Adult Rat Dorsal Root Ganglion Neurons Research Articles

Piezo1 Regulates Stiffness-Dependent DRG Axon Regeneration via Modifying Cytoskeletal Dynamics.

Despite medical interventions, the regenerative capacity of the peripheral nervous system is limited. Dorsal root ganglion (DRG) neurons possess the capacity to detect mechanical signals from their microenvironment, but the impact and mechanism by which these signals regulate axon regrowth and even regeneration in DRG neurons remain unclear. In this study, DRG neurons from newborn rats are cultured on substrates with varying degrees of stiffness in vitro to investigate the role of mechanical signals in axon regrowth. The findings reveal that substrate stiffness plays a crucial role in regulating axon regrowth, with an optimal stiffness required for this process. In addition, the data demonstrate that Piezo1, a mechanosensitive cation channel, detects substrate stiffness at the growth cone and regulates axon regrowth through activating downstream Ca2+-CaMKII-FAK-actin cascade signaling pathway. Interestingly, knocking down Piezo1 in adult rat DRG neurons leads to enhanced axon regeneration and accelerated recovery of sensory function after sciatic nerve injury. Overall, these findings contribute to the understanding of the role of mechanical signals in axon regeneration and highlight microenvironmental stiffness as a promising therapeutic target for repairing nerve injuries.

A Live Imaging Assay for Regenerating Peripheral Neurons.

The cell intrinsic mechanisms directing peripheral nerve regeneration have remained largely understudied, thus limiting our understanding of these processes and constraining the advancement of novel clinical therapeutics. The use of primary adult rat dorsal root ganglion (DRG) neurons cultured in vitro is well established. Despite this, these cells can be challenging to culture and have so far not been amenable to robust transfection or live-cell imaging. The ability to transfect these cells with fluorescent plasmid constructs to label subcellular structures, combined with high resolution time-lapse imaging has the potential to provide invaluable insight into how peripheral neurons coordinate their regenerative response, and which specific cellular structures are involved in this process. Here we describe a protocol that facilitates transfection and subsequent live-imaging of adult rat DRG neurons.

TSLP in DRG neurons causes the development of neuropathic pain through T cells

BackgroundPeripheral nerve injury to dorsal root ganglion (DRG) neurons develops intractable neuropathic pain via induction of neuroinflammation. However, neuropathic pain is rare in the early life of rodents. Here, we aimed to identify a novel therapeutic target for neuropathic pain in adults by comprehensively analyzing the difference of gene expression changes between infant and adult rats after nerve injury.MethodsA neuropathic pain model was produced in neonatal and young adult rats by spared nerve injury. Nerve injury-induced gene expression changes in the dorsal root ganglion (DRG) were examined using RNA sequencing. Thymic stromal lymphopoietin (TSLP) and its siRNA were intrathecally injected. T cells were examined using immunofluorescence and were reduced by systemic administration of FTY720.ResultsDifferences in changes in the transcriptome in injured DRG between infant and adult rats were most associated with immunological functions. Notably, TSLP was markedly upregulated in DRG neurons in adult rats, but not in infant rats. TSLP caused mechanical allodynia in adult rats, whereas TSLP knockdown suppressed the development of neuropathic pain. TSLP promoted the infiltration of T cells into the injured DRG and organized the expressions of multiple factors that regulate T cells. Accordingly, TSLP caused mechanical allodynia through T cells in the DRG.ConclusionThis study demonstrated that TSLP is causally involved in the development of neuropathic pain through T cell recruitment.

The Endocannabinoid Analgesic Entourage Effect: Investigations in Cultured DRG Neurons.

The endocannabinoid 2-Arachidonyl glycerol (2-AG) exerts dose-related anti-nociceptive effects, which are potentiated by the related but inactive 2-palmitoyl glycerol (2-PG) and 2-linoleoyl glycerol (2-LG). This potentiation of analgesia and other in vivo measures was described as the "entourage effect". We investigated this effect on TRPV1 signalling in cultured dorsal root ganglion (DRG) nociceptors. Adult rat DRG neurons were cultured in medium containing NGF and GDNF at 37°C. 48 h later cultures were loaded with 2 µM Fura2AM for calcium imaging, and treated with 2-AG, 2-PG and 2-LG, individually or combined, for 5 min, followed by 1 µMol capsaicin. The amplitude and latency of capsaicin responses were measured (N=3-7 rats, controls N=16), and analysed. In controls, 1 µMol capsaicin elicited immediate calcium influx in a subset of neurons, with average latency of 1.27 ± 0.2 s and amplitude of 0.15 ± 0.01 Units. 2-AG (10-100 µMol) elicited calcium influx in some neurons. In the presence of 2-AG (0.001-100 µMol), capsaicin responses were markedly delayed in 64% neurons by up to 320 s (P<0.001). 2-PG increased capsaicin response latency at 0.1 nMol-100 µMol (P<0.001), in 60% neurons, as did 2-LG at 0.1-100 µMol (P<0.001), in 76% neurons. Increased capsaicin response latency due to 2-AG and 2-PG was sensitive to the CB2 but not to the CB1 receptor antagonist. Combined application of 1 µMol 2-AG, 5 µMol 2-PG and 10 µMol 2-LG, also resulted in significantly increased capsaicin response latency up to 281.5 ± 41.5 s (P<0.001), in 96% neurons, that was partially restored by the CB2, but not the CB1 antagonist. 2-AG, 2-LG and 2-PG significantly delayed TRPV1 signalling in the majority of capsaicin-sensitive DRG neurons, that was markedly increased following combined application. Further studies of these endocannabinoids are required to identify the underlying mechanisms.

Pretreatment with Zonisamide Mitigates Oxaliplatin-Induced Toxicity in Rat DRG Neurons and DRG Neuron–Schwann Cell Co-Cultures

Oxaliplatin (OHP) is a platinum-based agent that can cause peripheral neuropathy, an adverse effect in which the dorsal root ganglion (DRG) neurons are targeted. Zonisamide has exhibited neuroprotective activities toward adult rat DRG neurons in vitro and therefore, we aimed to assess its potential efficacy against OHP-induced neurotoxicity. Pretreatment with zonisamide (100 μM) alleviated the DRG neuronal death caused by OHP (75 μM) and the protective effects were attenuated by a co-incubation with 25 μM of the mitogen-activated protein kinase (MAPK; MEK/ERK) inhibitor, U0126, or the phosphatidyl inositol-3′-phosphate-kinase (PI3K) inhibitor, LY294002. Pretreatment with zonisamide also suppressed the OHP-induced p38 MAPK phosphorylation in lined DRG neurons, ND7/23, while the OHP-induced DRG neuronal death was alleviated by pretreatment with the p38 MAPK inhibitor, SB239063 (25 μM). Although zonisamide failed to protect the immortalized rat Schwann cells IFRS1 from OHP-induced cell death, it prevented neurite degeneration and demyelination-like changes, as well as the reduction of the serine/threonine-specific protein kinase (AKT) phosphorylation in DRG neuron–IFRS1 co-cultures exposed to OHP. Zonisamide’s neuroprotection against the OHP-induced peripheral sensory neuropathy is possibly mediated by a stimulation of the MEK/ERK and PI3K/AKT signaling pathways and suppression of the p38 MAPK pathway in DRG neurons. Future studies will allow us to solidify zonisamide as a promising remedy against the neurotoxic adverse effects of OHP.

5-HT3 Receptors in Rat Dorsal Root Ganglion Neurons: Ca2+ Entry and Modulation of Neurotransmitter Release.

Rat dorsal root ganglion (DRG) neurons express 5-hydroxytryptamine receptors (5-HT3Rs). To elucidate their physiological role in the modulation of sensory signaling, we aimed to quantify their functional expression in newborn and adult rat DRG neurons, as well as their ability to modulate the Ca2+-dependent neurotransmitter release, by means of electrophysiological techniques combined with fluorescence-based Ca2+ imaging. The selective 5-HT3R agonist mCPBG (10 μM) elicited whole-cell currents in 92.5% of adult DRG neurons with a significantly higher density current than in responding newborn cells (52.2%), suggesting an increasing serotoninergic modulation on primary afferent cells during development. Briefly, 5-HT3Rs expressed by adult DRG neurons are permeable to Ca2+ ions, with a measured fractional Ca2+ current (i.e., the percentage of total current carried by Ca2+ ions, Pf) of 1.0%, similar to the value measured for the human heteromeric 5-HT3A/B receptor (Pf = 1.1%), but lower than that of the human homomeric 5-HT3A receptor (Pf = 3.5%). mCPBG applied to co-cultures of newborn DRG and spinal neurons significantly increased the miniature excitatory postsynaptic currents (mEPSCs) frequency in a subset of recorded spinal neurons, even in the presence of Cd2+, a voltage-activated Ca2+ channel blocker. Considered together, our findings indicate that the Ca2+ influx through heteromeric 5-HT3Rs is sufficient to increase the spontaneous neurotransmitter release from DRG to spinal neurons.

Glycolaldehyde induces sensory neuron death through activation of the c-Jun N-terminal kinase and p-38 MAP kinase pathways.

Glycolaldehyde (GA) is a highly reactive hydroxyaldehyde and one of the glycolytic metabolites producing advanced glycation endproducts (AGEs), but its toxicity toward neurons and Schwann cells remains unclear. In the present study, we found that GA exhibited more potent toxicity than other AGE precursors (glyceraldehyde, glyoxal, methylglyoxal and 3-deoxyglucosone) against immortalized IFRS1 adult rat Schwann cells and ND7/23 neuroblastoma × neonatal rat dorsal root ganglion (DRG) neuron hybrid cells. GA affected adult rat DRG neurons and ND7/23 cells more severely than GA-derived AGEs, and exhibited concentration- and time-dependent toxicity toward ND7/23 cells (10 < 100 < 250 < 500µM; 6h < 24h). Treatment with 500µM GA significantly up-regulated the phosphorylation of c-jun N-terminal kinase (JNK) and p-38 mitogen-activated kinase (p-38 MAPK) in ND7/23 cells. Furthermore, GA-induced ND7/23 cell death was significantly inhibited due to co-treatment with 10µM of the JNK inhibitor SP600125 or the p-38 MAPK inhibitor SB239063. These findings suggest the involvement of JNK and p-38 MAPK-signaling pathways in GA-induced neuronal cell death and that enhanced GA production under diabetic conditions might be involved in the pathogenesis of diabetic neuropathy.

Granulocyte-macrophage colony-stimulating factor receptor expression in clinical pain disorder tissues and role in neuronal sensitization.

Granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) is highly expressed in peripheral macrophages and microglia, and is involved in arthritis and cancer pain in animal models. However, there is limited information on GM-CSFR expression in human central nervous system (CNS), peripheral nerves, or dorsal root ganglia (DRG), particularly in chronic pain conditions. Immunohistochemistry was used to quantify GM-CSFR expression levels in human tissues, and functional sensory effects of GM-CSF were studied in cultured DRG neurons. Granulocyte-macrophage colony-stimulating factor receptor was markedly increased in microglia at lesional sites of multiple sclerosis spinal cords (P = 0.01), which co-localised with macrophage marker CD68 (P = 0.009). In human DRG, GM-CSFR was expressed in a subset of small/medium diameter cells (30%) and few large cells (10%), with no significant change in avulsion-injured DRG. In peripheral nerves, there was a marked decrease in axonal GM-CSFR after chronic painful nerve injury (P = 0.004) and in painful neuromas (P = 0.0043); CD-68-positive macrophages were increased (P = 0.017) but did not appear to express GM-CSFR. Although control synovium showed absent GM-CSFR immunostaining, this was markedly increased in macrophages of painful osteoarthritis knee synovium. Granulocyte-macrophage colony-stimulating factor receptor was expressed in 17 ± 1.7% of small-/medium-sized cultured adult rat DRG neurons, and in 27 ± 3.3% of TRPV1-positive neurons. Granulocyte-macrophage colony-stimulating factor treatment sensitized capsaicin responses in vitro, which were diminished by p38 MAPK or TrkA inhibitors. Our findings support GM-CSFR as a therapeutic target for pain and hypersensitivity in clinical CNS and peripheral inflammatory conditions. Although GM-CSFR was decreased in chronic painful injured peripheral nerves, it could mediate CNS neuroinflammatory effects, which deserves study.

Peripheral nerve injury induces loss of nociceptive neuron-specific Gαi-interacting protein in neuropathic pain rat.

BackgroundGαi-interacting protein (GINIP) is expressed specifically in dorsal root ganglion (DRG) neurons and functions in modulation of peripheral gamma-aminobutyric acid B receptor (GBR). Genetic deletion of GINIP leads to impaired responsiveness to GBR agonist-mediated analgesia in rodent. It is, however, not defined whether nerve injury changes GINIP expression.ResultsImmunolabeling with validated antibody revealed GINIP expression in ∼40% of total lumbar DRG neurons in normal adult rats. GINIP immunoreactivity was detected in ∼80% of IB4-positive (nonpeptidergic) and ∼30% of CGRP-positive (peptidergic) neurons. GINIP immunoreactivity in the spinal cord dorsal horn was colabeled with IB4 and partially with CGRP. In addition, GINIP was expressed in DRG neurons immunopositive for GBR1, GBR2, Gαi(s), and Gαo and was also extensively colabeled with multiple nociceptive neuronal markers, including Trpv1, NaV1.7, CaV2.2α1b, CaV3.2α1b, TrkA, and Trek2. Peripheral nerve injury by L5 spinal nerve ligation significantly decreased the proportion of GINIP immunoreactivity-positive neurons from 40 ± 8.4% to 0.8 ± 0.1% (p < 0.01, mean ± SD, four weeks after spinal nerve ligation) and the total GINIP protein to 1.3% ± 0.04% of its basal level (p < 0.01, n = 6 animals in each group, two weeks after spinal nerve ligation) in the ipsilateral L5 DRGs.ConclusionOur results show that GINIP is predominantly expressed by small nonpeptidergic nociceptive neurons and that nerve injury triggers loss of GINIP expression. Signal transduction roles of GINIP may be diverse as it colabeled with various subgroups of nociceptive neurons. Future studies may investigate details of the signaling mechanism engaged by GINIP, as well as the pathophysiological significance of lost expression of GINIP in neuropathic pain.

Full length talin stimulates integrin activation and axon regeneration.

Integrin function is regulated by activation involving conformational changes that modulate ligand-binding affinity and downstream signaling. Activation is regulated through inside-out signaling which is controlled by many signaling pathways via a final common pathway through kindlin and talin, which bind to the intracellular tail of beta integrins. Previous studies have shown that the axon growth inhibitory molecules NogoA and chondroitin sulfate proteoglycans (CSPGs) inactivate integrins. Overexpressing kindlin-1 in dorsal root ganglion (DRG) neurons activates integrins, enabling their axons to overcome inhibitory molecules in the environment, and promoting regeneration in vivo following dorsal root crush. Other studies have indicated that expression of the talin head alone or with kindlin can enhance integrin activation. Here, using adult rat DRG neurons, we investigate the effects of overexpressing various forms of talin on axon growth and integrin signaling. We found that overexpression of the talin head activated axonal integrins but inhibited downstream signaling via FAK, and did not promote axon growth. Similarly, co-expression of the talin head and kindlin-1 prevented the growth-promoting effect of kindlin-1, suggesting that the talin head acts as a form of dominant negative for integrin function. Using full-length talin constructs in PC12 cells we observed that neurite growth was enhanced by the expression of wild-type talin and more so by two ‘activated’ forms of talin produced by point mutation (on laminin and aggrecan–laminin substrates). Nevertheless, co-expression of full-length talin with kindlin did not promote neurite growth more than either molecule alone. In vivo, we find that talin is present in PNS axons (sciatic nerve), and also in CNS axons of the corticospinal tract.

Efficacy of glucagon-like peptide-1 mimetics for neural regeneration.

Glucagon-like peptide 1 (GLP-1) is secreted from enteroendocrine L cells in response to nutrient ingestion and exhibits insulinotropic properties by stimulating specific G protein-linked receptors (GLP-1Rs) on pancreatic β cells. Several GLP-1 mimetics, such as exenatide (exendin-4 (Ex-4)), liraglutide, and lixisenatide, have been developed and approved as treatments for patients with type 2 diabetes. These peptides show bioactivities almost identical to those of GLP-1 and have a substantially longer plasma half-life than GLP-1 because of their resistance to dipeptidyl peptidase-4, a GLP-1 degrading enzyme. GLP-1Rs are found in not only the pancreas but also the extrapancreatic tissues, including the nervous tissues (Harkavyi and Whitton, 2010). It is important to note that GLP-1 mimetics can cross the blood brain barrier and directly act on neurons in the central nervous system. In addition to the inhibition of appetite, the neuroprotective properties of GLP-1 have been receiving increasing attention. Recent studies have suggested that GLP-1 mimetics confer beneficial effects in neurodegenerative disorders, such as Parkinson's disease (PD), Alzheimer's disease, amyotrophic lateral sclerosis, ischemia and stroke, and multiple sclerosis (Holscher, 2014). In particular, the neuroprotective properties of Ex-4 have been demonstrated in animal and cell culture models of PD. A single-blinded clinical trial with 45 PD patients revealed that the treatment with Ex-4 significantly improved the cognition and memory of patients (Aviles-Olmos et al., 2013). The beneficial effects of GLP-1 mimetics on the peripheral nervous system (PNS) have also been reported. Both GLP-1 and Ex-4 delivered via osmotic minipumps prevented axonal degeneration in a rat model of pyridoxine-induced neuropathy (Perry et al., 2007). Treatment of streptozotocin (STZ)-induced diabetic mice with Ex-4 for 4 weeks restored motor and sensory nerve conduction velocities and hypoalgesia without normalizing blood glucose levels (Himeno et al., 2011). In addition, repeated intraperitoneal injections of Ex-4 significantly promoted axonal regeneration and functional recovery following sciatic nerve crush injury in normal adult rats (Yamamoto et al., 2013). These findings are in agreement with in vitro studies that revealed that GLP-1 and Ex-4 promoted neurite outgrowth of rat pheochromocytoma-derived PC12 cells (Perry et al., 2002) and adult mouse dorsal root ganglion (DRG) neurons (Himeno et al., 2011). Together these results provide further evidence of the direct actions of Ex-4 on the PNS; however, the underlying mechanisms remain unclear. Our recent study (Tsukamoto et al., 2015) aimed to elucidate the precise localization of GLP-1R in adult rat DRG in vivo and in vitro as well as to determine the neurotrophic and neuroprotective properties of Ex-4 in adult rat DRG neurons.

SrGAP3 promotes neurite outgrowth of dorsal root ganglion neurons by inactivating RAC1

ObjectiveTo explore effect of srGAP3 promotes neurite outgrowth of dorsal root ganglion neurons. MethodsIn this study, expression of Slit1 was observed predominantly in the glia, while expression of Robo2 and srGAP3 was detected in sensory neurons of postnatal rat cultured dorsal root ganglion (DRG). Furthermore, upregulation of srGAP3 following sciatic nerve transection was detected by immunohistochemistry and Western blotting. ResultsIt was observed that inhibition of neurite outgrowth in cultured adult DRG neurons following treatment with anti-srGAP3 or anti-Robo2 was more effectively (1.5-fold higher) than that following treatment with an anti-BDNF positive control antibody. It demonstrated that srGAP3 interacted with Robo2 and Slit1 protein to decrease Rac1-GTP activity in cultured adult rat DRG neurons and the opposite effect on Rac1-GTP activity was detected by co-immunoprecipitation and immunoblotting analyses following treatment with anti-Robo2 or anti-srGAP3. These data demonstrated a role for srGAP3 in neurite outgrowth of DRG sensory neurons. ConclusionsOur observations suggest that srGAP3 promotes neurite outgrowth and filopodial growth cones by interacting with Robo2 to inactivate Rac1 in mammalian DRG neurons.

Down-regulation of A-type potassium channel in gastric-specific DRG neurons in a rat model of functional dyspepsia.

Although without evidence of organic structural abnormalities, pain or discomfort is a prominent symptom of functional dyspepsia and considered to reflect visceral hypersensitivity whose underlying mechanism is poorly understood. Here, we studied electrophysiological properties and expression of voltage-gated potassium channels in dorsal root ganglion (DRG) neurons in a rat model of functional dyspepsia induced by neonatal gastric irritation. Male Sprague-Dawley rat pups at 10-day old received 0.1% iodoacetamide (IA) or vehicle by oral gavage for 6 days and studied at adulthood. Retrograde tracer-labeled gastric-specific T8 -T12 DRG neurons were harvested for the patch-clamp study in voltage and current-clamp modes and protein expression of K(+) channel in T8 -T12 DRGs was examined by western blotting. (1) Gastric specific but not non-gastric DRG neurons showed an enhanced excitability in neonatal IA-treated rats compared to the control: depolarized resting membrane potentials, a lower current threshold for action potential (AP) activation, and an increase in the number of APs in response to current stimulation. (2) The current density of tetraethylammonium insensitive (transiently inactivating A-type current), but not the tetraethylammonium sensitive (slow-inactivating delayed rectifier K(+) currents), was significantly smaller in IA-treated rats (65.4 ± 6.9 pA/pF), compared to that of control (93.1 ± 8.3 pA/pF). (3) Protein expression of KV 4.3 was down-regulated in IA-treated rats. A-type potassium channels are significantly down-regulated in the gastric-specific DRG neurons in adult rats with mild neonatal gastric irritation, which in part contribute to the enhanced DRG neuron excitabilities that leads to the development of gastric hypersensitivity.

Despite Differences in Cytosolic Calcium Regulation, Lidocaine Toxicity Is Similar in Adult and Neonatal Rat Dorsal Root Ganglia In Vitro

Neuraxial local anesthetics may have neurological complications thought to be due to neurotoxicity. A primary site of action of local anesthetics is the dorsal root ganglia (DRG) neuron. Physiologic differences have been noted between young and adult DRG neurons; hence, the authors examined whether there were any differences in lidocaine-induced changes in calcium and lidocaine toxicity in neonatal and adult rat DRG neurons. DRG neurons were cultured from postnatal day 7 (P7) and adult rats. Lidocaine-induced changes in cytosolic calcium were examined with the calcium indicator Fluo-4. Cells were incubated with varying concentrations of lidocaine and examined for viability using calcein AM and ethidium homodimer-1 staining. Live imaging of caspase-3/7 activation was performed after incubation with lidocaine. The mean KCl-induced calcium transient was greater in P7 neurons (P < 0.05), and lidocaine significantly inhibited KCl-induced calcium responses in both ages (P < 0.05). Frequency distribution histograms of KCl-evoked calcium increases were more heterogeneous in P7 than in adult neurons. With lidocaine, KCl-induced calcium transients in both ages became more homogeneous but remained different between the groups. Interestingly, cell viability was decreased by lidocaine in a dose-dependent manner similarly in both ages. Lidocaine treatment also activated caspase-3/7 in a dose- and time-dependent manner similarly in both ages. Despite physiological differences in P7 and adult DRG neurons, lidocaine cytotoxicity is similar in P7 and adult DRG neurons in vitro. Differences in lidocaine- and KCl-evoked calcium responses suggest the similarity in lidocaine cytotoxicity involves other actions in addition to lidocaine-evoked effects on cytosolic calcium responses.

Nerve growth factor acts through the TrkA receptor to protect sensory neurons from the damaging effects of the HIV-1 viral protein, Vpr

Distal sensory polyneuropathy (DSP) with associated neuropathic pain is the most common neurological disorder affecting patients with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). Viral protein R (Vpr) is a neurotoxic protein encoded by HIV-1 and secreted by infected macrophages. Vpr reduces neuronal viability, increases cytosolic calcium and membrane excitability of cultured dorsal root ganglion (DRG) sensory neurons, and is associated with mechanical allodynia in vivo. A clinical trial with HIV/AIDS patients demonstrated that nerve growth factor (NGF) reduced the severity of DSP-associated neuropathic pain, a problem linked to damage to small diameter, potentially NGF-responsive fibers. Herein, the actions of NGF were investigated in our Vpr model of DSP and we demonstrated that NGF significantly protected sensory neurons from the effects of Vpr. Footpads of immunodeficient Vpr transgenic (vpr/RAG1−/−) mice displayed allodynia (p<0.05), diminished epidermalinnervation (p<0.01) and reduced NGF mRNA expression (p<0.001) compared to immunodeficient (wildtype/RAG1−/−) littermate control mice. Compartmented cultures confirmed recombinant Vpr exposure to the DRG neuronal perikarya decreased distal neurite extension (p<0.01), whereas NGF exposure at these distal axons protected the DRG neurons from the Vpr-induced effect on their cell bodies. NGF prevented Vpr-induced attenuation of the phosphorylated glycogen synthase-3 axon extension pathway and tropomyosin-related kinase A (TrkA) receptor expression in DRG neurons (p<0.05) and it directly counteracted the cytosolic calcium burst caused by Vpr exposure to DRG neurons (p<0.01). TrkA receptor agonist indicated that NGFacted through the TrkA receptor to block the Vpr-mediated decrease in axon outgrowth in neonatal and adult rat and fetal human DRG neurons (p<0.05). Similarly, inhibiting the lower affinity NGF receptor, p75, blocked Vpr’s effect on DRG neurons. Overall, NGF/TrkA signaling or p75 receptor inhibition protects somatic sensory neurons exposed to Vpr, thus laying the groundwork for potential therapeutic options for HIV/AIDS patients suffering from DSP.

GDNF promotes neurite outgrowth and upregulates galectin-1 through the RET/PI3K signaling in cultured adult rat dorsal root ganglion neurons

Galectin-1 (GAL-1), a member of a family of β-galactoside binding animal lectins, is predominantly expressed in isolectin B4 (IB4)-binding small non-peptidergic (glial cell line-derived neurotrophic factor (GDNF)-responsive) sensory neurons in the sections of adult rat dorsal root ganglia (DRG), but its functional role and the regulatory mechanisms of its expression in the peripheral nervous system remain unclear. In the present study, both recombinant nerve growth factor (NGF) and GDNF (50ng/ml) promoted neurite outgrowth from cultured adult rat DRG neurons, whereas GDNF, but not NGF, significantly increased the number of IB4-binding neurons and the relative protein expression of GAL-1 in the neuron-enriched culture of DRG. The GAL-1 expression in immortalized adult rat Schwann cells IFRS1 and DRG neuron-IFRS1 cocultures was unaltered by treatment with GDNF, which suggests that GDNF/GAL-1 signaling axis is more related to neurite outgrowth, rather than neuron-Schwann cell interactions. The GDNF-induced neurite outgrowth and GAL-1 upregulation were attenuated by anti-GDNF family receptor (RET) antibody and phosphatidyl inositol-3′-phosphate-kinase (PI3K) inhibitor LY294002, suggesting that the neurite-outgrowth promoting activity of GDNF may be attributable, at least partially, to the upregulation of GAL-1 through RET-PI3K pathway. On the contrary, no significant differences were observed between GAL-1 knockout and wild-type mice in DRG neurite outgrowth in the presence or absence of GDNF. Considerable immunohistochemical colocalization of GAL-3 with GAL-1 in DRG sections and GDNF-induced upregulation of GAL-3 in cultured DRG neurons imply the functional redundancy between these galectins.

シュワン細胞株をもちいた末梢神経再生機構の解析

We have established spontaneously immortalized Schwann cell lines from adult ICR mice [IMS32] and Fischer344 rats [IFRS1]. IMS32 cells display distinct Schwann cell phenotypes such as a spindle-shaped morphology and the expression of glial cell markers (e.g. S100, glial fibrillary acidic protein (GFAP), p75 low-affinity neurotrophin receptor (p75(NTR))) and neurotrophic factors. In addition, conditioned medium obtained from IMS32 cells enhances neurite elongation of PC12 cells and mouse dorsal root ganglion (DRG) neurons. IMS32 cells have been utilized to investigate the action mechanisms of various molecules that accelerate peripheral nerve regeneration (e.g. ciliary neurotrophic factor, sonic hedgehog, galectin-1). Like IMS32 cells, IFRS1 cells retain the characteristic features of mature Schwann cells as described above. Furthermore, IFRS1 cells have been shown to myelinate neurites in coculture with adult rat DRG neurons and PC12 cells. Our current investigation with IFRS1 cells focuses on the molecular mechanisms of myelination-inducible factors, such as soluble neuregulin-1 type III and exendin-4. These Schwann cell lines can be valuable tools for exploring neuron-Schwann cell interactions, pathobiology of axonal degeneration and regeneration in the peripheral nervous system, and novel therapeutic approaches against neurological disorders in patients with relevant diseases.

Axotomy-induced miR-21 promotes axon growth in adult dorsal root ganglion neurons.

Following injury, dorsal root ganglion (DRG) neurons undergo transcriptional changes so as to adopt phenotypic changes that promote cell survival and axonal regeneration. Here we used a microarray approach to profile changes in a population of small noncoding RNAs known as microRNAs (miRNAs) in the L4 and L5 DRG following sciatic nerve transection. Results showed that 20 miRNA transcripts displayed a significant change in expression levels, with 8 miRNAs transcripts being altered by more than 1.5-fold. Using quantitative reverse transcription PCR, we demonstrated that one of these miRNAs, miR-21, was upregulated by 7-fold in the DRG at 7 days post-axotomy. In dissociated adult rat DRG neurons lentiviral vector-mediated overexpression of miR-21 promoted neurite outgrowth on a reduced laminin substrate. miR-21 directly downregulated expression of Sprouty2 protein, as confirmed by Western blot analysis and 3′ untranslated region (UTR) luciferase assays. Our data show that miR-21 is an axotomy-induced miRNA that enhances axon growth, and suggest that miRNAs are important players in regulating growth pathways following peripheral nerve injury.

Upregulation of Nav1.3 Channel Induced by rrTNF in Cultured Adult Rat DRG Neurons via p38 MAPK and JNK Pathways

Activation of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal protein kinase (JNK) in the dorsal root ganglia (DRG) is critical for the development of neuropathic pain. Tetraodontoxin-sensitive Nav1.3 channel, expressed at a very low level in the adult nervous system, is up-regulated in DRG neurons after peripheral nerve injury or peri-sciatic administration of rat recombinant tumour necrosis factor-alpha (rrTNF-α). To test if activation of p38 MAPK and JNK is required for the re-expression of Nav1.3 channel in cultured adult rat DRG neurons, we administrated rrTNF to cultured adult rat DRG neurons to induce Nav1.3 re-expression, and pre-treated with p38 MAPK inhibitor (SB203580 at 2.65, 26.5 and 265 μM) or JNK inhibitor (SP600125 at 1, 10 and 100 μM) 2 h before rrTNF to observe changes of Nav1.3-immunoreactivity. Compared with the DMSO vehicle pre-treatment group, SB203580 at 2.65 μM partially blocked the re-expression of Nav1.3 (P<0.001), and at 26.5 and 265 μM completely blocked Nav1.3 (P<0.001). Similarly, SP600125 at the concentration of 1 μM blocked the re-expression of Nav1.3 partially (P<0.001), and at 10 and 100 μM blocked Nav1.3 completely (P<0.001). These data show that the activation of both p38 MAPK and JNK in DRG neurons was involved in the re-expression of Nav1.3 channel triggered by TNF-α, which might contribute to neuropathic pain.

Na+ Channel Scn1b Gene Regulates Dorsal Root Ganglion Nociceptor Excitability in Vivo

Nociceptive dorsal root ganglion (DRG) neurons express tetrodotoxin-sensitive (TTX-S) and -resistant (TTX-R) Na(+) current (I(Na)) mediated by voltage-gated Na(+) channels (VGSCs). In nociceptive DRG neurons, VGSC β2 subunits, encoded by Scn2b, selectively regulate TTX-S α subunit mRNA and protein expression, ultimately resulting in changes in pain sensitivity. We hypothesized that VGSCs in nociceptive DRG neurons may also be regulated by β1 subunits, encoded by Scn1b. Scn1b null mice are models of Dravet Syndrome, a severe pediatric encephalopathy. Many physiological effects of Scn1b deletion on CNS neurons have been described. In contrast, little is known about the role of Scn1b in peripheral neurons in vivo. Here we demonstrate that Scn1b null DRG neurons exhibit a depolarizing shift in the voltage dependence of TTX-S I(Na) inactivation, reduced persistent TTX-R I(Na), a prolonged rate of recovery of TTX-R I(Na) from inactivation, and reduced cell surface expression of Na(v)1.9 compared with their WT littermates. Investigation of action potential firing shows that Scn1b null DRG neurons are hyperexcitable compared with WT. Consistent with this, transient outward K(+) current (I(to)) is significantly reduced in null DRG neurons. We conclude that Scn1b regulates the electrical excitability of nociceptive DRG neurons in vivo by modulating both I(Na) and I(K).