Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory sys... more Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system. It is usually chronic and challenging to treat. In order to study neuropathic pain and its treatments, different models have been developed in rodents. These models derive from known etiologies, thus reproducing peripheral nerve injuries, central injuries, and metabolic-, infectious- or chemotherapy-related neuropathies. Murine models of peripheral nerve injury often target the sciatic nerve which is easy to access and allows nociceptive tests on the hind paw. These models rely on a compression and/or a section. Here, the detailed surgery procedure for the "cuff model " of neuropathic pain in mice is described. In this model, a cuff of PE-20 polyethylene tubing of standardized length (2 mm) is unilaterally implanted around th...
Les antidepresseurs ont un effet antiallodynique qui depend de la stimulation des recepteurs β2-a... more Les antidepresseurs ont un effet antiallodynique qui depend de la stimulation des recepteurs β2-adrenergiques. Ceux-ci stimulent la production d’adenosine monophosphate cyclique (AMPc) regule par les phosphodiesterases de type 4 (PDE4). Nous avons ici etudie l’effet d’inhibiteurs de PDE (iPDE) sur la douleur neuropathique, grâce a des approches de pharmacologie comportementale chez la souris completees par de l’imagerie calcium et des approches moleculaires. Nos resultats montrent un effet antiallodynique des iPDE4 et des iPDE5. L’action des iPDE4 est liee a une diminution d’expression du TNFα dans le ganglion rachidien et au recrutement des recepteurs delta des opioides. Celle des iPDE5 necessite a la fois les recepteurs mu et delta. Nous montrons aussi que l’action d’un iPDE4 depend de la dose, l’activation de cellules gliales semblant correlee a l’effet antiallodynique a faible dose, alors que celle des neurones a forte dose a un effet pronociceptif via les recepteurs TRPV1.
Translational regulation of mRNA permeates nociception. The identities of transcripts subject to ... more Translational regulation of mRNA permeates nociception. The identities of transcripts subject to translational control are almost entirely unknown. To address this problem, we examine the landscape of nascent translation in DRG neurons treated with inflammatory mediators using ribosome profiling. We identify and validate two targets, the immediate early genes Arc and Fos, as targets of induced translation. Mechanistically, we demonstrate that the ribosomal protein S6 kinase (S6K1) is required for their biosynthesis. Pharmacologic blockade of either S6K1 or Fos attenuates mechanical and thermal hyperalgesia triggered by inflammatory stimuli. Genetic disruption of Arc did not alter pain associated behaviors. However, Arc deficient mice display exaggerated paw temperatures and vasodilation in response to an inflammatory challenge. Since Arc has recently been shown to be released from neurons in extracellular vesicles, we asked if intercellular Arc signaling regulates the inflammatory r...
Title: Nociceptor translational profiling reveals the RagA-mTORC1 network as a critical generator... more Title: Nociceptor translational profiling reveals the RagA-mTORC1 network as a critical generator of neuropathic pain Short Title: Nociceptor translational profiling Salim Megat1, Pradipta R. Ray1, Jamie K. Moy1, Tzu-Fang Lou2, Paulino Barragan-Iglesias1, Yan Li3, Grishma Pradhan1, Andi Wanghzou1, Ayesha Ahmad1, Robert Y. North3, Patrick M. Dougherty3, Arkady Khoutorsky4, Nahum Sonenberg5, Kevin R. Webster6, Gregory Dussor1 , Zachary T. Campbell2* and Theodore J. Price1,* 1 University of Texas at Dallas, School of Behavioral and Brain Sciences. 2 University of Texas at Dallas, Department of Biological Sciences. 3 University of Texas Health Science Center, Department of Anesthesia and Pain Medicine. 4 McGill University, Department of Anesthesia 5 McGill University, Goodman Cancer Research Center, Department of Biochemistry 6 eFFECTOR Therapeutics
Mutations disrupting the nuclear localization of the RNA-binding protein FUS characterize a subse... more Mutations disrupting the nuclear localization of the RNA-binding protein FUS characterize a subset of amyotrophic lateral sclerosis patients (ALS-FUS). FUS regulates nuclear RNAs, but its role at the synapse is poorly understood. Using super-resolution imaging we determined that the localization of FUS within synapses occurs predominantly near the vesicle reserve pool of presynaptic sites. Using CLIP-seq on synaptoneurosomes, we identified synaptic FUS RNA targets, encoding proteins associated with synapse organization and plasticity. Significant increase of synaptic FUS during early disease in a mouse model of ALS was accompanied by alterations in density and size of GABAergic synapses. mRNAs abnormally accumulated at the synapses of 6-month-old ALS-FUS mice were enriched for FUS targets and correlated with those depicting increased short-term mRNA stability via binding primarily on multiple exonic sites. Our study indicates that synaptic FUS accumulation in early disease leads to ...
Mutations in FUS, an RNA-binding protein involved in multiple steps of RNA metabolism, are associ... more Mutations in FUS, an RNA-binding protein involved in multiple steps of RNA metabolism, are associated with the most severe forms of amyotrophic lateral sclerosis (ALS). Accumulation of cytoplasmic FUS is likely to be a major culprit in the toxicity of FUS mutations. Thus, preventing cytoplasmic mislocalization of the FUS protein may represent a valuable therapeutic strategy. FUS binds to its own pre-mRNA creating an autoregulatory loop efficiently buffering FUS excess through multiple proposed mechanisms including retention of introns 6 and/or 7. Here, we introduced a wild-type FUS gene allele, retaining all intronic sequences, in mice whose heterozygous or homozygous expression of a cytoplasmically retained FUS protein (Fus∆NLS) was previously shown to provoke ALS-like disease or postnatal lethality, respectively. Wild-type FUS completely rescued the early lethality caused by the two Fus∆NLS alleles, and improved the age-dependent motor deficits and reduced lifespan caused by heter...
Gene mutations causing cytoplasmic mislocalization of the RNA-binding protein FUS lead to severe ... more Gene mutations causing cytoplasmic mislocalization of the RNA-binding protein FUS lead to severe forms of amyotrophic lateral sclerosis (ALS). Cytoplasmic accumulation of FUS is also observed in other diseases, with unknown consequences. Here, we show that cytoplasmic mislocalization of FUS drives behavioral abnormalities in knock-in mice, including locomotor hyperactivity and alterations in social interactions, in the absence of widespread neuronal loss. Mechanistically, we identified a progressive increase in neuronal activity in the frontal cortex of Fus knock-in mice in vivo, associated with altered synaptic gene expression. Synaptic ultrastructural and morphological defects were more pronounced in inhibitory than excitatory synapses and associated with increased synaptosomal levels of FUS and its RNA targets. Thus, cytoplasmic FUS triggers synaptic deficits, which is leading to increased neuronal activity in frontal cortex and causing related behavioral phenotypes. These result...
The genetic basis of amyotrophic lateral sclerosis (ALS) is still incompletely understood. Using ... more The genetic basis of amyotrophic lateral sclerosis (ALS) is still incompletely understood. Using two independent genetic strategies, we show here that a large part of ALS heritability lies in genes expressed in inhibitory and excitatory neurons, especially at splicing sites regulated by a defined set of RNA binding proteins including TDP-43 and FUS. We conducted a transcriptome wide association study (TWAS) and identified 59 loci associated with ALS, including 14 previously identified genes, some of them not previously reaching significance in genome wide association studies. Among the 45 novel genes, several genes are involved in pathways known to be affected in ALS such as mitochondrial metabolism (including ATP5H, ATP5D, BCS1L), proteostasis (including COPS7A, G2E3, TMEM175, USP35) or gene expression and RNA metabolism (including ARID1B, ATXN3, PTBP2, TAF10). Interestingly, decreased expression of NUP50, a constrained gene encoding a nuclear pore basket protein, was associated wi...
ABSTRACTBackgroundThere are clinically relevant sex differences in acute and chronic pain mechani... more ABSTRACTBackgroundThere are clinically relevant sex differences in acute and chronic pain mechanisms, but we are only beginning to understand their mechanistic basis. Transcriptome analyses of rodent whole dorsal root ganglion (DRG) have revealed sex differences, mostly in immune cells. We examined the transcriptome and translatome of the mouse DRG with the goal of identifying sex differences.MethodsWe used Translating Ribosome Affinity Purification (TRAP) sequencing and behavioral pharmacology to test the hypothesis that nociceptor (Nav1.8 expressing neurons) translatomes would differ by sex.ResultsWe found 66 genes whose mRNA were sex-differentially bound to nociceptor ribosomes. Many of these genes have known neuronal functions but have not been explored in sex differences in pain. We focused on Ptgds, which was increased in female mice. The mRNA encodes the prostaglandin D2 (PGD2) synthesizing enzyme. We observed increased Ptgds protein and PGD2 in female mouse DRG. The Ptgds in...
ABSTRACTInjury responses require communication between different cell types in the skin. Sensory ... more ABSTRACTInjury responses require communication between different cell types in the skin. Sensory neurons contribute to inflammation and can secrete signaling molecules that affect non-neuronal cells. Despite the pervasive role of translational regulation in nociception, the contribution of activity-dependent protein synthesis to inflammation is not well understood. To address this problem, we examined the landscape of nascent translation in DRG neurons treated with inflammatory mediators using ribosome profiling. We identified the activity-dependent gene, Arc, as a target of privileged translation in vitro and in vivo. Inflammatory cues promote local translation of Arc in the skin. Arc deficient mice display exaggerated paw temperatures and vasodilation in response to an inflammatory challenge. Since Arc has recently been shown to be released from neurons in extracellular vesicles, we hypothesized that intercellular Arc signaling regulates the inflammatory response in skin. We found...
Gene mutations causing cytoplasmic mislocalization of the RNA-binding protein FUS, lead to severe... more Gene mutations causing cytoplasmic mislocalization of the RNA-binding protein FUS, lead to severe forms of amyotrophic lateral sclerosis (ALS). Cytoplasmic accumulation of FUS is also observed in other diseases, with unknown consequences. Here, we show that cytoplasmic mislocalization of FUS drives behavioral abnormalities in knock-in mice, including locomotor hyperactivity and alterations in social interactions, in the absence of widespread neuronal loss. Mechanistically, we identified a profound increase in neuronal activity in the frontal cortex of Fus knock-in mice in vivo. Importantly, RNAseq analysis suggested involvement of defects in inhibitory neurons, that was confirmed by ultrastructural and morphological defects of inhibitory synapses and increased synaptosomal levels of mRNAs involved in inhibitory neurotransmission. Thus, cytoplasmic FUS triggers inhibitory synaptic deficits, leading to increased neuronal activity and behavioral phenotypes. FUS mislocalization may trig...
Pain sensing neurons, nociceptors, are key drivers of neuropathic pain. We used translating ribos... more Pain sensing neurons, nociceptors, are key drivers of neuropathic pain. We used translating ribosome affinity purification (TRAP) to comprehensively characterize up-and down-regulated mRNA translation in Scn10a-positive nociceptors in chemotherapy-induced neuropathic pain. We provide evidence that an underlying mechanism driving these changes in gene expression is a sustained mTORC1 activation driven by MNK1-eIF4E signaling. RagA, a GTPase controlling mTORC1 activity, is identified as a novel target of MNK1-eIF4E signaling, demonstrating a new link between these distinct signaling pathways. Neuropathic pain and RagA translation are strongly attenuated by genetic ablation of eIF4E phosphorylation, MNK1 elimination or treatment with the MNK inhibitor eFT508. We reveal a novel translational circuit for the genesis of neuropathic pain with important implications for next generation neuropathic pain therapeutics.One Sentence SummaryCell-specific sequencing of translating mRNAs elucidates...
Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory sys... more Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system. It is usually chronic and challenging to treat. In order to study neuropathic pain and its treatments, different models have been developed in rodents. These models derive from known etiologies, thus reproducing peripheral nerve injuries, central injuries, and metabolic-, infectious- or chemotherapy-related neuropathies. Murine models of peripheral nerve injury often target the sciatic nerve which is easy to access and allows nociceptive tests on the hind paw. These models rely on a compression and/or a section. Here, the detailed surgery procedure for the "cuff model " of neuropathic pain in mice is described. In this model, a cuff of PE-20 polyethylene tubing of standardized length (2 mm) is unilaterally implanted around th...
Les antidepresseurs ont un effet antiallodynique qui depend de la stimulation des recepteurs β2-a... more Les antidepresseurs ont un effet antiallodynique qui depend de la stimulation des recepteurs β2-adrenergiques. Ceux-ci stimulent la production d’adenosine monophosphate cyclique (AMPc) regule par les phosphodiesterases de type 4 (PDE4). Nous avons ici etudie l’effet d’inhibiteurs de PDE (iPDE) sur la douleur neuropathique, grâce a des approches de pharmacologie comportementale chez la souris completees par de l’imagerie calcium et des approches moleculaires. Nos resultats montrent un effet antiallodynique des iPDE4 et des iPDE5. L’action des iPDE4 est liee a une diminution d’expression du TNFα dans le ganglion rachidien et au recrutement des recepteurs delta des opioides. Celle des iPDE5 necessite a la fois les recepteurs mu et delta. Nous montrons aussi que l’action d’un iPDE4 depend de la dose, l’activation de cellules gliales semblant correlee a l’effet antiallodynique a faible dose, alors que celle des neurones a forte dose a un effet pronociceptif via les recepteurs TRPV1.
Translational regulation of mRNA permeates nociception. The identities of transcripts subject to ... more Translational regulation of mRNA permeates nociception. The identities of transcripts subject to translational control are almost entirely unknown. To address this problem, we examine the landscape of nascent translation in DRG neurons treated with inflammatory mediators using ribosome profiling. We identify and validate two targets, the immediate early genes Arc and Fos, as targets of induced translation. Mechanistically, we demonstrate that the ribosomal protein S6 kinase (S6K1) is required for their biosynthesis. Pharmacologic blockade of either S6K1 or Fos attenuates mechanical and thermal hyperalgesia triggered by inflammatory stimuli. Genetic disruption of Arc did not alter pain associated behaviors. However, Arc deficient mice display exaggerated paw temperatures and vasodilation in response to an inflammatory challenge. Since Arc has recently been shown to be released from neurons in extracellular vesicles, we asked if intercellular Arc signaling regulates the inflammatory r...
Title: Nociceptor translational profiling reveals the RagA-mTORC1 network as a critical generator... more Title: Nociceptor translational profiling reveals the RagA-mTORC1 network as a critical generator of neuropathic pain Short Title: Nociceptor translational profiling Salim Megat1, Pradipta R. Ray1, Jamie K. Moy1, Tzu-Fang Lou2, Paulino Barragan-Iglesias1, Yan Li3, Grishma Pradhan1, Andi Wanghzou1, Ayesha Ahmad1, Robert Y. North3, Patrick M. Dougherty3, Arkady Khoutorsky4, Nahum Sonenberg5, Kevin R. Webster6, Gregory Dussor1 , Zachary T. Campbell2* and Theodore J. Price1,* 1 University of Texas at Dallas, School of Behavioral and Brain Sciences. 2 University of Texas at Dallas, Department of Biological Sciences. 3 University of Texas Health Science Center, Department of Anesthesia and Pain Medicine. 4 McGill University, Department of Anesthesia 5 McGill University, Goodman Cancer Research Center, Department of Biochemistry 6 eFFECTOR Therapeutics
Mutations disrupting the nuclear localization of the RNA-binding protein FUS characterize a subse... more Mutations disrupting the nuclear localization of the RNA-binding protein FUS characterize a subset of amyotrophic lateral sclerosis patients (ALS-FUS). FUS regulates nuclear RNAs, but its role at the synapse is poorly understood. Using super-resolution imaging we determined that the localization of FUS within synapses occurs predominantly near the vesicle reserve pool of presynaptic sites. Using CLIP-seq on synaptoneurosomes, we identified synaptic FUS RNA targets, encoding proteins associated with synapse organization and plasticity. Significant increase of synaptic FUS during early disease in a mouse model of ALS was accompanied by alterations in density and size of GABAergic synapses. mRNAs abnormally accumulated at the synapses of 6-month-old ALS-FUS mice were enriched for FUS targets and correlated with those depicting increased short-term mRNA stability via binding primarily on multiple exonic sites. Our study indicates that synaptic FUS accumulation in early disease leads to ...
Mutations in FUS, an RNA-binding protein involved in multiple steps of RNA metabolism, are associ... more Mutations in FUS, an RNA-binding protein involved in multiple steps of RNA metabolism, are associated with the most severe forms of amyotrophic lateral sclerosis (ALS). Accumulation of cytoplasmic FUS is likely to be a major culprit in the toxicity of FUS mutations. Thus, preventing cytoplasmic mislocalization of the FUS protein may represent a valuable therapeutic strategy. FUS binds to its own pre-mRNA creating an autoregulatory loop efficiently buffering FUS excess through multiple proposed mechanisms including retention of introns 6 and/or 7. Here, we introduced a wild-type FUS gene allele, retaining all intronic sequences, in mice whose heterozygous or homozygous expression of a cytoplasmically retained FUS protein (Fus∆NLS) was previously shown to provoke ALS-like disease or postnatal lethality, respectively. Wild-type FUS completely rescued the early lethality caused by the two Fus∆NLS alleles, and improved the age-dependent motor deficits and reduced lifespan caused by heter...
Gene mutations causing cytoplasmic mislocalization of the RNA-binding protein FUS lead to severe ... more Gene mutations causing cytoplasmic mislocalization of the RNA-binding protein FUS lead to severe forms of amyotrophic lateral sclerosis (ALS). Cytoplasmic accumulation of FUS is also observed in other diseases, with unknown consequences. Here, we show that cytoplasmic mislocalization of FUS drives behavioral abnormalities in knock-in mice, including locomotor hyperactivity and alterations in social interactions, in the absence of widespread neuronal loss. Mechanistically, we identified a progressive increase in neuronal activity in the frontal cortex of Fus knock-in mice in vivo, associated with altered synaptic gene expression. Synaptic ultrastructural and morphological defects were more pronounced in inhibitory than excitatory synapses and associated with increased synaptosomal levels of FUS and its RNA targets. Thus, cytoplasmic FUS triggers synaptic deficits, which is leading to increased neuronal activity in frontal cortex and causing related behavioral phenotypes. These result...
The genetic basis of amyotrophic lateral sclerosis (ALS) is still incompletely understood. Using ... more The genetic basis of amyotrophic lateral sclerosis (ALS) is still incompletely understood. Using two independent genetic strategies, we show here that a large part of ALS heritability lies in genes expressed in inhibitory and excitatory neurons, especially at splicing sites regulated by a defined set of RNA binding proteins including TDP-43 and FUS. We conducted a transcriptome wide association study (TWAS) and identified 59 loci associated with ALS, including 14 previously identified genes, some of them not previously reaching significance in genome wide association studies. Among the 45 novel genes, several genes are involved in pathways known to be affected in ALS such as mitochondrial metabolism (including ATP5H, ATP5D, BCS1L), proteostasis (including COPS7A, G2E3, TMEM175, USP35) or gene expression and RNA metabolism (including ARID1B, ATXN3, PTBP2, TAF10). Interestingly, decreased expression of NUP50, a constrained gene encoding a nuclear pore basket protein, was associated wi...
ABSTRACTBackgroundThere are clinically relevant sex differences in acute and chronic pain mechani... more ABSTRACTBackgroundThere are clinically relevant sex differences in acute and chronic pain mechanisms, but we are only beginning to understand their mechanistic basis. Transcriptome analyses of rodent whole dorsal root ganglion (DRG) have revealed sex differences, mostly in immune cells. We examined the transcriptome and translatome of the mouse DRG with the goal of identifying sex differences.MethodsWe used Translating Ribosome Affinity Purification (TRAP) sequencing and behavioral pharmacology to test the hypothesis that nociceptor (Nav1.8 expressing neurons) translatomes would differ by sex.ResultsWe found 66 genes whose mRNA were sex-differentially bound to nociceptor ribosomes. Many of these genes have known neuronal functions but have not been explored in sex differences in pain. We focused on Ptgds, which was increased in female mice. The mRNA encodes the prostaglandin D2 (PGD2) synthesizing enzyme. We observed increased Ptgds protein and PGD2 in female mouse DRG. The Ptgds in...
ABSTRACTInjury responses require communication between different cell types in the skin. Sensory ... more ABSTRACTInjury responses require communication between different cell types in the skin. Sensory neurons contribute to inflammation and can secrete signaling molecules that affect non-neuronal cells. Despite the pervasive role of translational regulation in nociception, the contribution of activity-dependent protein synthesis to inflammation is not well understood. To address this problem, we examined the landscape of nascent translation in DRG neurons treated with inflammatory mediators using ribosome profiling. We identified the activity-dependent gene, Arc, as a target of privileged translation in vitro and in vivo. Inflammatory cues promote local translation of Arc in the skin. Arc deficient mice display exaggerated paw temperatures and vasodilation in response to an inflammatory challenge. Since Arc has recently been shown to be released from neurons in extracellular vesicles, we hypothesized that intercellular Arc signaling regulates the inflammatory response in skin. We found...
Gene mutations causing cytoplasmic mislocalization of the RNA-binding protein FUS, lead to severe... more Gene mutations causing cytoplasmic mislocalization of the RNA-binding protein FUS, lead to severe forms of amyotrophic lateral sclerosis (ALS). Cytoplasmic accumulation of FUS is also observed in other diseases, with unknown consequences. Here, we show that cytoplasmic mislocalization of FUS drives behavioral abnormalities in knock-in mice, including locomotor hyperactivity and alterations in social interactions, in the absence of widespread neuronal loss. Mechanistically, we identified a profound increase in neuronal activity in the frontal cortex of Fus knock-in mice in vivo. Importantly, RNAseq analysis suggested involvement of defects in inhibitory neurons, that was confirmed by ultrastructural and morphological defects of inhibitory synapses and increased synaptosomal levels of mRNAs involved in inhibitory neurotransmission. Thus, cytoplasmic FUS triggers inhibitory synaptic deficits, leading to increased neuronal activity and behavioral phenotypes. FUS mislocalization may trig...
Pain sensing neurons, nociceptors, are key drivers of neuropathic pain. We used translating ribos... more Pain sensing neurons, nociceptors, are key drivers of neuropathic pain. We used translating ribosome affinity purification (TRAP) to comprehensively characterize up-and down-regulated mRNA translation in Scn10a-positive nociceptors in chemotherapy-induced neuropathic pain. We provide evidence that an underlying mechanism driving these changes in gene expression is a sustained mTORC1 activation driven by MNK1-eIF4E signaling. RagA, a GTPase controlling mTORC1 activity, is identified as a novel target of MNK1-eIF4E signaling, demonstrating a new link between these distinct signaling pathways. Neuropathic pain and RagA translation are strongly attenuated by genetic ablation of eIF4E phosphorylation, MNK1 elimination or treatment with the MNK inhibitor eFT508. We reveal a novel translational circuit for the genesis of neuropathic pain with important implications for next generation neuropathic pain therapeutics.One Sentence SummaryCell-specific sequencing of translating mRNAs elucidates...
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