Monocyte chemoattractant protein-1 functions as a neuromodulator in dorsal root ganglia neurons

It has previously been observed that expression of chemokine monocyte chemoattractant protein-1 (MCP-1/CC chemokine ligand 2 (CCL2)) and its receptor CC chemokine receptor 2 (CCR2) is up-regulated by dorsal root ganglion (DRG) neurons in association with rodent models of neuropathic pain. MCP-1 increases the excitability of nociceptive neurons after a peripheral nerve injury, while disruption of MCP-1/CCR2 signaling blocks the development of neuropathic pain, suggesting MCP-1 signaling is responsible for heightened pain sensitivity. To define the mechanisms of MCP-1 signaling in DRG, we studied intracellular processing, release, and receptor-mediated signaling of MCP-1 in DRG neurons. We found that in a focal demyelination model of neuropathic pain both MCP-1 and CCR2 were up-regulated by the same neurons including transient receptor potential vanilloid receptor subtype 1 (TRPV1) expressing nociceptors. MCP-1 expressed by DRG neurons was packaged into large dense-core vesicles whose release could be induced from the soma by depolarization in a Ca2+-dependent manner. Activation of CCR2 by MCP-1 could sensitize nociceptors via transactivation of transient receptor potential channels. Our results suggest that MCP-1 and CCR2, up-regulated by sensory neurons following peripheral nerve injury, might participate in neural signal processing which contributes to sustained excitability of primary afferent neurons.ope

Local protein synthesis in neuronal axons: why and how we study

Adaptive brain function and synaptic plasticity rely on dynamic regulation of local proteome. One way for the neuron to introduce new proteins to the axon terminal is to transport those from the cell body, which had long been thought as the only source of axonal proteins. Another way, which is the topic of this review, is synthesizing proteins on site by local mRNA translation. Recent evidence indicates that the axon stores a reservoir of translationally silent mRNAs and regulates their expression solely by translational control. Different stimuli to axons, such as guidance cues, growth factors, and nerve injury, promote translation of selective mRNAs, a process required for the axon's ability to respond to these cues. One of the critical questions in the field of axonal protein synthesis is how mRNA-specific local translation is regulated by extracellular cues. Here, we review current experimental techniques that can be used to answer this question. Furthermore, we discuss how new technologies can help us understand what biological processes are regulated by axonal protein synthesis in vivo.ope

Excision of the first intron from the gonadotropin-releasing hormone (GnRH) transcript serves as a key regulatory step for GnRH biosynthesis

The mammalian gonadotropin-releasing hormone (GnRH) gene consists of four short exons (denoted as 1, 2, 3, and 4) and three intervening introns (A, B, and C). Recently, we demonstrated that excision of the first intron (intron A) from the GnRH transcript is regulated in a tissue- and developmental stage-specific fashion and is severely attenuated in hypogonadal (hpg) mouse because of its lack of exonic splicing enhancers (ESE) 3 and 4. In the present study, we examined the influence of intron A on translational efficiency, thereby establishing a post-transcriptional control over GnRH biosynthesis. First, we verified that an intron A-retained GnRH transcript is a splicing variant but not a splicing intermediate. Intron A-retained transcripts can be transported to the cytoplasm in contrast to intron B-containing transcripts, which are restricted to the nucleus. This result implicates the intron A-retained GnRH transcript as a splicing variant; it has a long 5'-untranslated region, as the GnRH prohormone open reading frame (ORF) begins on exon 2. We investigated whether an intron A-retained GnRH transcript can properly initiate translation at the appropriate start codon and found that intron A completely blocks the translation initiation of its downstream reporter ORF both in vivo and in vitro. The inhibition of translation initiation appears to be due to the presence of a tandem repeat of ATG sequences within intron A. Constructs bearing mutations of ATGs to AAGs restored translation initiation at the downstream start codon; the extent of this restoration correlated with the number of mutated ATGs. Besides the failure in the translation initiation of GnRH-coding region in the intron A-containing variant, the present study also suggests that the interference between mature GnRH mRNA and intron A-retained splicing variant could occur to lower the efficiency of GnRH biosynthesis in the GT1-1-immortalized GnRH-producing cell line. Therefore, our results indicate that the precise and efficient excision of intron A and the joining of adjacent exons may be a critical regulatory step for the post-transcriptional regulation of GnRH biosynthesis.ope

The chemokine stromal cell-derived factor-1 regulates GABAergic inputs to neural progenitors in the postnatal dentate gyrus

Stromal cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) are important regulators of the development of the dentate gyrus (DG). Both SDF-1 and CXCR4 are also highly expressed in the adult DG. We observed that CXCR4 receptors were expressed by dividing neural progenitor cells located in the subgranular zone (SGZ) as well as their derivatives including doublecortin-expressing neuroblasts and immature granule cells. SDF-1 was located in DG neurons and in endothelial cells associated with DG blood vessels. SDF-1-expressing neurons included parvalbumin-containing GABAergic interneurons known as basket cells. Using transgenic mice expressing an SDF-1-mRFP1 (monomeric red fluorescence protein 1) fusion protein we observed that SDF-1 was localized in synaptic vesicles in the terminals of basket cells together with GABA-containing vesicles. These terminals were often observed to be in close proximity to dividing nestin-expressing neural progenitors in the SGZ. Electrophysiological recordings from slices of the DG demonstrated that neural progenitors received both tonic and phasic GABAergic inputs and that SDF-1 enhanced GABAergic transmission, probably by a postsynaptic mechanism. We also demonstrated that, like GABA, SDF-1 was tonically released in the DG and that GABAergic transmission was partially dependent on coreleased SDF-1. These data demonstrate that SDF-1 plays a novel role as a neurotransmitter in the DG and regulates the strength of GABAergic inputs to the pool of dividing neural progenitors. Hence, SDF-1/CXCR4 signaling is likely to be an important regulator of adult neurogenesis in the DG.ope

Visualization of chemokine receptor activation in transgenic mice reveals peripheral activation of CCR2 receptors in states of neuropathic pain.

CCR2 chemokine receptor signaling has been implicated in the generation of diverse types of neuropathology, including neuropathic pain. For example, ccr2 knock-out mice are resistant to the establishment of neuropathic pain, and mice overexpressing its ligand, monocyte chemoattractant protein-1 (MCP1; also known as CCL2), show enhanced pain sensitivity. However, whether CCR2 receptor activation occurs in the central or peripheral nervous system in states of neuropathic pain has not been clear. We developed a novel method for visualizing CCR2 receptor activation in vivo by generating bitransgenic reporter mice in which the chemokine receptor CCR2 and its ligand MCP1 were labeled by the fluorescent proteins enhanced green fluorescent protein and monomeric red fluorescent protein-1, respectively. CCR2 receptor activation under conditions such as acute inflammation and experimental autoimmune encephalomyelitis could be faithfully visualized by using these mice. We examined the status of CCR2 receptor activation in a demyelination injury model of neuropathic pain and found that MCP1-induced CCR2 receptor activation mainly occurred in the peripheral nervous system, including the injured peripheral nerve and dorsal root ganglia. These data explain the rapid antinociceptive effects of peripherally administered CCR2 antagonists under these circumstances, suggesting that CCR2 antagonists may ameliorate pain by inhibiting CCR2 receptor activation in the periphery. The method developed here for visualizing CCR2 receptor activation in vivo may be extended to G-protein-coupled receptors (GPCRs) in general and will be valuable for studying intercellular GPCR-mediated communication in vivo.ope

Local mRNA translation in long-term maintenance of axon health and function

Distal axons, remote from their cell bodies and nuclei, must survive the lifetime of an organism. Recent studies have provided compelling evidence that proteins are locally synthesized in healthy, mature central nervous system axons and presynaptic terminals in vivo. Presynaptic, mitochondrial and ribosomal proteins are locally synthesized in most adult axons of diverse cell types, linking local translation to axon function and survival. Accordingly, inhibiting the intra-axonal translation of key mRNAs or the function of their translational regulators causes dying-back axon degeneration, and human mutations in RNA metabolic pathways are increasingly being associated with neurodegenerative diseases that accompany axon degeneration. Here, we summarize recent relevant findings in a highly simplified 'RNA operon'-based model and discuss open questions and future directions.ope

Altered neurotransmitter release machinery in mice deficient for the deubiquitinating enzyme Usp14

Homozygous ataxic mice (ax(J)) express reduced levels of the deubiquitinating enzyme Usp14. They develop severe tremors by 2-3 wk of age, followed by hindlimb paralysis, and death by 6-8 wk. While changes in the ubiquitin proteasome system often result in the accumulation of ubiquitin protein aggregates and neuronal loss, these pathological markers are not observed in the ax(J) mice. Instead, defects in neurotransmission were observed in both the central and peripheral nervous systems of ax(J) mice. We have now identified several new alterations in peripheral neurotransmission in the ax(J) mice. Using the two-microelectrode voltage clamp technique on diaphragm muscles of ax(J) mice, we observed that under normal neurotransmitter release conditions ax(J) mice lacked paired-pulse facilitation and exhibited a frequency-dependent increase in rundown of the end plate current at high-frequency stimulation (HFS). Combined electrophysiology and styryl dye staining revealed a significant reduction in quantal content during the initial and plateau portions of the HFS train. In addition, uptake of styryl dyes (FM dye) during HFS demonstrated that the size of the readily releasable vesicle pool was significantly reduced. Destaining rates for styryl dyes suggested that ax(J) neuromuscular junctions are unable to mobilize a sufficient number of vesicles during times of intense activity. These results imply that ax(J) nerve terminals are unable to recruit a sufficient number of vesicles to keep pace with physiological rates of transmitter release. Therefore, ubiquitination of synaptic proteins appears to play an important role in the normal operation of the neurotransmitter release machinery and in regulating the size of pools of synaptic vesicles.ope

Translational regulation in growth cones.

Axonal growth cones (GCs) steer in response to extrinsic cues using mechanisms that include local protein synthesis. This adaptive form of gene regulation occurs with spatial precision and depends on subcellular mRNA localisation. Recent genome-wide studies have shown unexpectedly complex and dynamically changing mRNA repertoires in growing axons and GCs. Axonal targeting of some transcripts seems to be highly selective and involves sequence diversity in non-coding regions generated by transcriptional and/or post-transcriptional mechanisms. New evidence reports direct coupling of a guidance receptor to the protein synthesis machinery and other findings demonstrate that some guidance cues can repress translation. The recent findings shed further light on the exquisitely regulated process that enables distant cellular compartments to respond to local stimuli.ope

Axonal mRNA localization and local protein synthesis in nervous system assembly, maintenance and repair

mRNAs can be targeted to specific neuronal subcellular domains, which enables rapid changes in the local proteome through local translation. This mRNA-based mechanism links extrinsic signals to spatially restricted cellular responses and can mediate stimulus-driven adaptive responses such as dendritic plasticity. Local mRNA translation also occurs in growing axons where it can mediate directional responses to guidance signals. Recent profiling studies have revealed that both growing and mature axons possess surprisingly complex and dynamic transcriptomes, thereby suggesting that axonal mRNA localization is highly regulated and has a role in a broad range of processes, a view that is increasingly being supported by new experimental evidence. Here, we review current knowledge on the roles and regulatory mechanisms of axonal mRNA translation and discuss emerging links to axon guidance, survival, regeneration and neurological disorders.ope

Local translation of mRNAs in neural development.

Growing axons encounter numerous developmental signals to which they must promptly respond in order to properly form complex neural circuitry. In the axons, these signals are often transduced into a local increase or decrease in protein levels. Contrary to the traditional view that the cell bodies are the exclusive source of axonal proteins, it is becoming increasingly clear not only that de novo protein synthesis takes place in axons, but also that it is required for the axons to respond to certain signals. Here we review the current knowledge of local mRNA translation in developing neurons with a special focus on protein synthesis occurring in axons and growth cones.ope