Angiotensin Converting Enzyme (ACE) expression in microglia reduces amyloid ß deposition and neurodegeneration by increasing SYK signaling and endolysosomal trafficking.

Genome-wide association studies (GWAS) have identified many gene polymorphisms associated with an increased risk of developing Late Onset Alzheimer's Disease (LOAD). Many of these LOAD risk-associated alleles alter disease pathogenesis by influencing microglia innate immune responses and lipid metabolism. Angiotensin Converting Enzyme (ACE), a GWAS LOAD risk-associated gene best known for its role in regulating systemic blood pressure, also enhances innate immunity and lipid processing in peripheral myeloid cells, but a role for ACE in modulating the function of myeloid-derived microglia remains unexplored. Using novel mice engineered to express ACE in microglia and CNS associated macrophages (CAMs), we find that ACE expression in microglia reduces Aß plaque load, preserves vulnerable neurons and excitatory synapses, and greatly reduces learning and memory abnormalities in the 5xFAD amyloid mouse model of Alzheimer's Disease (AD). ACE-expressing microglia show enhanced Aß phagocytosis and endolysosomal trafficking, increased clustering around amyloid plaques, and increased SYK tyrosine kinase activation downstream of the major Aß receptors, TREM2 and CLEC7A. Single microglia sequencing and digital spatial profiling identifies downstream SYK signaling modules that are expressed by ACE expression in microglia that mediate endolysosomal biogenesis and trafficking, mTOR and PI3K/AKT signaling, and increased oxidative phosphorylation, while gene silencing or pharmacologic inhibition of SYK activity in ACE-expressing microglia abrogates the potentiated Aß engulfment and endolysosomal trafficking. These findings establish a role for ACE in enhancing microglial immune function and they identify a potential use for ACE-expressing microglia as a cell-based therapy to augment endogenous microglial responses to Aß in AD.

Repopulated spinal cord microglia exhibit a unique transcriptome and contribute to pain resolution.

Microglia are implicated as primarily detrimental in pain models; however, they exist across a continuum of states that contribute to homeostasis or pathology depending on timing and context. To clarify the specific contribution of microglia to pain progression, we take advantage of a temporally controlled transgenic approach to transiently deplete microglia. Unexpectedly, we observe complete resolution of pain coinciding with microglial repopulation rather than depletion. We find that repopulated mouse spinal cord microglia are morphologically distinct from control microglia and exhibit a unique transcriptome. Repopulated microglia from males and females express overlapping networks of genes related to phagocytosis and response to stress. We intersect the identified mouse genes with a single-nuclei microglial dataset from human spinal cord to identify human-relevant genes that may ultimately promote pain resolution after injury. This work presents a comprehensive approach to gene discovery in pain and provides datasets for the development of future microglial-targeted therapeutics.

RALY participates in nerve trauma-induced nociceptive hypersensitivity through triggering Eif4g2 gene expression in primary sensory neurons.

BACKGROUND AND PURPOSE: Peripheral nerve trauma-induced dysregulation of pain-associated genes in the primary sensory neurons of dorsal root ganglion (DRG) contributes to neuropathic pain genesis. RNA-binding proteins participate in gene transcription. We hypothesized that RALY, an RNA-binding protein, participated in nerve trauma-induced dysregulation of DRG pain-associated genes and nociceptive hypersensitivity. METHODS AND RESULTS: Immunohistochemistry staining showed that RALY was expressed exclusively in the nuclei of DRG neurons. Peripheral nerve trauma caused by chronic constriction injury (CCI) of unilateral sciatic nerve produced time-dependent increases in the levels of Raly mRNA and RALY protein in injured DRG. Blocking this increase through DRG microinjection of adeno-associated virus 5 (AAV5)-expressing Raly shRNA reduced the CCI-induced elevation in the amount of eukaryotic initiation factor 4 gamma 2 (Eif4g2) mRNA and Eif4g2 protein in injured DRG and mitigated the development and maintenance of CCI-induced nociceptive hypersensitivity, without altering basal (acute) response to noxious stimuli and locomotor activity. Mimicking DRG increased RALY through DRG microinjection of AAV5 expressing Raly mRNA up-regulated the expression of Eif4g2 mRNA and Eif4g2 protein in the DRG and led to hypersensitive responses to noxious stimuli in the absence of nerve trauma. Mechanistically, CCI promoted the binding of RALY to the promoter of Eif4g2 gene and triggered its transcriptional activity. CONCLUSION AND IMPLICATIONS: Our findings indicate that RALY participates in nerve trauma-induced nociceptive hypersensitivity likely through transcriptionally triggering Eif4g2 expression in the DRG. RALY may be a potential target in neuropathic pain management.

A sensory neuron-specific long non-coding RNA reduces neuropathic pain by rescuing KCNN1 expression.

Nerve injury to peripheral somatosensory system causes refractory neuropathic pain. Maladaptive changes of gene expression in primary sensory neurons are considered molecular basis of this disorder. Long non-coding RNAs (lncRNAs) are key regulators of gene transcription; however, their significance in neuropathic pain remains largely elusive.Here, we reported a novel lncRNA, named sensory neuron-specific lncRNA (SS-lncRNA), for its expression exclusively in dorsal root ganglion (DRG) and trigeminal ganglion. SS-lncRNA was predominantly expressed in small DRG neurons and significantly downregulated due to a reduction of early B cell transcription factor 1 in injured DRG after nerve injury. Rescuing this downregulation reversed a decrease of the calcium-activated potassium channel subfamily N member 1 (KCNN1) in injured DRG and alleviated nerve injury-induced nociceptive hypersensitivity. Conversely, DRG downregulation of SS-lncRNA reduced the expression of KCNN1, decreased total potassium currents and afterhyperpolarization currents and increased excitability in DRG neurons and produced neuropathic pain symptoms.Mechanistically, downregulated SS-lncRNA resulted in the reductions of its binding to Kcnn1 promoter and heterogeneous nuclear ribonucleoprotein M (hnRNPM), consequent recruitment of less hnRNPM to the Kcnn1 promoter and silence of Kcnn1 gene transcription in injured DRG.These findings indicate that SS-lncRNA may relieve neuropathic pain through hnRNPM-mediated KCNN1 rescue in injured DRG and offer a novel therapeutic strategy specific for this disorder.

FUS Contributes to Nerve Injury-Induced Nociceptive Hypersensitivity by Activating NF-κB Pathway in Primary Sensory Neurons.

Dysregulation of pain-associated genes in the dorsal root ganglion (DRG) is considered to be a molecular basis of neuropathic pain genesis. Fused in sarcoma (FUS), a DNA/RNA-binding protein, is a critical regulator of gene expression. However, whether it contributes to neuropathic pain is unknown. This study showed that peripheral nerve injury caused by the fourth lumbar (L4) spinal nerve ligation (SNL) or chronic constriction injury (CCI) of the sciatic nerve produced a marked increase in the expression of FUS protein in injured DRG neurons. Blocking this increase through microinjection of the adeno-associated virus (AAV) 5-expressing Fus shRNA into the ipsilateral L4 DRG mitigated the SNL-induced nociceptive hypersensitivities in both male and female mice. This microinjection also alleviated the SNL-induced increases in the levels of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and glial fibrillary acidic protein (GFAP) in the ipsilateral L4 dorsal horn. Furthermore, mimicking this increase through microinjection of AAV5 expressing full-length Fus mRNA into unilateral L3/4 DRGs produced the elevations in the levels of p-ERK1/2 and GFAP in the dorsal horn, enhanced responses to mechanical, heat and cold stimuli, and induced the spontaneous pain on the ipsilateral side of both male and female mice in the absence of SNL. Mechanistically, the increased FUS activated the NF-κB signaling pathway by promoting the translocation of p65 into the nucleus and phosphorylation of p65 in the nucleus from injured DRG neurons. Our results indicate that DRG FUS contributes to neuropathic pain likely through the activation of NF-κB in primary sensory neurons.SIGNIFICANCE STATEMENT In the present study, we reported that fused in sarcoma (FUS), a DNA/RNA-binding protein, is upregulated in injured dorsal root ganglion (DRG) following peripheral nerve injury. This upregulation is responsible for nerve injury-induced translocation of p65 into the nucleus and phosphorylation of p65 in the nucleus from injured DRG neurons. Because blocking this upregulation alleviates nerve injury-induced nociceptive hypersensitivity, DRG FUS participates in neuropathic pain likely through the activation of NF-κB in primary sensory neurons. FUS may be a potential target for neuropathic pain management.

E74-like factor 1 contributes to nerve trauma-induced nociceptive hypersensitivity through transcriptionally activating matrix metalloprotein-9 in dorsal root ganglion neurons.

ABSTRACT: Nerve trauma-induced alternations of gene expression in the neurons of dorsal root ganglion (DRG) participate in nerve trauma-caused nociceptive hypersensitivity. Transcription factors regulate gene expression. Whether the transcription factor E74-like factor 1 (ELF1) in the DRG contributes to neuropathic pain is unknown. We report here that peripheral nerve trauma caused by chronic constriction injury (CCI) of unilateral sciatic nerve or unilateral fourth lumbar spinal nerve ligation led to the time-dependent increases in the levels of Elf1 mRNA and ELF1 protein in injured DRG, but not in the spinal cord. Preventing this increase through DRG microinjection of adeno-associated virus 5 expressing Elf1 shRNA attenuated the CCI-induced upregulation of matrix metallopeptidase 9 (MMP9) in injured DRG and induction and maintenance of nociceptive hypersensitivities, without changing locomotor functions and basal responses to acute mechanical, heat, and cold stimuli. Mimicking this increase through DRG microinjection of AAV5 expressing full-length Elf1 upregulated DRG MMP9 and produced enhanced responses to mechanical, heat, and cold stimuli in naive mice. Mechanistically, more ELF1 directly bond to and activated Mmp9 promoter in injured DRG neurons after CCI. Our data indicate that ELF1 participates in nerve trauma-caused nociceptive hypersensitivity likely through upregulating MMP9 in injured DRG. E74-like factor 1 may be a new target for management of neuropathic pain.

A nerve injury-specific long noncoding RNA promotes neuropathic pain by increasing Ccl2 expression.

Maladaptive changes of nerve injury-associated genes in dorsal root ganglia (DRGs) are critical for neuropathic pain genesis. Emerging evidence supports the role of long noncoding RNAs (lncRNAs) in regulating gene transcription. Here we identified a conserved lncRNA, named nerve injury-specific lncRNA (NIS-lncRNA) for its upregulation in injured DRGs exclusively in response to nerve injury. This upregulation was triggered by nerve injury-induced increase in DRG ELF1, a transcription factor that bound to the NIS-lncRNA promoter. Blocking this upregulation attenuated nerve injury-induced CCL2 increase in injured DRGs and nociceptive hypersensitivity during the development and maintenance periods of neuropathic pain. Mimicking NIS-lncRNA upregulation elevated CCL2 expression, increased CCL2-mediated excitability in DRG neurons, and produced neuropathic pain symptoms. Mechanistically, NIS-lncRNA recruited more binding of the RNA-interacting protein FUS to the Ccl2 promoter and augmented Ccl2 transcription in injured DRGs. Thus, NIS-lncRNA participates in neuropathic pain likely by promoting FUS-triggered DRG Ccl2 expression and may be a potential target in neuropathic pain management.

Downregulation of a Dorsal Root Ganglion-Specifically Enriched Long Noncoding RNA is Required for Neuropathic Pain by Negatively Regulating RALY-Triggered Ehmt2 Expression.

Nerve injury-induced maladaptive changes of gene expression in dorsal root ganglion (DRG) neurons contribute to neuropathic pain. Long non-coding RNAs (lncRNAs) are emerging as key regulators of gene expression. Here, a conserved lncRNA is reported, named DRG-specifically enriched lncRNA (DS-lncRNA) for its high expression in DRG neurons. Peripheral nerve injury downregulates DS-lncRNA in injured DRG due, in part, to silencing of POU domain, class 4, transcription factor 3, a transcription factor that interacts with the DS-lncRNA gene promoter. Rescuing DS-lncRNA downregulation blocks nerve injury-induced increases in the transcriptional cofactor RALY-triggered DRG Ehmt2 mRNA and its encoding G9a protein, reverses the G9a-controlled downregulation of opioid receptors and Kcna2 in injured DRG, and attenuates nerve injury-induced pain hypersensitivities in male mice. Conversely, DS-lncRNA downregulation increases RALY-triggered Ehmt2/G9a expression and correspondingly decreases opioid receptor and Kcna2 expression in DRG, leading to neuropathic pain symptoms in male mice in the absence of nerve injury. Mechanistically, downregulated DS-lncRNA promotes more binding of increased RALY to RNA polymerase II and the Ehmt2 gene promoter and enhances Ehmt2 transcription in injured DRG. Thus, downregulation of DS-lncRNA likely contributes to neuropathic pain by negatively regulating the expression of RALY-triggered Ehmt2/G9a, a key neuropathic pain player, in DRG neurons.

FTO (Fat-Mass and Obesity-Associated Protein) Participates in Hemorrhage-Induced Thalamic Pain by Stabilizing Toll-Like Receptor 4 Expression in Thalamic Neurons.

Background and Purpose: Hemorrhage-caused gene changes in the thalamus likely contribute to thalamic pain genesis. RNA N6-methyladenosine modification is an additional layer of gene regulation. Whether FTO (fat-mass and obesity-associated protein), an N6-methyladenosine demethylase, participates in hemorrhage-induced thalamic pain is unknown. Methods: Expression of Fto mRNA and protein was assessed in mouse thalamus after hemorrhage caused by microinjection of Coll IV (type IV collagenase) into unilateral thalamus. Effect of intraperitoneal administration of meclofenamic acid (a FTO inhibitor) or microinjection of adeno-associated virus 5 (AAV5) expressing Cre into the thalamus of Ftofl/fl mice on the Coll IV microinjection­induced TLR4 (Toll-like receptor 4) upregulation and nociceptive hypersensitivity was examined. Effect of thalamic microinjection of AAV5 expressing Fto (AAV5-Fto) on basal thalamic TLR4 expression and nociceptive thresholds was also analyzed. Additionally, level of N6-methyladenosine in Tlr4 mRNA and its binding to FTO or YTHDF2 (YTH N6-methyladenosine RNA binding protein 2) were observed. Results: FTO was detected in neuronal nuclei of thalamus. Level of FTO protein, but not mRNA, was time-dependently increased in the ipsilateral thalamus on days 1 to 14 after Coll IV microinjection. Intraperitoneal injection of meclofenamic acid or adeno-associated virus-5 expressing Cre microinjection into Ftofl/fl mouse thalamus attenuated the Coll IV microinjection­induced TLR4 upregulation and tissue damage in the ipsilateral thalamus and development and maintenance of nociceptive hypersensitivities on the contralateral side. Thalamic microinjection of AAV5-Fto increased TLR4 expression and elicited hypersensitivities to mechanical, heat and cold stimuli. Mechanistically, Coll IV microinjection produced an increase in FTO binding to Tlr4 mRNA, an FTO-dependent loss of N6-methyladenosine sites in Tlr4 mRNA and a reduction in the binding of YTHDF2 to Tlr4 mRNA in the ipsilateral thalamus. Conclusions: Our findings suggest that FTO participates in hemorrhage-induced thalamic pain by stabilizing TLR4 upregulation in thalamic neurons. FTO may be a potential target for the treatment of this disorder.

Temporal Contribution of Myeloid-Lineage TLR4 to the Transition to Chronic Pain: A Focus on Sex Differences.

Complex regional pain syndrome (CRPS) is a chronic pain disorder with a clear acute-to-chronic transition. Preclinical studies demonstrate that toll-like receptor 4 (TLR4), expressed by myeloid-lineage cells, astrocytes, and neurons, mediates a sex-dependent transition to chronic pain; however, evidence is lacking on which exact TLR4-expressing cells are responsible. We used complementary pharmacologic and transgenic approaches in mice to more specifically manipulate myeloid-lineage TLR4 and outline its contribution to the transition from acute-to-chronic CRPS based on three key variables: location (peripheral vs central), timing (prevention vs treatment), and sex (male vs female). We demonstrate that systemic TLR4 antagonism is more effective at improving chronic allodynia trajectory when administered at the time of injury (early) in the tibial fracture model of CRPS in both sexes. In order to clarify the contribution of myeloid-lineage cells peripherally (macrophages) or centrally (microglia), we rigorously characterize a novel spatiotemporal transgenic mouse line, Cx3CR1-CreERT2-eYFP;TLR4fl/fl (TLR4 cKO) to specifically knock out TLR4 only in microglia and no other myeloid-lineage cells. Using this transgenic mouse, we find that early TLR4 cKO results in profound improvement in chronic, but not acute, allodynia in males, with a significant but less robust effect in females. In contrast, late TLR4 cKO results in partial improvement in allodynia in both sexes, suggesting that downstream cellular or molecular TLR4-independent events may have already been triggered. Overall, we find that the contribution of TLR4 is time- and microglia-dependent in both sexes; however, females also rely on peripheral myeloid-lineage (or other TLR4 expressing) cells to trigger chronic pain.SIGNIFICANCE STATEMENT The contribution of myeloid cell TLR4 to sex-specific pain progression remains controversial. We used complementary pharmacologic and transgenic approaches to specifically manipulate TLR4 based on three key variables: location (peripheral vs central), timing (prevention vs treatment), and sex (male vs female). We discovered that microglial TLR4 contributes to early pain progression in males, and to a lesser extent in females. We further found that maintenance of chronic pain likely occurs through myeloid TLR4-independent mechanisms in both sexes. Together, we define a more nuanced contribution of this receptor to the acute-to-chronic pain transition in a mouse model of complex regional pain syndrome.

Eukaryotic initiation factor 4 gamma 2 contributes to neuropathic pain through down-regulation of Kv1.2 and the mu opioid receptor in mouse primary sensory neurones.

BACKGROUND: Nerve injury-induced changes in gene expression in the dorsal root ganglion (DRG) contribute to neuropathic pain genesis. Eukaryotic initiation factor 4 gamma 2 (eIF4G2) is a general repressor of cap-dependent mRNA translation. Whether DRG eIF4G2 participates in nerve injury-induced alternations in gene expression and nociceptive hypersensitivity is unknown. METHODS: The expression and distribution of eIF4G2 mRNA and protein in mouse DRG after spinal nerve ligation (SNL) were assessed. Effects of eIF4G2 siRNA microinjected through a glass micropipette into the injured DRG on the SNL-induced DRG mu opioid receptor (MOR) and Kv1.2 downregulation and nociceptive hypersensitivity were examined. In addition, effects of DRG microinjection of adeno-associated virus 5-expressing eIF4G2 (AAV5-eIF4G2) on basal DRG MOR and Kv1.2 expression and nociceptive thresholds were analysed. RESULTS: eIF4G2 protein co-expressed with Kv1.2 and MOR in DRG neurones. Levels of eIF4G2 mRNA (1.7 [0.24] to 2.3 [0.14]-fold of sham, P<0.01) and protein (1.6 [0.14] to 2.5 [0.22]-fold of sham, P<0.01) in injured DRG were time-dependently increased on days 3-14 after SNL. Blocking increased eIF4G2 through microinjection of eIF4G2 siRNA into the injured DRG attenuated SNL-induced downregulation of DRG MOR and Kv1.2 and development and maintenance of nociceptive hypersensitivities. DRG microinjection of AAV5-eIF4G2 reduced DRG MOR and Kv1.2 expression and elicited hypersensitivities to mechanical, heat and cold stimuli in naïve mice. CONCLUSIONS: eIF4G2 contributes to neuropathic pain through participation in downregulation of Kv1.2 and MOR in injured DRG and is a potential target for treatment of this disorder.

CREB Participates in Paclitaxel-Induced Neuropathic Pain Genesis Through Transcriptional Activation of Dnmt3a in Primary Sensory Neurons.

Chemotherapy-induced peripheral neuropathic pain (CIPNP) often occurs in cancer patients treated with antineoplastic drugs. Therapeutic management of CIPNP is very limited, at least in part due to the largely unknown mechanisms that underlie CIPNP genesis. Here, we showed that systemic administration of the chemotherapeutic drug paclitaxel significantly and time-dependently increased the levels of cyclic AMP response element-binding protein (CREB) in dorsal root ganglion (DRG) neurons. Blocking this increase through DRG microinjection of Creb siRNA attenuated paclitaxel-induced mechanical, heat, and cold nociceptive hypersensitivities. Mimicking this increase through DRG microinjection of the adeno-associated virus 5 expressing full-length Creb mRNA led to enhanced responses to basal mechanical, heat, and cold stimuli in mice in absence of paclitaxel treatment. Mechanically, paclitaxel-induced increase of DRG CREB protein augmented Dnmt3a promoter activity and participated in the paclitaxel-induced upregulation of DNMT3a protein in the DRG. CREB overexpression also elevated the expression of DNMT3a in in vivo and in vitro DRG neurons of naïve mice. Given that DNMT3a is an endogenous instigator of CIPNP and that CREB co-expresses with DNMT3a in DRG neurons, CREB may be a key player in CIPNP through transcriptional activation of the Dnmt3a gene in primary sensory neurons. CREB is thus a likely potential target for the therapeutic management of this disorder.

Corrigendum: Role of MicroRNA-143 in Nerve Injury-Induced Upregulation of Dnmt3a Expression in Primary Sensory Neurons.

[This corrects the article DOI: 10.3389/fnmol.2017.00350.].

Fgr contributes to hemorrhage-induced thalamic pain by activating NF-κB/ERK1/2 pathways.

Thalamic pain, a type of central poststroke pain, frequently occurs following ischemia/hemorrhage in the thalamus. Current treatment of this disorder is often ineffective, at least in part due to largely unknown mechanisms that underlie thalamic pain genesis. Here, we report that hemorrhage caused by microinjection of type IV collagenase or autologous whole blood into unilateral ventral posterior lateral nucleus and ventral posterior medial nucleus of the thalamus increased the expression of Fgr, a member of the Src family nonreceptor tyrosine kinases, at both mRNA and protein levels in thalamic microglia. Pharmacological inhibition or genetic knockdown of thalamic Fgr attenuated the hemorrhage-induced thalamic injury on the ipsilateral side and the development and maintenance of mechanical, heat, and cold pain hypersensitivities on the contralateral side. Mechanistically, the increased Fgr participated in hemorrhage-induced microglial activation and subsequent production of TNF-α likely through activation of both NF-κB and ERK1/2 pathways in thalamic microglia. Our findings suggest that Fgr is a key player in thalamic pain and a potential target for the therapeutic management of this disorder.

Author Correction: DNA methyltransferase DNMT3a contributes to neuropathic pain by repressing Kcna2 in primary afferent neurons.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

N6-Methyladenosine Demethylase FTO Contributes to Neuropathic Pain by Stabilizing G9a Expression in Primary Sensory Neurons.

Nerve injury-induced change in gene expression in primary sensory neurons of dorsal root ganglion (DRG) is critical for neuropathic pain genesis. N6-methyladenosine (m6A) modification of RNA represents an additional layer of gene regulation. Here, it is reported that peripheral nerve injury increases the expression of the m6A demethylase fat-mass and obesity-associated proteins (FTO) in the injured DRG via the activation of Runx1, a transcription factor that binds to the Fto gene promoter. Mimicking this increase erases m6A in euchromatic histone lysine methyltransferase 2 (Ehmt2) mRNA (encoding the histone methyltransferase G9a) and elevates the level of G9a in DRG and leads to neuropathic pain symptoms. Conversely, blocking this increase reverses a loss of m6A sites in Ehmt2 mRNA and destabilizes the nerve injury-induced G9a upregulation in the injured DRG and alleviates nerve injury-associated pain hypersensitivities. FTO contributes to neuropathic pain likely through stabilizing nerve injury-induced upregulation of G9a, a neuropathic pain initiator, in primary sensory neurons.

A Guide to Understanding "State-of-the-Art" Basic Research Techniques in Anesthesiology.

Perioperative medicine is changing from a "protocol-based" approach to a progressively personalized care model. New molecular techniques and comprehensive perioperative medical records allow for detection of patient-specific phenotypes that may better explain, or even predict, a patient's response to perioperative stress and anesthetic care. Basic science technology has significantly evolved in recent years with the advent of powerful approaches that have translational relevance. It is incumbent on us as a primarily clinical specialty to have an in-depth understanding of rapidly evolving underlying basic science techniques to incorporate such approaches into our own research, critically interpret the literature, and improve future anesthesia patient care. This review focuses on 3 important and most likely practice-changing basic science techniques: next-generation sequencing (NGS), clustered regularly interspaced short palindromic repeat (CRISPR) modulations, and inducible pluripotent stem cells (iPSCs). Each technique will be described, potential advantages and limitations discussed, open questions and challenges addressed, and future developments outlined. We hope to provide insight for practicing physicians when confronted with basic science articles and encourage investigators to apply "state-of-the-art" technology to their future experiments.

Toll-like receptor 7 contributes to neuropathic pain by activating NF-κB in primary sensory neurons.

Toll like receptor 7 (TLR7) is expressed in neurons of the dorsal root ganglion (DRG), but whether it contributes to neuropathic pain is elusive. We found that peripheral nerve injury caused by ligation of the fourth lumbar (L4) spinal nerve (SNL) or chronic constriction injury of sciatic nerve led to a significant increase in the expression of TLR7 at mRNA and protein levels in mouse injured DRG. Blocking this increase through microinjection of the adeno-associated virus (AAV) 5 expressing TLR7 shRNA into the ipsilateral L4 DRG alleviated the SNL-induced mechanical, thermal and cold pain hypersensitivities in both male and female mice. This microinjection also attenuated the SNL-induced increases in the levels of phosphorylated extracellular signal-regulated kinase ½ (p-ERK1/2) and glial fibrillary acidic protein (GFAP) in L4 dorsal horn on the ipsilateral side during both development and maintenance periods. Conversely, mimicking this increase through microinjection of AAV5 expressing full-length TLR7 into unilateral L3/4 DRGs led to elevations in the amounts of p-ERK1/2 and GFAP in the dorsal horn, augmented responses to mechanical, thermal and cold stimuli, and induced the spontaneous pain on the ipsilateral side in the absence of SNL. Mechanistically, the increased TLR7 activated the NF-κB signaling pathway through promoting the translocation of p65 into the nucleus and phosphorylation of p65 in the nucleus from the injured DRG neurons. Our findings suggest that DRG TLR7 contributes to neuropathic pain by activating NF-κB in primary sensory neurons. TLR7 may be a potential target for therapeutic treatment of this disorder.

Long noncoding RNA H19 in the injured dorsal root ganglion contributes to peripheral nerve injury-induced pain hypersensitivity.

Peripheral nerve injury-induced changes in gene transcription and translation in the dorsal root ganglion (DRG) play a critical role in the development and maintenance of neuropathic pain. Long noncoding RNAs (lncRNAs) regulate gene expression. Here, we report that peripheral nerve injury caused by ligation of the fourth spinal nerve (SNL) led to a time-dependent increase in the expression in H19, an lncRNA, in the injured DRG. Microinjection of a specific H19 siRNA, but not negative control scrambled siRNA, into the injured DRG 4 days before SNL alleviated mechanical allodynia and thermal hyperalgesia on days 3 and 5 post-SNL. Additionally, DRG microinjection of the H19 siRNA on day 7 after SNL reduced mechanical allodynia and thermal hyperalgesia on days 10 and 12 post-SNL. DRG microinjection of neither siRNA affected locomotor activity and acute basal responses to mechanical and thermal stimuli. Our findings suggest that H19 participates in the peripheral mechanism underlying the development and maintenance of neuropathic pain. H19 may be a potential target for treatment of this disorder.

Alternative Splicing of Nrcam Gene in Dorsal Root Ganglion Contributes to Neuropathic Pain.

NrCAM, a neuronal cell adhesion molecule in the L1 family of the immunoglobulin superfamily, is subjected to extensively alternative splicing and involved in neural development and some disorders. The aim of this study was to explore the role of Nrcam mRNA alternative splicing in neuropathic pain. A next generation RNA sequencing analysis of dorsal root ganglions (DRGs) showed the differential expression of two splicing variants of Nrcam, Nrcam+10 and Nrcam-10, in the injured DRG after the fourth lumbar spinal nerve ligation (SNL) in mice. SNL increased the exon 10 insertion, resulting in an increase in the amount of Nrcam+10 and a corresponding decrease in the level of Nrcam-10 in the injured DRG. An antisense oligonucleotide (ASO) that specifically targeted exon 10 of Nrcam gene (Nrcam ASO) repressed RNA expression of Nrcam+10 and increased RNA expression of Nrcam-10 in in vitro DRG cell culture. Either DRG microinjection or intrathecal injection of Nrcam ASO attenuated SNL-induced the development of mechanical allodynia, thermal hyperalgesia, or cold allodynia. Nrcam ASO also relieved SNL- or chronic compression of DRG (CCD)-induced the maintenance of pain hypersensitivities in male and female mice. PERSPECTIVE: We conclude that the relative levels of alternatively spliced Nrcam variants are critical for neuropathic pain genesis. Targeting Nrcam alternative splicing via the antisense oligonucleotides may be a new potential avenue in neuropathic pain management.