Neuronal Culture System Research Articles

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility. Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA(+) neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor(1). A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

April 2012: This Month in JoVE

The April issue of the Journal of Visualized Experiments (JoVE) includes an article that follows a lab from the University of Miami of Ohio to Lake Bonney - a permanently ice-covered saline lake in Antarctica. Few organisms can survive the harsh conditions under the ice, where light, warmth, and carbon sources are in very short supply. Single celled microorganisms called protists are the major class of microbes living beneath Lake Bonney, and are capable of bringing carbon, via photosynthesis, into the circle of life. After determining the appropriate sampling site, our authors drill and melt through the ice so that they can collect samples from various depths within the lake’s chemically stratified water column. Once collected, samples are taken to a field laboratory for filtration and stabilization. After processing, microbes present in the water sample are cultured for further experimentation. For example, our authors show that their enrichment cultures retain photosynthetic activity, and future experiments should help better understand protist physiology, as well as how such organisms can affect global geochemical cycling. In Clinical and Translational Medicine, JoVE turns its attention to stereotactic body radiotherapy, or SBRT, for the treatment of gynecological tumors. SBRT is a minimally invasive form of cancer treatment that relies on radiation, delivered by a linear accelerator mounted on a robotic arm, to biologically inactivate tumors. Our authors, from Case Medical Center, explain how gold soft tissue fiducial markers are placed around the cancer target at various depths based on computed tomography imaging. During a treatment planning session, CT and PET images are taken and then co-registered. Guided by these images, both radiation and gynecological oncologists designate the cancer volumes that will be targeted during therapy as well as contour surrounding normal structures that should be left unharmed. At the start of treatment, a target localizing system detects the fiducial markers via cross-plane radiographic imaging and marker location is compared with previously obtained imaging data. During treatment, light emitting diodes track a patient’s movement, in order to correlate their breathing pattern to the location of the fiducial markers, so that radiation can be delivered accurately. Overcoming the complications associated with patient immobilization, our authors show that their procedure for stereotaxic body radiotherapy can precisely deliver radiation to the target volume while limiting dosage to nearby structures. In our Bioengineering section, JoVE presents an article, which involves making biocompatible substrates from silkworm cocoons. First, cocoons are screened for contamination and processed so that silk extract is produced. Extract is placed atop a PDMS mold, which has been designed to create a pattern in the silk film drying on its surface. Once dried, the silk film is subjected to a process referred to as water annealing, which renders the film transparent and perfect for microscopy. Following cell seeding on the films, our authors show that tracking cell migration on the micro-patterned silk substrate is just as easy as on more conventional tissue culture surfaces. The biocompatibility of silk films as well as their potential to be chemically and structurally modified makes them attractive candidate materials for in vivo applications, like medical device design. Surprisingly, primary neuronal culture makes its way into our Immunology and Infection section, because this April, JoVE documents an innovative primary sympathetic neuron culture system used to study herpes simplex virus latency and activation. Peripheral neurons act like reservoirs for the herpes virus that are the source of reactivation events. To establish a peripheral neuron culture, superior cervical ganglia are isolated from embryonic mice and following digestion and dissociation are plated in 96 well plates. At around day 6 in vitro, herpes viruses encoding a viral-GFP fusion protein are added to the cells along with an inhibitor of lytic replication. Upon inhibitor withdrawal, latent virus will reactivate and GFP expression will be observable via fluorescent microscopy. By counting the number of wells exhibiting GFP fluorescence, the ability of small molecules or gene silencing oligonucleotides to either suppress or induce viral reactivation can be tested, making this assay a potential tool to uncover new therapeutics to help manage HSV-1 infection. This brief summary describes only four of the fifty articles JoVE will release in April. Other notable releases include methods for quantifying obesity in rats using x-ray computed tomography, inducing myocardial infarction in the zebrafish heart, and retrograde labeling of the excised spinal cord. Stay tuned.

Locust primary neuronal culture for the study of synaptic transmission

We have designed a cell culture system for thoracic neurons of adult Locusta migratoria that enables the establishment of functional synapses in vitro. Patch-clamp recordings revealed three different neuron classes. About half of the neurons (47%) had unexcitable somata with outward and no inward conductance. The other half generated either single (37%) or multiple action potentials (18%) and differed mainly in lower outward conductance. Selectively stained motor neurons were analyzed to demonstrate varied physiological properties due to culture conditions. Using paired patch clamp recordings we demonstrate directly synaptic transmission in morphologically connected neurons in vitro. Presynaptic stimulation resulted in postsynaptic potentials in 42 pairs of neurons tested, independent of the type of neuron. According to pharmacological experiments most of these synapses were either glutamatergic or GABAergic. In addition to these chemical synapses, electrical synapses were found. With the demonstration of synapse formation in cell culture of adult locust neurons, this study provides the basis for the future analysis of more defined insect neuronal circuits in culture.

Valproic acid mediates the synaptic excitatory/inhibitory balance through astrocytes — A preliminary study

Valproic acid (VPA) is one of the most widely used anticonvulsant and mood-stabilizing agents for the treatment of epilepsy and bipolar disorder. However, the underlying therapeutic mechanisms of the treatment of each disease remain unclear. Recently, the anti-epileptic effect of VPA has been found to lead to modulation of the synaptic excitatory/inhibitory balance. In addition, the therapeutic action of VPA has been linked to its effect on astrocytes by regulating gene expression at the molecular level, perhaps through an epigenetic mechanism as a histone deacetylase (HDAC) inhibitor. To provide insight into the mechanisms underlying the actions of VPA, this study investigated whether the synaptic excitatory/inhibitory (E/I) balance could be mediated by VPA through astrocytes. First, using the primary rat neuronal, astroglial, and neuro-glial mixed culture systems, we demonstrated that VPA treatment could regulate the mRNA levels of two post-synaptic cell adhesion molecules(neuroligin-1 and neuregulin-1) and two extracellular matrices (neuronal pentraxin-1and thrombospondin-3) in primary rat astrocyte cultures in a time- and concentration-dependent manner. Moreover, the up-regulation effect of VPA was noted in astrocytes, but not in neurons. In addition, these regulatory effects could be mimicked by sodium butyrate, a HDAC inhibitor, but not by lithium or two other glycogen synthase kinase-3 beta inhibitors. With the known role of these four proteins in regulating the synaptic E/I balance, we further demonstrated that VPA increased excitatory post-synaptic protein (postsynaptic density 95) and inhibitory post-synaptic protein (Gephyrin) in cortical neuro-glial mixed cultures. Our results suggested that VPA might affect the synaptic excitatory/inhibitory balance through its effect on astrocytes. This work provides the basis for future evaluation of the role of astroglial cell adhesion molecules and the extracellular matrix on the control of excitatory and inhibitory synapse formation.

A Modified Technique for Culturing Primary Fetal Rat Cortical Neurons

The study explored a modified primary culture system for fetal rat cortical neurons. Day E18 embryos from pregnant Sprague Dawley rats were microdissected under a stereoscope. To minimize enzymatic damage to the cultured neurons, we applied a sequential digestion protocol using papain and Dnase I. The resulting sifted cell suspension was seeded at a density of 50,000 cells per cm2 onto 0.1 mg/mL L-PLL-covered vessels. After a four-hour incubation in high-glucose Dulbecco's Modified Eagle's Medium (HG-DMEM) to allow the neurons to adhere, the media was changed to neurobasal medium that was refreshed by changing half of the volume after three days followed by a complete medium change every week. The cells displayed progressively robust neurite extension, and nonneuronal-like cells could barely be detected by five days in vitro (DIV); cell growth was still substantial at 14 DIV. Neurons were identified by β-tubulin III immunofluorescence, and neuronal purity within the cultures was assessed at over 95% by both flow cytometry and by dark-field counting of β-tubulin III-positive cells. These results suggest that the protocol was successful and that the high purity of neurons in this system could be used as the basis for generating various cell models of neurological disease.

Therapeutic Approaches to Neuroprotective Activity by Complementary and Alternative Medicines

Therapeutic Approaches to Neuroprotective Activity by Complementary and Alternative Medicines

HIF活性化・Akt活性化を標的とした新規脳保護薬の開発

We here presented two new neuroprotective agents; a HIF activator and an Akt activator. Hypoxia inducible factor (HIF) plays a pivotal role in the adaptation to ischemic conditions, and its activity is modulated by an oxygen-dependent hydroxylation of proline residues by prolyl hydroxylases (PHD). We discovered a unique compound TM6008, which inhibited PHD and stabilized HIF activity in vitro. Oral administration of TM6008 protected neurons in forebrain as well as focal ischemia. The protection was associated with amelioration of apoptosis but independent of enhanced angiogenesis. Next, elevating Akt activation is an obvious clinical strategy to prevent progressive neuronal death in neurological diseases. We identified a small molecule SC79 that inhibits Akt membrane translocation, but paradoxically activates Akt in the cytosol. SC79 specifically binds to the PH domain of Akt. SC79-bound Akt adopts a conformation favorable for phosphorylation by upstream protein kinases. In a hippocampal neuronal culture system and a mouse model for ischemic stroke, the cytosolic activation of Akt by SC79 is sufficient to recapitulate the primary cellular function of Akt signaling, resulting in augmented neuronal survival. Thus, HIF and Akt activators may be the new therapeutic strategy against cerebral infarction, and be expanded the therapeutic time window for hemolysis.

NeuriteQuant: An open source toolkit for high content screens of neuronal Morphogenesis

BackgroundTo date, some of the most useful and physiologically relevant neuronal cell culture systems, such as high density co-cultures of astrocytes and primary hippocampal neurons, or differentiated stem cell-derived cultures, are characterized by high cell density and partially overlapping cellular structures. Efficient analytical strategies are required to enable rapid, reliable, quantitative analysis of neuronal morphology in these valuable model systems.ResultsHere we present the development and validation of a novel bioinformatics pipeline called NeuriteQuant. This tool enables fully automated morphological analysis of large-scale image data from neuronal cultures or brain sections that display a high degree of complexity and overlap of neuronal outgrowths. It also provides an efficient web-based tool to review and evaluate the analysis process. In addition to its built-in functionality, NeuriteQuant can be readily extended based on the rich toolset offered by ImageJ and its associated community of developers. As proof of concept we performed automated screens for modulators of neuronal development in cultures of primary neurons and neuronally differentiated P19 stem cells, which demonstrated specific dose-dependent effects on neuronal morphology.ConclusionsNeuriteQuant is a freely available open-source tool for the automated analysis and effective review of large-scale high-content screens. It is especially well suited to quantify the effect of experimental manipulations on physiologically relevant neuronal cultures or brain sections that display a high degree of complexity and overlap among neurites or other cellular structures.

Matrix Metalloproteinase-8 Is Involved in Dermal Nerve Growth: Implications for Possible Application to Pruritus from In Vitro Models

Cutaneous nerve density is related to abnormal itch perception in dermatoses, such as atopic dermatitis and xerosis. However, the mechanisms underlying the elongation of dermal nerve fibers within the interstitial collagen (CoL) matrix are poorly understood. In this study, a culture system of rat dorsal root ganglion neurons consisting of type I CoL and a Boyden chamber containing a nerve growth factor (NGF) concentration gradient was used. Nerve fibers penetrating into type I CoL gel were observed in the presence of the NGF concentration gradient. Levels of matrix metalloproteinase-8 (MMP-8) mRNA and protein were increased in the cultured neurons and the conditioned medium, respectively. The nerve fiber penetration was dose dependently inhibited by MMP-8 blockers. Moreover, MMP-8 immunoreactivity was partially localized at growth cones in NGF-responsive nerve fibers. Semaphorin 3A stimulation also showed the opposite effects on these NGF-dependent events. Intriguingly, MMP-8 expression was upregulated by type I and III CoLs, which are substrates for this enzyme. These results suggested that MMP-8 is involved in sensory nerve growth within the interstitial CoL matrix through modulation by the axonal guidance molecules and/or extracellular matrix components. These findings provide insight into the development of pruritus involving skin nerve density.

A Lysosomal Culprit

Neurobiology Parkinson's disease (PD) has been clinically linked to a rare lysosomal storage disease known as Gaucher disease (GD). Patients with GD lack the enzyme glucocerebrosidase (GCase), which leads to the accumulation of the glycolipid glucosylceramide. In PD, intracellular accumulation and aggregation of the α-synuclein protein in neurons is a key event in disease pathogenesis. Mazzulli et al. wanted to elucidate the mechanistic link between these two disorders. In neuronal tissue culture systems and in mouse disease models, intracellular accumulation of glucosylceramide compromised lysosomal proteolysis and led to the accumulation of cytotoxic α-synuclein and neurodegeneration. Furthermore, glucosylceramide directly promoted the aggregation of purified α-synuclein in vitro. The accumulation of the α-synuclein itself in neurons also compromised the production of mature, active lysosomal GCase, generating a pathogenic cycle. Analysis of postmortem brain samples from human patients also suggested that GCase deficiencies were often linked to pathological α-synuclein accumulation. Intervention in this pathogenic cycle by increasing the efficiency of GCase targeting to lysosomes may thus represent a future approach toward ameliorating PD and other related diseases. Cell 146 , 37 (2011).

Modeling the functional genomics of autism using human neurons

Human neural progenitors from a variety of sources present new opportunities to model aspects of human neuropsychiatric disease in vitro. Such in vitro models provide the advantages of a human genetic background, combined with rapid and easy manipulation, making them highly useful adjuncts to animal models. Here, we examined whether a human neuronal culture system could be utilized to assess the transcriptional program involved in human neural differentiation and in modeling some of the molecular features of a neurodevelopmental disorder, such as autism. Primary normal human neuronal progenitors (NHNPs) were differentiated into a post-mitotic neuronal state through addition of specific growth factors and whole-genome gene expression was examined throughout a time course of neuronal differentiation. After four weeks of differentiation, a significant number of genes associated with autism spectrum disorders (ASD) are either induced or repressed. This includes the ASD susceptibility gene neurexin 1, which showed a distinct pattern from neurexin 3 in vitro, and which we validated in vivo in fetal human brain. Using weighted gene co-expression network analysis (WGCNA), we visualized the network structure of transcriptional regulation, demonstrating via this unbiased analysis that a significant number of ASD candidate genes are coordinately regulated during the differentiation process. Since NHNPs are genetically tractable and manipulable, they can be used to study both the effects of mutations in multiple ASD candidate genes on neuronal differentiation and gene expression in combination with the effects of potential therapeutic molecules. These data also provide a step towards better understanding of the signaling pathways disrupted in ASD.

Repetitive magnetic stimulation of human-derived neuron-like cells activates cAMP-CREB pathway

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive neurostimulatory technique widely used in research, diagnostics, and neuro-psychiatric therapy. Despite its growing popularity, basic molecular mechanisms underlying the clinical effects of rTMS have remained largely under-researched. Here, we present a human-derived neuronal cell culture system responsive to rTMS effects. SH-SY5Y neuroblastoma cells were differentiated by retinoic acid treatment for 10days, resulting in a neuronal phenotype characterized by upregulation of neuronal marker proteins and generation of an action potential in response to depolarizing current step injection. Repetitive magnetic stimulation of these cells resulted in increased intracellular cAMP levels and increased phosphorylation of transcription factor CREB. Pretreatment with ketamine (1μM) potentiated, while pretreatment with lithium (2mM) attenuated this cellular response to repetitive magnetic stimulation. In conclusion, we introduce here a novel in vitro system responding to rTMS at the level of second messenger signaling. The use of human-derived cells with neuron-like properties will prove useful for further studies on the cellular effects of rTMS.

Neuronal chromatin dynamics of imprinting in development and disease

Epigenetic mechanisms play essential roles in mammalian neurodevelopment and genetic mutations or chromosomal deletions or duplications of epigenetically regulated loci or pathways result in several important human neurodevelopmental disorders. Postnatal mammalian neurons have among the most structured and dynamic nuclear organization of any cell type. Human chromosome 15q11-13 is an imprinted locus required for normal neurodevelopment and is regulated by a plethora of epigenetic mechanisms in neurons, including multiple noncoding RNAs, parentally imprinted transcription and histone modifications, large-scale chromatin decondensation, and homologous pairing in mature neurons of the mammalian brain. Here, we describe the multiple epigenetic layers regulating 15q11-13 gene expression and chromatin dynamics in neurons and propose a model of how noncoding RNAs may influence the unusual neuronal chromatin structure and dynamics at this locus. We also discuss the need for improved neuronal cell culture systems that model human 15q11-13 and other neurodevelopmental disorders with epigenetic bases in order to test the mechanisms of chromatin dynamics and nuclear organization in neurons. Induced pluripotent stem cells and other stem cell technologies hold promise for improved understanding of and therapeutic interventions for multiple human neurodevelopmental disorders.

Nature and Duration of Growth Factor Signaling through Receptor Tyrosine Kinases Regulates HSV-1 Latency in Neurons

Herpes simplex virus-1 (HSV-1) establishes life-long latency in peripheral neurons where productive replication is suppressed. While periodic reactivation results in virus production, the molecular basis of neuronal latency remains incompletely understood. Using a primary neuronal culture system of HSV-1 latency and reactivation, we show that continuous signaling through the phosphatidylinositol 3-kinase (PI3-K) pathway triggered by nerve growth factor (NGF)-binding to the TrkA receptor tyrosine kinase (RTK) is instrumental in maintaining latent HSV-1. The PI3-K p110α catalytic subunit, but not the β or δ isoforms, is specifically required to activate 3-phosphoinositide-dependent protein kinase-1 (PDK1) and sustain latency. Disrupting this pathway leads to virus reactivation. EGF and GDNF, two other growth factors capable of activating PI3-K and PDK1 but that differ from NGF in their ability to persistently activate Akt, do not fully support HSV-1 latency. Thus, the nature of RTK signaling is a critical host parameter that regulates the HSV-1 latent-lytic switch.

ChemInform Abstract: Neurotrophic Sesquiterpene-Neolignans from Magnolia obovata: Structure and Neurotrophic Activity.

Abstract Novel sesquiterpene-neolignans, eudesobovatos A (1) and B (2), eudesmagnolol (3), eudeshonokiols A (4) and B (5), clovanemagnolol (6), and caryolanemagnolol (7), have been isolated from the bark of Magnolia obovata. Their structures were elucidated to be sesquiterpenes (eudesmol, 4,4,8-trimethyltricyclo [6.3.1.02,5] dodecane-1,9-diol, and clovanediol) combined through ether bond with neolignans such as obovatol, honokiol, and magnolol on the basis of spectral data, degradation, and/or synthesis. Compounds 1, 6, and 7 were found to exhibit interesting neurotrophic activity on a neuronal cell culture system derived from fetal rat hemisphere.

Enhancing the GLP‐1 receptor signaling pathway leads to proliferation and neuroprotection in human neuroblastoma cells

Increasing evidence suggests that glucagon-like peptide-1 (GLP-1), an incretin hormone of current interest in type 2 diabetes, is neuroprotective in both cell culture and animal models. To characterize the neuroprotective properties of GLP-1 and associated underlying mechanisms, we over-expressed the GLP-1 receptor (GLP-1R) on human neuroblastoma SH-SY5Y cells to generate a neuronal culture system featuring enhanced GLP-1R signaling. In GLP-1R over-expressing SH-SY5Y (SH-hGLP-1R#9) cells, GLP-1 and the long-acting agonist exendin-4 stimulated cell proliferation and increased cell viability by 2-fold at 24 h at physiologically relevant concentrations. This GLP-1R-dependent action was mediated via the protein kinase A and phosphoinositide 3-kinase signaling pathways, with the MAPK pathway playing a minor role. GLP-1 and exendin-4 pretreatment dose-dependently protected SH-hGLP-1R#9 cells from hydrogen peroxide (H(2)O(2))- and 6-hydroxydopamine-induced cell death. This involved amelioration of elevated caspase 3 activity, down-regulation of pro-apoptotic Bax and up-regulation of anti-apoptotic Bcl-2 protein. In the presence of 6-hydroxydopamine, GLP-1's ability to lower caspase-3 activity was abolished with the phosphoinositide 3-kinase inhibitor, LY2940002, and partly reduced with the protein kinase A inhibitor, H89. Hence, GLP-1R mediated neurotrophic and anti-apoptotic actions co-contribute to the neuroprotective property of GLP-1 in neuronal cell cultures, and reinforce the potential therapeutic value of GLP-1R agonists in neurodegenerative disorders involving oxidative stress.

Cannabinoid Receptor Agonist WIN-55,212-2 Protects Differentiated PC12 Cells From Organophosphorus- Induced Apoptosis

Cannabinoid neuroprotection is usually greater in vivo than in neuronal cell culture systems. To the authors' knowledge, a good in vitro culture model for the neuroprotective effects of cannabinoids does not exist. Therefore, a 3-dimensional (3D) culture system was developed to investigate the neuroprotective effects of the cannabinoid receptor agonist WIN-55,212-2 on apoptosis of differentiated PC12 cells, caused by the organophosphorus compounds paraoxon and diazinon. Cells pretreated with WIN-55,212-2 were exposed to a proapoptotic concentration of paraoxon and diazinon. TUNEL was used to detect apoptosis, and neurite length was assessed by morphometry. Both paraoxon and diazinon induced apoptosis, although the latter was more potent. WIN-55,212-2 also protected cells from neurite retraction and DNA fragmentation induced by the OPs. The results suggest that WIN-55,212-2 protects PC12 cells cultured under 3D conditions from organophosphorus-induced apoptosis. This 3D culture system may prove to be a useful tool for investigating the neuroprotective effects of cannabinoids.

Dorsal root ganglia sensory neuronal cultures: a tool for drug discovery for peripheral neuropathies

Background: Peripheral neuropathies affect many people worldwide and are caused by or associated with a wide range of conditions, both genetic and acquired. Current therapies are directed at symptomatic control because no effective regenerative treatment exists. The primary challenge is that mechanisms that lead to distal axonal degeneration, a common feature of all peripheral neuropathies, are largely unknown. Objective/methods: To address the role and specific characteristics of dorsal root ganglia (DRG) derived sensory neuron culture system as a useful model in evaluating the pathogenic mechanisms of peripheral neuropathies and examination and validation of potential therapeutic compounds. A thorough review of the recent literature was conducted and select examples of the use of DRG neurons in different peripheral neuropathy models were chosen to highlight the utility of these cultures. Conclusion: Many useful models of different peripheral neuropathies have been developed using DRG neuronal culture and potential therapeutic targets have been examined, but so far none of the potential therapeutic compounds have succeeded in clinical trials. In recent years, the focus has changed to evaluation of axon degeneration as the primary outcome measure advocating a drug development strategy starting with phenotypic drug screening, followed by validation in primary complex co-cultures and animal models.

Anterograde Spread of Herpes Simplex Virus Type 1 Requires Glycoprotein E and Glycoprotein I but Not Us9

Anterograde neuronal spread (i.e., spread from the neuron cell body toward the axon terminus) is a critical component of the alphaherpesvirus life cycle. Three viral proteins, gE, gI, and Us9, have been implicated in alphaherpesvirus anterograde spread in several animal models and neuron culture systems. We sought to better define the roles of gE, gI, and Us9 in herpes simplex virus type 1 (HSV-1) anterograde spread using a compartmentalized primary neuron culture system. We found that no anterograde spread occurred in the absence of gE or gI, indicating that these proteins are essential for HSV-1 anterograde spread. However, we did detect anterograde spread in the absence of Us9 using two independent Us9-deleted viruses. We confirmed the Us9 finding in different murine models of neuronal spread. We examined viral transport into the optic nerve and spread to the brain after retinal infection; the production of zosteriform disease after flank inoculation; and viral spread to the spinal cord after flank inoculation. In all models, anterograde spread occurred in the absence of Us9, although in some cases at reduced levels. This finding contrasts with gE- and gI-deleted viruses, which displayed no anterograde spread in any animal model. Thus, gE and gI are essential for HSV-1 anterograde spread, while Us9 is dispensable.