Growth-suppressor microRNAs mediate synaptic overgrowth and behavioral deficits in Fragile X mental retardation protein deficiency.

Abnormal neuronal and synapse growth is a core pathology resulting from deficiency of the Fragile X mental retardation protein (FMRP), but molecular links underlying the excessive synthesis of key synaptic proteins remain incompletely defined. We find that basal brain levels of the growth suppressor let-7 microRNA (miRNA) family are selectively lowered in FMRP-deficient mice and activity-dependent let-7 downregulation is abrogated. Primary let-7 miRNA transcripts are not altered in FMRP-deficiency and posttranscriptional misregulation occurs downstream of MAPK pathway induction and elevation of Lin28a, a let-7 biogenesis inhibitor. Neonatal restoration of brain let-7 miRNAs corrects hallmarks of FMRP-deficiency, including dendritic spine overgrowth and social and cognitive behavioral deficits, in adult mice. Blockade of MAPK hyperactivation normalizes let-7 miRNA levels in both brain and peripheral blood plasma from Fmr1 KO mice. These results implicate dysregulated let-7 miRNA biogenesis in the pathogenesis of FMRP-deficiency, and highlight let-7 miRNA-based strategies for future biomarker and therapeutic development.

Computational Analysis Tutorial for Chimeric Small Noncoding RNA: Target RNA Sequencing Libraries.

An understanding of the in vivo gene regulatory interactions of small noncoding RNAs (sncRNAs), such as microRNAs (miRNAs), with their target RNAs has been advanced in recent years by biochemical approaches which use cross-linking followed by ligation to capture sncRNA:target RNA interactions through the formation of chimeric RNAs and subsequent sequencing libraries. While datasets from chimeric RNA sequencing provide genome-wide and substantially less ambiguous input than miRNA prediction software, distilling this data into meaningful and actionable information requires additional analyses and may dissuade investigators lacking a computational background. This report provides a tutorial to support entry-level computational biologists in installing and applying a recent open-source software tool: Small Chimeric RNA Analysis Pipeline (SCRAP). Platform requirements, updates, and an explanation of pipeline steps and manipulation of key user-input variables is provided. Reducing a barrier for biologists to gain insights from chimeric RNA sequencing approaches has the potential to springboard discovery-based investigations of regulatory sncRNA:target RNA interactions in multiple biological contexts.

Regulation by noncoding RNAs of local translation, injury responses, and pain in the peripheral nervous system.

Neuropathic pain is a chronic condition arising from damage to somatosensory pathways that results in pathological hypersensitivity. Persistent pain can be viewed as a consequence of maladaptive plasticity which, like most enduring forms of cellular plasticity, requires altered expression of specific gene programs. Control of gene expression at the level of protein synthesis is broadly utilized to directly modulate changes in activity and responsiveness in nociceptive pathways and provides an effective mechanism for compartmentalized regulation of the proteome in peripheral nerves through local translation. Levels of noncoding RNAs (ncRNAs) are commonly impacted by peripheral nerve injury leading to persistent pain. NcRNAs exert spatiotemporal regulation of local proteomes and affect signaling cascades supporting altered sensory responses that contribute to hyperalgesia. This review discusses ncRNAs found in the peripheral nervous system (PNS) that are dysregulated following nerve injury and the current understanding of their roles in pathophysiological pain-related responses including neuroimmune interactions, neuronal survival and axon regeneration, Schwann cell dedifferentiation and proliferation, intercellular communication, and the generation of ectopic action potentials in primary afferents. We review progress in the field beyond cataloging, with a focus on the relevant target transcripts and mechanisms underlying pain modulation by ncRNAs.

SCRAP: a bioinformatic pipeline for the analysis of small chimeric RNA-seq data.

MicroRNAs (miRNAs) are small non-coding RNAs (sncRNAs) that function in post-transcriptional gene regulation through imperfect base pairing with mRNA targets which results in inhibition of translation and typically destabilization of bound transcripts. Sequence-based algorithms historically used to predict miRNA targets face inherent challenges in reliably reflecting in vivo interactions. Recent strategies have directly profiled miRNA-target interactions by crosslinking and ligation of sncRNAs to their targets within the RNA-induced silencing complex (RISC), followed by high throughput sequencing of the chimeric sncRNA:target RNAs. Despite the strength of these direct profiling approaches, standardized pipelines for effectively analyzing the resulting chimeric sncRNA:target RNA sequencing data are not readily available. Here we present SCRAP, a robust Small Chimeric RNA Analysis Pipeline for the bioinformatic processing of chimeric sncRNA:target RNA sequencing data. SCRAP consists of two parts, each of which are specifically optimized for the distinctive characteristics of chimeric small RNA sequencing reads: first, read processing and alignment and second, peak calling and annotation. We apply SCRAP to benchmark chimeric sncRNA:target RNA sequencing datasets generated by distinct molecular approaches, and compare SCRAP to existing chimeric RNA analysis pipelines. SCRAP has minimal hardware requirements, is cross-platform, and contains extensive annotation to broaden accessibility for processing small chimeric RNA sequencing data and enable insights about the targets of small non-coding RNAs in regulating diverse biological systems.

Combined single-molecule fluorescence in situ hybridization and immunohistochemistry analysis in intact murine dorsal root ganglia and sciatic nerve.

Single-molecule fluorescence in situ hybridization (smFISH) allows spatial mapping of gene expression. This protocol presents advances in smFISH fidelity and flexibility in intact murine sensory nervous system tissue. An approach using RNAscope probes allows multiplexing, enhanced target specificity, and immunohistochemistry compatibility. Computational strategies increase quantification accuracy of mRNA puncta with a point spread function for clustered transcripts in the dorsal root ganglion and 3D masking for intermingled sciatic nerve cell types. Approaches are validated for mRNAs of modest (Lin28a) and medium (Ppib) steady-state abundance in neurons.

Multi-Level Regulatory Interactions between NF-κB and the Pluripotency Factor Lin28.

An appreciation for the complex interactions between the NF-κB transcription factor and the Lin28 RNA binding protein/let-7 microRNA pathways has grown substantially over the past decade. Both the NF-κB and Lin28/let-7 pathways are master regulators impacting cell survival, growth and proliferation, and an understanding of how interfaces between these pathways participate in governing pluripotency, progenitor differentiation, and neuroplastic responses remains an emerging area of research. In this review, we provide a concise summary of the respective pathways and focus on the function of signaling interactions at both the transcriptional and post-transcriptional levels. Regulatory loops capable of providing both reinforcing and extinguishing feedback have been described. We highlight convergent findings in disparate biological systems and indicate future directions for investigation.

Cellular Specificity of NF-κB Function in the Nervous System.

Nuclear Factor Kappa B (NF-κB) is a ubiquitously expressed transcription factor with key functions in a wide array of biological systems. While the role of NF-κB in processes, such as host immunity and oncogenesis has been more clearly defined, an understanding of the basic functions of NF-κB in the nervous system has lagged behind. The vast cell-type heterogeneity within the central nervous system (CNS) and the interplay between cell-type specific roles of NF-κB contributes to the complexity of understanding NF-κB functions in the brain. In this review, we will focus on the emerging understanding of cell-autonomous regulation of NF-κB signaling as well as the non-cell-autonomous functional impacts of NF-κB activation in the mammalian nervous system. We will focus on recent work which is unlocking the pleiotropic roles of NF-κB in neurons and glial cells (including astrocytes and microglia). Normal physiology as well as disorders of the CNS in which NF-κB signaling has been implicated will be discussed with reference to the lens of cell-type specific responses.

A biosensor for MAPK-dependent Lin28 signaling.

Intracellular levels of the RNA-binding protein and pluripotency factor, Lin28a, are tightly controlled to govern cellular and organismal growth. Lin28a is extensively regulated at the posttranscriptional level, and can undergo mitogen-activated protein kinase (MAPK)-mediated elevation from low basal levels in differentiated cells by phosphorylation-dependent stabilizing interaction with the RNA-silencing factor HIV TAR RNA-binding protein (TRBP). However, molecular and spatiotemporal details of this critical control mechanism remained unknown. In this work, we dissect the interacting regions of Lin28a and TRBP proteins and develop biosensors to visualize this interaction. We identify truncated domains of Lin28a and of TRBP that are sufficient to support coassociation and mutual elevation of protein levels, and a requirement for MAPK-dependent phosphorylation of TRBP at putative Erk-target serine 152, as well as Lin28a serine 200 phosphorylation, in mediating the increase of Lin28a protein by TRBP. The phosphorylation-dependent association of Lin28a and TRBP truncated constructs is leveraged to develop fluorescence resonance energy transfer (FRET)-based sensors for dynamic monitoring of Lin28a and TRBP interaction. We demonstrate the response of bimolecular and unimolecular FRET sensors to growth factor stimulation in living cells, with coimaging of Erk activation to achieve further understanding of the role of MAPK signaling in Lin28a regulation.

Targeting of NF-κB to Dendritic Spines Is Required for Synaptic Signaling and Spine Development.

Long-term forms of brain plasticity share a requirement for changes in gene expression induced by neuronal activity. Mechanisms that determine how the distinct and overlapping functions of multiple activity-responsive transcription factors, including nuclear factor κB (NF-κB), give rise to stimulus-appropriate neuronal responses remain unclear. We report that the p65/RelA subunit of NF-κB confers subcellular enrichment at neuronal dendritic spines and engineer a p65 mutant that lacks spine enrichment (p65ΔSE) but retains inherent transcriptional activity equivalent to wild-type p65. Wild-type p65 or p65ΔSE both rescue NF-κB-dependent gene expression in p65-deficient murine hippocampal neurons responding to diffuse (PMA/ionomycin) stimulation. In contrast, neurons lacking spine-enriched NF-κB are selectively impaired in NF-κB-dependent gene expression induced by elevated excitatory synaptic stimulation (bicuculline or glycine). We used the setting of excitatory synaptic activity during development that produces NF-κB-dependent growth of dendritic spines to test physiological function of spine-enriched NF-κB in an activity-dependent response. Expression of wild-type p65, but not p65ΔSE, is capable of rescuing spine density to normal levels in p65-deficient pyramidal neurons. Collectively, these data reveal that spatial localization in dendritic spines contributes unique capacities to the NF-κB transcription factor in synaptic activity-dependent responses.SIGNIFICANCE STATEMENT Extensive research has established a model in which the regulation of neuronal gene expression enables enduring forms of plasticity and learning. However, mechanisms imparting stimulus specificity to gene regulation, ensuring biologically appropriate responses, remain incompletely understood. NF-κB is a potent transcription factor with evolutionarily conserved functions in learning and the growth of excitatory synaptic contacts. Neuronal NF-κB is localized in both synapse and somatic compartments, but whether the synaptic pool of NF-κB has discrete functions is unknown. This study reveals that NF-κB enriched in dendritic spines (the postsynaptic sites of excitatory contacts) is selectively required for NF-κB activation by synaptic stimulation and normal dendritic spine development. These results support spatial localization at synapses as a key variable mediating selective stimulus-response coupling.

A Rapid Induction Mechanism for Lin28a in Trophic Responses.

Environmental cues provoke rapid transitions in gene expression to support growth and cellular plasticity through incompletely understood mechanisms. Lin28 RNA-binding proteins have evolutionarily conserved roles in post-transcriptional coordination of pro-growth gene expression, but signaling pathways allowing trophic stimuli to induce Lin28 have remained uncharacterized. We find that Lin28a protein exhibits rapid basal turnover in neurons and that mitogen-activated protein kinase (MAPK)-dependent phosphorylation of the RNA-silencing factor HIV TAR-RNA-binding protein (TRBP) promotes binding and stabilization of Lin28a, but not Lin28b, with an accompanying reduction in Lin28-regulated miRNAs, downstream of brain-derived neurotrophic factor (BDNF). Binding of Lin28a to TRBP in vitro is also enhanced by phospho-mimic TRBP. Further, phospho-TRBP recapitulates BDNF-induced neuronal dendritic spine growth in a Lin28a-dependent manner. Finally, we demonstrate MAPK-dependent TRBP and Lin28a induction, with physiological function in growth and survival, downstream of diverse growth factors in multiple primary cell types, supporting a broad role for this pathway in trophic responses.

Characterizing autism spectrum disorders by key biochemical pathways.

The genetic and phenotypic heterogeneity of autism spectrum disorders (ASD) presents a substantial challenge for diagnosis, classification, research, and treatment. Investigations into the underlying molecular etiology of ASD have often yielded mixed and at times opposing findings. Defining the molecular and biochemical underpinnings of heterogeneity in ASD is crucial to our understanding of the pathophysiological development of the disorder, and has the potential to assist in diagnosis and the rational design of clinical trials. In this review, we propose that genetically diverse forms of ASD may be usefully parsed into entities resulting from converse patterns of growth regulation at the molecular level, which lead to the correlates of general synaptic and neural overgrowth or undergrowth. Abnormal brain growth during development is a characteristic feature that has been observed both in children with autism and in mouse models of autism. We review evidence from syndromic and non-syndromic ASD to suggest that entities currently classified as autism may fundamentally differ by underlying pro- or anti-growth abnormalities in key biochemical pathways, giving rise to either excessive or reduced synaptic connectivity in affected brain regions. We posit that this classification strategy has the potential not only to aid research efforts, but also to ultimately facilitate early diagnosis and direct appropriate therapeutic interventions.

IKK kinase assay for assessment of canonical NF-κB activation in neurons.

Nuclear factor kappa B (NF-κB) is a potent transcription factor highly expressed in the central nervous system (CNS) where it has been shown to be required for multiple behavioral paradigms of learning and memory in both mammalian and invertebrate systems. NF-κB dimers are found in neuronal cell bodies, are also present at synapses, and can participate in the activity-dependent regulation of gene expression in response to excitatory neurotransmission. Multiple serine-directed phosphorylation events are critical in the canonical NF-κB activation pathway, including activation of the IκB kinase complex (IKK) and phosphorylation and degradation of the inhibitor of NF-κB (IκB). In this chapter, we describe methods for immunoprecipitation (IP) of the IKK complex from dissociated cultured murine hippocampal neurons, followed by in vitro kinase assay to evaluate excitatory neurotransmission-induced IKK activation by monitoring phosphorylation of a GST-IκBα substrate. These methods can also be successfully implemented in subcellular-reduced brain preparations, such as biochemically isolated synapses.

Transcript specificity in BDNF-regulated protein synthesis.

Brain-derived neurotrophic factor (BDNF) is a critical activity-dependent modulator of gene expression, which can regulate both transcription and translation. Several functions of BDNF, including the induction of dendrite outgrowth and long-term synaptic plasticity, are known to depend, in particular, upon the ability of BDNF to regulate protein synthesis. Although BDNF modestly increases total neuronal protein synthesis, substantial evidence indicates that BDNF induces the translation of only a small subset of expressed mRNAs and demonstrates an extraordinary degree of transcript specificity. The mechanism by which BDNF selectively upregulates the translation of only a discrete group of mRNAs is of intrinsic importance to its trophic function in promoting neuronal growth and plasticity, and is the focus of this review. This article is part of the Special Issue entitled 'BDNF Regulation of Synaptic Structure, Function, and Plasticity'.

Opposing action of nuclear factor κB and Polo-like kinases determines a homeostatic end point for excitatory synaptic adaptation.

Homeostatic responses critically adjust synaptic strengths to maintain stability in neuronal networks. Compensatory adaptations to prolonged excitation include induction of Polo-like kinases (Plks) and degradation of spine-associated Rap GTPase-activating protein (SPAR) to reduce synaptic excitation, but mechanisms that limit overshooting and allow refinement of homeostatic adjustments remain poorly understood. We report that Plks produce canonical pathway-mediated activation of the nuclear factor κB (NF-κB) transcription factor in a process that requires the kinase activity of Plks. Chronic elevated activity, which induces Plk expression, also produces Plk-dependent activation of NF-κB. Deficiency of NF-κB, in the context of exogenous Plk2 expression or chronic elevated neuronal excitation, produces exaggerated homeostatic reductions in the size and density of dendritic spines, synaptic AMPA glutamate receptor levels, and excitatory synaptic currents. During the homeostatic response to chronic elevated activity, NF-κB activation by Plks subsequently opposes Plk-mediated SPAR degradation by transcriptionally upregulating SPAR in mouse hippocampal neurons in vitro and in vivo. Exogenous SPAR expression can rescue the overshooting of homeostatic reductions at excitatory synapses in NF-κB-deficient neurons responding to elevated activity. Our data establish an integral feedback loop involving NF-κB, Plks, and SPAR that regulates the end point of homeostatic synaptic adaptation to elevated activity and are the first to implicate a transcription factor in the regulation of homeostatic synaptic responses.

Dual regulation of miRNA biogenesis generates target specificity in neurotrophin-induced protein synthesis.

Control of translation is a fundamental source of regulation in gene expression. The induction of protein synthesis by brain-derived neurotrophic factor (BDNF) critically contributes to enduring modifications of synaptic function, but how BDNF selectively affects only a minority of expressed mRNAs is poorly understood. We report that BDNF rapidly elevates Dicer, increasing mature miRNA levels and inducing RNA processing bodies in neurons. BDNF also rapidly induces Lin28, causing selective loss of Lin28-regulated miRNAs and a corresponding upregulation in translation of their target mRNAs. Binding sites for Lin28-regulated miRNAs are necessary and sufficient to confer BDNF responsiveness to a transcript. Lin28 deficiency, or expression of a Lin28-resistant Let-7 precursor miRNA, inhibits BDNF translation specificity and BDNF-dependent dendrite arborization. Our data establish that specificity in BDNF-regulated translation depends upon a two-part posttranscriptional control of miRNA biogenesis that generally enhances mRNA repression in association with GW182 while selectively derepressing and increasing translation of specific mRNAs.

A requirement for nuclear factor-kappaB in developmental and plasticity-associated synaptogenesis.

Structural plasticity of dendritic spines and synapses is a fundamental mechanism governing neuronal circuits and may form an enduring basis for information storage in the brain. We find that the p65 subunit of the nuclear factor-κB (NF-κB) transcription factor, which is required for learning and memory, controls excitatory synapse and dendritic spine formation and morphology in murine hippocampal neurons. Endogenous NF-κB activity is elevated by excitatory transmission during periods of rapid spine and synapse development. During in vitro synaptogenesis, NF-κB enhances dendritic spine and excitatory synapse density and loss of endogenous p65 decreases spine density and spine head volume. Cell-autonomous function of NF-κB within the postsynaptic neuron is sufficient to regulate the formation of both presynaptic and postsynaptic elements. During synapse development in vivo, loss of NF-κB similarly reduces spine density and also diminishes the amplitude of synaptic responses. In contrast, after developmental synaptogenesis has plateaued, endogenous NF-κB activity is low and p65 deficiency no longer attenuates basal spine density. Instead, NF-κB in mature neurons is activated by stimuli that induce demand for new synapses, including estrogen and short-term bicuculline, and is essential for upregulating spine density in response to these stimuli. p65 is enriched in dendritic spines making local protein-protein interactions possible; however, the effects of NF-κB on spine density require transcription and the NF-κB-dependent regulation of PSD-95, a critical postsynaptic component. Collectively, our data define a distinct role for NF-κB in imparting transcriptional regulation required for the induction of changes to, but not maintenance of, excitatory synapse and spine density.

Stimulated nuclear translocation of NF-kappaB and shuttling differentially depend on dynein and the dynactin complex.

Translocation from the cytoplasm to the nucleus is required for the regulation of gene expression by transcription factors of the nuclear factor kappa B (NF-kappaB) family. The p65:p50 NF-kappaB heterodimer that predominates in many cell types can undergo stimulated movement, following degradation of the IkappaB inhibitor, as well as shuttling in the absence of stimulation with IkappaB bound. Disruption of the dynactin complex and knockdown of endogenous dynein were used to investigate the nuclear translocation requirements for stimulated and shuttling movement of NF-kappaB. A differential dependence of these two modes of transport on the dynein molecular motor and dynactin was found. NF-kappaB used active dynein-dependent transport following stimulation while translocation during shuttling was mediated by a dynein-independent pathway that could be potentiated by dynactin disruption, consistent with a process of facilitated diffusion. Nuclear translocation and activation of NF-kappaB-dependent gene expression showed a dependence on endogenous dynein in a variety of cell types and in response to diverse activating stimuli, suggesting that dynein-dependent transport of NF-kappaB may be a conserved mechanism in the NF-kappaB activation pathway and could represent a potential point of regulation.

Novel roles for the NF-kappaB signaling pathway in regulating neuronal function.

Two new reports offer exciting evidence of novel roles for components of the nuclear factor kappaB (NF-kappaB) pathway in the nervous system. Transcriptional activation by NF-kappaB and chromatin remodeling by inhibitor of kappaB (IkappaB) kinase complex (IKK) have been linked to recall and reconsolidation of conditioned fear memories in the mammalian central nervous system. In the Drosophila neuromuscular junction, a member of the NF-kappaB family has been reported to regulate glutamate receptor clustering. Both reports could have important implications for the function of the NF-kappaB signaling pathway in neuronal plasticity.

Physiological functions for brain NF-kappaB.

Members of the nuclear factor kappaB (NF-kappaB) family of transcription factors are activated within the CNS in pathological settings of apoptosis and neurological disease. Recent work using several model systems provides accumulating evidence that these transcription factors also participate in the regulation of neuronal activity-dependent transcription and behavior under physiological conditions. This review highlights advances in our understanding of the mechanisms of Ca(2+)-responsive activation and synaptic signaling to the nucleus by NF-kappaB transcription factors within the CNS, and the relevance of this transcription factor family for learning and memory.