Agonists acting at alpha(2)-adrenergic and opioid receptors (alpha(2)ARs and ORs, respectively) inhibit pain transmission in the spinal cord. When coadministered, agonists activating these receptors interact in a synergistic manner. Although the existence of alpha(2)AR/OR synergy has been well characterized, its mechanism remains poorly understood. The formation of heterooligomers has been proposed as a molecular basis for interactions between neuronal G-protein-coupled receptors. The relevance of heterooligomer formation to spinal analgesic synergy requires demonstration of the expression of both receptors within the same neuron as well as the localization of both receptors in the same neuronal compartment. We used immunohistochemistry to investigate the spatial relationship between alpha(2)ARs and ORs in the rat spinal cord to determine whether coexpression could be demonstrated between these receptors. We observed extensive colocalization between alpha(2A)-adrenergic and delta-opioid receptors (DOP) on substance P (SP)-immunoreactive (-ir) varicosities in the superficial dorsal horn of the spinal cord and in peripheral nerve terminals in the skin. alpha(2A)AR- and DOP-ir elements were colocalized in subcellular structures of 0.5 mum or less in diameter in isolated nerve terminals. Furthermore, coincubation of isolated synaptosomes with alpha(2)AR and DOP agonists resulted in a greater-than-additive increase in the inhibition of K(+)-stimulated neuropeptide release. These findings suggest that coexpression of the synergistic receptor pair alpha(2A)AR-DOP on primary afferent nociceptive fibers may represent an anatomical substrate for analgesic synergy, perhaps as a result of protein-protein interactions such as heterooligomerization.

Oro-facial pain following injury and infection is frequently observed in dental clinics. While neuropathic pain evoked by injury associated with nerve lesion has an involvement of glia/immune cells, inflammatory hyperalgesia has an exaggerated sensitization mediated by local and circulating immune mediators. To better understand the contribution of central nervous system (CNS) glial cells in these different pathological conditions, in this study we sought to characterize functional phenotypes of glial cells in response to trigeminal nerve injury (loose ligation of the mental branch), infection (subcutaneous injection of lipopolysaccharide--LPS) and to sterile inflammation (subcutaneous injection of complete Freund's adjuvant--CFA) on the lower lip. Each of the three insults triggered a specific pattern of mechanical allodynia. In parallel with changes in sensory response, CNS glial cells reacted distinctively to the challenges. Following ligation of the mental nerve, both microglia and astrocytes in the trigeminal nuclear complex were highly activated, more prominent in the principal sensory nucleus (Pr5) and subnucleus caudalis (Sp5C) area. Microglial response was initiated early (days 3-14), followed by delayed astrocytes activation (days 7-28). Although the temporal profile of microglial and astrocyte reaction corresponded respectively to the initiation and chronic stage of neuropathic pain, these activated glial cells exhibited a low profile of cytokine expression. Local injection of LPS in the lower lip skin also triggered a microglial reaction in the brain, which started in the circumventricular organs (CVOs) at 5 hours post-injection and diffused progressively into the brain parenchyma at 48 hours. This LPS-induced microglial reaction was accompanied by a robust induction of IκB-α mRNA and pro-inflammatory cytokines within the CVOs. However, LPS induced microglial activation did not specifically occur along the pain signaling pathway. In contrast, CFA injection led to minor microglial morphological changes and an induction of IκB-α mRNA in the CVO regions; a significant increase in IL-1β and IL-6 mRNA started only at 48 hours post-injection, when the induced pain-related behavior started to resolve. Our detailed analysis of CNS glial response clearly revealed that both nerve injury and oro-facial infection/inflammation induced CNS glial activation, but in a completely different pattern, which suggests a remarkable plasticity of glial cells in response to dynamic changes in their microenvironment and different potential involvement of this non-neuronal cell population in pathological pain development.

The lesion bypass pathway, which is regulated by monoubiquitination of proliferating cell nuclear antigen (PCNA), is essential for resolving replication stalling due to DNA lesions. This process is important for preventing genomic instability and cancer development. Previously, it was shown that cells deficient in tumour suppressor p33ING1 (ING1b) are hypersensitive to DNA damaging agents via unknown mechanism. In this study, we demonstrated a novel tumour suppressive function of ING1b in preserving genomic stability upon replication stress through regulating PCNA monoubiquitination. We found that ING1b knockdown cells are more sensitive to UV due to defects in recovering from UV-induced replication blockage, leading to enhanced genomic instability. We revealed that ING1b is required for the E3 ligase Rad18-mediated PCNA monoubiquitination in lesion bypass. Interestingly, ING1b-mediated PCNA monoubiquitination is associated with the regulation of histone H4 acetylation. Results indicate that chromatin remodelling contributes to the stabilization of stalled replication fork and to the regulation of PCNA monoubiquitination during lesion bypass.

Sex differences in humans on virtual water maze navigation are well established when overall performance is measured, e.g., by the total time taken to find the hidden platform, total path length, or quadrant dwell time during probe trials. Currently, it is unknown whether males are better spatial learners than females, or if overall performance differences reflect other aspects of the task unrelated to spatial memory. Here, males and females were tested on a virtual analogue of the Morris water maze. We devised a novel method of analysis in which each trial was divided into an initial trajectory phase and search phase. We also implemented a new measure of spatial learning during early and late training, by including trials in which subjects were only required to indicate where they thought the hidden target zone was located. Consistent with previous reports, males outperformed females on overall measures of task performance. Males also performed significantly better on all initial trajectory phase variables. Interestingly, only small (non-significant) differences were observed during the search phase and when spatial learning was tested without the constraints of a typical water maze trial. Our results suggest that spatial knowledge regarding the location of the hidden target zone is not the main factor responsible for overall sex differences in virtual water maze performance. Instead, the largest sex differences were observed during the initial trajectory phase of the trial, which is thought to depend on effective processing of distal features of the environment.

Neuropeptide Y (NPY) has been implicated in the modulation of pain. Under normal conditions, NPY is found in interneurons in the dorsal horn of the spinal cord and in sympathetic postganglionic neurons but is absent from the cell bodies of sensory neurons. Following peripheral nerve injury NPY is dramatically upregulated in the sensory ganglia. How NPY expression is altered in the peripheral nervous system, distal to a site of nerve lesion, remains unknown. To address this question, NPY expression was investigated using immunohistochemistry at the level of the trigeminal ganglion, the mental nerve and in the skin of the lower lip in relation to markers of sensory and sympathetic fibers in a rat model of trigeminal neuropathic pain.

Fragile X syndrome (FXS) is the leading genetic cause of autism. Mutations in Fmr1 (fragile X mental retardation 1 gene) engender exaggerated translation resulting in dendritic spine dysmorphogenesis, synaptic plasticity alterations, and behavioral deficits in mice, which are reminiscent of FXS phenotypes. Using postmortem brains from FXS patients and Fmr1 knockout mice (Fmr1(-/y)), we show that phosphorylation of the mRNA 5' cap binding protein, eukaryotic initiation factor 4E (eIF4E), is elevated concomitant with increased expression of matrix metalloproteinase 9 (MMP-9) protein. Genetic or pharmacological reduction of eIF4E phosphorylation rescued core behavioral deficits, synaptic plasticity alterations, and dendritic spine morphology defects via reducing exaggerated translation of Mmp9 mRNA in Fmr1(-/y) mice, whereas MMP-9 overexpression produced several FXS-like phenotypes. These results uncover a mechanism of regulation of synaptic function by translational control of Mmp-9 in FXS, which opens the possibility of new treatment avenues for the diverse neurological and psychiatric aspects of FXS.

Activation of the mechanistic/mammalian target of rapamycin (mTOR) kinase in models of acute and chronic pain is strongly implicated in mediating enhanced translation and hyperalgesia. However, the molecular mechanisms by which mTOR regulates nociception remain unclear. Here we show that deletion of the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), a major mTOR downstream effector, which represses eIF4E activity and cap-dependent translation, leads to mechanical, but not thermal pain hypersensitivity. Mice lacking 4E-BP1 exhibit enhanced spinal cord expression of neuroligin 1, a cell-adhesion postsynaptic protein regulating excitatory synapse function, and show increased excitatory synaptic input into spinal neurons, and a lowered threshold for induction of synaptic potentiation. Pharmacological inhibition of eIF4E or genetic reduction of neuroligin 1 levels normalizes the increased excitatory synaptic activity and reverses mechanical hypersensitivity. Thus, translational control by 4E-BP1 downstream of mTOR effects the expression of neuroligin 1 and excitatory synaptic transmission in the spinal cord, and thereby contributes to enhanced mechanical nociception.

Neuropathic pain is a chronic debilitating disease that results from nerve damage, persists long after the injury has subsided, and is characterized by spontaneous pain and mechanical hypersensitivity. Although loss of inhibitory tone in the dorsal horn of the spinal cord is a major contributor to neuropathic pain, the molecular and cellular mechanisms underlying this disinhibition are unclear. Here, we combined pharmacogenetic activation and selective ablation approaches in mice to define the contribution of spinal cord parvalbumin (PV)-expressing inhibitory interneurons in naive and neuropathic pain conditions. Ablating PV neurons in naive mice produce neuropathic pain-like mechanical allodynia via disinhibition of PKCγ excitatory interneurons. Conversely, activating PV neurons in nerve-injured mice alleviates mechanical hypersensitivity. These findings indicate that PV interneurons are modality-specific filters that gate mechanical but not thermal inputs to the dorsal horn and that increasing PV interneuron activity can ameliorate the mechanical hypersensitivity that develops following nerve injury.

Non-peptidergic nociceptive neurons are a sub-population of small diameter primary sensory neurons that comprise approximately 50 % of the C fiber population. Together with the peptidergic sub-population, they transmit nociceptive information from the periphery to the superficial dorsal horn of the spinal cord. Despite the numerous studies investigating the role of the non-peptidergic primary afferents, their role in normal nociception and in pain remains poorly understood. Our lab has previously demonstrated that, in rat models of neuropathic and inflammatory pain, there is a de novo expression of substance P receptors (NK-1r) by lamina I pyramidal projection neurons, a neuronal population that normally does not express these receptors.

Platelet-activating factor (PAF) is a pleiotropic phospholipid with proinflammatory, procoagulant and angiogenic actions on the vasculature. We and others have reported the presence of PAF receptor (Ptafr) at intracellular sites such as the nucleus. However, mechanisms of localization and physiologic functions of intracellular Ptafr remain poorly understood. We hereby identify the importance of C-terminal motif of the receptor and uncover novel roles of Rab11a GTPase and importin-5 in nuclear translocation of Ptafr in primary human retinal microvascular endothelial cells. Nuclear localization of Ptafr is independent of exogenous PAF stimulation as well as intracellular PAF biosynthesis. Moreover, nuclear Ptafr is responsible for the upregulation of unique set of growth factors, including vascular endothelial growth factor, in vitro and ex vivo. We further corroborate the intracrine PAF signaling, resulting in angiogenesis in vivo, using Ptafr antagonists with distinct plasma membrane permeability. Collectively, our findings show that nuclear Ptafr translocates in an agonist-independent manner, and distinctive functions of Ptafr based on its cellular localization point to another dimension needed for pharmacologic selectivity of drugs.

Pain-related (nociceptive) information is carried from the periphery to the dorsal horn of the spinal cord mostly by two populations of small diameter primary afferents, the peptidergic and the non-peptidergic. The peptidergic population expresses neuropeptides, such as substance P and calcitonin gene-related peptide, while the non-peptidergic fibers are devoid of neuropeptides, express the purinergic receptor P2X3, and bind the isolectin B4 (IB4). Although it has been known for some time that in rat the peptidergic afferents terminate mostly in lamina I and outer lamina II and non-peptidergic afferents in inner lamina II, the extent of the termination of the latter population in lamina I was never investigated as it was considered as very minor. Because our preliminary evidence suggested otherwise, we decided to re-examine the termination of non-peptidergic afferents in lamina I, in particular with regards to their innervation of projection neurons expressing substance P receptors (NK-1r). We used retrograde labeling of neurons from the parabrachial nucleus combined with lectin IB4 binding and immunocytochemistry. Samples were examined by confocal and electron microscopy.

Kisspeptin has been shown to stimulate prolactin secretion. We investigated whether kisspeptin acts through the Kiss1 receptor (Kiss1r) to regulate dopamine and prolactin. Initially, we evaluated prolactin response in a Kiss1r-deficient mouse line, in which Kiss1r had been knocked into GnRH neurons (Kiss1r-/-R). Intracerebroventricular kisspeptin-10 (Kp-10) increased prolactin release in wild-type but not in Kiss1r-/-R female mice. In ovariectomized, estradiol-treated rats, the Kiss1r antagonist kisspeptin-234 abolished the Kp-10-induced increase in prolactin release but failed to prevent the concomitant reduction in the activity of tuberoinfundibular dopaminergic (TIDA) neurons, as determined by the 3,4-dihydroxyphenylacetic acid/dopamine ratio in the median eminence. Using whole-cell patch clamp recordings in juvenile male rats, we found no direct effect of Kp-10 on the electrical activity of TIDA neurons. In addition, dual-label in situ hybridization in the hypothalamus of female rats showed that Kiss1r is expressed in the periventricular nucleus of the hypothalamus (Pe) and arcuate nucleus of the hypothalamus (ARC) but not in tyrosine hydroxylase (Th)-expressing neurons. Kisspeptin also has affinity for the neuropeptide FF receptor 1 (Npffr1), which was expressed in the majority of Pe dopaminergic neurons but only in a low proportion of TIDA neurons in the ARC. Our findings demonstrate that Kiss1r is necessary to the effect of kisspeptin on prolactin secretion, although TIDA neurons lack Kiss1r and are electrically unresponsive to kisspeptin. Thus, kisspeptin is likely to stimulate prolactin secretion via Kiss1r in nondopaminergic neurons, whereas the colocalization of Npffr1 and Th suggests that Pe dopaminergic neurons may play a role in the kisspeptin-induced inhibition of dopamine release.

The MAPK/ERK (mitogen-activated protein kinases/extracellular signal-regulated kinase) pathway is a cardinal regulator of synaptic plasticity, learning, and memory in the hippocampus. One of major endpoints of this signaling cascade is the 5' mRNA cap binding protein eIF4E (eukaryotic Initiation Factor 4E), which is phosphorylated on Ser 209 by MNK (MAPK-interacting protein kinases) and controls mRNA translation. The precise role of phospho-eIF4E in the brain is yet to be determined. Herein, we demonstrate that ablation of eIF4E phosphorylation in male mice (4Eki mice) does not impair long-term spatial or contextual fear memory, or the late phase of LTP. Using unbiased translational profiling in mouse brain, we show that phospho-eIF4E differentially regulates the translation of a subset of mRNAs linked to inflammation, the extracellular matrix, pituitary hormones, and the serotonin pathway. Consequently, 4Eki male mice display exaggerated inflammatory responses and reduced levels of serotonin, concomitant with depression and anxiety-like behaviors. Remarkably, eIF4E phosphorylation is required for the chronic antidepressant action of the selective serotonin reuptake inhibitor fluoxetine. Finally, we propose a novel phospho-eIF4E-dependent translational control mechanism in the brain, via the GAIT complex (gamma IFN activated inhibitor of translation). In summary, our work proposes a novel translational control mechanism involved in the regulation of inflammation and depression, which could be exploited to design novel therapeutics.SIGNIFICANCE STATEMENT We demonstrate that downstream of the MAPK (mitogen-activated protein kinase) pathway, eukaryotic Initiation Factor 4E (eIF4E) Ser209 phosphorylation is not required for classical forms of hippocampal LTP and memory. We reveal a novel role for eIF4E phosphorylation in inflammatory responses and depression-like behaviors. eIF4E phosphorylation is required for the chronic action of antidepressants, such as fluoxetine in mice. These phenotypes are accompanied by selective translation of extracellular matrix, pituitary hormones, and serotonin pathway genes, in eIF4E phospho-mutant mice. We also describe a previously unidentified translational control mechanism in the brain, whereby eIF4E phosphorylation is required for inhibiting the translation of gamma IFN activated inhibitor of translation element-containing mRNAs. These findings can be used to design novel therapeutics for depression.

The aim of this study was to develop a method to study genome-wide local translation in biochemically isolated synaptic fractions (synaptoneurosomes). This methodology is of particular interest for neurons, due to the cardinal role of local translational control in neuronal sub-compartments, such as dendrites, for plasticity, learning, memory, and for disorders of the nervous system.

Contextual fear conditioning (CFC) in rodents is the most widely used behavioural paradigm in neuroscience research to elucidate the neurobiological mechanisms underlying learning and memory. It is based on the pairing of an aversive unconditioned stimulus (US; e.g. mild footshock) with a neutral conditioned stimulus (CS; e.g. context of the test chamber) in order to acquire associative long-term memory (LTM), which persists for days and even months. Using genome-wide analysis, several studies have generated lists of genes modulated in response to CFC in an attempt to identify the "memory genes", which orchestrate memory formation. Yet, most studies use naïve animals as a baseline for assessing gene-expression changes, while only few studies have examined the effect of the US alone, without pairing to context, using genome-wide analysis of gene-expression. Herein, using the ribosome profiling methodology, we show that in male mice an immediate shock, which does not lead to LTM formation, elicits pervasive translational and transcriptional changes in the expression of Immediate Early Genes (IEGs) in dorsal hippocampus (such as Fos and Arc), a fact which has been disregarded by the majority of CFC studies. By removing the effect of the immediate shock, we identify and validate a new set of genes, which are translationally and transcriptionally responsive to the association of context-to-footshock in CFC, and thus constitute salient "memory genes".

Neurons in anterior cingulate cortex (aCC) project to dorsomedial striatum (DMS) as part of a corticostriatal circuit with putative roles in learning and other cognitive functions. In the present study, the spatial-cognitive importance of aCC and DMS was assessed in the hidden-platform version of the Morris water maze (MWM). Brain lesion experiments that focused on areas of connectivity between these regions indicated their involvement in spatial cognition. MWM learning curves were markedly delayed in DMS-lesioned mice in the absence of other major functional impairments, whereas there was a more subtle, but still significant influence of aCC lesions. Lesioned mice displayed impaired abilities to use spatial search strategies, increased thigmotaxic swimming, and decreased searching in the proximity of the escape platform. Additionally, aCC and DMS activity was compared in mice between the early acquisition phase (2 and 3 days of training) and the over-trained high-proficiency phase (after 30 days of training). Neuroplasticity-related expression of the immediate early gene Arc implicated both regions during the goal-directed, early phases of spatial learning. These results suggest the functional involvement of aCC and DMS in processes of spatial cognition that model associative cortex-dependent, human episodic memory abilities.

Mechanistic target of rapamycin complex 1 (mTORC1) controls cell growth and proliferation by sensing fluctuations in environmental cues such as nutrients, growth factors, and energy levels. The Rag GTPases (Rags) serve as a critical module that signals amino acid (AA) availability to modulate mTORC1 localization and activity. Recent studies have demonstrated how AAs regulate mTORC1 activity through Rags. Here, we uncover an unconventional pathway that activates mTORC1 in response to variations in threonine (Thr) levels via mitochondrial threonyl-tRNA synthetase TARS2. TARS2 interacts with inactive Rags, particularly GTP-RagC, leading to increased GTP loading of RagA. mTORC1 activity in cells lacking TARS2 is resistant to Thr repletion, showing that TARS2 is necessary for Thr-dependent mTORC1 activation. The requirement of TARS2, but not cytoplasmic threonyl-tRNA synthetase TARS, for this effect demonstrates an additional layer of complexity in the regulation of mTORC1 activity.

Tendons are specialized tissues composed primarily of load-responsive fibroblasts (tenocytes) embedded in a collagen-rich extracellular matrix. Habitual mechanical loading or targeted exercise causes tendon cells to increase the stiffness of the extracellular matrix; this adaptation may occur in part through collagen synthesis or remodeling. Integrins are likely to play an important role in transmitting mechanical stimuli from the extracellular matrix to tendon cells, thereby triggering cell signaling pathways which lead to adaptive regulation of mRNA translation and protein synthesis. In this study, we discovered that mechanical stimulation of integrin β1 leads to the phosphorylation of AKT, an event which required the presence of integrin-linked kinase (ILK). Repetitive stretching of tendon cells activates the AKT and mTOR pathways, which in turn regulates mRNA translation and collagen expression. These results support a model in which integrins are an upstream component of the mechanosensory cellular apparatus, regulating fundamental tendon cell functions relevant to exercise-induced adaptation and mechanotherapy.

Long-lasting cognitive impairment in juveniles undergoing repeated general anesthesia has been observed in numerous preclinical and clinical studies, yet, the underlying mechanisms remain unknown and no preventive treatment is available. We found that daily intranasal insulin administration to juvenile mice for 7 days prior to repeated isoflurane anesthesia rescues deficits in hippocampus-dependent memory and synaptic plasticity in adulthood. Moreover, intranasal insulin prevented anesthesia-induced apoptosis of hippocampal cells, which is thought to underlie cognitive impairment. Inhibition of the mechanistic target of rapamycin complex 1 (mTORC1), a major intracellular effector of insulin receptor, blocked the beneficial effects of intranasal insulin on anesthesia-induced apoptosis. Consistent with this finding, mice lacking mTORC1 downstream translational repressor 4E-BP2 showed no induction of repeated anesthesia-induced apoptosis. Our study demonstrates that intranasal insulin prevents general anesthesia-induced apoptosis of hippocampal cells, and deficits in synaptic plasticity and memory, and suggests that the rescue effect is mediated via mTORC1/4E-BP2 signaling.

Mutations in HPRT1, a gene encoding a rate-limiting enzyme for purine salvage, cause Lesch-Nyhan disease which is characterized by self-injury and motor impairments. We leveraged stem cell and genetic engineering technologies to model the disease in isogenic and patient-derived forebrain and midbrain cell types. Dopaminergic progenitor cells deficient in HPRT showed decreased intensity of all developmental cell-fate markers measured. Metabolic analyses revealed significant loss of all purine derivatives, except hypoxanthine, and impaired glycolysis and oxidative phosphorylation. real-time glucose tracing demonstrated increased shunting to the pentose phosphate pathway for de novo purine synthesis at the expense of ATP production. Purine depletion in dopaminergic progenitor cells resulted in loss of RHEB, impairing mTORC1 activation. These data demonstrate dopaminergic-specific effects of purine salvage deficiency and unexpectedly reveal that dopaminergic progenitor cells are programmed to a high-energy state prior to higher energy demands of terminally differentiated cells.