe SVC1's ability to deliver short hairpin RNA (shRNA), localized SVC1 administration to various tissues, and its minimal immunogenicity. To validate the therapeutic potential of SVC1, we used it to deliver influenza-targeting antiviral shRNAs to respiratory tissues in vivo. These data are the first to establish the safety and efficacy of this bacteria-based delivery platform for use in multiple tissue types and as an antiviral in the mammalian respiratory tract. We expect that this optimized delivery platform will enable a variety of advanced therapeutic approaches.

baz-2 enhances systemic proteostasis in vivo by regulating acetylcholine metabolism.

Neurodegenerative disorders, such as Alzheimer's disease (AD) or Huntington's disease (HD), are linked to protein aggregate neurotoxicity. According to the "cholinergic hypothesis," loss of acetylcholine (ACh) signaling contributes to the AD pathology, and therapeutic restoration of ACh signaling is a common treatment strategy. How disease causation and the effect of ACh are linked to protein aggregation and neurotoxicity remains incompletely understood, thus limiting the development of more effective therapies. Here, we show that BAZ-2, the Caenorhabditis elegans ortholog of human BAZ2B, limits ACh signaling. baz-2 mutations reverse aggregation and toxicity of amyloid-beta as well as polyglutamine peptides, thereby restoring health and lifespan in nematode models of AD and HD, respectively. The neuroprotective effect of Δbaz-2 is mediated by choline acetyltransferase, phenocopied by ACh-esterase depletion, and dependent on ACh receptors. baz-2 reduction or ectopic ACh treatment augments proteostasis via induction of the endoplasmic reticulum unfolded protein response and the ubiquitin proteasome system.

Memory-Based Prediction Deficits and Dorsolateral Prefrontal Dysfunction in Schizophrenia.

BACKGROUND: Theories suggest that people with schizophrenia (SZ) have problems generating predictions based on past experiences. The dorsolateral prefrontal cortex (DLPFC) and hippocampus participate in memory-based prediction. We used functional magnetic resonance imaging to investigate DLPFC and hippocampal function in healthy control (HC) subjects and people with SZ during memory-based prediction. METHODS: Prior to scanning, HC subjects (n = 54) and people with SZ (n = 31) learned 5-object sequences presented in fixed or random orders on each repetition. During scanning, participants made semantic decisions (e.g., "Can this object fit in a shoebox?") on a continuous stream of objects from fixed and random sequences. Sequence prediction was demonstrated by faster semantic decisions for objects in fixed versus random sequences because memory could be used to anticipate and more efficiently process semantic information about upcoming objects in fixed sequences. Representational similarity analyses were used to determine how each sequence type was represented in the posterior hippocampus and DLPFC. RESULTS: Sequence predictions were reduced in individuals with SZ relative to HC subjects. Representational similarity analyses revealed stronger memory-based predictions in the DLPFC of HC subjects than people with SZ, and DLPFC representations correlated with more successful predictions in HC subjects only. For the posterior hippocampus, voxel pattern similarity was increased for fixed versus random sequences in HC subjects only, but no significant between-group differences or correlations with prediction success were observed. CONCLUSIONS: Individuals with SZ are capable of learning temporal sequences; however, they are impaired using memory to predict upcoming events as efficiently as HC subjects. This deficit appears related to disrupted neural representation of sequence information in the DLPFC.

Retrieval practice facilitation of family psychoeducation in people with early psychosis.

BACKGROUND: Providing early psychosis (EP) individuals with family psychoeducation (FPE) can reduce symptoms and improve clinical outcomes. However, relational memory problems may limit prospective utilization of FPE information. This study examines whether memory for FPE can be improved by testing participants during the initial FPE workshop presentation. METHOD: Data were obtained from 20 people with EP and 20 demographically matched healthy comparison subjects (HC). During session one, FPE was presented in small group workshops, with half of the information re-studied twice (re-study condition) and the remaining information tested twice using cued recall tasks (retrieval practice condition). One week later (session two), delayed cued recall was tested for all FPE information. "Testing effects" (i.e., better memory following retrieval practice versus re-study) were examined across all items (standard analysis) and also limited to items successfully retrieved during session one (conditionalized analysis). RESULTS: HC had better initial recall and learned more over the two retrieval practice trials than EP. However, HC also lost more information than EP over the one-week delay. Both groups produced a significant testing effect. This effect was smaller in EP versus HC across all test items, but did not differ for the conditionalized analysis. Negative symptoms were inversely correlated with delayed cued recall in EP. CONCLUSIONS: EP participants benefit from retrieval practice, with participants with less severe negative symptoms showing the greatest benefit. These results encourage use of memory tests during group psychoeducation to improve subsequent long-term recall of clinically relevant information.

Restoration of Proteostasis in the Endoplasmic Reticulum Reverses an Inflammation-Like Response to Cytoplasmic DNA in Caenorhabditis elegans.

Innate immune responses protect organisms against various insults, but may lead to tissue damage when aberrantly activated. In higher organisms, cytoplasmic DNA can trigger inflammatory responses that can lead to tissue degeneration. Simpler metazoan models could shed new mechanistic light on how inflammatory responses to cytoplasmic DNA lead to pathologies. Here, we show that in a DNase II-defective Caenorhabditis elegans strain, persistent cytoplasmic DNA leads to systemic tissue degeneration and loss of tissue functionality due to impaired proteostasis. These pathological outcomes can be therapeutically alleviated by restoring protein homeostasis, either via ectopic induction of the ER unfolded protein response or N-acetylglucosamine treatment. Our results establish C. elegans as an ancestral metazoan model for studying the outcomes of inflammation-like conditions caused by persistent cytoplasmic DNA and provide insight into potential therapies for human conditions involving chronic inflammation.

The Escherichia coli histone-like protein HU has a role in stationary phase adaptive mutation.

Stationary phase adaptive mutation in Escherichia coli is thought to be a mechanism by which mutation rates are increased during stressful conditions, increasing the possibility that fitness-enhancing mutations arise. Here we present data showing that the histone-like protein, HU, has a role in the molecular pathway by which adaptive Lac(+) mutants arise in E. coli strain FC40. Adaptive Lac(+) mutations are largely but not entirely due to error-prone DNA polymerase IV (Pol IV). Mutations in either of the HU subunits, HUalpha or HUbeta, decrease adaptive mutation to Lac(+) by both Pol IV-dependent and Pol IV-independent pathways. Additionally, HU mutations inhibit growth-dependent mutations without a reduction in the level of Pol IV. These effects of HU mutations on adaptive mutation and on growth-dependent mutations reveal novel functions for HU in mutagenesis.

DNA damage responses and stress resistance: Concepts from bacterial SOS to metazoan immunity.

The critical need for species preservation has driven the evolution of mechanisms that integrate stress signals from both exogenous and endogenous sources. Past research has been largely focused on cell-autonomous stress responses; however, recently their systemic outcomes within an organism and their implications at the ecological and species levels have emerged. Maintenance of species depends on the high fidelity transmission of the genome over infinite generations; thus, many pathways exist to monitor and restore the integrity of the genome and to coordinate DNA repair with other cellular processes, such as cell division and growth. The specifics of these DNA damage responses (DDRs) vary vastly but some general themes are conserved from ancient organisms, such as bacteria and archaea, to humans. Despite decades of research, however, DDRs still have many layers of complexity and some surprises left to be discovered. One of the most interesting current research topics is the link between DNA damage and stress resistance: the outcomes of DDRs can protect the organism from other secondary challenges. At this time, these types of responses are best characterized in bacteria and the simple metazoan model, Caenorhabditis elegans, but it is becoming clear that similar processes also exist in higher organisms.

p53 in the DNA-Damage-Repair Process.

The cells in the human body are continuously challenged by a variety of genotoxic attacks. Erroneous repair of the DNA can lead to mutations and chromosomal aberrations that can alter the functions of tumor suppressor genes or oncogenes, thus causing cancer development. As a central tumor suppressor, p53 guards the genome by orchestrating a variety of DNA-damage-response (DDR) mechanisms. Already early in metazoan evolution, p53 started controlling the apoptotic demise of genomically compromised cells. p53 plays a prominent role as a facilitator of DNA repair by halting the cell cycle to allow time for the repair machineries to restore genome stability. In addition, p53 took on diverse roles to also directly impact the activity of various DNA-repair systems. It thus appears as if p53 is multitasking in providing protection from cancer development by maintaining genome stability.

Cell-Type Specific Responses to DNA Replication Stress in Early C. elegans Embryos.

To better understand how the cellular response to DNA replication stress is regulated during embryonic development, we and others have established the early C. elegans embryo as a model system to study this important problem. As is the case in most eukaryotic cell types, the replication stress response is controlled by the ATR kinase in early worm embryos. In this report we use RNAi to systematically characterize ATR pathway components for roles in promoting cell cycle delay during a replication stress response, and we find that these genetic requirements vary, depending on the source of stress. We also examine how individual cell types within the embryo respond to replication stress, and we find that the strength of the response, as defined by duration of cell cycle delay, varies dramatically within blastomeres of the early embryo. Our studies shed light on how the replication stress response is managed in the context of embryonic development and show that this pathway is subject to developmental regulation.

Zygotic Genome Activation Triggers Chromosome Damage and Checkpoint Signaling in C. elegans Primordial Germ Cells.

Recent findings have identified highly transcribed genes as a source of genome instability; however, the degree to which large-scale shifts in transcriptional activity cause DNA damage was not known. One example of a large-scale shift in transcriptional activity occurs during development, when maternal regulators are destroyed and zygotic genome activation (ZGA) occurs. Here, we show that ZGA triggers widespread chromosome damage in the primordial germ cells of the nematode C. elegans. We show that ZGA-induced DNA damage activates a checkpoint response, the damage is repaired by factors required for inter-sister homologous recombination, and topoisomerase II plays a role in generating the damage. These findings identify ZGA as a source of intrinsic genome instability in the germline and suggest that genome destabilization may be a general consequence of extreme shifts in cellular transcriptional load.

Spontaneous mutation rates come into focus in Escherichia coli.

Although the long-term outcome of mutagenesis is evolution by natural selection, it can also have profound immediate effects even on the level of individual organisms. In humans, the accumulation of mutations can cause many types of cancer; in bacteria, mutations can lead to dangerous antibiotic resistance and other phenotypic changes; and in viruses, mutations can cause drastic changes in the pathogenesis or modes of transfer. For these reasons, among others, a thorough understanding of mutagenesis is extremely important. One of the fundamental properties of the mutagenesis is its rate-the probability of a mutation occurring within a defined time frame. Despite the lengthy history of studies on mutagenesis and mutation rates, new and exciting findings continue to emerge. This review briefly summarizes the state-of-the-art in mutation rate analysis and continues with a discussion of some recent compelling discoveries on the mutational topology of the E. coli chromosome.

DAF-16/FOXO and EGL-27/GATA promote developmental growth in response to persistent somatic DNA damage.

Genome maintenance defects cause complex disease phenotypes characterized by developmental failure, cancer susceptibility and premature ageing. It remains poorly understood how DNA damage responses function during organismal development and maintain tissue functionality when DNA damage accumulates with ageing. Here we show that the FOXO transcription factor DAF-16 is activated in response to DNA damage during development, whereas the DNA damage responsiveness of DAF-16 declines with ageing. We find that in contrast to its established role in mediating starvation arrest, DAF-16 alleviates DNA-damage-induced developmental arrest and even in the absence of DNA repair promotes developmental growth and enhances somatic tissue functionality. We demonstrate that the GATA transcription factor EGL-27 co-regulates DAF-16 target genes in response to DNA damage and together with DAF-16 promotes developmental growth. We propose that EGL-27/GATA activity specifies DAF-16-mediated DNA damage responses to enable developmental progression and to prolong tissue functioning when DNA damage persists.

Stress-Induced Mutagenesis.

Early research on the origins and mechanisms of mutation led to the establishment of the dogma that, in the absence of external forces, spontaneous mutation rates are constant. However, recent results from a variety of experimental systems suggest that mutation rates can increase in response to selective pressures. This chapter summarizes data demonstrating that,under stressful conditions, Escherichia coli and Salmonella can increase the likelihood of beneficial mutations by modulating their potential for genetic change.Several experimental systems used to study stress-induced mutagenesis are discussed, with special emphasison the Foster-Cairns system for "adaptive mutation" in E. coli and Salmonella. Examples from other model systems are given to illustrate that stress-induced mutagenesis is a natural and general phenomenon that is not confined to enteric bacteria. Finally, some of the controversy in the field of stress-induced mutagenesis is summarized and discussed, and a perspective on the current state of the field is provided.

Double-Strand Break Repair and Holliday Junction Processing Are Required for Chromosome Processing in Stationary-Phase Escherichia coli Cells.

As nutrients are depleted and cell division ceases in batch cultures of bacteria, active processes are required to ensure that each cell has a complete copy of its genome. How chromosome number is manipulated and maintained in nondividing bacterial cells is not fully understood. Using flow cytometric analysis of cells from different growth phases, we show that the Holliday junction-processing enzymes RuvABC and RecG, as well as RecBCD, the enzyme complex that initiates DNA double-strand break repair, are required to establish the normal distribution of fluorescent peaks, which is commonly accepted to reflect the distribution of chromosome numbers. Our results reveal that these proteins are required for the proper processing of chromosomes in stationary phase.

Interplay of DNA repair, homologous recombination, and DNA polymerases in resistance to the DNA damaging agent 4-nitroquinoline-1-oxide in Escherichia coli.

Escherichia coli has three DNA damage-inducible DNA polymerases: DNA polymerase II (Pol II), DNA polymerase IV (Pol IV), and DNA polymerase V (Pol V). While the in vivo function of Pol V is well understood, the precise roles of Pol IV and Pol II in DNA replication and repair are not as clear. Study of these polymerases has largely focused on their participation in the recovery of failed replication forks, translesion DNA synthesis, and origin-independent DNA replication. However, their roles in other repair and recombination pathways in E. coli have not been extensively examined. This study investigated how E. coli's inducible DNA polymerases and various DNA repair and recombination pathways function together to convey resistance to 4-nitroquinoline-1-oxide (NQO), a DNA damaging agent that produces replication blocking DNA base adducts. The data suggest that full resistance to this compound depends upon an intricate interplay among the activities of the inducible DNA polymerases and recombination. The data also suggest new relationships between the different pathways that process recombination intermediates.