The NLR gene family: from discovery to present day.

The mammalian NLR gene family was first reported over 20 years ago, although several genes that were later grouped into the family were already known at that time. Although it is widely known that NLRs include inflammasome receptors and/or sensors that promote the maturation of caspase 1, IL-1ß, IL-18 and gasdermin D to drive inflammation and cell death, the other functions of NLR family members are less well appreciated by the scientific community. Examples include MHC class II transactivator (CIITA), a master transcriptional activator of MHC class II genes, which was the first mammalian NBD-LRR-containing protein to be identified, and NLRC5, which regulates the expression of MHC class I genes. Other NLRs govern key inflammatory signalling pathways or interferon responses, and several NLR family members serve as negative regulators of innate immune responses. Multiple NLRs regulate the balance of cell death, cell survival, autophagy, mitophagy and even cellular metabolism. Perhaps the least discussed group of NLRs are those with functions in the mammalian reproductive system. The focus of this Review is to provide a synopsis of the NLR family, including both the intensively studied and the underappreciated members. We focus on the function, structure and disease relevance of NLRs and highlight issues that have received less attention in the NLR field. We hope this may serve as an impetus for future research on the conventional and non-conventional roles of NLRs within and beyond the immune system.

The accessible chromatin landscape of the murine hippocampus at single-cell resolution.

Here we present a comprehensive map of the accessible chromatin landscape of the mouse hippocampus at single-cell resolution. Substantial advances of this work include the optimization of a single-cell combinatorial indexing assay for transposase accessible chromatin (sci-ATAC-seq); a software suite, scitools, for the rapid processing and visualization of single-cell combinatorial indexing data sets; and a valuable resource of hippocampal regulatory networks at single-cell resolution. We used sci-ATAC-seq to produce 2346 high-quality single-cell chromatin accessibility maps with a mean unique read count per cell of 29,201 from both fresh and frozen hippocampi, observing little difference in accessibility patterns between the preparations. By using this data set, we identified eight distinct major clusters of cells representing both neuronal and nonneuronal cell types and characterized the driving regulatory factors and differentially accessible loci that define each cluster. Within pyramidal neurons, we identified four major clusters, including CA1 and CA3 neurons, and three additional subclusters. We then applied a recently described coaccessibility framework, Cicero, which identified 146,818 links between promoters and putative distal regulatory DNA. Identified coaccessibility networks showed cell-type specificity, shedding light on key dynamic loci that reconfigure to specify hippocampal cell lineages. Lastly, we performed an additional sci-ATAC-seq preparation from cultured hippocampal neurons (899 high-quality cells, 43,532 mean unique reads) that revealed substantial alterations in their epigenetic landscape compared with nuclei from hippocampal tissue. This data set and accompanying analysis tools provide a new resource that can guide subsequent studies of the hippocampus.

IgSF21 promotes differentiation of inhibitory synapses via binding to neurexin2α.

Coordinated development of excitatory and inhibitory synapses is essential for higher brain function, and impairment in this development is associated with neuropsychiatric disorders. In contrast to the large body of accumulated evidence regarding excitatory synapse development, little is known about synaptic adhesion and organization mechanisms underlying inhibitory synapse development. Through unbiased expression screens and proteomics, we identified immunoglobulin superfamily member 21 (IgSF21) as a neurexin2α-interacting membrane protein that selectively induces inhibitory presynaptic differentiation. IgSF21 localizes postsynaptically and recruits axonal neurexin2α in a trans-interaction manner. Deleting IgSF21 in mice impairs inhibitory presynaptic organization, especially in the hippocampal CA1 stratum radiatum, and also diminishes GABA-mediated synaptic transmission in hippocampal CA1 neurons without affecting their excitatory synapses. Finally, mice lacking IgSF21 show a sensorimotor gating deficit. These findings suggest that IgSF21 selectively regulates inhibitory presynaptic differentiation through interacting with presynaptic neurexin2α and plays a crucial role in synaptic inhibition in the brain.Molecular mechanisms regulating the development of inhibitory synapses are poorly understood. Here the authors show that IgSF21 interacts with neurexin2α to induce presynaptic differentiation of inhibitory synapses, and that mice lacking IgSF21 exhibit deficits in inhibitory synaptic transmission.

A high-resolution imaging approach to investigate chromatin architecture in complex tissues.

We present ChromATin, a quantitative high-resolution imaging approach for investigating chromatin organization in complex tissues. This method combines analysis of epigenetic modifications by immunostaining, localization of specific DNA sequences by FISH, and high-resolution segregation of nuclear compartments using array tomography (AT) imaging. We then apply this approach to examine how the genome is organized in the mammalian brain using female Rett syndrome mice, which are a mosaic of normal and Mecp2-null cells. Side-by-side comparisons within the same field reveal distinct heterochromatin territories in wild-type neurons that are altered in Mecp2-null nuclei. Mutant neurons exhibit increased chromatin compaction and a striking redistribution of the H4K20me3 histone modification into pericentromeric heterochromatin, a territory occupied normally by MeCP2. These events are not observed in every neuronal cell type, highlighting ChromATin as a powerful in situ method for examining cell-type-specific differences in chromatin architecture in complex tissues.

Synchronous and asynchronous modes of synaptic transmission utilize different calcium sources.

Asynchronous transmission plays a prominent role at certain synapses but lacks the mechanistic insights of its synchronous counterpart. The current view posits that triggering of asynchronous release during repetitive stimulation involves expansion of the same calcium domains underlying synchronous transmission. In this study, live imaging and paired patch clamp recording at the zebrafish neuromuscular synapse reveal contributions by spatially distinct calcium sources. Synchronous release is tied to calcium entry into synaptic boutons via P/Q type calcium channels, whereas asynchronous release is boosted by a propagating intracellular calcium source initiated at off-synaptic locations in the axon and axonal branch points. This secondary calcium source fully accounts for the persistence following termination of the stimulus and sensitivity to slow calcium buffers reported for asynchronous release. The neuromuscular junction and CNS neurons share these features, raising the possibility that secondary calcium sources are common among synapses with prominent asynchronous release. DOI: http://dx.doi.org/10.7554/eLife.01206.001.

The specific α-neurexin interactor calsyntenin-3 promotes excitatory and inhibitory synapse development.

Perturbations of cell surface synapse-organizing proteins, particularly α-neurexins, contribute to neurodevelopmental and psychiatric disorders. From an unbiased screen, we identify calsyntenin-3 (alcadein-ß) as a synapse-organizing protein unique in binding and recruiting α-neurexins, but not ß-neurexins. Calsyntenin-3 is present in many pyramidal neurons throughout cortex and hippocampus but is most highly expressed in interneurons. The transmembrane form of calsyntenin-3 can trigger excitatory and inhibitory presynapse differentiation in contacting axons. However, calsyntenin-3-shed ectodomain, which represents about half the calsyntenin-3 pool in brain, suppresses the ability of multiple α-neurexin partners including neuroligin 2 and LRRTM2 to induce presynapse differentiation. Clstn3⁻/⁻ mice show reductions in excitatory and inhibitory synapse density by confocal and electron microscopy and corresponding deficits in synaptic transmission. These results identify calsyntenin-3 as an α-neurexin-specific binding partner required for normal functional GABAergic and glutamatergic synapse development.

Zebrafish calls for reinterpretation for the roles of P/Q calcium channels in neuromuscular transmission.

A long-held tenet of neuromuscular transmission is that calcium-dependent neurotransmitter release is mediated by N-type calcium channels in frog but P/Q-type channels in mammals. The N-type assignment in frog is based principally on pharmacological sensitivity to ω-conotoxin GVIA. Our studies show that zebrafish neuromuscular transmission is also sensitive to ω-conotoxin GVIA. However, positional cloning of a mutant line with compromised neuromuscular function identified a mutation in a P/Q- rather than N-type channel. Cloning and heterologous expression of this P/Q-type channel confirmed a block by ω-conotoxin GVIA raising the likelihood that all vertebrates, including frog, use the P/Q-type calcium channel for neuromuscular transmission. In addition, our P/Q defective mutant line offered a means of testing the ability of roscovitine, known to potentiate frog neuromuscular transmission, to mediate behavioral and functional rescue. Acute treatment led to rapid improvement of both, pointing to potential therapeutic benefit for myasthenic disorders involving calcium channel dysfunction.

Distinct roles for two synaptotagmin isoforms in synchronous and asynchronous transmitter release at zebrafish neuromuscular junction.

An obligatory role for the calcium sensor synaptotagmins in stimulus-coupled release of neurotransmitter is well established, but a role for synaptotagmin isoform involvement in asynchronous release remains conjecture. We show, at the zebrafish neuromuscular synapse, that two separate synaptotagmins underlie these processes. Specifically, knockdown of synaptotagmin 2 (syt2) reduces synchronous release, whereas knockdown of synaptotagmin 7 (syt7) reduces the asynchronous component of release. The zebrafish neuromuscular junction is unique in having a very small quantal content and a high release probability under conditions of either low-frequency stimulation or high-frequency augmentation. Through these features, we further determined that during the height of shared synchronous and asynchronous transmission these two modes compete for the same release sites.

An unbiased expression screen for synaptogenic proteins identifies the LRRTM protein family as synaptic organizers.

Delineating the molecular basis of synapse development is crucial for understanding brain function. Cocultures of neurons with transfected fibroblasts have demonstrated the synapse-promoting activity of candidate molecules. Here, we performed an unbiased expression screen for synaptogenic proteins in the coculture assay using custom-made cDNA libraries. Reisolation of NGL-3/LRRC4B and neuroligin-2 accounts for a minority of positive clones, indicating that current understanding of mammalian synaptogenic proteins is incomplete. We identify LRRTM1 as a transmembrane protein that induces presynaptic differentiation in contacting axons. All four LRRTM family members exhibit synaptogenic activity, LRRTMs localize to excitatory synapses, and artificially induced clustering of LRRTMs mediates postsynaptic differentiation. We generate LRRTM1(-/-) mice and reveal altered distribution of the vesicular glutamate transporter VGLUT1, confirming an in vivo synaptic function. These results suggest a prevalence of LRR domain proteins in trans-synaptic signaling and provide a cellular basis for the reported linkage of LRRTM1 to handedness and schizophrenia.

How to build a central synapse: clues from cell culture.

Central neurons develop and maintain molecularly distinct synaptic specializations for excitatory and inhibitory transmitters, often only microns apart on their dendritic arbor. Progress towards understanding the molecular basis of synaptogenesis has come from several recent studies using a coculture system of non-neuronal cells expressing molecules that generate presynaptic or postsynaptic "hemi-synapses" on contacting neurons. Together with molecular properties of these protein families, such studies have yielded interesting clues to how glutamatergic and GABAergic synapses are assembled. Other clues come from heterochronic cultures, manipulations of activity in subsets of neurons in a network, and of course many in vivo studies. Taking into account these data, we consider here how basic parameters of synapses--competence, placement, composition, size and longevity--might be determined.

Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins.

Formation of synaptic connections requires alignment of neurotransmitter receptors on postsynaptic dendrites opposite matching transmitter release sites on presynaptic axons. beta-neurexins and neuroligins form a trans-synaptic link at glutamate synapses. We show here that neurexin alone is sufficient to induce glutamate postsynaptic differentiation in contacting dendrites. Surprisingly, neurexin also induces GABA postsynaptic differentiation. Conversely, neuroligins induce presynaptic differentiation in both glutamate and GABA axons. Whereas neuroligins-1, -3, and -4 localize to glutamate postsynaptic sites, neuroligin-2 localizes primarily to GABA synapses. Direct aggregation of neuroligins reveals a linkage of neuroligin-2 to GABA and glutamate postsynaptic proteins, but the other neuroligins only to glutamate postsynaptic proteins. Furthermore, mislocalized expression of neuroligin-2 disperses postsynaptic proteins and disrupts synaptic transmission. Our findings indicate that the neurexin-neuroligin link is a core component mediating both GABAergic and glutamatergic synaptogenesis, and differences in isoform localization and binding affinities may contribute to appropriate differentiation and specificity.

Decreased apoptosome activity with neuronal differentiation sets the threshold for strict IAP regulation of apoptosis.

Despite the potential of the inhibitor of apoptosis proteins (IAPs) to block cytochrome c-dependent caspase activation, the critical function of IAPs in regulating mammalian apoptosis remains unclear. We report that the ability of endogenous IAPs to effectively regulate caspase activation depends on the differentiation state of the cell. Despite being expressed at equivalent levels, endogenous IAPs afforded no protection against cytochrome c-induced apoptosis in naive pheochromocytoma (PC12) cells, but were remarkably effective in doing so in neuronally differentiated cells. Neuronal differentiation was also accompanied with a marked reduction in Apaf-1, resulting in a significant decrease in apoptosome activity. Importantly, this decrease in Apaf-1 protein was directly linked to the increased ability of IAPs to stringently regulate apoptosis in neuronally differentiated PC12 and primary cells. These data illustrate specifically how the apoptotic pathway acquires increased regulation with cellular differentiation, and are the first to show that IAP function and apoptosome activity are coupled in cells.

FGF22 and its close relatives are presynaptic organizing molecules in the mammalian brain.

Target-derived cues promote local differentiation of axons into nerve terminals at sites of synaptic contact. Using clustering of synaptic vesicles in cultured neurons as an assay, we purified putative target-derived presynaptic organizing molecules from mouse brain and identified FGF22 as a major active species. FGF7 and FGF10, the closest relatives of FGF22, share this activity; other FGFs have distinct effects. FGF22 is expressed by cerebellar granule cells during the period when they receive synapses. Its receptor, FGFR2, is expressed by pontine and vestibular neurons when their axons (mossy fibers) are making synapses on granule cells. Neutralization of FGF7, -10, and -22 inhibits presynaptic differentiation of mossy fibers at sites of contact with granule cells in vivo. Inactivation of FGFR2 has similar effects. These results indicate that FGF22 and its relatives are presynaptic organizing molecules in the mammalian brain and suggest new functions for this family of signaling molecules.

Serine residues 286, 288, and 293 within the CIITA: a mechanism for down-regulating CIITA activity through phosphorylation.

CIITA is the primary factor activating the expression of the class II MHC genes necessary for the exogenous pathway of Ag processing and presentation. Strict control of CIITA is necessary to regulate MHC class II gene expression and induction of an immune response. We show in this study that the nuclear localized form of CIITA is a predominantly phosphorylated form of the protein, whereas cytoplasmic CIITA is predominantly unphosphorylated. Novel phosphorylation sites were determined to be located within a region that contains serine residues 286, 288, and 293. Double mutations of these residues increased nuclear CIITA, indicating that these sites are not required for nuclear import. CIITA-bearing mutations of these serine residues significantly increased endogenous MHC class II expression, but did not significantly enhance trans-activation from a MHC class II promoter, indicating that these phosphorylation sites may be important for gene activation from intact chromatin rather than artificial plasmid-based promoters. These data suggest a model for CIITA function in which phosphorylation of these specific sites in CIITA in the nucleus serves to down-regulate CIITA activity.

Cutting edge: CIAS1/cryopyrin/PYPAF1/NALP3/CATERPILLER 1.1 is an inducible inflammatory mediator with NF-kappa B suppressive properties.

Mutations in the cold-induced autoinflammatory syndrome 1 (CIAS1) gene have been recently linked to three chronic autoinflammatory disorders. These observations point to an important role for CIAS1 in regulating inflammatory processes. We report that TNF-alpha and ligands recognized by multiple Toll-like receptors rapidly induce CIAS1 gene expression in primary human monocytes. Transfection of full-length CIAS1 or either of two shorter, naturally occurring isoforms dramatically inhibited TNF-alpha-induced activation of NF-kappaB reporter activity. Furthermore, CIAS1 suppressed TNF-alpha-induced nuclear translocation of endogenous p65. Transcriptional activity of exogenous NF-kappaB p65 was also blocked by CIAS1. The nucleotide-binding and leucine-rich repeat regions, but not the pyrin domain of CIAS1, are responsible for this inhibition. These data suggest CIAS1/cryopyrin may act as a key regulator of inflammation, induced to dampen NF-kappaB-dependent proinflammatory signals.

Stability and apoptotic activity of recombinant human cytochrome c.

An efficient system for producing human cytochrome c variants is important to help us understand the roles of this protein in biological processes relevant to human diseases including apoptosis and oxidative stress. Here, we describe an Escherichia coli expression system for producing recombinant human cytochrome c. We also characterize the structure, stability, and function of the protein and show its utility for studying apoptosis. Yields of greater than 8 mg of pure protein per liter culture were attained. Circular dichroism spectropolarimetry studies show that the secondary and tertiary structures of the human protein are nearly identical to those of the horse protein, but the human protein is more stable than other eukaryotic cytochromes c. Furthermore, recombinant human cytochrome c is capable of inducing caspase-3 activity in a cell-free caspase activation assay. We use data from this assay along with data from the literature to define the apaf-1 binding site on human cytochrome c.

Cutting edge: Monarch-1: a pyrin/nucleotide-binding domain/leucine-rich repeat protein that controls classical and nonclassical MHC class I genes.

Proteins containing a limited number of N-terminal motifs followed by nucleotide-binding domain and leucine-rich repeat regions are emerging as important regulators for immunity. A search of human genome scaffold databases has identified a large family of known and unknown genes, which we have recently called the CATERPILLER (caspase recruitment domain, transcription enhancer, r(purine)-binding, pyrin, lots of leucine repeats) gene family. This work describes the characterization of a new member, Monarch-1. Monarch-1 has four different splice forms due to the differential splicing of leucine-rich repeat motifs. It is expressed in cells of myeloid-monocytic origin. Affymetrix microarrays and small interfering RNA were used to elucidate the downstream effects of Monarch-1 expression in cells including those of myeloid-monocytic origin. These analyses show that Monarch-1 enhances nonclassical and classical MHC class I expression at the level of the promoter, RNA, and protein expression.

Cutting edge: CATERPILLER: a large family of mammalian genes containing CARD, pyrin, nucleotide-binding, and leucine-rich repeat domains.

Large mammalian proteins containing a nucleotide-binding domain (NBD) and C-terminal leucine-rich repeats (LRR) similar in structure to plant disease resistance proteins have been suggested as critical in innate immunity. Our interest in CIITA, a NBD/LRR protein, and recent reports linking mutations in two other NBD/LRR proteins to inflammatory disorders have prompted us to perform a search for other members. Twenty-two known and novel NBD/LRR genes are spread across eight human chromosomes, with multigene clusters occurring on 11, 16, and 19. Most of these are telomeric. Their N termini vary, but most have a pyrin domain. The genomic organization demonstrates a high degree of conservation of the NBD- and LRR-encoding exons. Except for CIITA, all the predicted NBD/LRR proteins are likely ATP-binding proteins. Some have broad tissue expression, whereas others are restricted to myeloid cells. The implications of these data on origins, expression, and function of these genes are discussed.

Leucine-rich repeats of the class II transactivator control its rate of nuclear accumulation.

Activation of class II major histocompatibility complex (MHC) gene expression is regulated by a master regulator, class II transcriptional activator (CIITA). Transactivation by CIITA requires its nuclear import. This study will address a mechanistic role for the leucine-rich repeats (LRR) of CIITA in regulating nuclear translocation by mutating 12 individual consensus-motif "leucine" residues in both its alpha-motifs and beta-motifs. While some leucine mutations in the LRR motif of CIITA cause congruent loss of transactivation function and nuclear import, other alanine substitutions in both the alpha-helices and the beta-sheets have normal transactivation function but a loss of nuclear accumulation (i.e., functional mutants). This seeming paradox is resolved by the observations that nuclear accumulation of these functional mutants does occur but is significantly less than wild-type. This difference is revealed only in the presence of leptomycin B and actinomycin D, which permit examination of nuclear accumulation unencumbered by nuclear export and new CIITA synthesis. Further analysis of these mutants reveals that at limiting concentrations of CIITA, a dramatic difference in transactivation function between mutants and wild-type CIITA is easily detected, in agreement with their lowered nuclear accumulation. These experiments reveal an interesting aspect of LRR in controlling the amount of nuclear accumulation.