P. Joel Ross

The inflammasome recognizes cytosolic microbial and host DNA and triggers an innate immune response

The innate immune system recognizes nucleic acids during infection and tissue damage. Whereas viral RNA is detected by endosomal toll-like receptors (TLR3, TLR7, TLR8) and cytoplasmic RIG-I and MDA5, endosomal TLR9 and cytoplasmic DAI bind DNA, resulting in the activation of nuclear factor-κB and interferon regulatory factor transcription factors. However, viruses also trigger pro-inflammatory responses, which remain poorly defined. Here we show that internalized adenoviral DNA induces maturation of pro-interleukin-1β in macrophages, which is dependent on NALP3 and ASC, components of the innate cytosolic molecular complex termed the inflammasome. Correspondingly, NALP3- and ASC-deficient mice display reduced innate inflammatory responses to adenovirus particles. Inflammasome activation also occurs as a result of transfected cytosolic bacterial, viral and mammalian (host) DNA, but in this case sensing is dependent on ASC but not NALP3. The DNA-sensing pro-inflammatory pathway functions independently of TLRs and interferon regulatory factors. Thus, in addition to viral and bacterial components or danger signals in general, inflammasomes sense potentially dangerous cytoplasmic DNA, strengthening their central role in innate immunity.

MBNL proteins repress ES-cell-specific alternative splicing and reprogramming

Previous investigations of the core gene regulatory circuitry that controls the pluripotency of embryonic stem (ES) cells have largely focused on the roles of transcription, chromatin and non-coding RNA regulators. Alternative splicing represents a widely acting mode of gene regulation, yet its role in regulating ES-cell pluripotency and differentiation is poorly understood. Here we identify the muscleblind-like RNA binding proteins, MBNL1 and MBNL2, as conserved and direct negative regulators of a large program of cassette exon alternative splicing events that are differentially regulated between ES cells and other cell types. Knockdown of MBNL proteins in differentiated cells causes switching to an ES-cell-like alternative splicing pattern for approximately half of these events, whereas overexpression of MBNL proteins in ES cells promotes differentiated-cell-like alternative splicing patterns. Among the MBNL-regulated events is an ES-cell-specific alternative splicing switch in the forkhead family transcription factor FOXP1 that controls pluripotency. Consistent with a central and negative regulatory role for MBNL proteins in pluripotency, their knockdown significantly enhances the expression of key pluripotency genes and the formation of induced pluripotent stem cells during somatic cell reprogramming.

Spine growth in the anterior cingulate cortex is necessary for the consolidation of contextual fear memory

Remodeling of cortical connectivity is thought to allow initially hippocampus-dependent memories to be expressed independently of the hippocampus at remote time points. Consistent with this, consolidation of a contextual fear memory is associated with dendritic spine growth in neurons of the anterior cingulate cortex (aCC). To directly test whether such cortical structural remodeling is necessary for memory consolidation, we disrupted spine growth in the aCC at different times following contextual fear conditioning in mice. We took advantage of previous studies showing that the transcription factor myocyte enhancer factor 2 (MEF2) negatively regulates spinogenesis both in vitro and in vivo. We found that increasing MEF2-dependent transcription in the aCC during a critical posttraining window (but not at later time points) blocked both the consolidation-associated dendritic spine growth and subsequent memory expression. Together, these data strengthen the causal link between cortical structural remodeling and memory consolidation and, further, identify MEF2 as a key regulator of these processes.

MEF2 negatively regulates learning-induced structural plasticity and memory formation

Memory formation is thought to be mediated by dendritic-spine growth and restructuring. Myocyte enhancer factor 2 (MEF2) restricts spine growth in vitro, suggesting that this transcription factor negatively regulates the spine remodeling necessary for memory formation. Here we show that memory formation in adult mice was associated with changes in endogenous MEF2 levels and function. Locally and acutely increasing MEF2 function in the dentate gyrus blocked both learning-induced increases in spine density and spatial-memory formation. Increasing MEF2 function in amygdala disrupted fear-memory formation. We rescued MEF2-induced memory disruption by interfering with AMPA receptor endocytosis, suggesting that AMPA receptor trafficking is a key mechanism underlying the effects of MEF2. In contrast, decreasing MEF2 function in dentate gyrus and amygdala facilitated the formation of spatial and fear memory, respectively. These bidirectional effects indicate that MEF2 is a key regulator of plasticity and that relieving the suppressive effects of MEF2-mediated transcription permits memory formation.

MECP2e1 isoform mutation affects the form and function of neurons derived from Rett syndrome patient iPS cells

MECP2 mutations cause the X-linked neurodevelopmental disorder Rett Syndrome (RTT) by consistently altering the protein encoded by the MECP2e1 alternative transcript. While mutations that simultaneously affect both MECP2e1 and MECP2e2 isoforms have been widely studied, the consequence of MECP2e1 deficiency on human neurons remains unknown. Here we report the first isoform-specific patient induced pluripotent stem cell (iPSC) model of RTT. RTTe1 patient iPS cell-derived neurons retain an inactive X-chromosome and express only the mutant allele. Single-cell mRNA analysis demonstrated they have a molecular signature of cortical neurons. Mutant neurons exhibited a decrease in soma size, reduced dendritic complexity and decreased cell capacitance, consistent with impaired neuronal maturation. The soma size phenotype was rescued cell-autonomously by MECP2e1 transduction in a level-dependent manner but not by MECP2e2 gene transfer. Importantly, MECP2e1 mutant neurons showed dysfunction in action potential generation, voltage-gated Na+ currents, and miniature excitatory synaptic current frequency and amplitude. We conclude that MECP2e1 mutation affects soma size, information encoding properties and synaptic connectivity in human neurons that are defective in RTT.

Optimizing neuronal differentiation from induced pluripotent stem cells to model ASD

Autism spectrum disorder (ASD) is an early-onset neurodevelopmental disorder characterized by deficits in social communication, and restricted and repetitive patterns of behavior. Despite its high prevalence, discovery of pathophysiological mechanisms underlying ASD has lagged due to a lack of appropriate model systems. Recent advances in induced pluripotent stem cell (iPSC) technology and neural differentiation techniques allow for detailed functional analyses of neurons generated from living individuals with ASD. Refinement of cortical neuron differentiation methods from iPSCs will enable mechanistic studies of specific neuronal subpopulations that may be preferentially impaired in ASD. In this review, we summarize recent accomplishments in differentiation of cortical neurons from human pluripotent stems cells and efforts to establish in vitro model systems to study ASD using personalized neurons.

Host Cell Detection of Noncoding Stuffer DNA Contained in Helper-Dependent Adenovirus Vectors Leads to Epigenetic Repression of Transgene Expression

ABSTRACT Helper-dependent adenovirus (hdAd) vectors have shown great promise as therapeutic gene delivery vehicles in gene therapy applications. However, the level and duration of gene expression from hdAd can differ considerably depending on the nature of the noncoding stuffer DNA contained within the vector. For example, an hdAd containing 22 kb of prokaryotic DNA (hdAd-prok) expresses its transgene 60-fold less efficiently than a similar vector containing eukaryotic DNA (hdAd-euk). Here we have determined the mechanistic basis of this phenomenon. Although neither vector was subjected to CpG methylation and both genomes associated with cellular histones to similar degrees, hdAd-prok chromatin was actively deacetylated. Insertion of an insulator element between the transgene and the bacterial DNA derepressed hdAd-prok, suggesting that foreign DNA nucleates repressive chromatin structures that spread to the transgene. We found that Sp100B/Sp100HMG and Daxx play a role in repressing transgene expression from hdAd and act independently of PML bodies. Thus, we have identified nuclear factors involved in recognizing foreign DNA and have determined the mechanism by which associated genes are repressed.

Assembly of Helper-Dependent Adenovirus DNA into Chromatin Promotes Efficient Gene Expression

ABSTRACT Helper-dependent adenovirus (hdAd) vectors have shown tremendous potential in animal models of human disease in numerous preclinical studies. Expression of a therapeutic transgene can be maintained for several years after a single administration of the hdAd vector. However, despite the long-term persistence of hdAd DNA in the transduced cell, little is known of the fate and structure of hdAd DNA within the host nucleus. In this study, we have characterized the assembly of hdAd DNA into chromatin in tissue culture. Eviction of the Ad DNA-packaging protein VII, histone deposition, and vector-associated gene expression all began within 2 to 6 h of host cell transduction. Inhibition of transcription elongation through the vector DNA template had no effect on the loss of VII, suggesting that transcription was not necessary for removal of the majority of protein VII. Vector DNA assembled into physiologically spaced nucleosomes within 6 h. hdAd vectors incorporated the histone H3 variant H3.3, which was dependent on the histone chaperone HIRA. Knockdown of HIRA reduced hdAd association with histones and reduced expression of the vector-carried transgene by 2- to 3-fold. Our study elucidates an essential role for hdAd DNA chromatinization for optimal vector gene expression.

Construction and Characterization of Adenovirus Vectors

Genetically modified adenoviruses (Ads) make attractive vectors for the delivery of exogenous DNA to mammalian cells for basic science and gene therapy applications. Ad vector production consists of (1) cloning a transgene into an infectious plasmid by in vivo recombination in bacteria, (2) rescuing and propagating the vector in complementing cells, and (3) purifying the vector. All of this can be accomplished using commercially available reagents, plasmids, and cell lines. First-generation Ads have a large cloning capacity (5-14 kbp) and efficiently transduce a wide range of both quiescent and proliferating cell types. They are readily propagated to produce high-titer stocks (10-10 vector particles from a 3-L culture). Furthermore, Ads rarely integrate into the host genome and are relatively safe. However, Ad vector production typically takes 4-6 wk, and promiscuous host-cell transduction can occur in vivo. Furthermore, immune responses against viral proteins encoded by the vector backbone can occur, which limits the duration of transgene expression in vivo. Regardless of these limitations, Ad remains one of the more versatile and efficient gene delivery systems. Here, we discuss methods for the generation, propagation, purification, and characterization of first-generation Ad vectors.

Use of a murine secreted alkaline phosphatase as a non-immunogenic reporter gene in mice

The development of any vector system as a gene delivery system requires its optimization in vitro and in vivo. Preliminary studies frequently involve the use of a reporter gene, which allows for the rapid and simple assay of vector function through monitoring expression levels of the reporter gene. However, evaluation of vector efficacy can be compromised by immune responses directed against immunogenic reporter proteins.