Modifying the antiviral innate immune response by selective writing, erasing, and reading of m6A on viral and cellular RNA.

Viral infection and the antiviral innate immune response are regulated by the RNA modification m6A. m6A directs nearly all aspects of RNA metabolism by recruiting RNA-binding proteins that mediate the fate of m6A-containing RNA. m6A controls the antiviral innate immune response in diverse ways, including shielding viral RNA from detection by antiviral sensors and influencing the expression of cellular mRNAs encoding antiviral signaling proteins, cytokines, and effector proteins. While m6A and the m6A machinery are important for the antiviral response, the precise mechanisms that determine how the m6A machinery selects specific viral or cellular RNA molecules for modification during infection are not fully understood. In this review, we highlight recent findings that shed light on how viral infection redirects the m6A machinery during the antiviral response. A better understanding of m6A targeting during viral infection could lead to new immunomodulatory and therapeutic strategies against viral infection.

Integrin ß3 regulates apical dendritic morphology of pyramidal neurons throughout hippocampal CA3.

In excitatory hippocampal pyramidal neurons, integrin ß3 is critical for synaptic maturation and plasticity in vitro. Itgb3 is a potential autism susceptibility gene that regulates dendritic morphology in the cerebral cortex in a cell-specific manner. However, it is unknown what role Itgb3 could have in regulating hippocampal pyramidal dendritic morphology in vivo, a key feature that is aberrant in many forms of autism and intellectual disability. We found that Itgb3 mRNA is expressed in the stratum pyramidale of CA3. We examined the apical dendritic morphology of CA3 hippocampal pyramidal neurons in conditional Itgb3 knockouts and controls, utilizing the Thy1-GFP-M line. We fully reconstructed the apical dendrite of each neuron and determined each neuron's precise location along the dorsoventral, proximodistal, and radial axes of the stratum pyramidale. We found a very strong effect for Itgb3 expression on CA3 apical dendritic morphology: neurons from conditional Itgb3 knockouts had longer and thinner apical dendrites than controls, particularly in higher branch orders. We also assessed potential relationships between pairs of topographic or morphological variables, finding that most variable pairs were free from any linear relationships to each other. We also found that some neurons from controls, but not conditional Itgb3 knockouts, had a graded pattern of overall diameter along the dorsoventral and proximodistal axes of the stratum pyramidale of CA3. Taken together, Itgb3 is essential for constructing normal dendritic morphology in pyramidal neurons throughout CA3.

Simultaneous 3D cellular positioning and apical dendritic morphology of transgenic fluorescent mouse CA3 hippocampal pyramidal neurons.

BACKGROUND: Pyramidal neurons throughout hippocampal CA3 are diverse in their dendritic morphology, and CA3 is not homogenous in its structure or function. Nonetheless, few structural studies have captured the precise 3D somatic position and the 3D dendritic morphology of CA3 pyramidal neurons simultaneously. NEW METHOD: Here, we present a simple approach to reconstruct the apical dendritic morphology of CA3 pyramidal neurons using the transgenic fluorescent Thy1-GFP-M line. The approach simultaneously tracks the dorsoventral, tangential, and radial positions of reconstructed neurons within the hippocampus. It is especially designed for use with transgenic fluorescent mouse lines, which are commonly used in genetic studies of neuronal morphology and development. RESULTS: We demonstrate how topographic and morphological data are captured from transgenic fluorescent mouse CA3 pyramidal neurons. COMPARISON WITH EXISTING METHODS: There is no need to select and label CA3 pyramidal neurons with the transgenic fluorescent Thy1-GFP-M line. By taking transverse (not coronal) serial sections, we preserve fine dorsoventral, tangential, and radial somatic positioning of 3D-reconstructed neurons. Because CA2 is well defined by PCP4 immunohistochemistry, we use that technique here to to increase precision in defining tangential position along CA3. CONCLUSIONS: We developed a method for simultaneously collecting precise somatic positioning as well as 3D morphological data among transgenic fluorescent mouse hippocampal pyramidal neurons. This fluorescent method should be compatible with many other transgenic fluorescent reporter lines and immunohistochemical methods, facilitating the capture of topographic and morphological data from a wide variety of genetic experiments in mouse hippocampus.

WTAP Targets the METTL3 m6A-Methyltransferase Complex to Cytoplasmic Hepatitis C Virus RNA to Regulate Infection.

Modification of the hepatitis C virus (HCV) positive-strand RNA genome by N6-methyladenosine (m6A) regulates the viral life cycle. This life cycle takes place solely in the cytoplasm, while m6A addition on cellular mRNA takes place in the nucleus. Thus, the mechanisms by which m6A is deposited on the viral RNA have been unclear. In this work, we find that m6A modification of HCV RNA by the m6A-methyltransferase proteins methyltransferase-like 3 and 14 (METTL3 and METTL14) is regulated by Wilms' tumor 1-associating protein (WTAP). WTAP, a predominantly nuclear protein, is an essential member of the cellular mRNA m6A-methyltransferase complex and known to target METTL3 to mRNA. We found that HCV infection induces localization of WTAP to the cytoplasm. Importantly, we found that WTAP is required for both METTL3 interaction with HCV RNA and m6A modification across the viral RNA genome. Further, we found that WTAP, like METTL3 and METTL14, negatively regulates the production of infectious HCV virions, a process that we have previously shown is regulated by m6A. Excitingly, WTAP regulation of both HCV RNA m6A modification and virion production was independent of its ability to localize to the nucleus. Together, these results reveal that WTAP is critical for HCV RNA m6A modification by METTL3 and METTL14 in the cytoplasm. IMPORTANCE Positive-strand RNA viruses such as HCV represent a significant global health burden. Previous work has described that HCV RNA contains the RNA modification m6A and how this modification regulates viral infection. Yet, how this modification is targeted to HCV RNA has remained unclear due to the incompatibility of the nuclear cellular processes that drive m6A modification with the cytoplasmic HCV life cycle. In this study, we present evidence for how m6A modification is targeted to HCV RNA in the cytoplasm by a mechanism in which WTAP recruits the m6A-methyltransferase METTL3 to HCV RNA. This targeting strategy for m6A modification of cytoplasmic RNA viruses is likely relevant for other m6A-modified positive-strand RNA viruses with cytoplasmic life cycles such as enterovirus 71 and SARS-CoV-2 and provides an exciting new target for potential antiviral therapies.

WTAP targets the METTL3 m 6 A-methyltransferase complex to cytoplasmic hepatitis C virus RNA to regulate infection.

Modification of the hepatitis C virus (HCV) positive-strand RNA genome by N6-methyladenosine (m 6 A) regulates the viral lifecycle. This lifecycle takes place solely in the cytoplasm, while m 6 A addition on cellular mRNA takes place in the nucleus. Thus, the mechanisms by which m 6 A is deposited on the viral RNA have been unclear. In this work, we find that m 6 A modification of HCV RNA by the m 6 A-methyltransferase proteins METTL3 and METTL14 is regulated by WTAP. WTAP, a predominantly nuclear protein, is an essential member of the cellular mRNA m 6 A-methyltransferase complex and known to target METTL3 to mRNA. We found that HCV infection induces localization of WTAP to the cytoplasm. Importantly, we found that WTAP is required for both METTL3 interaction with HCV RNA and for m 6 A modification across the viral RNA genome. Further, we found that WTAP, like METTL3 and METTL14, negatively regulates the production of infectious HCV virions, a process that we have previously shown is regulated by m 6 A. Excitingly, WTAP regulation of both HCV RNA m 6 A modification and virion production were independent of its ability to localize to the nucleus. Together, these results reveal that WTAP is critical for HCV RNA m 6 A modification by METTL3 and METTL14 in the cytoplasm. IMPORTANCE: Positive-strand RNA viruses such as HCV represent a significant global health burden. Previous work has described how HCV RNA contains the RNA modification m 6 A and how this modification regulates viral infection. Yet, how this modification is targeted to HCV RNA has remained unclear due to the incompatibility of the nuclear cellular processes that drive m 6 A modification with the cytoplasmic HCV lifecycle. In this study, we present evidence for how m 6 A modification is targeted to HCV RNA in the cytoplasm by a mechanism in which WTAP recruits the m 6 A-methyltransferase METTL3 to HCV RNA. This targeting strategy for m 6 A modification of cytoplasmic RNA viruses is likely relevant for other m 6 A-modified positive-strand RNA viruses with cytoplasmic lifecycles such as enterovirus 71 and SARS-CoV-2 and provides an exciting new target for potential antiviral therapies.

Integrin ß3 in forebrain Emx1-expressing cells regulates repetitive self-grooming and sociability in mice.

BACKGROUND: Autism spectrum disorder (ASD) is characterized by repetitive behaviors, deficits in communication, and overall impaired social interaction. Of all the integrin subunit mutations, mutations in integrin ß3 (Itgb3) may be the most closely associated with ASD. Integrin ß3 is required for normal structural plasticity of dendrites and synapses specifically in excitatory cortical and hippocampal circuitry. However, the behavioral consequences of Itgb3 function in the forebrain have not been assessed. We tested the hypothesis that behaviors that are typically abnormal in ASD-such as self-grooming and sociability behaviors-are disrupted with conditional Itgb3 loss of function in forebrain circuitry in male and female mice. METHODS: We generated male and female conditional knockouts (cKO) and conditional heterozygotes (cHET) of Itgb3 in excitatory neurons and glia that were derived from Emx1-expressing forebrain cells during development. We used several different assays to determine whether male and female cKO and cHET mice have repetitive self-grooming behaviors, anxiety-like behaviors, abnormal locomotion, compulsive-like behaviors, or abnormal social behaviors, when compared to male and female wildtype (WT) mice. RESULTS: Our findings indicate that only self-grooming and sociability are altered in cKO, but not cHET or WT mice, suggesting that Itgb3 is specifically required in forebrain Emx1-expressing cells for normal repetitive self-grooming and social behaviors. Furthermore, in cKO (but not cHET or WT), we observed an interaction effect for sex and self-grooming environment and an interaction effect for sex and sociability test chamber. LIMITATIONS: While this study demonstrated a role for forebrain Itgb3 in specific repetitive and social behaviors, it was unable to determine whether forebrain Itgb3 is required for a preference for social novelty, whether cHET are haploinsufficient with respect to repetitive self-grooming and social behaviors, or the nature of the interaction effect for sex and environment/chamber in affected behaviors of cKO. CONCLUSIONS: Together, these findings strengthen the idea that Itgb3 has a specific role in shaping forebrain circuitry that is relevant to endophenotypes of autism spectrum disorder.

FMRP regulates the subcellular distribution of cortical dendritic spine density in a non-cell-autonomous manner.

Fragile X syndrome (FXS) is the most common form of intellectual disability that arises from the dysfunction of a single gene-Fmr1. The main neuroanatomical correlate of FXS is elevated dendritic spine density on cortical pyramidal neurons, which has been modeled in Fmr1-/Y mice. However, the cell-autonomous contribution of Fmr1 on cortical dendritic spine density has not been assessed. Even less is known about the role of Fmr1 in heterozygous female mosaic mice, which are a putative model for human Fmr1 full mutation carriers (i.e., are heterozygous for the full Fmr1-silencing mutation). In this neuroanatomical study, spine density in cortical pyramidal neurons of Fmr1+/- and Fmr1-/Y mice was studied at multiple subcellular compartments, layers, and brain regions. Spine density in Fmr1+/- mice is higher than WT but lower than Fmr1-/Y. Not all subcellular compartments in layer V Fmr1+/- and Fmr1-/Y cortical pyramidal neurons are equally affected: the apical dendrite, a key subcellular compartment, is principally affected over basal dendrites. Within apical dendrites, spine density is differentially affected across branch orders. Finally, identification of FMRP-positive and FMRP-negative neurons within Fmr1+/- permitted the study of the cell-autonomous effect of Fmr1 on spine density. Surprisingly, layer V cortical pyramidal spine density between FMRP-positive and FMRP-negative neurons does not differ, suggesting that the regulation of the primary neuroanatomical defect of FXS-elevated spine density-is non-cell-autonomous.

Integrin ß3 organizes dendritic complexity of cerebral cortical pyramidal neurons along a tangential gradient.

Dysfunctional dendritic arborization is a key feature of many developmental neurological disorders. Across various human brain regions, basal dendritic complexity is known to increase along a caudal-to-rostral gradient. We recently discovered that basal dendritic complexity of layer II/III cortical pyramidal neurons in the mouse increases along a caudomedial-to-rostrolateral gradient spanning multiple regions, but at the time, no molecules were known to regulate that exquisite pattern. Integrin subunits have been implicated in dendritic development, and the subunit with the strongest associations with autism spectrum disorder and intellectual disability is integrin ß3 (Itgb3). In mice, global knockout of Itgb3 leads to autistic-like neuroanatomy and behavior. Here, we tested the hypothesis that Itgb3 is required for increasing dendritic complexity along the recently discovered tangential gradient among layer II/III cortical pyramidal neurons. We targeted a subset of layer II/III cortical pyramidal neurons for Itgb3 loss-of-function via Cre-loxP-mediated excision of Itgb3. We tracked the rostrocaudal and mediolateral position of the targeted neurons and reconstructed their dendritic arbors. In contrast to controls, the basal dendritic complexity of Itgb3 mutant neurons was not related to their cortical position. Basal dendritic complexity of mutant and control neurons differed because of overall changes in branch number across multiple branch orders (primary, secondary, etc.), rather than any changes in the average length at those branch orders. Furthermore, dendritic spine density was related to cortical position in control but not mutant neurons. Thus, the autism susceptibility gene Itgb3 is required for establishing a tangential pattern of basal dendritic complexity among layer II/III cortical pyramidal neurons, suggesting an early role for this molecule in the developing brain.

Cross-Regional Gradient of Dendritic Morphology in Isochronically-Sourced Mouse Supragranular Pyramidal Neurons.

Architectonic heterogeneity in neurons is thought to be important for equipping the mammalian cerebral cortex with an adaptable network that can organize the manifold totality of information it receives. To this end, the dendritic arbors of supragranular pyramidal neurons, even those of the same class, are known to vary substantially. This diversity of dendritic morphology appears to have a rostrocaudal configuration in some brain regions of various species. For example, in humans and non-human primates, neurons in more rostral visual association areas (e.g., V4) tend to have more complex dendritic arbors than those in the caudal primary visual cortex. A rostrocaudal configuration is not so clear in any region of the mouse, which is increasingly being used as a model for neurodevelopmental disorders that arise from dysfunctional cerebral cortical circuits. Therefore, in this study we investigated the complexity of dendritic arbors of neurons distributed throughout a broad area of the mouse cerebral cortex. We reduced selection bias by labeling neurons restricted to become supragranular pyramidal neurons using in utero electroporation. While we observed that the simple rostrocaudal position, cortical depth, or even functional region of a neuron was not directly related to its dendritic morphology, a model that instead included a caudomedial-to-rostrolateral gradient accounted for a significant amount of the observed dendritic morphological variance. In other words, rostrolateral neurons from our data set were generally more complex when compared to caudomedial neurons. Furthermore, dividing the cortex into a visual area and a non-visual area maintained the power of the relationship between caudomedial-to-rostrolateral position and dendritic complexity. Our observations therefore support the idea that dendritic morphology of mouse supragranular excitatory pyramidal neurons across much of the tangential plane of the cerebral cortex is partly shaped by a developmental gradient spanning several functional regions.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation.

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.