Diversity in responses to oncolytic Lassa-vesicular stomatitis virus in patient-derived glioblastoma cells.

The difficulty of glioblastoma treatment makes it a good candidate for novel therapies, such as oncolytic viruses. Vesicular stomatitis virus expressing Lassa virus glycoprotein (Lassa-VSV) showed significant promise in animal models using established glioblastoma cell lines. These experiments were to determine the susceptibility of low-passage, patient-derived cell lines to Lassa-VSV oncolysis. Four patient-derived glioblastoma cell lines were infected with Lassa-VSV that expresses green fluorescent protein (GFP) and analyzed by fluorescence microscopy, flow cytometry, and cell viability assays. Cells were also analyzed as tumorspheres containing primarily glioma stem-like cells. Three low-passage, patient-derived cells were further analyzed with RNA sequencing (RNA-seq). Individual cell lines varied somewhat in their levels of viral gene expression and time course of Lassa-VSV-induced cell death, but each was susceptible to Lassa-VSV. Brain Tumor Center of Excellence (BTCOE) 4765 cells had the highest level of expression of interferon-stimulated genes but were most susceptible to Lassa-VSV-induced cell death, indicating that more susceptible cells do not necessarily have lower interferon pathway activation. Cells cultured as tumorspheres and infected with Lassa-VSV also showed variable susceptibility to Lassa-VSV, but BTCOE 4765 cells were least susceptible. Thus, patient-derived brain tumor cells show variable responses to Lassa-VSV infection, but each of the lines was susceptible to VSV oncolysis.

Immune cells enhance Zika virus-mediated neurologic dysfunction in brain of mice with humanized immune systems.

Zika virus (ZIKV) can generate a number of neurological dysfunctions in infected humans. Here, we tested the potential of human immune cells to protect against ZIKV infection in genetically humanized MISTRG mice. FACS analysis showed robust reconstitution of the mouse spleen with human T cells. Peripheral ZIKV inoculation resulted in infection within the brains of MISTRG mice. Mice that were reconstituted with human peripheral blood mononuclear cells (PBMC) showed a more rapid lethal response to ZIKV than the control mice lacking these immune cells. Immunocytochemical analysis of T cell markers CD3, CD45, or CD8 showed strong T cell presence in the brain, together with robust infection by ZIKV particularly in the excitatory pyramidal and granule neurons of the hippocampus. Infection was also found in cortex, striatum, the dopamine neurons of the substantia nigra, and other brain loci. Infection was considerably less in other regions such as the septum and hypothalamus. These data support the perspective that, rather than exerting a protective function, T cells may underlie some ZIKV-mediated neuropathology in the brain.

Mucin-Like Domain of Ebola Virus Glycoprotein Enhances Selective Oncolytic Actions against Brain Tumors.

Given that the Ebola virus (EBOV) infects a wide array of organs and cells yet displays a relative lack of neurotropism, we asked whether a chimeric vesicular stomatitis virus (VSV) expressing the EBOV glycoprotein (GP) might selectively target brain tumors. The mucin-like domain (MLD) of the EBOV GP may enhance virus immune system evasion. Here, we compared chimeric VSVs in which EBOV GP replaces the VSV glycoprotein, thereby reducing the neurotoxicity associated with wild-type VSV. A chimeric VSV expressing the full-length EBOV GP (VSV-EBOV) containing the MLD was substantially more effective and safer than a parallel construct with an EBOV GP lacking the MLD (VSV-EBOVΔMLD). One-step growth, reverse transcription-quantitative PCR, and Western blotting assessments showed that VSV-EBOVΔMLD produced substantially more progeny faster than VSV-EBOV. Using immunodeficient SCID mice, we focused on targeting human brain tumors with these VSV-EBOVs. Similar to the findings of our previous study in which we used an attenuated VSV-EBOV with no MLD that expressed green fluorescent protein (GFP) (VSV-EBOVΔMLD-GFP), VSV-EBOVΔMLD without GFP targeted glioma but yielded only a modest extension of survival. In contrast, VSV-EBOV containing the MLD showed substantially better targeting and elimination of brain tumors after intravenous delivery and increased the survival of brain tumor-bearing mice. Despite the apparent destruction of most tumor cells by VSV-EBOVΔMLD, the virus remained active within the SCID mouse brain and showed widespread infection of normal brain cells. In contrast, VSV-EBOV eliminated the tumors and showed relatively little infection of normal brain cells. Parallel experiments with direct intracranial virus infection generated similar results. Neither VSV-EBOV nor VSV-EBOVΔMLD showed substantive infection of the brains of normal immunocompetent mice.IMPORTANCE The Ebola virus glycoprotein contains a mucin-like domain which may play a role in immune evasion. Chimeric vesicular stomatitis viruses with the EBOV glycoprotein substituted for the VSV glycoprotein show greater safety and efficacy in targeting brain tumors in immunodeficient mice when the MLD was expressed within the EBOV glycoprotein than when EBOV lacked the mucin-like domain.

Dynamic Network Activation of Hypothalamic MCH Neurons in REM Sleep and Exploratory Behavior.

Most brain neurons are active in waking, but hypothalamic neurons that synthesize the neuropeptide melanin-concentrating hormone (MCH) are claimed to be active only during sleep, particularly rapid eye movement (REM) sleep. Here we use deep-brain imaging to identify changes in fluorescence of the genetically encoded calcium (Ca2+) indicator GCaMP6 in individual hypothalamic neurons that contain MCH. An in vitro electrophysiology study determined a strong relationship between depolarization and Ca2+ fluorescence in MCH neurons. In 10 freely behaving MCH-cre mice (male and female), the highest fluorescence occurred in all recorded neurons (n = 106) in REM sleep relative to quiet waking or non-REM sleep. Unexpectedly, 70% of the MCH neurons had strong fluorescence activity when the mice explored novel objects. Spatial and temporal mapping of the change in fluorescence between pairs of MCH neurons revealed dynamic activation of MCH neurons during REM sleep and activation of a subset of the same neurons during exploratory behavior. Functional network activity maps will facilitate comparisons of not only single-neuron activity, but also network responses in different conditions and disease.SIGNIFICANCE STATEMENT Functional activity maps identify brain circuits responding to specific behaviors, including rapid eye movement sleep (REM sleep), a sleep phase when the brain is as active as in waking. To provide the first activity map of individual neurons during REM sleep, we use deep-brain calcium imaging in unrestrained mice to map the activity of hypothalamic melanin-concentrating hormone (MCH) neurons. MCH neurons were found to be synchronously active during REM sleep, and also during the exploration of novel objects. Spatial mapping revealed dynamic network activation during REM sleep and activation of a subset of the neurons during exploratory behavior. Functional activity maps at the cellular level in specific behaviors, including sleep, are needed to establish a brain connectome.

Defining the caudal hypothalamic arcuate nucleus with a focus on anorexic excitatory neurons.

KEY POINTS: Excitatory glutamate neurons are sparse in the rostral hypothalamic arcuate nucleus (ARC), the subregion that has received the most attention in the past. In striking contrast, excitatory neurons are far more common (by a factor of 10) in the caudal ARC, an area which has received relatively little attention. These glutamate cells may play a negative role in energy balance and food intake. They can show an increase in phosphorylated Stat-3 in the presence of leptin, are electrically excited by the anorectic neuromodulator cholecystokinin, and inhibited by orexigenic neuromodulators neuropeptide Y, met-enkephalin, dynorphin and the catecholamine dopamine. The neurons project local axonal connections that excite other ARC neurons including proopiomelanocortin neurons that can play an important role in obesity. These data are consistent with models suggesting that the ARC glutamatergic neurons may play both a rapid and a slower role in acting as anorectic neurons in CNS control of food intake and energy homeostasis. ABSTRACT: Here we interrogate a unique class of excitatory neurons in the hypothalamic arcuate nucleus (ARC) that utilizes glutamate as a fast neurotransmitter using mice expressing GFP under control of the vesicular glutamate transporter 2 (vGluT2) promoter. These neurons show a unique distribution, synaptic characterization, cellular physiology and response to neuropeptides involved in energy homeostasis. Although apparently not previously appreciated, the caudal ARC showed a far greater density of vGluT2 cells than the rostral ARC, as seen in transgenic vGluT2-GFP mice and mRNA analysis. After food deprivation, leptin induced an increase in phosphorylated Stat-3 in vGluT2-positive neurons, indicating a response to hormonal cues of energy state. Based on whole-cell recording electrophysiology in brain slices, vGluT2 neurons were spontaneously active with a spike frequency around 2 Hz. vGluT2 cells were responsive to a number of neuropeptides related to energy homeostasis; they were excited by the anorectic peptide cholecystokinin, but inhibited by orexigenic neuropeptide Y, dynorphin and met-enkephalin, consistent with an anorexic role in energy homeostasis. Dopamine, associated with the hedonic aspect of enhancing food intake, inhibited vGluT2 neurons. Optogenetic excitation of vGluT2 cells evoked EPSCs in neighbouring neurons, indicating local synaptic excitation of other ARC neurons. Microdrop excitation of ARC glutamate cells in brain slices rapidly increased excitatory synaptic activity in anorexigenic proopiomelanocortin neurons. Together these data support the perspective that vGluT2 cells may be more prevalent in the ARC than previously appreciated, and play predominantly an anorectic role in energy metabolism.

Chikungunya-vesicular stomatitis chimeric virus targets and eliminates brain tumors.

Vesicular stomatitis virus (VSV) shows potential for targeting and killing cancer cells, but can be dangerous in the brain due to its neurotropic glycoprotein. Here we test a chimeric virus in which the VSV glycoprotein is replaced with the Chikungunya polyprotein E3-E2-6K-E1 (VSVΔG-CHIKV). Control mice with brain tumors survived a mean of 40 days after tumor implant. VSVΔG-CHIKV selectively infected and eliminated the tumor, and extended survival substantially in all tumor-bearing mice to over 100 days. VSVΔG-CHIKV also targeted intracranial primary patient derived melanoma xenografts. Virus injected into one melanoma spread to other melanomas within the same brain with little detectable infection of normal cells. Intravenous VSVΔG-CHIKV infected tumor cells but not normal tissue. In immunocompetent mice, VSVΔG-CHIKV selectively infected mouse melanoma cells within the brain. These data suggest VSVΔG-CHIKV can target and destroy brain tumors in multiple animal models without the neurotropism associated with the wild type VSV glycoprotein.

Valnoctamide Inhibits Cytomegalovirus Infection in Developing Brain and Attenuates Neurobehavioral Dysfunctions and Brain Abnormalities.

Cytomegalovirus (CMV) is the most common infectious cause of brain defects and neurological dysfunction in developing human babies. Due to the teratogenicity and toxicity of available CMV antiviral agents, treatment options during early development are markedly limited. Valnoctamide (VCD), a neuroactive mood stabilizer with no known teratogenic activity, was recently demonstrated to have anti-CMV potential. However, it is not known whether this can be translated into an efficacious therapeutic effect to improve CMV-induced adverse neurological outcomes. Using multiple models of CMV infection in the developing mouse brain, we show that subcutaneous low-dose VCD suppresses CMV by reducing the level of virus available for entry into the brain and by acting directly within the brain to block virus replication and dispersal. VCD during the first 3 weeks of life restored timely acquisition of neurological milestones in neonatal male and female mice and rescued long-term motor and behavioral outcomes in juvenile male mice. CMV-mediated brain defects, including decreased brain size, cerebellar hypoplasia, and neuronal loss, were substantially attenuated by VCD. No adverse side effects on neurodevelopment of uninfected control mice receiving VCD were detected. Treatment of CMV-infected human fetal astrocytes with VCD reduced both viral infectivity and replication by blocking viral particle attachment to the cell, a mechanism that differs from available anti-CMV drugs. These data suggest that VCD during critical periods of neurodevelopment can effectively suppress CMV replication in the brain and safely improve both immediate and long-term neurological outcomes.SIGNIFICANCE STATEMENT Cytomegalovirus (CMV) can irreversibly damage the developing brain. No anti-CMV drugs are available for use during fetal development, and treatment during the neonatal period has substantial limitations. We studied the anti-CMV actions of valnoctamide (VCD), a psychiatric sedative that appears to lack teratogenicity and toxicity, in the newborn mouse brain, a developmental period that parallels that of an early second-trimester human fetus. In infected mice, subcutaneous VCD reaches the brain and suppresses viral replication within the CNS, rescuing the animals from CMV-induced brain defects and neurological problems. Treatment of uninfected control animals exerts no detectable adverse effects. VCD also blocks CMV replication in human fetal brain cells.

Rapid binge-like eating and body weight gain driven by zona incerta GABA neuron activation.

The neuronal substrate for binge eating, which can at times lead to obesity, is not clear. We find that optogenetic stimulation of mouse zona incerta (ZI) γ-aminobutyric acid (GABA) neurons or their axonal projections to paraventricular thalamus (PVT) excitatory neurons immediately (in 2 to 3 seconds) evoked binge-like eating. Minimal intermittent stimulation led to body weight gain; ZI GABA neuron ablation reduced weight. ZI stimulation generated 35% of normal 24-hour food intake in just 10 minutes. The ZI cells were excited by food deprivation and the gut hunger signal ghrelin. In contrast, stimulation of excitatory axons from the parasubthalamic nucleus to PVT or direct stimulation of PVT glutamate neurons reduced food intake. These data suggest an unexpected robust orexigenic potential for the ZI GABA neurons.

Zika Virus Targeting in the Developing Brain.

Zika virus (ZIKV), a positive-sense RNA flavivirus, has attracted considerable attention recently for its potential to cause serious neurological problems, including microcephaly, cortical thinning, and blindness during early development. Recent findings suggest that ZIKV infection of the brain can occur not only during very early stages of development, but also in later fetal/early neonatal stages of maturation. Surprisingly, after peripheral inoculation of immunocompetent mice on the day of birth, the first cells targeted throughout the brain were isolated astrocytes. At later stages, more neurons showed ZIKV immunoreactivity, in part potentially due to ZIKV release from infected astrocytes. In all developing mice studied, we detected infection of retinal neurons; in many mice, this was also associated with infection of the lateral geniculate, suprachiasmatic nuclei, and superior colliculus, suggesting a commonality for the virus to infect cells of the visual system. Interestingly, in mature mice lacking a Type 1 interferon response (IFNR-/-), after inoculation of the eye, the initial majority of infected cells in the visual system were glial cells along the optic tract. ZIKV microinjection into the somatosensory cortex on one side of the normal mouse brain resulted in mirror infection restricted to the contralateral somatosensory cortex without any infection of midline brain regions, indicating the virus can move by axonal transport to synaptically coupled brain loci. These data support the view that ZIKV shows considerable complexity in targeting the CNS and may target different cells at different stages of brain development.SIGNIFICANCE STATEMENT Zika virus (ZIKV) can cause substantial damage to the developing human brain. Here we examine a developmental mouse model of ZIKV infection in the newborn mouse in which the brain is developmentally similar to a second-trimester human fetus. After peripheral inoculation, the virus entered the CNS in all mice tested and initially targeted astrocytes throughout the brain. Infections of the retina were detected in all mice, and infection of CNS visual system nuclei in the brain was common. We find that ZIKV can be transported axonally, thereby enhancing virus spread within the brain. These data suggest that ZIKV infects multiple cell types within the brain and that astrocyte infection may play a more important role in initial infection than previously appreciated.

Integrated Control of Predatory Hunting by the Central Nucleus of the Amygdala.

Superior predatory skills led to the evolutionary triumph of jawed vertebrates. However, the mechanisms by which the vertebrate brain controls predation remain largely unknown. Here, we reveal a critical role for the central nucleus of the amygdala in predatory hunting. Both optogenetic and chemogenetic stimulation of central amygdala of mice elicited predatory-like attacks upon both insect and artificial prey. Coordinated control of cervical and mandibular musculatures, which is necessary for accurately positioning lethal bites on prey, was mediated by a central amygdala projection to the reticular formation in the brainstem. In contrast, prey pursuit was mediated by projections to the midbrain periaqueductal gray matter. Targeted lesions to these two pathways separately disrupted biting attacks upon prey versus the initiation of prey pursuit. Our findings delineate a neural network that integrates distinct behavioral modules and suggest that central amygdala neurons instruct predatory hunting across jawed vertebrates.

Chikungunya, Influenza, Nipah, and Semliki Forest Chimeric Viruses with Vesicular Stomatitis Virus: Actions in the Brain.

Recombinant vesicular stomatitis virus (VSV)-based chimeric viruses that include genes from other viruses show promise as vaccines and oncolytic viruses. However, the critical safety concern is the neurotropic nature conveyed by the VSV glycoprotein. VSVs that include the VSV glycoprotein (G) gene, even in most recombinant attenuated strains, can still show substantial adverse or lethal actions in the brain. Here, we test 4 chimeric viruses in the brain, including those in which glycoprotein genes from Nipah, chikungunya (CHIKV), and influenza H5N1 viruses were substituted for the VSV glycoprotein gene. We also test a virus-like vesicle (VLV) in which the VSV glycoprotein gene is expressed from a replicon encoding the nonstructural proteins of Semliki Forest virus. VSVΔG-CHIKV, VSVΔG-H5N1, and VLV were all safe in the adult mouse brain, as were VSVΔG viruses expressing either the Nipah F or G glycoprotein. In contrast, a complementing pair of VSVΔG viruses expressing Nipah G and F glycoproteins were lethal within the brain within a surprisingly short time frame of 2 days. Intranasal inoculation in postnatal day 14 mice with VSVΔG-CHIKV or VLV evoked no adverse response, whereas VSVΔG-H5N1 by this route was lethal in most mice. A key immune mechanism underlying the safety of VSVΔG-CHIKV, VSVΔG-H5N1, and VLV in the adult brain was the type I interferon response; all three viruses were lethal in the brains of adult mice lacking the interferon receptor, suggesting that the viruses can infect and replicate and spread in brain cells if not blocked by interferon-stimulated genes within the brain.IMPORTANCE Vesicular stomatitis virus (VSV) shows considerable promise both as a vaccine vector and as an oncolytic virus. The greatest limitation of VSV is that it is highly neurotropic and can be lethal within the brain. The neurotropism can be mostly attributed to the VSV G glycoprotein. Here, we test 4 chimeric viruses of VSV with glycoprotein genes from Nipah, chikungunya, and influenza viruses and nonstructural genes from Semliki Forest virus. Two of the four, VSVΔG-CHIKV and VLV, show substantially attenuated neurotropism and were safe in the healthy adult mouse brain. VSVΔG-H5N1 was safe in the adult brain but lethal in the younger brain. VSVΔG Nipah F+G was even more neurotropic than wild-type VSV, evoking a rapid lethal response in the adult brain. These results suggest that while chimeric VSVs show promise, each must be tested with both intranasal and intracranial administration to ensure the absence of lethal neurotropism.

Optogenetic activation of melanin-concentrating hormone neurons increases non-rapid eye movement and rapid eye movement sleep during the night in rats.

Neurons containing melanin-concentrating hormone (MCH) are located in the hypothalamus. In mice, optogenetic activation of the MCH neurons induces both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep at night, the normal wake-active period for nocturnal rodents [R. R. Konadhode et al. (2013) J. Neurosci., 33, 10257-10263]. Here we selectively activate these neurons in rats to test the validity of the sleep network hypothesis in another species. Channelrhodopsin-2 (ChR2) driven by the MCH promoter was selectively expressed by MCH neurons after injection of rAAV-MCHp-ChR2-EYFP into the hypothalamus of Long-Evans rats. An in vitro study confirmed that the optogenetic activation of MCH neurons faithfully triggered action potentials. In the second study, in Long-Evans rats, rAAV-MCH-ChR2, or the control vector, rAAV-MCH-EYFP, were delivered into the hypothalamus. Three weeks later, baseline sleep was recorded for 48 h without optogenetic stimulation (0 Hz). Subsequently, at the start of the lights-off cycle, the MCH neurons were stimulated at 5, 10, or 30 Hz (1 mW at tip; 1 min on - 4 min off) for 24 h. Sleep was recorded during the 24-h stimulation period. Optogenetic activation of MCH neurons increased both REM and NREM sleep at night, whereas during the day cycle, only REM sleep was increased. Delta power, an indicator of sleep intensity, was also increased. In control rats without ChR2, optogenetic stimulation did not increase sleep or delta power. These results lend further support to the view that sleep-active MCH neurons contribute to drive sleep in mammals.

Mood stabilizers inhibit cytomegalovirus infection.

Cytomegalovirus (CMV) infection can generate debilitating disease in immunocompromised individuals and neonates. It is also the most common infectious cause of congenital birth defects in infected fetuses. Available anti-CMV drugs are partially effective but are limited by some toxicity, potential viral resistance, and are not recommended for fetal exposure. Valproate, valpromide, and valnoctamide have been used for many years to treat epilepsy and mood disorders. We report for the first time that, in contrast to the virus-enhancing actions of valproate, structurally related valpromide and valnoctamide evoke a substantial and specific inhibition of mouse and human CMV in vitro. In vivo, both drugs safely attenuate mouse CMV, improving survival, body weight, and developmental maturation of infected newborns. The compounds appear to act by a novel mechanism that interferes with CMV attachment to the cell. Our work provides a novel potential direction for CMV therapeutics through repositioning of agents already approved for use in psychiatric disorders.

Hypothalamic arcuate nucleus tyrosine hydroxylase neurons play orexigenic role in energy homeostasis.

Energy homeostasis, food intake, and body weight are regulated by specific brain circuits. Here we introduce an unexpected neuron, the tyrosine hydroxylase (TH) neuron of the arcuate nucleus (ARC), that we show makes an orexigenic contribution. Optogenetic stimulation of mouse ARC TH neurons increased food intake; attenuating transmitter release reduced body weight. Optogenetic stimulation of ARC TH cells inhibited pro-opiomelanocortin (POMC) neurons through synaptic mechanisms. ARC TH cells project to the hypothalamic paraventricular nucleus; optogenetic stimulation of ARC TH axons inhibited paraventricular nucleus neurons by dopamine and GABA co-release. Dopamine excited orexigenic neurons that synthesize agouti-related peptide and neuropeptide Y but inhibited anorexigenic neurons that synthesize POMC, as determined by whole cell recording. Food deprivation increased c-fos expression and spike frequency in ARC TH neurons. The gut peptide ghrelin evoked direct excitatory effects, suggesting these neurons monitor metabolic cues. Together these data support the view that ARC TH cells play an unrecognized and influential positive role in energy homeostasis.

Separate circuitries encode the hedonic and nutritional values of sugar.

Sugar exerts its potent reinforcing effects via both gustatory and post-ingestive pathways. It is, however, unknown whether sweetness and nutritional signals engage segregated brain networks to motivate ingestion. We found in mice that separate basal ganglia circuitries mediated the hedonic and nutritional actions of sugar. During sugar intake, suppressing hedonic value inhibited dopamine release in ventral, but not dorsal, striatum, whereas suppressing nutritional value inhibited dopamine release in dorsal, but not ventral, striatum. Consistently, cell-specific ablation of dopamine-excitable cells in dorsal, but not ventral, striatum inhibited sugar's ability to drive the ingestion of unpalatable solutions. Conversely, optogenetic stimulation of dopamine-excitable cells in dorsal, but not ventral, striatum substituted for sugar in its ability to drive the ingestion of unpalatable solutions. Our data indicate that sugar recruits a distributed dopamine-excitable striatal circuitry that acts to prioritize energy-seeking over taste quality.

Dopamine/Tyrosine Hydroxylase Neurons of the Hypothalamic Arcuate Nucleus Release GABA, Communicate with Dopaminergic and Other Arcuate Neurons, and Respond to Dynorphin, Met-Enkephalin, and Oxytocin.

We employ transgenic mice with selective expression of tdTomato or cre recombinase together with optogenetics to investigate whether hypothalamic arcuate (ARC) dopamine/tyrosine hydroxylase (TH) neurons interact with other ARC neurons, how they respond to hypothalamic neuropeptides, and to test whether these cells constitute a single homogeneous population. Immunostaining with dopamine and TH antisera was used to corroborate targeted transgene expression. Using whole-cell recording on a large number of neurons (n = 483), two types of neurons with different electrophysiological properties were identified in the dorsomedial ARC where 94% of TH neurons contained immunoreactive dopamine: bursting and nonbursting neurons. In contrast to rat, the regular oscillations of mouse bursting neurons depend on a mechanism involving both T-type calcium and A-type potassium channel activation, but are independent of gap junction coupling. Optogenetic stimulation using cre recombinase-dependent ChIEF-AAV-DJ expressed in ARC TH neurons evoked postsynaptic GABA currents in the majority of neighboring dopamine and nondopamine neurons, suggesting for the first time substantial synaptic projections from ARC TH cells to other ARC neurons. Numerous met-enkephalin (mENK) and dynorphin-immunoreactive boutons appeared to contact ARC TH neurons. mENK inhibited both types of TH neuron through G-protein coupled inwardly rectifying potassium currents mediated by δ and µ opioid receptors. Dynorphin-A inhibited both bursting and nonbursting TH neurons by activating κ receptors. Oxytocin excited both bursting and nonbursting neurons. These results reveal a complexity of TH neurons that communicate extensively with neurons within the ARC. SIGNIFICANCE STATEMENT: Here, we show that the great majority of mouse hypothalamic arcuate nucleus (ARC) neurons that synthesize TH in the dorsomedial ARC also contain immunoreactive dopamine, and show either bursting or nonbursting electrical activity. Unlike rats, the mechanism underlying bursting was not dependent on gap junctions but required T-type calcium and A-type potassium channel activation. Neuropeptides dynorphin and met-enkephalin inhibited dopamine neurons, whereas oxytocin excited them. Most ventrolateral ARC TH cells did not contain dopamine and did not show bursting electrical activity. TH-containing neurons appeared to release synaptic GABA within the ARC onto dopamine neurons and unidentified neurons, suggesting that the cells not only control pituitary hormones but also may modulate nearby neurons.

Lassa-vesicular stomatitis chimeric virus safely destroys brain tumors.

UNLABELLED: High-grade tumors in the brain are among the deadliest of cancers. Here, we took a promising oncolytic virus, vesicular stomatitis virus (VSV), and tested the hypothesis that the neurotoxicity associated with the virus could be eliminated without blocking its oncolytic potential in the brain by replacing the neurotropic VSV glycoprotein with the glycoprotein from one of five different viruses, including Ebola virus, Marburg virus, lymphocytic choriomeningitis virus (LCMV), rabies virus, and Lassa virus. Based on in vitro infections of normal and tumor cells, we selected two viruses to test in vivo. Wild-type VSV was lethal when injected directly into the brain. In contrast, a novel chimeric virus (VSV-LASV-GPC) containing genes from both the Lassa virus glycoprotein precursor (GPC) and VSV showed no adverse actions within or outside the brain and targeted and completely destroyed brain cancer, including high-grade glioblastoma and melanoma, even in metastatic cancer models. When mice had two brain tumors, intratumoral VSV-LASV-GPC injection in one tumor (glioma or melanoma) led to complete tumor destruction; importantly, the virus moved contralaterally within the brain to selectively infect the second noninjected tumor. A chimeric virus combining VSV genes with the gene coding for the Ebola virus glycoprotein was safe in the brain and also selectively targeted brain tumors but was substantially less effective in destroying brain tumors and prolonging survival of tumor-bearing mice. A tropism for multiple cancer types combined with an exquisite tumor specificity opens a new door to widespread application of VSV-LASV-GPC as a safe and efficacious oncolytic chimeric virus within the brain. IMPORTANCE: Many viruses have been tested for their ability to target and kill cancer cells. Vesicular stomatitis virus (VSV) has shown substantial promise, but a key problem is that if it enters the brain, it can generate adverse neurologic consequences, including death. We tested a series of chimeric viruses containing genes coding for VSV, together with a gene coding for the glycoprotein from other viruses, including Ebola virus, Lassa virus, LCMV, rabies virus, and Marburg virus, which was substituted for the VSV glycoprotein gene. Ebola and Lassa chimeric viruses were safe in the brain and targeted brain tumors. Lassa-VSV was particularly effective, showed no adverse side effects even when injected directly into the brain, and targeted and destroyed two different types of deadly brain cancer, including glioblastoma and melanoma.

Attenuation of vesicular stomatitis virus infection of brain using antiviral drugs and an adeno-associated virus-interferon vector.

Vesicular stomatitis virus (VSV) shows promise as a vaccine-vector and oncolytic virus. However, reports of neurotoxicity of VSV remain a concern. We compared 12 antiviral compounds to control infection of VSV-CT9-M51 and VSV-rp30 using murine and human brain cultures, and in vivo mouse models. Inhibition of replication, cytotoxicity and infectivity was strongest with ribavirin and IFN-α and to some extent with mycophenolic acid, chloroquine, and adenine 9-ß-d-arabinofuranoside. To generate continuous IFN exposure, we made an adeno-associated virus vector expressing murine IFN; AAV-mIFN-ß protected mouse brain cells from VSV, as did a combination of IFN, ribavirin and chloroquine. Intracranial AAV-mIFN-ß protected the brain against VSV-CT9-M51. In SCID mice bearing human glioblastoma, AAV-mIFN-ß moderately enhanced survival. VSV-CT9-M51 doubled median survival when administered after AAV-mIFN-ß; some surviving mice showed complete tumor destruction. Together, these data suggest that AAV-IFN or IFN with ribavirin and chloroquine provide an optimal anti-virus combination against VSV in the brain.

Autonomous parvoviruses neither stimulate nor are inhibited by the type I interferon response in human normal or cancer cells.

UNLABELLED: Members of the genus Parvovirus are small, nonenveloped single-stranded DNA viruses that are nonpathogenic in humans but have potential utility as cancer therapeutics. Because the innate immune response to parvoviruses has received relatively little attention, we compared the response to parvoviruses to that of several other types of viruses in human cells. In normal human glia, fibroblasts, or melanocytes, vesicular stomatitis virus evoked robust beta interferon (IFN-ß) responses. Cytomegalovirus, pseudorabies virus, and Sindbis virus all evoked a 2-log-unit or greater upregulation of IFN-ß in glia; in contrast, LuIII and MVMp parvoviruses did not evoke a detectable IFN-ß or interferon-stimulated gene (ISG; MX1, oligoadenylate synthetase [OAS], IFIT-1) response in the same cell types. The lack of response raised the question of whether parvoviral infection can be attenuated by IFN; interestingly, we found that IFN did not decrease parvovirus (MVMp, LuIII, and H-1) infectivity in normal human glia, fibroblasts, or melanocytes. The same was true in human cancers, including glioma, sarcoma, and melanoma. Similarly, IFN failed to attenuate transduction by the dependovirus vector adeno-associated virus type 2. Progeny production of parvoviruses was also unimpaired by IFN in both glioma and melanoma, whereas vesicular stomatitis virus replication was blocked. Sarcoma cells with upregulated IFN signaling that show high levels of resistance to other viruses showed strong infection by LuIII. Unlike many other oncolytic viruses, we found no evidence that impairment of innate immunity in cancer cells plays a role in the oncoselectivity of parvoviruses in human cells. Parvoviral resistance to the effects of IFN in cancer cells may constitute an advantage in the virotherapy of some tumors. IMPORTANCE: Understanding the interactions between oncolytic viruses and the innate immune system will facilitate employing these viruses as therapeutic agents in cancer patients. The cancer-selective nature of some oncolytic viruses is based on the impaired innate immunity of many cancer cells. The parvoviruses H-1, LuIII, and MVM target cancer cells; however, their relationship with the innate immune system is relatively uncharacterized. Surprisingly, we found that these parvoviruses do not evoke an interferon response in normal human fibroblasts, glia, or melanocytes. Furthermore, unlike most other types of virus, we found that parvovirus infectivity is unaffected by interferon treatment of human normal or tumor cells. Finally, parvoviral replication was unimpaired by interferon in four human tumor types, including those with residual interferon functionality. We conclude that deficits in the interferon antiviral response of cancer cells do not contribute to parvoviral oncoselectivity in human cells. The interferon-resistant phenotype of parvoviruses may give them an advantage over interferon-sensitive oncolytic viruses in tumors showing residual interferon functionality.

Long-distance interferon signaling within the brain blocks virus spread.

UNLABELLED: Serious permanent neurological or psychiatric dysfunction may result from virus infections in the central nervous system (CNS). Olfactory sensory neurons are in direct contact with the external environment, making them susceptible to infection by viruses that can enter the brain via the olfactory nerve. The rarity of full brain viral infections raises the important question of whether unique immune defense mechanisms protect the brain. Here we show that both RNA (vesicular stomatitis virus [VSV]) and DNA (cytomegalovirus [CMV]) virus inoculations of the nasal mucosa leading to olfactory bulb (OB) infection activate long-distance signaling that upregulates antiviral interferon (IFN)-stimulated gene (ISG) expression in uninfected remote regions of the brain. This signaling mechanism is dependent on IFN-α/ß receptors deep within the brain, leading to the activation of a distant antiviral state that prevents infection of the caudal brain. In normal mice, VSV replication is limited to the OB, and these animals typically survive the infection. In contrast, mice lacking the IFN-α/ß receptor succumbed to the infection, with VSV spreading throughout the brain. Chemical destruction of the olfactory sensory neurons blocked both virus trafficking into the OB and the IFN response in the caudal brain, indicating a direct signaling within the brain after intranasal infection. Most signaling within the brain occurs across the 20-nm synaptic cleft. The unique long-distance IFN signaling described here occurs across many millimeters within the brain and is critical for survival and normal brain function. IMPORTANCE: The olfactory mucosa can serve as a conduit for a number of viruses to enter the brain. Yet infections in the CNS rarely occur. The mechanism responsible for protecting the brain from viruses that successfully invade the OB, the first site of infection subsequent to infection of the nasal mucosa, remains elusive. Here we demonstrate that the protection is mediated by a long-distance interferon signaling, particularly IFN-ß released by infected neurons in the OB. Strikingly, in the absence of neurotropic virus infection, ISGs are induced in the posterior regions of the brain, activating an antiviral state and preventing further virus invasion.