Establishing a Herpesvirus Quiescent Infection in Differentiated Human Dorsal Root Ganglion Neuronal Cell Line Mediated by Micro-RNA Overexpression
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ABSTRACT:
SUBMITTER: Chen Y
PROVIDER: S-EPMC9317301 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The molecular mechanisms of pain associated with alphaherpesvirus latency are not clear. We hypothesize that the voltage-gated sodium channels (VGSC) on the dorsal root ganglion (DRG) neurons controlling electrical impulses may have abnormal activity during latent viral infection and reactivation. We used herpes simplex virus 1 (HSV-1) to infect the human DRG-derived neuronal cell line HD10.6 in order to study the establishment and maintenance of viral latency, viral reactivation, and changes in the functional expression of VGSCs. Differentiated cells exhibited robust tetrodotoxin (TTX)-sensitive sodium currents, and acute infection significantly reduced the functional expression of VGSCs within 24 h and completely abolished VGSC activity within 3 days. A quiescent state of infection mimicking latency can be achieved in the presence of acyclovir (ACV) for 7 days followed by 5 days of ACV washout, and then the viruses can remain dormant for another 3 weeks. It was noted that during the establishment of HSV-1 latency, the loss of VGSC activity caused by HSV-1 infection could not be blocked by ACV treatment. However, neurons with continued ACV treatment for another 4 days showed a gradual recovery of VGSC functional expression. Furthermore, the latently infected neurons exhibited higher VGSC activity than controls. The overall regulation of VGSCs by HSV-1 during quiescent infection was proved by increased transcription and possible translation of Nav1.7. Together, these observations demonstrated a very complex pattern of electrophysiological changes during HSV infection of DRG neurons, which may have implications for understanding of the mechanisms of virus-mediated pain linked to latency and reactivation.IMPORTANCE The reactivation of herpesviruses, most commonly varicella-zoster virus (VZV) and pseudorabies virus (PRV), may cause cranial nerve disorder and unbearable pain. Clinical studies have also reported that HSV-1 causes postherpetic neuralgia and chronic occipital neuralgia in humans. The current work meticulously studies the functional expression profile changes of VGSCs during the processes of HSV-1 latency establishment and reactivation using human dorsal root ganglion-derived neuronal HD10.6 cells as an in vitro model. Our results indicated that VGSC activity was eliminated upon infection but steadily recovered during latency establishment and that latent neurons exhibited even higher VGSC activity. This finding advances our knowledge of how ganglion neurons generate uncharacteristic electrical impulses due to abnormal VGSC functional expression influenced by the latent virus.
| S-EPMC7000965 | biostudies-literature
Project description:Our study focuses on characterization of dorsal root ganglion (DRG) neurons cultured on silicon micro-pillar substrates (MPS) with the ultimate goal of designing micro-electrode arrays (MEAs) for successful electrophysiological recordings of DRG neurons. Adult and neonatal DRG neurons were cultured on MPS and glass coverslips for 7 days in vitro. DRG neuronal distribution and morphometric analysis, including neurite alignment and length, was performed on MPS areas with different pillar width and spacing. We showed that MPS provide an environment for growth of adult and neonatal DRG neurons as permissive as control glass surfaces. Neonatal DRG neurons were present on MPS areas with narrow pillar spacing, while adult neurons preferred wider pillar spacing. Compared to the control glass surfaces the neonatal and adult DRG neurons in regions with narrow pillar spacing range developed a smaller number of longer neurites. In the same area, neurites were preferentially oriented along three directional axes at 30°, 90° and 150°. MPS architecture influenced growth directionality of all main DRG neuronal subtypes. We can conclude that specific micro-pillar substrate topography affects the morphology of DRG neurons. This knowledge can enable development of MEAs with precisely defined physical features for various neuroscience applications.
| S-EPMC5180168 | biostudies-literature
Project description:The isolation and culture of dorsal root ganglion (DRG) neurons cause adaptive changes in the expression and regulation of ion channels, with consequences on neuronal excitability. Considering that not all neurons survive the isolation and that DRG neurons are heterogeneous, it is difficult to find the cellular subtype of interest. For this reason, researchers opt for DRG-derived immortal cell lines to investigate endogenous properties. The F-11 cell line is a hybridoma of embryonic rat DRG neurons fused with the mouse neuroblastoma line N18TG2. In the proliferative condition, F-11 cells do not display a gene expression profile correspondent with specific subclasses of sensory neurons, but the most significant differences when compared with DRGs are the reduction of voltage-gated sodium, potassium and calcium channels, and the small amounts of TRPV1 transcripts. To investigate if functional properties of mature F-11 cells showed more similarities with those of isolated DRG neurons, we differentiated them by serum deprivation. Potassium and sodium currents significantly increased with differentiation, and biophysical properties of tetrodotoxin (TTX)-sensitive currents were similar to those characterized in small DRG neurons. The analysis of the voltage-dependence of calcium currents demonstrated the lack of low threshold activated components. The exclusive expression of high threshold activated Ca2+ currents and of TTX-sensitive Na+ currents correlated with the generation of a regular tonic electrical activity, which was recorded in the majority of the cells (80%) and was closely related to the activity of afferent TTX-sensitive A fibers of the proximal urethra and the bladder. Responses to capsaicin and substance P were also recorded in ~20% and ~80% of cells, respectively. The percentage of cells responsive to acetylcholine was consistent with the percentage referred for rat DRG primary neurons and cell electrical activity was modified by activation of non-NMDA receptors as for embryonic DRG neurons. These properties and the algesic profile (responses to pH5 and sensitivity to both ATP and capsaicin), proposed in literature to define a sub-classification of acutely dissociated rat DRG neurons, suggest that differentiated F-11 cells express receptors and ion channels that are also present in sensory neurons.
| S-EPMC6825413 | biostudies-literature
Project description:N-arachidonoyl ethanolamine (AEA) and 2-arachidonoyl glycerol (2-AG) are endogenous cannabinoids binding to the cannabinoid receptors CB1 and CB2 to modulate neuronal excitability and synaptic transmission in primary afferent neurons. To investigate the compartmentalization of the machinery for AEA and 2-AG signalling, we studied their partitioning into lipid raft fractions isolated from a dorsal root ganglion X neuroblastoma cell line (F-11).F-11 cells were homogenized and fractionated using a detergent-free OptiPrep density gradient. All lipids were partially purified from methanolic extracts of the fractions on solid phase cartridges and quantified using liquid chromatography tandem mass spectrometry (LC/MS/MS). Protein distribution was determined by Western blotting.Under basal conditions, the endogenous cannabinoid AEA was present in both lipid raft and specific non-lipid raft fractions as was one of its biosynthetic enzymes, NAPE-PLD. The 2-AG precursor 1-stearoyl-2-arachidonoyl-sn-glycerol (DAG), diacylglycerol lipase alpha (DAGLalpha), which cleaves DAG to form 2-AG, and 2-AG were all co-localized with lipid raft markers. CB1 receptors, previously reported to partition into lipid raft fractions, were not detected in F-11 membranes, but CB2 receptors were detected at high levels and partitioned into non-lipid raft fractions.The biochemical machinery for the production of 2-AG via the putative diacylglycerol pathway is localized within lipid rafts, suggesting that 2-AG synthesis via DAG occurs within these microdomains. The observed co-localization of AEA, 2-AG, and their synthetic enzymes with the reported localization of CB1 raises the possibility of intrinsic-autocrine signalling within lipid raft domains and/or retrograde-paracrine signalling.
| S-EPMC2219527 | biostudies-other
Project description:Corneal tissue displays the highest peripheral nerve density in the human body. Engineering of biomaterials to promote interactions between neurons and corneal tissue could provide tissue models for nerve/cornea development, platforms for drug screening, as well as innovative opportunities to regenerate cornea tissue. The focus of this study was to develop a coculture system for differentiated human corneal stromal stem cells (dhCSSCs) and dorsal root ganglion neurons (DRG) to mimic the human cornea tissue interactions. Axon extension, connectivity, and neuron cell viability were studied. DRG neurons developed longer axons when cocultured with dhCSSCs in comparison to neuron cultures alone. To assess the mechanism involved in the coculture response, nerve growth factors (NGF) secreted by dhCSSCs including NGF, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and neurotrophin-3 were characterized with greater focus on BDNF secretion. DhCSSCs also secreted collagen type I, an extracellular matrix molecule favorable for neuronal outgrowth. This coculture system provides a slowly degrading silk matrix to study neuronal responses in concert with hCSSCs related to innervation of corneal tissue with utility toward human corneal nerve regeneration and associated diseases.
| S-EPMC4552610 | biostudies-literature
Project description:Osteopontin expression has previously been demonstrated in the adult rat dorsal root ganglion, although its function remains unclear. Here, we demonstrate, using real-time reverse transcription-polymerase (RT-PCR) chain reaction, that osteopontin mRNA expression is increased 1 and 3 weeks following sciatic nerve section (axotomy). Further, immunohistochemical staining suggests that this increase is restricted to neurons already expressing the protein. Osteopontin knock-out animals have significantly increased mechanosensory thresholds in the intact adult compared with the wild-type controls; however no differences in allodynia are noted between genotypes using a model of neuropathic pain. Lastly, exogenous recombinant osteopontin has no effect on neurite outgrowth from adult wild-type sensory neurons, nor were differences in neurite outgrowth observed in osteopontin knock-out animals compared with wild-type controls.
| S-EPMC2725313 | biostudies-literature
Project description:The molecular identity of touch sensation is still largely unknown. We choose an extrem and very specific example, touch sensation of pennis glan, to study this problem. It's known from previous retro-grade labeling result that only one DRG in rat innervate the pennis glan, which makes it idea for microarry analysis. To clone the mechanosensory receptor specifically or highly expressed in the primary sensory neurons innervating pennis glan. There is a specific mechanosensory receptor in the primary sensory neurons innervating pennis glan. 1. Retrograde labeling the innervating nerve by DiI; 2. Dissect DRGs, dissociate neurons and pick up the fluorescent cells. 3. Pool around 20 positive cells and 50 control cells (big diameter neuron and small to medium diameter neuron respectively), purify total RNA. 4. Two rounds of amplification; 5. Affymetrix analysis
2008-06-11 | E-GEOD-11207 | biostudies-arrayexpress
Project description:There are only a few DRG specific genes which have been cloned, but almost no study about level specific gene expression. Clone different expressed genes in different level of DRGs; There are genes specifically expressed in one level of DRG but not the other. 1. dissect different level of DRGs2. extract total RNA
2008-06-13 | E-GEOD-4413 | biostudies-arrayexpress
Project description:Neurological disorders in ruminants have an important impact on veterinary health, but very few host-specific in vitro models have been established to study diseases affecting the nervous system. Here we describe a primary neuronal dorsal root ganglia (DRG) culture derived from calves after being conventionally slaughtered for food consumption. The study focuses on the in vitro characterization of bovine DRG cell populations by immunofluorescence analysis. The effects of various growth factors on neuron viability, neurite outgrowth and arborisation were evaluated by morphological analysis. Bovine DRG neurons are able to survive for more than 4 weeks in culture. GF supplementation is not required for neuronal survival and neurite outgrowth. However, exogenously added growth factors promote neurite outgrowth. DRG cultures from regularly slaughtered calves represent a promising and sustainable host specific model for the investigation of pain and neurological diseases in bovines.
| S-EPMC5148591 | biostudies-literature
Project description:Available evidence indicates voltage-gated Na+ channels (VGSCs) in peripheral sensory neurons are essential for the pain and hypersensitivity associated with tissue injury. However, our understanding of the biophysical and pharmacological properties of the channels in sensory neurons is largely based on the study of heterologous systems or rodent tissue, despite evidence that both expression systems and species differences influence these properties. Therefore, we sought to determine the extent to which the biophysical and pharmacological properties of VGSCs were comparable in rat and human sensory neurons. Whole cell patch clamp techniques were used to study Na+ currents in acutely dissociated neurons from human and rat. Our results indicate that while the two major current types, generally referred to as tetrodotoxin (TTX)-sensitive and TTX-resistant were qualitatively similar in neurons from rats and humans, there were several differences that have important implications for drug development as well as our understanding of pain mechanisms.
| S-EPMC5433841 | biostudies-literature